Practical Part of Spectral Analysis and Applied Spectroscopy (CP508) 2024-2025 PDF

Cairo University Faculty of Science Chemistry Department Practical Part of Spectral Analysis and Applied Spectroscopy (CP508) For Biochemistry Diploma Students 1st Semester 2024-2025...

Cairo University Faculty of Science Chemistry Department Practical Part of Spectral Analysis and Applied Spectroscopy (CP508) For Biochemistry Diploma Students 1st Semester 2024-2025 1  Biochemical Lab Instruments Contents Electrophoresis Techniques HPLC Spectrophotometer ELISA Flow Cytometry Electrophoresis Techniques Electrophoresis may be defined as the migration of the charged particle (such as: DNA, RNA, or protein molecules) through a solution under the influence of an external electrical field. Ions that are suspended between two electrodes tends to travel towards the electrodes that bears opposite charges. The rate of migration of an ion in electrical field depend on: 1. Net charge of molecule 2. Size and shape of particle 3. Strength of electrical field 4. Properties of supporting medium 5. Temperature of operation Mobility of the particles Size and shape of the particle decide the velocity with which it will migrate under the given electrical field and the medium. 2 Strength of electrical field It determined by the force exerted on the particle, and the charge the particle carrying. When force is exerted on the particle it start moving, however the movement is restricted by the experience of the frictional force because of the viscosity. Effect of pH on Mobility As the molecule exist as amphoteric, they will carry the charges based on the solvent pH. Their overall net charge is NEUTRAL when it is at zwitterion state. And hence the mobility is retarded to zero. Mobility is directly proportional to the magnitude of the charge, which is functional of the pH of solvent. The pH is maintained by the use of Buffers of different pH. Types of electrophoresis Zone electrophoresis a) Paper electrophoresis b) Gel electrophoresis c) Thin layer electrophoresis d) Cellulose acetate electrophoresis Moving Boundary Electrophoresis a) Capillary Electrophoresis b) Isotachophoresis c) Isoelectric Focusing Zone electrophoresis It involves the migration of the charged particle on the supporting media (Paper, cellulose acetate membrane, starch gel, poly acrylamide). The separated components are distributed into discrete zone on the support media. Supporting media is saturated with buffer solution, small volume of the sample is applied as narrow band. 3 Paper electrophoresis Filter paper such as Whatmann no.1 and no.3 in strip of 3mm or 5cm wide have been used to good effect. Separation takes place in 12 to 14 hrs. Advantages It is economical Easy to use Disadvantages Certain compounds such as proteins, hydrophilic molecules cannot be resolved due to the adsorptive and ionogenic properties of paper Gel electrophoresis Separation is brought about through molecular sieving technique, based on the molecular size of the substances. Gel material acts as a “molecular sieve” Gel is a colloid in a solid form (99% is water). It is important that the support media is electrically neutral. Different types of gels which can be used are; Agar and Agarose gel, Polyacrylamide gels, Starch. A porous gel acts as a sieve by retarding or, in some cases, by completely obstructing the movement of macromolecules while allowing smaller molecules to migrate freely. 4 Agar and agarose gel Agar is a mixture of polysaccharides extracted from sea weeds. Agarose is a highly purified uncharged polysaccharide derived from agar. Agarose is chemically basic disaccharide repeating units of 3,6- anhydro-L-galactose. Agarose dissolves when added to boiling liquid. It remains in a liquid state until the temperature is lowered to about 40°C at which point it gels. The pore size may be predetermined by adjusting the concentration of agarose in the gel. Agarose gels are fragile. They are actually hydrocolloids, and they are held together by the formation of weak hydrogen and hydrophobic bonds. The pores of an agarose gel are large, agarose is used to separate macromolecules such as nucleic acids, large proteins and protein complexes. Advantages Easy to prepare and small concentration