In Vitro Diagnostics PDF

Contents IN VITRO DIAGNOSTICS – GOOD NOTES VERSION............................................................................................................................................................... 2 FACTORS AFFECTING ASSAY SENSITIVITY...................................................................................................................................................................... 3 METHODS FOR AFFINITY DETERMINATION..................................................................................................................................................................... 4 EFFECT OF ANTIBODY AFFINITY AND NONSPECIFIC BINDING ON IMMUNOASSAY........................................................................................................... 5 CHAPTER 2.1 REPORTER SYSTEMS...................................................................................................................................................................................... 6 REPORTER SYSTEMS BASED ON SCATTERING AND ABSORPTION OF LIGHT..................................................................................................................... 6 LIGHT ABSORBING PARTICLES...................................................................................................................................................................................... 6 ELISA........................................................................................................................................................................................................................... 7 COLORIMETRIC ELISA................................................................................................................................................................................................... 7 TECHNOLOGIES BASED ON PRODUCTION OF LIGHT (luminescence, fluorescence, phosphorescence)............................................................................. 8 FLUOROPHORES PRODUCING SIGNAL.......................................................................................................................................................................... 8 FLUORESCENT LABELS................................................................................................................................................................................................. 8 FLUOROMETRIC ELISA.................................................................................................................................................................................................. 9 LANTHANIDE CHELATES............................................................................................................................................................................................... 9 DELFIA (dissociation enhanced fluoroimmuno assay)..................................................................................................................................................... 9 CHAPTER 2.2 REPORTER SYSTEMS..................................................................................................................................................................................... 10 PARTICLE-BASED FLUORESCENT REPORTERS............................................................................................................................................................... 10 EUROPIUM NANOPARTICLES....................................................................................................................................................................................... 10 QUANTUM DOTS......................................................................................................................................................................................................... 11 UPCONVERTING NANOPARTICLES (UCNP)................................................................................................................................................................... 11 DISASVANTAGES OF PARTICLE BASED REPORTERS....................................................................................................................................................... 11 PHOTOBLEACHING..................................................................................................................................................................................................... 12 CHAPTER 2.3 REPORTER SYSTEMS..................................................................................................................................................................................... 12 CHEMILUMINESCENCE............................................................................................................................................................................................... 12 ELECTROCHEMILUMINESCNECE................................................................................................................................................................................. 12 IMMUNO-PCR............................................................................................................................................................................................................. 13 DIRECT ELISA.............................................................................................................................................................................................................. 13 ELECTROCHEMICAL IMMUNOASSAY............................................................................................................................................................................ 14 BIO-FET...................................................................................................................................................................................................................... 15 CHAPTER 3: BINDERS........................................................................................................................................................................................................ 16 IMMUNOASSAY INTERFERENCE................................................................................................................................................................................... 16 ALTERNATIVE BINDERS................................................................................................................................................................................................ 17 ALTERNATIVE PROTEIN SCAFFOLDS............................................................................................................................................................................. 17 AFFIBODIES................................................................................................................................................................................................................ 17 APTAMERS.................................................................................................................................................................................................................. 17 PLASTIC ANTIBODIES (MOLECULAR IMPRINTED POLYMERS).......................................................................................................................................... 18 LECTINS...................................................................................................................................................................................................................... 18 CHAPTER 4: SOLID PHASES AND IMMOBILISATION............................................................................................................................................................. 19 PLASTIC AS SOLID SURFACE MATERIAL......................................................................................................................................................................... 19 PASSIVE COATING....................................................................................................................................................................................................... 19 POLYSTERENE FOR PROTEIN COATING......................................................................................................................................................................... 20 INDIRECT COATING..................................................................................................................................................................................................... 20 BIOTIN AND STREPTAVIDIN.......................................................................................................................................................................................... 20 CHAPTER 5: MULTIPLEX ASSAYS........................................................................................................................................................................................ 21 SPECTRAL RESOLUTION.............................................................................................................................................................................................. 21 SINGLE MOLECULES COUNTING.................................................................................................................................................................................. 21 SPATIAL RESOLUTION.................................................................................................................................................................................................. 21 RESOLUTION BY SEQUENCE........................................................................................................................................................................................ 21 SOMAScan.................................................................................................................................................................................................................. 22 CHAPTER 6: HOMOGENOUS ASSAYS................................................................................................................................................................................. 23 AGGLUTINATION BASED ASSAYS.................................................................................................................................................................................. 23 EMIT = ENZYME MULTIPLE IMMUNOASSAY TECHNIQUE................................................................................................................................................. 23 FLUORESCENCE POLARIZATION IMMUNOASSAY (FPIA)................................................................................................................................................. 23 FRET-BASED ASSAYS.................................................................................................................................................................................................... 23 FETI- FLUORESCENCE ENERGY TANSFER IMMUNOASSAY.............................................................................................................................................. 24 TR-FRET...................................................................................................................................................................................................................... 24 LOCI (LUMINESCENCE OXYGEN CHANNELLING IMMUNOASSAYS)................................................................................................................................. 24 CHAPTER 7: LATERAL FLOW ASSAYS.................................................................................................................................................................................. 25 SAMPLE PAD............................................................................................................................................................................................................... 25 CONJUGATE PAD......................................................................................................................................................................................................... 25 WICKING PAD (absorbent pad)..................................................................................................................................................................................... 25 BACKING MATERIAL..................................................................................................................................................................................................... 25 ANALYTICAL MEMBRANE............................................................................................................................................................................................. 26 BINDERS..................................................................................................................................................................................................................... 26 LABELS....................................................................................................................................................................................................................... 26 CHAPTER 8: NUCLEIC ACIDS ASSAY................................................................................................................................................................................... 27 SYBR GREEN............................................................................................................................................................................................................... 27 qPCR.......................................................................................................................................................................................................................... 27 TaqMan PROBES.......................................................................................................................................................................................................... 27 PRIMER DESIGN.......................................................................................................................................................................................................... 27 ALTERNATIVE PROBE TYPES......................................................................................................................................................................................... 27 DROPLET DIGITAL PCR................................................................................................................................................................................................. 28 ISOTHERMAL AMPLIFICATION METHODS...................................................................................................................................................................... 28 EXTRACTION AND PURIFICATION OF NUCLEIC ACIDS................................................................................................................................................... 28 CONTAMINATION CONCERNS..................................................................................................................................................................................... 28 CONTROLS................................................................................................................................................................................................................. 28 IN VITRO DIAGNOSTICS – GOOD NOTES VERSION An important parameter to determine efficacy of an immunoassay is its sensitivity, or in other words, the smallest quantity of analyte that can reliably be analysed. The Immunoassay Sensitivity (IA Se from now on) is affected by single dose-response and precision. With the term “single dose-response” it means the slope created by plotting on a y axis the signal received and on a x axis the analyte concentration. Larger is the response to the analyte, better is the sensitivity of the assay. Steeper the slope, better the sensitivity* *This is only true if the precision is high. For precision we mean the smaller variation possible between results. The precision improves the sensitivity. Sensitivity is ALWAYS expressed as concentration levels, but there are different ways to describe sensitivity, related to different methods. These parameters CANNOT be compared together because of the different methods used to quantify the sensitivity, thus the values obtained are different and not comparable. Analytical Sensitivity is used in labs to test new assay as they are developed, this assay uses gaussian distribution and the concept that the background signal is defines as either 2SD (standard deviations) or 3SD. The comprehensive definition of analytical sensitivity is “The analyte concentration at which the signal level equals background mean signal + 3SD (or 2SD) of background signal”. Since this is a very basic way to determine sensitivity for more complex samples we need more information, thus us possible to sue guidelines to give a general way to determine sensitivity. This is based on three parameters: 1. LoB: Is limit of blank, it is determined as background signal + 1.65 SD, meaning that 95% of the results would be considered positive, reducing risks of false positive only at 5%. It must take into account the day-to-day variation and it requires multiple batches to reduce risk of error. 2. LoD: Limit of detection, it considers the patient samples and determines the level at which the patients’ samples are very unlikely to contain the analyte. It is determined by using LoB +1.65 SD, where the SD is the one of the patient samples. We perform more measurements to increase the reliability. Moreover, we use pool of different patients due to problems related with single patient sample size. 3. LoQ: Limit of Quantitation, it uses the coefficient of variation (%), which is obtained by computing SD /mean. We accept a cv of 10%, but it still depends on the test ultimately. Again, we repat multiple times the test and use patients pool to increase the reliability. The tests are repeated over different days to decrease the day-to-day variations. We use pooling of the sample since plasma of different patients has different composition, the process of pooling, overall, mimics variations given by sample and day to day variations given by diet or technique of the assay. FACTORS AFFECTING ASSAY SENSITIVITY As stated before, the sensitivity is determined by the signal dose/response and the precision. There are different factors that affect these two parameters: Factors affecting the signal dose-response Specific activity of the label -> Is the strength of the signal that label produces, the higher the activity of the label then stronger the signal. Is possible to produce a higher signal by stacking labels on top of each other. Fluorescence labels produce the highest quantity of signal followed by enzyme labels. Binder affinity -> it means how strongly the antibody binds to the analyte. Basically, the 3D structures of the analyte and the Antibody binding site have to “fit” together. This means also that their chemical groups have to interact together through weak interactions like electrostatic, hydrogen bonds, Van der Waals forces and hydrophobic interactions. In some cases, the antibody and antigen 3D structures do not match but some interactions can still be seen thanks to chemical affinity of the groups. Antibody cross-reactivity : is defined as the binding dependent on the surface structure meaning that if similar structures are found also on nontarget molecule, then the binding will still happen. Nonspecific Binding -> Antibodies can be defined as sticky, meaning that they can bind or attach to almost everything, including non-target analytes, this means that nonspecific binding creates background signal. This nonspecific binding cannot be washed away, thus, to prevent it from happening we block the free surface, reducing the background signal. Variations and errors-> errors and variations can be caused by multiple different things such as accuracy in pipetting, but also precision in distribution of volumes in the wells and precision in the different steps of the assay. For example, an incorrect or incomplete washing can lead to false results due to unbound antibodies still present. Antibody affinity-> It regards the paratope-epitope interactions. A higher affinity means higher “on rate” or Kon, which means the concentration of bound complexes. Specific binding is calculated through the formula: but also which is the law of mass action. Bigger is Ka value, then better the affinity. It is measured in L/mol. Along with Ka we have also the dissociation constant that is Kd, calculated as 1/Ka. Smaller Kd means better affinity. It is measured in nmol/L or just nM. A scatchard plot can be created by “playing around” with the formula. We plot on the x axis the concentration of the bound AB and on the y axis the value for [bound Ab]/ [free Ab]. eg. Usage of scatchard plot for tracer antibody. The specific binding cannot increase over a certain number, if we continue with the reaction, we will see the increase in nonspecific binding due to saturation of all the available analyte, thus the Antibody will start binding nontarget molecules. METHODS FOR AFFINITY DETERMINATION There are three different system to determine the affinity, they do not require any label, and they report also the Koff, Kon and Kd values. The technologies are: -SRP, is the acronym for surface plasmon resonance. It is a label free technology used to detect bind of molecules thus meaning that is useful for determination of affinity but also epitope mapping and immunoassays. It uses a glass chip covered with gold film attached to flow system. The system requires a light source that bounce on the gold surface through a prism. At a specific resonance angle light absorbed by the film to produce surface plasmons meaning that the intensity of reflected light decreased at a specific angle. The binding of molecules (formation of AbAg complex) to the chip changes the resonance angle, so in practice no light is absorbed. The change in resonance is recorded by a sensorgram which yield the value. -BLI, acronym for bio-layer interferometry. The sensor is a tip, it has Ab embedded on the end of it through a biocompatible matrix. The matrix is just below the optical sensor. By inserting the tip in two different buffers, one with analyte and one without, it is able to determine Ka and Kd. This system analyses the interference pattern of white light reflected by the surfaces but the binding of molecules, so the analyte, causes shift in the pattern. The shift is measured in real time. This assay can be automated. -ITC, isothermal titration calorimetry. It can detect reactions between molecules by observing temperature changes between two solutions, one with analytes and one without. No binding surface is required. The ligand is added in small quantities until the temperature change is no longer observe thus is possible to determine the maximum saturation. No Kon/Koff values are obtained. EFFECT OF ANTIBODY AFFINITY AND NONSPECIFIC BINDING ON IMMUNOASSAY Noncompetitive assay: we use an excess of capture antibody (which makes the assay quite expensive). Higher the affinity of the antibody, better the dose-response. the tracer antibody has to have a lower affinity respect to the primary antibody but if the affinity is too low, the quantity required is higher. We try to use to the antibodies with the lowest nonspecific binding to optimize the protocol. Dynamic range is the range of antigen concentration that can be measured accurately by the assay. Is calculated as cv%. Is possible to optimize a sandwich assay by using high affinity or high specific activity reporter, which gives in turn a wider dynamic range, but could also impact the precision. It all depends on the type of the assay. If the assay is done in one step, then it must be taken into account the concentration needed to be measured in a clinical setting. If the analyte concentration is too high, it would mean no sandwich formation due to independent saturation of both capture Ab and tracer Ab. The non-creation of sandwich is called high-dose hook effect, and it gives falsely low results. To avoid this effect, is better to carry out the assay in two steps or to dilute the analyte sample before performing the assay. Competitive assay: In this type of assay, the signal depends on both the capture antibody and tracer antibody. Is possible to modify competitive assay to make them more sensitive by using less capture antibody. NOTE: the dynamic range changes too consequently, becoming smaller. At low analyte concentrations, the high end of the signal is highly imprecise. To improve sensitivity is possible also to increase the affinity of the antibody, increase the specific activity of the label or decrease the nonspecific binding. Even using the same antibody, the noncompetitive assay may be more sensitive than the competitive ones. When labelling an antibody, the affinity of the latter is changed. It may decrease but also the labelling can lead to antibody aggregation and denaturation, thus making it no more utilizable. Moreover, also immobilization of the antibody represents a problem since the antibodies are not organized and they do not all bind in their optimal position, also shape of the antibody matters too. CHAPTER 2.1 REPORTER SYSTEMS The first ever technology use in immunoassays was the radiotechnology, a competitive type of immune assays where the analytes were labelled with radioactive isotopes. The isotopes, when decaying, produces ionizing radiations that are translated as flashes of light. The signal was measured with a scintillation counter, which is what translated the ionizing radiations into flashes of light). Another type of assay developed after the first one is the immunoradiometric assay, which is noncompetitive assay. In both cases the labels where small but had stability problems due to the fact that the labels decay. They are no more the preferred way to perform an immunoassay because safer and stronger labelling technologies exist. Technologies based on “change of light”. For changes in light, it can be meant: - Loss of light, basically scattering and absorption - Change of light direction, so scattering (we measure the change in angle of the light) - Change in wavelength, represented by fluorescence. - Production of light, which can be both Chemiluminescence and Electrochemiluminescence REPORTER SYSTEMS BASED ON SCATTERING AND ABSORPTION OF LIGHT In this category we have represented different technologies such as turbidimetry and nephelometry. They are used for the detection of any particle and are based on the principle that particles in solution scatter the light that passes through the solution. It is possible to create immune assays, thus immunoturbidimetry and immunonephelometric, thanks to antibody cross-linking with the analytes and creating a mesh network that acts as light scattering complexes. This network can be formed if the antibodies used are all equal and if they bind the same epitope of the target antigen. If the concentration of antibody is low, the network is not formed If the concentration of antigen is too high, no network is formed because every single binding site is saturated This is not a very sensitive methos and is more commonly used for the quantification of serum proteins found at high concentrations. Nevertheless, it’s a homogenous technology, thus it has lees steps and do not require any separation since no label is present. To measure the scattering is possible to use a spectrophotometer. The light intensity is measured in a straight line, measure the loss of light intensity, so how much light is transmitted through the solution. In principle this theory is less sensitive than nephelometry, moreover here the sensitivity is highly dependent on the accuracy and sensitivity of the instrument used. Nephelometry, instead, measure the intensity of scattered light at specific angles when light hits the particles in solution. Different light angles are produced for different analyte sizes. It is a more sensitive method respect to turbidimetry but is also more complicated and requires expensive and fancy instrumentation. LIGHT ABSORBING PARTICLES They are the label of choice for assays, mainly lateral flow assay. The most common option is the gold particle. The particularity of gold particles is that strong absorption of light is at wavelength specified by the size of the particles. Other options for light absorbing particles can be coloured latex beads. It has to be noted that lateral flow assays are not very sensitive but is possible to use some devices to measure the intensity of the reflected light. To measure the reflectance of a particle, we direct light on them and measure the decrease of light at a determined wavelength. Reflectance spectroscopy is one of the technologies used to measure signal produce in POC tests based on immunochromatography. It has some advantages like requirement for simple instrumentation that will quantify the visible band, thus making the LFIA from a qualitative test to a semi-quantitative or quantitative test. It has also some disadvantages, like being difficult to standardize due to the variation of angle of incidence of the light, how the surface area variates and also if the surface area is uneven there are different variations caused by it. There are devices that provide not only the LFIA strip test but also the portable spectrophotometer to interpret the results. ELISA It means enzyme-linked immunosorbent assay, it entitles an enzyme attached to the tracer antibody. The label is the enzyme, if there isn’t the enzyme, then is not ELISA. In ELISA, the enzyme converts a non-detectable substrate to a detectable product, the product can be colourful in the sense of light- absorbing, fluorescent or chemiluminescence. The addition of the substrate to be converted into the visible product is an extra step in the assay, moreover it has to be considered the best conditions in which the enzyme works and if the assay contains interfering agents with the enzyme activity. While these two steps seem easy enough, they are few of the drawbacks of the ELISA assay. There are also many advantages to this method, for example the signal amplification, meaning that the enzyme will convert substrate into product as long as the reaction is allowed to go or until the substrate runs out. NOTE: when comparing the reaction in each well, the reaction must be stopped at the same time, otherwise the comparison is not fair because different in product quantities cannot be assessed. There are also some challenges related to the assay like the fact that the specific activity is dependent on the number of active enzymes per antibody, the time of enzyme reaction and the detectability of the product. But also, the reaction involved to link the enzyme to the antibody, which can result in lower affinity of the latter; the multiple steps involved in the assay; instability of the enzyme and interfering factors. COLORIMETRIC ELISA in case of colorimetric ELISA, the assay is not very sensitive, and it range due to the absorbance of the product produced. The absorbance is not measured, it is mathematically calculated from the transmittance measurements. In the case of low absorbance, we don’t have high detection sensitivity. If the product is light absorbing, we also have some problems, since too much of it would mean that the concentration cannot be detected due to the light not passing through. There are different enzymes and substrate that can be used for colorimetric ELISA, like HRP or Alkaline Phosphatase. They each have a different stopper depending on the type of substrate, the stopper can be SDS, sulfuric acids or NaOH. The dynamic range of the assay is the concentration range at which the response of the assay is directly proportional to the concentration of the target molecule, allowing the quantity of analyte present to be determined. TECHNOLOGIES BASED ON PRODUCTION OF LIGHT (luminescence, fluorescence, phosphorescence) Luminescence is the emission of light or photons; it is not due to heat. It is subdivided in photoluminescence, which means absorption of photons, as subbranches we have fluorescence and phosphorescence; chemiluminescence instead produces light or photons due to a chemical reaction, subclasses are biochemiluminescence and electrochemiluminescence. Regarding fluorescence and phosphorescence, the principle is that when a molecule absorbs a photon, it goes from ground state to excited singlet state, when the energy returns to ground state it’s at lower values (due to some loss given by heat and collision of the photon with the molecule), when the energy goes back to ground state a photon is emitted, thus producing light referred to as fluorescence. The light is measured at a longer wavelength than the absorbed one. Phosphorescence is due to excited triplet state, it is a rare case in which electrons are in parallel spin. When the molecule returns to ground state it will emit a photon, but since it has to change the spin of the electron it will take a longer time, moreover the photon emitted has a longer wavelength, thus lower energy, than the absorbed one. In comparison with fluorescence, the phosphorescence has even longer wavelengths. FLUOROPHORES PRODUCING SIGNAL Fluorescence can be detected with UV light, regarding the characteristics of fluorophore, they have a molar absorption coefficient, a quantum yield, stoke shift and a defined fluorescence lifetime. The light intensity produced by the label is determined through a formula that defines epsilon as the molar absorption coefficient, c the molar concentration and l as the pathlength. It describes the absorbance. Another law can be used to define the quantum yield, indicated as phi.. If the quantum yield is high, then the fluorescence route is favoured, while if it is low, the radiation less energy transfer rout is preferred. With low quantum yield we have to find the precise energy levels to increase probability of producing fluorescence. Each fluorophore is also defined by an excitation and emission spectra, the maximum points of this spectra are the wavelengths at which maximum excitation and emission happens. The difference between these two maximum points is called stoke shift. A small stoke shift means difficulty in dividing the signal between excitation and emission light, thus sometimes we do not use the maximum emission and excitation wavelength, this improves slightly the recognition of the signal. FLUORESCENT LABELS Organic fluorophores, all organic fluorophores contain aromatic groups and conjugated bonds. The aromatic groups are easily excited, moreover each organic fluorophore has a specific emission wavelength which makes it easier to used them for multiplex assays. Fluorescent proteins, there is also the possibility to use proteins as fluorophores, given that they have natural bioluminescence. They are mainly used for microscopic techniques, used as fusion proteins mainly. They can be GFP from jellyfish, which can also be mutated and give other colours than green, but also proteins coming from red algae (APC or PE) FLUOROMETRIC ELISA The enzyme converts a non-fluorescent substrate into a fluorescence product, there are different options available for excitation and emission profiles along with sensitivity of the assay. Regarding the enzymes that can be used, the options can be beta galactosidase, HRP or alkaline phosphatase, they convert their substrates into different products with defined spectra. The typical feature of this label is that they have a quite narrow stroke shift, and the fluorescence signal has a short half-life of few nanoseconds, meaning that the signal has to be measured instantaneously. Moreover, these labels have always some kind of background signal going on, either due to other molecules or due to the plastic backing of the assay. Another drawback of these labels is the self-quenching, a dampening of the signal that happens when the label is used at high concentrations-. LANTHANIDE CHELATES Are the rare earth elements, the most common ones used in immunoassays are the Europium (Eu), terbium (Tb), samarium (Sm) and dysprosium (Dy). The particularity of lanthanides is that they for ions (La 3+) with aqueous quenching of fluorescence signal. The fluorescence signal can be observed only when firmly bound to an organic ligand able to deliver energy to the ion and shield it from water. Moreover, this ligand can be used for reporter technologies to attach the lanthanide ion to a molecule, which could be either an antigen, an antibody or a nuclei acid. Labels containing lanthanides ions have multiple advantages such as: intense fluorescence, wide stoke shift with no overlapping between excitation and emission spectra, sharp emission bands and long- lived fluorescence, permitting measuring of signal even after few instants. There are two types of chelating structures: 1. Non-fluorescent chelates. a. They are just used as linkers; to obtain the signal produced by the ion, we have to separate them through a development step. 2. Inherently fluorescent chelate. a. Since they are inherently fluorescence, no development step is needed. DELFIA (dissociation enhanced fluoroimmuno assay) It is an assay procedure that allows two-site immune reaction to happen on a solid phase. It has high sensitivity. It involves an enhancement solution that create the fluorescence lanthanide chelate. The solution contains an acidic buffer, whose purpose is to dissociate the ions; beta diketone to form new fluorescent chelates by creating two resonance bonds with the lanthanide ion; a Lewis base to displace the water molecules thus enhancing fluorescence and prolonging its half-life; lastly, triton x-100 to stabilize the chelates and creates micelles, thus hydrophobic pockets where the chelate can be shielded from water. The criteria for a good lanthanide chelate reporter are: -high fluorescence, good protection of the lanthanide, -high energy absorption -highest quantum yield as possible for an efficient energy transfer. -good stability, -good thermodynamics, kinetics and conditional releasing of ion when needed (DELFIA) -coupling properties such as hydrophobicity, -efficient coupling -mild coupling reaction (Doesn’t have a huge impact on antibody functionality and affinity). Drawbacks: after the final wash of the well. The enhancement solution has to be added, left to develop for 30 minutes and then loaded on the machine for the instrument to measure the signal, the time to measure the well had to be precise otherwise the measurement would be wrong. The chelates structure are expensive and we cannot use this method with blood plasma, since EDTA would interfere with the assay. A newer assay using lanthanide chelates is the direct trf, which exploits the inherently fluorescent chelates, while they are better in the sense it doesn’t require any washing step, it also has significant water quenching, thus measurement must happen in dried wells, The dry ells can be measured even after half a year, since the signal is still maintained. The four lanthanides used in immunoassay have energy levels that allow them to be used for time- resolved fluorometry*, moreover they also have all different wavelength, thus allowing multiplex assay. *Time-resolved fluorescence means long lived fluorescence. In this case is characterized by a very short-lived background signal, followed by a counting window where we count the protons released by the sample. They have large stoke shifts, thus no self-quenching is observable, it is also easy to separate the excitation light form the emission light, just by suing specific filter that select the correct wavelengths. Some general drawbacks are represented by the instrument needed to measure the signal, which is quite complex. It provides a light source (pulse light) and a wavelength filter to filter for the emission light. The basic components are: 1. pulsed light source(xenon) with suitable excitation wavelength 2. spectral system to choose excitation band 3. optical system to collect and direct excitation light to the sample 4. Sample compartment and sample changer 5. optical system to collect emission from the sample into the detection system 6. spectral selection system to separate the specific emission from unwanted interference 7. Detector allowing time resolution 8. Read out system to collect data and save it. The excitation light comes from above while the emission light passes through the bottom. The benefit of this method is that the dynamic range has a wide range, but if the sample has high concentration, the dynamic range is reduced. CHAPTER 2.2 REPORTER SYSTEMS PARTICLE-BASED FLUORESCENT REPORTERS In the category of particle-based fluorescent reporters we can find three types of labels: -europium nanoparticles -quantum dots -upconverting nanoparticles (UCNPs) EUROPIUM NANOPARTICLES A nanoparticle full of europium is attached to an antibody, labelling it. The nanoparticles are filled with high numbers of europium chelates, the nanoparticle has the role to shield the europium chelates from water, avoiding quenching phenomenon. The nanoparticle used is quite big, of a size of 107nm. As a nanoparticle, it gives an intense fluorescence signal with large stokes shift and long-lasting fluorescence with time-resolved fluorescence measurement, providing grounds for a highly sensitive assay! There are two ways to obtain a noncompetitive immunoassay using europium nanoparticles: 1. Particles coated with streptavidin: the nanoparticles are coated with streptavidin; they react with a biotinylated antibody. The binding between antibody and streptavidin particle is in one step, but to add the Eu-labelled nanoparticles. 2. Particles coated with tracer antibody (tAb): is a two-step assays, the particle is coated with antibodies, making it highly polyvalent. Avidity effect is obtained, thus stronger binding and better sensitivity. QUANTUM DOTS Semiconductor materials composed by nanocrystals. They do not contain lanthanides. They are smaller molecules than europium nanoparticles, they stand at 2-10 nm in diameter. Since they have a small size, the materials act differently so having unprecedented tunability, thus also good for multiplex. Their core is made up of CdSe, and ZnS shelters the core. CdSe can be excited, but the sheltering restricts the excitation and eliminating the loss of energy through non- radiative pathway and photochemical degradation. Drawback of this technology: the materials used are toxic, moreover they have a narrow emission spectrum but a broad excitation one, making it difficult to find the optimum wavelengths. It must be said that the spectra produced by the quantum dots are both size and material dependent, thus both excitation and emission spectrum can be engineered. UPCONVERTING NANOPARTICLES (UCNP) At the start, this technology was called “upconverting phosphorous” but was a confusing term since phosphorous was not present, it was just a term to define presence of light. They used nanoparticles with a size inferior to 100nm, the nanoparticle is a lattice structure contain inorganic crystals and certain lanthanide ions, called dopants ions. The lattice network is thus made of multiple ions, in some place replaced by the lanthanide ones. The lanthanides have the ability of transferring energy, The upconverting nanoparticles are excited by two photons instead of one, the first photon makes the ion reach an intermediated state, only upon receiving the second photon, the ion will go back to ground state, producing only one emission photon whose wavelength is shorter than the excitation one. The UCNP are said to go against nature thus the nature of their spectra. (upconvertion is because a lower energy is converted to a higher one). Advantages: They have a long-lived fluorescence, meaning that time-resolved fluorescence measurement are possible, even though not used since there is no risk of autofluorescence. This is because the excitation wavelength has a longer wavelength respect to the emission one. Thanks to this particularity the assay has a higher sensitivity and uses a simpler device for detection of the signal. Emission wavelength is always in the visible UCNP can also be used in assays using whole blood, so no separation from plasma or serum. UCNP can be used for multiplexing assays this is due to dopant ions having different emission wavelengths but the same excitation ones. DISASVANTAGES OF PARTICLE BASED REPORTERS Coating of biomolecules is difficult since the particles have to be made both hydrophilic and they have to be covered in chemical groups that allow coupling with biomolecules. Another problem is represented by the particles aggregating with each other, giving rise to stability and variability problems, this is due to high avidity effect, which also enhances the nonspecific binding. In the case of large size of the particle, also the antibody option becomes limited, due steric hindrances. Also, a larger area will be occupied on the solid phase, limiting the assay range. PHOTOBLEACHING The fluorescent property of the fluorophore becomes destroyed by the same photons that are absorbed and emitted. Some fluorophores are more resistant to others to this phenomenon. The organic fluorophores are the most susceptible, while the TRF chelates, and the particle-based reporters are more resistant. CHAPTER 2.3 REPORTER SYSTEMS Lastly, we are going to inquire assays based on chemiluminescence, electrochemiluminescence, digital ELISA and reporter system based on electric current. CHEMILUMINESCENCE Chemiluminescence is defined as the emission of light that occurs as a result of excitation produced by certain chemical reactions. Is part of those assays that measure the change of light along with turbidimetry and fluorescence methods). The particularity of chemiluminescence assays is that there isn’t any initial absorption of light which, theoretically, would mean a higher sensitivity due to no radiation background. Moreover, it would also mean a less expensive assays since no technology is required to provide light for excitation. The disadvantage of this technology is that it is a one-time reaction. Still, chemiluminescence can be either long or short lived. In case of short-lived signal, a sample cuvette is needed close to the detector when the reaction is initiated, since measurement must be immediate. The signal is measured with luminometers, they are sophisticated instruments, but they can also be portable (less sensitive), or they can even be photographic films like in western blot. Chemiluminescence (CL) has to be carried in certain conditions: first, there must be sufficient energy produced from the chemical reaction to obtain excitation. The product must be capable of excitation. The reaction rate must be at least enough to produce an intense chemiluminescence; the reaction conditions must favour production of the excited state (otherwise there would be loss of energy as heat). Direct CL: requires a functional group for the antibody to be attached to the emitter, or the antigen to be attached to the emitter. The attachment reaction must be caried out in mild conditions, moreover the quantum yield of the label shouldn’t be affected by the coupling reaction itself. As emitters, often, acridinium esters are used, since they do not need any catalysis and oxygen peroxide in alkaline is what kick-starts the reaction. Moreover, it’s a stable compound that allows conjugation with protein. No quenching effect can be observed because reaction with oxygen peroxide makes the label dissociate form the antibody. Indirect CL: the enzyme is coupled to the antigen or antibody; it is the enzyme that converts the non- chemiluminescence precursor into an emitter. ELECTROCHEMILUMINESCNECE IS NOT AN ELECTROCHEMICAL ASSAY!!!!! As a label it uses Ruthenium complexes, that are attached with a direct method, meaning that no enzyme is required for direct attachment to antibody. To work the ruthenium bipyridine complex requires tripropylamine. The signal is initiated by luminescence and is enhanced through repeated electron cycling. The whole process is based on cycling reaction of oxidation and reduction (excitation trigger that makes light emission). How it works? The solution contains TPA, which releases a hydrogen ion which, by becoming a radical, reduces ruthenium, thus leads to the complex being excited and thus releasing light before going back to ground state and allowing the cycle to continue again. The light emitted is recorded at 620 nm. To make TPA release the hydrogen ion, the Ab sandwich is linked to an electrode, which activates the MSD sulfo tag linked to the tracer antibody. The MSD sulfo-tag is what triggers oxidation of TPA in solution. This is a highly sensitive and versatile technology, with wide dynamic ranges, small reporter molecules, no enzymes requires and simple signal amplification system (in this case represented by reporter recycling). Moreover, it uses cheap instrumentation since it only requires a functional electrode. IMMUNO-PCR It’s a qPCR reaction, with a sandwich type immunoassay that uses oligonucleotides as the label attached to the tAb. After the immunoassay is performed, the sequence is amplified and quantified by qPCR, the analyte concentration is directly related to the number of amplified sequences. DIRECT ELISA Also called SiMoA, or Single Molecule Array, or quanterix assay (commercial product). The principle of the assay is that ELISA is performed on a bead, linked to the antibody there is a beta-galactosidase enzyme, the link is obtained through streptavidin and biotinylated antibody. The beta-galactosidase converts resorufin beta-d-galactopyranosidase into a fluorescent resorufin. After the assay is performed, the beads are made to fall into an array thanks to gravity, theoretically in each well there will be only one bead. To the wells we add the substrate, and immediately seal them with oil or any other substance that avoids mixing of the liquid phase and thus leakage of the signal. We then measure the fluorescence emanated by the wells, if there is fluorescence then they assay detected the analyte. To measure the fluorescence, the solution is excited, and the light emitted is at specific wavelength. Not all beads will produce fluorescence, only does that do are said to be on beads. We count the number of on beads and we define the fraction of “on beads” as the number of “on beads” over the number of “total beads”. Unfortunately, it is utopistic to have only one analyte bound to just on bead, thus the average number of enzymes per bead (AEB) has to be considered. This is because as the analyte concentration increases, so does the likelihood of more than one analyte binding to the same bead. Too high AEB would that all beads are on. AEB is calculated thanks to Poisson distributions, which also allows us to determine the analyte concentration without counting each single bead. The Poisson distribution is defined as the likelihood of a number of possible events occurring if the average number of events is known. This equation will tell the actual number of enzymes if we suppose that there is an average of 0 enzymes per bead. Higher is the number of “on beads” then also the concentration of the analyte is higher, thus also the AEB analogue will get higher. At higher concentrations, also the signal strength has to be computed. Is also possible to determine the AEB digital in the case of low/medium concentrations of the analytes. The AEB digital is defined as the negative natural logarithm of 1 – “on beads”. Compared to the other assays, digital ELISA has the best sensitivity compared to chemiluminescence or analogue fluorescence. Indeed, the LOD of the digital ELISA is very low. Multiplex ELISA: It is possible to perform it by using different color-coded beads with fluorescence. Each bead is coated with the same antibody but different label. By exciting the wells at different wavelength, we will obtain the different signals only when we use the specific wavelength to excite the bead. Digital ELISA can also be used to detect miRNA or for single-Cell protein counting. DIGITAL ELISA troubleshooting: due to tracer binding to soldi phase, the tracer binds to a protein on solid phase, the tracer binds to capture antibody, the enzyme binds on-specifically, there is bridging with heterophile, there is cross reaction with target analyte. This will lead to background and false elevation. There can also be false suppression, this may be due to: heterophile blocking the cAb, the heterophile blocks the detection Ab, there is cross-reacting substance that blocks the detection Ab, there is cross- reacting substance blocking the cAb, or the target is blocked from reacting with the cAb. ELECTROCHEMICAL IMMUNOASSAY The binding event of the assay is detected as change in current due to a redox reaction. It uses biosensors, they have a binding surface with electrodes, to detect the change in electrical current. The requirement for the assay is that something must be reduced, and something must be oxidized. To be recorded, the reaction has to happen near the electrode. Unfortunately, for this assay we have problems of interference, thus washes are required. The reaction is measured by any instrument that can detect changes in electric current, thus cyclic voltammetry or amperometry. It is also possible to have an electrochemical ELISA, where the antibody is conjugated with HRP enzyme, the substrates must be oxidizable, otherwise the electrochemical part of the assay is not achievable. Cyclic voltammetry: the oxidation and reduction are detected by cyclic scanning of voltage; the current is registered and observable as peak correlated to the concentration of the molecule being oxidized or reduce. At optimal highest point, the target molecule is oxidized, while the decrease in current signals that the molecule is being reduced. Amperometry: the voltage is kept stable; thus, we observe only the change in current over time. The concentration of the molecule that is being oxidized or reduced is identifiable by looking at the peak value or by integrating the curve. Overall, the electrochemical immunoassay, while it has a promising future, doesn’t show a good sensitivity. BIO-FET Bio-fet is a biosensor based on field-effect transistor. When the analyte binds, the charge distribution at the sensor surface changes, thus having a detectable change in conductivity in the transistor. No sandwich is needed for this technology. They can also have high potential for example as POC devises, smartphone applications, paper-based diagnostics and wearable devices. CHAPTER 3: BINDERS Antibodies are not the only binders available in bio affinity assays. Regarding the Antibodies, we can have either monoclonals or polyclonal. For the alternative binders, the options are multiple, like alternative protein scaffold, aptamers, plastic antibodies and lectins. Production of monoclonal antibodies: the production of this type of antibody is quite tricky. We first inject the antigen of interest in the mouse and recover the B lymphocytes produced after the exposure to the antigen. We fuse the B cells with modified myeloma cells (unfused cells cannot grow in HAT medium) and recover only does cells that grew. These cells are called hybridomas. We test them for anti-antigen activity and recover only those that can bind with high affinity to the antigen of interest. Not all antibodies produced by the hybridoma cells are chosen, this is because the immune response is always POLYCLONAL. The solution that we recover from the immunized mouse is a polyclonal antiserum, with heterogenous properties of antibodies. They have different binding affinities, different isotypes, multiple paratopes, they are impure and there are batch-to-batch variations given by the natural process of antibody production. Pros and cons of polyclonal antiserum and mAb: the polyclonal antisera are quick and inexpensive to produce, it can yield antibodies with very high affinities, but it’s always a gamble. The polyclonality of the serum may be an advantage in detecting heterogenous antigens. On the other hand, it has limited stocks and batch-to-batch variations. Not always there is full antigen specificity, and for immunisation purposes the pure antigen is required. Regarding the monoclonal antibodies, they are always the same antibody thus there is a constant supply with same affinity and specificity, they have full antigen specificity, and the purity of the antigen is not required for immunizing. Unfortunately, sometimes they are too specific, thus not recognizing variants of the antigen, and they have often lower affinity respect to polyclonal. Theya re also difficult to produce due to presence of steroids, due to mouse immune reaction against antigen or if the target is toxic to the animal all together. IMMUNOASSAY INTERFERENCE Interference can be mediated by assay antibody interaction, the interfering antibodies can be anti- species, anti-subclass, anti-idiotypic (binding of two antibodies trough both of their paratopes), and rheumatoid factors (autoantibodies against antibodies). It must be considered also that antibodies are sticky and will bind to almost everything. There are different kinds of interference, one is the positive “false” interference, it is due to specific mAB- mAb combination, mainly due to things like anti-mouse Ab against either the Fc or Fab portion of the antibody. This phenomenon can be solved by changing the sandwiching antibody combination, or to remove the Fc portion either by using proteolytic enzymes or by recombination the Fab fragments. Removal of the Fc portion doesn’t solve the problem of anti-species and anti-idiotype interferences. It is also possible to neutralize IgG by using bovine immunoglobulins, using native IgG or denatured ones. Another mechanism of interference is the complement system, which generates negative interference. It might be contained in the serum, especially the C1q fragment that starts the classical pathway of the complement system, leading to destroy of the assay by binding to the antibody. To avoid the activation of the complement cascade, we do not use IgG type 2 and 2b, moreover, we can also use EDTA, which removes calcium ions thus removing an essential factor for the activation of the complement cascade. We could use heat to inactivate the C reactive proteins, but we would risk denaturation of our target protein. It is possible to use different fragments of the antibodies, but it would require more work for the separation, thus recombination of antibodies and their fragments is preferred. Recombination allows the avoidance of animal use and to engineer the desired properties. ALTERNATIVE BINDERS As said before, the antibodies cannot always be used, or even if we could, they are difficult to produce, they depend on disulfide bonds for stability, there is a low ration between the number of binding sites and the molecular mass, they tend to aggregate. Thus, is possible to use alternative binders, which could be alternative protein scaffold (such as affibodies), aptamers or plastic antibodies. ALTERNATIVE PROTEIN SCAFFOLDS A scaffold is any supporting structure that can accommodate an entirely novel binding activity, it can be rational designed thanks to computational analysis and use of combinatorial libraries of variants of natural proteins. The de novo synthesis of the novel protein uses computational analysis, producing nonnatural proteins, the problem is that the prediction of the novel protein folding is not easy. Here are some possible structures. AFFIBODIES Are small proteins of circa 58 aa, with a weight of 6kD. They have 3 alpha helices arranged in Z domain; no thiol bridges are present. They are stable in harsh conditions, and even allow inclusion of non-natural amino acids, moreover they are easily expressed in bacteria. APTAMERS Are oligonucleotides molecules, they can be either DNA or RNAA, they can act as binders because when they are in the single stranded form, they can achieve specific structures with binding domains. It is possible to obtain them through the SELEX method, so by producing a random sequence oligonucleotide library, screening for affinity and using PCRR to achieve the product, the product is found thanks to affinity chromatography, for each cycle is possible to increase the affinity until the desired product is achieved. Aptamers from the SELEX process is very small, they do not have unnecessary bits, and they can be functional even at 40 nucleotides or less. They can be created in 2 to 3 months, thus a shorter time respect to the production of mAb from mouse. SELEX aptamers have remarkable specificity, even able to detect the difference between a D- or L- enantiomers. Aptamers pros are that they are not animal dependent, they can have different selection conditions even non physiological, they can bind to toxins, they can be chemically synthesized thus purity is achievable, they are site specific and can be denatured and renatured in the original condition. Since aptamers have high specificity, in a two-site assay it would necessarily mean the masking of some epitopes of the antigen to allow multiple bindings. PLASTIC ANTIBODIES (MOLECULAR IMPRINTED POLYMERS) A polymer can be used to from a mould where the target molecule can fit inside. We take the molecule and assemble the plastic material around it, let it polymerize and remove the template, thus obtaining the imprinted material. The release of the template is the tricky part because it can go wrong in so many ways, for example we could have incomplete removal of the template, rapture of the cavity, collapse of the cavity and distortion of the binding sites. This means that the 3D structure must be maintained. Preparation of MIPs: 1. Reaction mixture: we us a template added to mixture of functional monomer / crosslinker / initiator in organic solvent. 2) Polymerization, done by using UV light or heat to initiate reaction. 3) Particle formation, it exploits mechanical grinding into µm particles or direct formation of uniform spheres in suspension polymerisation, it also uses magnetic particles with inclusion of iron oxide. 4) Extraction of the template, usually done with ethanol acetic acid, then methanol followed by drying. There aren’t many successful applications of MIPs. The pros of the MIPs are that they can be used against those compounds difficult to achieve through immunisation, they are small molecules that do not require a carrier, and they tolerate extreme conditions. The cons are that they can be used only in non-polar solvents, they have a limited binding strength, imprinting is difficult, and it has a high initial cost even though the MIP can be reused. LECTINS They are proteins that binds carbohydrates. They are found in multiple organisms such as animal, plants and microorganisms. They can be used for the specific detection of glycoproteins. Lectins have a weaker affinity respect to antibodies. There are a wide variety of lectins from different organisms, each with the specific target. They can be used in assays for the detection of cancer markers. It is possible to coat nanoparticles with lectins. CHAPTER 4: SOLID PHASES AND IMMOBILISATION An immunoassay has a solid phase, or solid support, on which the antibodies, or any other binder, is fixed on the surface. This allows easy separation between bound and free tracer molecules. The solid phase should have desirable characteristics, like low background signal, a large enough surface, easy to be coated, high capacity for protein binding, no leakage, easy handling, easy washing, low nonspecific binding, inexpensive and highly reproducible quality. The solid phase can have different formats, like microtitration plates, but also particles, beads, chips, channels, tips, membranes or tubes. The microtitration plates have various options, from the number of wells (even a single one alone up to 300 or more) to the bottom shape, which can be flat, conical, round, or flat with curved edges; the type of bottom is chosen depending on the type of assay, of the coating required, and the efficiency of mixing. Moreover, depending on the type of chosen reporter, they can be clear, black or white. Clear is for colorimetric assay, black is used in case of fluorescence, while white is used in case of luminescence. The advantage of microtitration plates is that they’re easy to wash, thus reducing to a minimum the background signal, but also, they provide high sensitivity. Nevertheless, they have limitations, such as providing a small area compared to the particle size, they have limited antibody binding, but also long diffusion distance, the coating takes time, and the kinetics are slow, because the molecules have to encounter each other. The coating procedure, moreover, it complex, it has to be done separately for each well, thus the reproducibility is an issue, the additional drying step must be done carefully cause the proteins must not be desiccated, otherwise their activity is compromised. Particles or beads, they do not require any drying for storing, they are available in different sizes. The difference between particles and beads is the size, the smaller ones are particles, larger ones are the beads. They can also have specific properties such as superparamagnetic by including iron oxide or chromium oxide, this simplifies washes, but also increases kinetics thanks to particle agitation. They have high binding capacity, a wide dynamic range, short diffusion distances with faster coating and rapid kinetics. The limitation of beads and particles is that they require a more time consuming and complex separation processes, moreover, nonspecific binding may be higher, with a direct proportion with the increase of surface area. Beads can be so sticky, that they have to be stored in continuous motion to avoid aggregation between each other. PLASTIC AS SOLID SURFACE MATERIAL Plastic polymers are the main material for solid surfaces, but there are also different polymers, the most common is polystyrene. While the polystyrene is well defined as a material, each commercial product behaves differently, due to raw material differences, due to different moulding forms and processes like heat and cooling, but also due to residual chemicals from the moulding process. Thus, by seeing the different variations that condition the material, the coating conditions must be separately determined for each polystyrene product. PASSIVE COATING It’s a method to attach proteins to the plastic surface. Passive coating, or passive adsorption, is the most common method to do so, it entitles just letting the protein sit on the surface for a while. It’s a simple and cheap technology but can only be applied with medium/large proteins. In case of small proteins, covalent coating is required, it is the active process of binding the protein on the surface, no leakage of the protein itself is seen. In general, the passive coating is affected by the hydrophobic/hydrophilic properties of both the protein and the plastic surface. The hydrophobic interactions are through to be the main factor in the protein adsorption. It has to be paid attention if the plastic has been treated during manufacture because it could enhance the hydrophilicity (negative). To improve coating is possible to perform different pre-treatments on the protein, like extreme pH, heat shock, partial chemical denaturation to expose hydrophobic bits. Overall, large proteins are way easier to immobilize due to high number of interaction points. Usually, the protein concentration used in the coating solution is 10-100 µg/mL, higher concentration would mean both less coating time but also different final conformation of the protein on the surface. Since protein do not bind in an organized manner, we define the binding capacity, with both the maximum number of proteins that could theoretically bind and the minimum number of proteins that could theoretically bind, we take the average and obtain the binding capacity, thus how much protein the surface can bind. POLYSTERENE FOR PROTEIN COATING Polystyrene contains some oxygen atoms near the surface, making it a hotspot site for interaction with the hydrophilic bits of the proteins. The polystyrene surface can also be modified through 𝛾-irradiation or plasma discharge, they increase the amount of surface oxygen. Other modification can also be chemical treatment to achieve covalent binding. INDIRECT COATING Can be used in the case in which the protein may lose its activity if coated through direct coating, moreover direct coating wastes lots of materials. The options are, anti-mouse IgG, Protein G and protein A (will bind the constant part of Igs, leakage is possible), and streptavidin-biotin linkage. BIOTIN AND STREPTAVIDIN Biotin is water soluble vitamin B7, it can be covalently coupled to proteins thanks to the -COOH tail. Streptavidin, instead, is a protein coming from Streptomyces avidinii. The biotin binds to streptavidin with high affinity, indeed is defined as the strongest non-covalent binding known in nature. It is possible to use the biotin-streptavidin linkages in immunoassay thanks to different options such as: streptavidin coated plates, biotinylated cAb. The coating doesn’t require overnight incubation (like for passive coating) but just one hour incubation. Still, for the assay to work, the sample must be low in biotin otherwise the results will be wrong. CHAPTER 5: MULTIPLEX ASSAYS Multiplex assays means that one reaction is performed in one test tube, but multiple analytes are screened. This is a time-efficient assay that allow analysis of volume-limited samples. There are some challenges with multiplexing, such as the quantity of antibodies in the assay, thus risking higher nonspecific interactions, moreover, to detect the signal has to be recorded with more sophisticated technologies, thus the cost must be justified. SPECTRAL RESOLUTION It is a way to perform multiplex assays. The name spectral refers to the usage of fluorescence labels, the fluorescence signal is emitted at different wavelength. Spectral resolution is characterized by binding of different analytes at the same physical location, the only thing that changes is the label. Spectral resolution could be applied to ELISA but is way more difficult. One example of spectral resolution is the multiplexed DELFIA assay: One application is to measure the free and total PSA in blood. The surface of the assay is coated with antibodies able to bind to the epitope of PSA. Different tracer antibodies are present, one that binds to a second epitope of PSA, always available even when bound with ACT, another tAb binds to an epitope usually masked by ACT. Both tAbs are linked with lanthanide chelates. This type of multiplex gives a clearer results respect to traditional ELISA but has some limitations like the signal of the labels must be clearly separated, thus more than two analytes cannot be screened with multiplex DELFIA. SINGLE MOLECULES COUNTING Is an ultra-sensitive immunoassay technology, it uses digital counting. Is used for research purposes. The assay entitles that after normal fluorescent label noncompetitive immunoassay labelled antibodies are detached, transferred to a detection well and scanned with a confocal laser to produce single molecules signals. The start point of the assay is the same to any other assay, so the analyte of interest is added to the solution, is captured by antibodies and then detected by a tAb, the solution is then eluted, where we obtain in solution only the tAb plus the analyte, at this point the solution is quantified thanks to a confocal laser. SPATIAL RESOLUTION The analyte concentration-dependent specific signal measured for different analytes at the same wavelength. The labels are physically at different locations in the sample solution and well. An example of the spatial resolution for multiplexed assays, are the spot arrays. The limit of this multiplex assay is the surface area, coupled with the sensitivity of detection technology. Examples: meso scale discovery, it uses electrochemiluminescence reporter technology, the detection happens through a CCD camera, the intensity of analyte yields a more intense light. The multiplexing is achieved by spatial resolution by spots coated with different antibodies. Lateral flow tests: the test lines have analyte-specific antibodies on the LF strips. The limit of this assay is by the time required for the complex to form. Bead arrays: uses fluorescence and tracer antibodies, there is an analyte-specific bead conjugated with an antibody, the tracer antibody is biotinylated and liked to a fluorescence label, the fluorescence is emitted at different wavelength, which is used to determine the analyte concentration. RESOLUTION BY SEQUENCE The label is an oligonucleotide that can be identified by its sequence. Is very similar to immunoPCR. Example: Proximity extension assay (PEA): allow usage of very low sample volumes. It is a homogenous immunoassay technology, with a sandwich type assay. Each analyte has a tail made of oligonucleotide; they have complementary sequences that can hybridize if near enough. The sequence is then extended by PCR or directly quantified by qPCR or NGS. For multiplexing, there are different antibodies with different oligonucleotides sequences. The interactions between multiple antibodies may interfere with the assay. The analytes are identified by identifying the specific sequence of the label. SOMAScan Uses aptamers as binders, allows the multiplexing of multiple molecules up to 7000 analytes at the same time. To the aptamer is possible toa attach a fluorescence molecule thanks to a linker, after the fluorescence dye, there is a spacer and a biotin molecule removable thanks to light. The biotin molecule allows attachment of the aptamer to the bead. The photo-cleavable biotin is cleaved releasing analyte- SOMAmer complexes to the solution, thus loosely bound molecules are released by a polyanionic competitor. Biotinylated analytes are bound to the surface of a new bead. SOMAmers are released and allowed to hybridize to DNA-microarray. Analyte concentration determined by the fluorescent signal at a specific site of the microarray. CHAPTER 6: HOMOGENOUS ASSAYS The key features of homogenous assays are that they do not require any separation between bound and unbound labels, the signal is produced only upon binding with the antigen. It has a simple protocol of mix and measure, thus easy also to automate because no washing is required. The assay has fast kinetics; thus, detection can be either continuous (real time) or can be detected after the reaction has reached an equilibrium. In homogenous assay, a specific device is needed for the detection of the specific signal that is created upon binding of antibody and antigen. The sensitivity and accuracy of homogeneous assays are limited by sample matrix and insufficient signal change even after antibody binding. Is an expensive but faster method, both the technologies and labels are sophisticated. AGGLUTINATION BASED ASSAYS It is the first homogenous tests ever performed, it has visible Ab-analyte complexes, the precipitating antibody. It is based on the same principle that happens in turbidimetry and nephelometry, used to identify c-reactive protein. The antibodies are attached to polystyrene beads, making it easier for the agglutination of the antibodies. The agglutinated beads are easy to see because the assay is carried on a black plate or sheet. EMIT = ENZYME MULTIPLE IMMUNOASSAY TECHNIQUE While it uses enzymes, IT IS NOT ELISA!!!! The principle of the assay is that is a competitive assay for small analytes. The system uses an enzyme with an active site, which becomes inactivated by binding with the antibody. This leads to no or low detectable signal. If the analyte is present, they will bind to the Antibody, which will dissociated form the enzyme leading to signal production because the active site is free and can convert the enzyme to signal. The signal increases with the analyte concentrations. FLUORESCENCE POLARIZATION IMMUNOASSAY (FPIA) Polarization means that the plane of light is changed through a polarizing filter, allowing the passage of light only in a determined orientation. If we excite a fluorescence molecule with a polarized light, it will emit a photon. Suppose that the molecule is immobile, then the photon emitted will have the same polarization of the light that excited the molecule. Molecules are never immobile when excited, they are always tumbling. Smaller the molecules, then higher it’s the tumbling rate, thus meaning rapid loss of polarization of the photon emitted. Large molecules will tumble slower, thus being more adept for this type of assay. By binding a small molecule to a larger one (like antibodies) it is possible to slow down the tumbling rate, thus the polarization is better preserved. To detect the signal, we use polarization emission filters thus only in case of slow tumbling molecules we will have the signal quite high, in case of no analyte is solution, the tumbling rate will still be high due to large complex being formed. This assay is not very sensitive, moreover it is a competitive assay. FRET-BASED ASSAYS FRET Fret can mean two things: fluorescence resonance energy transfer or Föster resonance energy transfer. The second name is the most used version in the scientific community. Energy is transferred through a radiationless way, meaning no photon is emitted. It is a distance- dependent assay, moreover it requires two different fluorescent molecules to make the assay work. One molecule will act as a DONOR, meaning that will be excited, and will pass the energy to the second nearby fluorescent molecules, defined as an ACCEPTOR. The acceptor must have an energetically accessible excited stated. The acceptor will emit light at a different wavelength with lower energy. The donor and the acceptor must have an overlapping spectrum of emission, moreover the distance must be in terms of 10-100 Åmstrongs, if the distance increases, then the efficiency of the assay dramatically decreases, due to problems with energy transfer. It is possible to use both conventional fluorescence labels, but also lanthanide chelates since they have long life-time fluorescence, but also chemiluminescent labels can be used. A third option is the use of quencher, where the signal is inhibited if the donor and acceptor are near each other, this means that only when the analyte is present, the signal will not be there and is seen in a decrease of overall signal emitted. With FRET we could either measure the decrease in donor emission or measure the increase in acceptor emission, which theoretically is a more sensitive approach since we go form a low to a high signal. FETI- FLUORESCENCE ENERGY TANSFER IMMUNOASSAY We link donor and acceptor to either two different antibodies that will perform sandwich immunoassay, or we label the antigen with the donor and the antibody with the acceptor, only if they bind the energy will be transferred to the acceptor, this is a competitive immunoassay. TR-FRET Fret technique that uses time-resolved measurement. In TR the donor is lanthanide-based chelates, while the acceptors are conventional fluorescent dyes. LANCE is an example of commercial application of TR FRET technology, when no analyte is present, we excite the label and observe normal fluorescence of the lanthanide chelate, which is europium. Since no complex is present, the acceptor will not be excited thus no emission is seen. If the complex is formed, thus the analyte is present, we will see emission of the acceptor molecule. TRACE = time resolved amplified cryptate emission. Uses lanthanide chelates, again europium, as principle of the assay. The lanthanide chelate is mounted on labelled probe. TR-FRET can is more sensitive than normal FRET techniques mainly because is less influenced by matrix effects. It can be automated as a technique and can be used to detected enzyme and drugs. The LANCE and TRANCE applications show that as sandwich assays they provide high sensitivity, but they cannot be used with whole blood samples thus are generally not used in clinical settings. LOCI (LUMINESCENCE OXYGEN CHANNELLING IMMUNOASSAYS) The principle is that the analyte, when binds to the antibody, will produce something that will be detected by the acceptor, making it release luminescence signal. In the case of LOCI sandwich assay, the “something” produces is a reactive oxygen species that, when activated by light, will excite the other antibody leading to luminescence. This generates an upconverting system because we use excitable beads at 680nm, but the emission will be at 550nm. To work, the beads must be near the chemiluminescence molecule, because the singlet reactive oxygen will be inactivated quickly. Multiplex is possible by using different chemiluminescence dyes. CHAPTER 7: LATERAL FLOW ASSAYS Later flow assays are immunochromatographic, they have series of advantages and disadvantages. As advantages they are portable, stable, user friendly, inexpensive, no washing required and provides visual results. The disadvantages are that it’s a qualitative/semi-quantitative assay, meaning that no information is given regarding the number of analytes. The reproducibility, sensitivity and specificity are all affected by variations. Moreover, some applications may require pretreatment of the sample. SAMPLE PAD Its role is to treat the sample, it contains different agents such as buffering of the pH, provides ionic strength. It also acts as a filter against red blood cells or any other material that could be filtered. It is made by glass fibres, cellulose and polyester. The sample pad is created by soaking it in buffer containing the buffering agent for the pH, proteins and polymers, non-ionic surfactants and salt. After the soaking, the pad is dried. CONJUGATE PAD Has the role of preserving the dried conjugated for the whole duration of the shelf life. It must also release efficiently and in a reproducible way the conjugate (antibody). Again, it can be made by glass fibres, polyesters or rayons. If the release of the conjugate is not efficient, then the sensitivity of the assay is affected. The conjugated pad is first created in the same way of the sample pad, but then it requires the additional steps of the addition of the conjugate, to add it we use a conjugate application buffer, which contains buffering agents, proteins, polymers, non-ionic surfactant, sugars and salt. The conjugate is then added through two methods: either by immersion or by non-contact dispersion. WICKING PAD (absorbent pad) Has the role of pulling the fluid upstream the strip and holding it there. It must not cause any back flow of fluid. It is the “engine” of the assay, since thanks to its highly absorbent capacity is able to pull the liquid from the sample pad through the strip. The wicking pad is generally made by high-density cellulose. Moreover, the wick length will vary depending on the total fluid added to the strip. BACKING MATERIAL The platform of the assay, it allows lamination of all the assay components in their place. It provides rigidity and easy handling of the strip. They are made of plastic, typically polystyrene, and is coated with pressure sensitive adhesive, something to consider is that the adhesive may be transferred to the strip cutter. ANALYTICAL MEMBRANE It’s an optimal membrane which must be allow the liquid to travel thanks to capillary forces, it must irreversibly bind proteins on test and control lines, it should not bind proteins outside test and control lines. The problem is that most material bind all proteins unspecifically or do not bind proteins at all. The most used polymers are nitrocellulose with electrostatic forces; polyvinylidene fluoride with hydrophobic forces, Nylon with ionic electrostatic force, and polyethersulfone that works through hydrophobic forces. Nitrocellulose membrane is the most common one, it has pros such as low manufacturing costs, it allows liquid flow thanks to capillary forces, it has high protein binding capacity, moreover there is a wide range of commercially available membranes. The cons are that the same high protein binding capacity, thus high nonspecific binding, it has imperfect reproducibility of performance, The membrane is fragile and flammable, moreover it varies due to environmental conditions. The nitrocellulose membrane can be manufactured with different pore sizes, larger pores mean faster flows, but it will compromise the sensitivity of the assay. For each membrane there is a defined flow time, which measures how many seconds it takes the liquid to cross 4 centimetres of the membrane. BINDERS They must be attached to the solid surface; they can be applied either by contact or noncontact printing (spraying). By contact it means that it is embedded by using pin spread. Test line must contain the primary antibodies, which will form complex with the analyte. Control line it has species-specific antibodies, to capture both the complex and antibodies without analyte, this is to confirm that the test is working. The nitrocellulose must be made hydrophilic by using different reactants, the antibody binds to the membrane thanks to electrostatic and hydrophobic forces, hydrogen bridges. It needs time to allow the binding to cure. The assay reproducibility and sensitivity depend on consistent immobilization of binders to the test and control lines. LABELS They are required for the detection of particles thus they must be detectable themselves and they must allow conjugation with other biomolecules. As labels it is pretty common to use colloidal gold, which is relatively inexpensive, easy to conjugate, intense in colour, doesn’t require development and detectable even by eye, unfortunately it has limited sensitivity. CHAPTER 8: NUCLEIC ACIDS ASSAY Diagnostic tests using biological molecules. These tests are designed for the detection of nucleic acid sequences to detect presence of bacterial or viral nucleic acids, or to detect the presence of specific gene like antibiotic resistance or for toxicity of cyanobacteria, but also to detect the presence of gene mutation or the number of specific sequences or check the expression levels of RNAs in a specific sample. At the base of nucleic acids tests there is the hybridization of complementary nucleic acids, in other words, the at and gc base pairs. Most test depend on PCR reactions and monitor the amplification with the usage of fluorescence labels, they are usually referred to as NAAT or nucleic acid amplification tests, they allow the detection of very small number of nucleic acid molecules. SYBR GREEN SYBR is an intercalating agent able to intercalate only in dsDNA, when it intercalates it produces fluorescence signal. The signal produced during the first cycle is low, only when it is above background signal is detectable. Once the signal is above background level, we have an exponential phase of growth of the signal followed by a long linear phase, ending with a plateau phase caused by exhaustion of materials. The exponential part is the only reliable one regarding the amplification efficiency. Not specific since it will create signal even for primer dimers or any other ds DNA that is not an amplicon. qPCR It is quantitative PCR or real time PCR. We have to consider the threshold signal level, meaning that the signal level must be distinguished from the background but still representing reactions at exponential phase. It is defined as threshold cycle, meaning the cycle at which the signal surpasses the background. By comparing the sample threshold cycle to a standard one, is possible to determine the starting quantity of analyte in the sample. Higher the quantity, faster the threshold cycle is approached. TaqMan PROBES TaqMan probes bind to target sequence in the middle of the amplicon during the annealing step, they are made of a fluorophore near a quencher. When the DNA polymerase approaches the probe, it will hydrolyse it, separating the fluorophore from the quencher. The sequence that links the fluorophore to the quencher MUST be specific. The specificity of the probe means that it will not bind to primer dimers. PRIMER DESIGN In the primer design we must consider the ration between at and gc pair, since they affect the melting temperature, moreover the sequence must be done in a way that it doesn’t favours any secondary structure. The primer length should be between 18 to 24 nucleotides and must ensure a tight enough binding. It is suggested to not use mononucleotide o dinucleotide repeats because they either lead to miss priming or to secondary structures. ALTERNATIVE PROBE TYPES Molecular beacons, no hydrolysis of the primer happens, it is found in a hairpin loop with reporter and quencher at the ends, during the annealing, the probe binds to the target sequence to separate reporter and quencher, thus signal is produced. Dual hybridization probes, FRET-based. WE have two probes, one with a donor, one with an acceptor, they usually have chelated. During annealing, the donor and acceptor will bind in a way that they are next to each other, thus based on FRET the donor will produce the signal at designed wavelength. Scorpion primers, they are found in hairpin conformation, and they consist of a reporter on one end and a quencher + PCR blocker on the other. They bind as hairpin loop during the annealing step, during the denaturation step they loop is open and the reporter can bind on the amplicon thanks to an internal target sequence that was masked by the loop. By binding on the amplicon, the reporter is far from the quencher, thus signal is produced. QZyme primers they are both reporters and primers, they encode for catalytic DNA. During annealing phase they will anneal to the target, during the extension, it will form catalytic DNA, which during the successive annealing steps will be bound by the reporting probe and will cleave it, thus separating the reporter form the quencher. DROPLET DIGITAL PCR The PCR reaction is confined in tiny droplets, it exploits the TaqMan principle of reporting system. The advantage of this assay is that each reaction is performed in a droplet in a water-in-oil droplet. If there was amplification, the droplet will be fluorescent even after the end of the reaction. This obliviates the problem of real-time monitoring. By measure each fluorescence intensity, is possible to calculate the concentration of the amplicon. ISOTHERMAL AMPLIFICATION METHODS The nucleic acid is amplified without the need of thermal cycling, meaning that DNA is not denature. This method requires a polymerase with intrinsic strand displacement activity. LAMP: practical example of isothermal amplification method, it is a loop-mediated isothermal amplification. The reaction is carried at 65°C for 30 minutes. Simple system but complex reaction. EXTRACTION AND PURIFICATION OF NUCLEIC ACIDS Nucleic acid tests require the extraction of the nucleic acids from the biological material containing it. The nucleic acids then must be purified from other substances that are inhibitors of the systems. One way to purify these nucleic acids is the use of commercial spin columns. CONTAMINATION CONCERNS Even just one contaminant molecules can have catastrophic results on the assay, this is because amplification tests are extremely sensitive, thus the contaminant may lead to false positive or false negative results. To avoid contamination, it is necessary to prepare the mixture carefully and to favour the close-tube detection of the signal. CONTROLS In each PCR we need different controls: NTC =” no template” control reaction. The result should be negative because they have to confirms that there is no contamination. Positive control reaction. It uses sample with known target concentration. The result should be positive and at the expected level thus confirming that the overall performance of the test is OK. IAC = internal amplification control Included in all amplification reactions Either specific amount of a sequence unrelated to the target added to the samples or some other nucleic acid target naturally found in all samples at stable concentration (e.g. ”house-keeping genes” Added sequence can be added before or after sample purification Amplified and detected with its own primers, probe and label– multiplexed detection required! Results should be the same for all reactions– confirms that result of the specific reaction is acceptable.

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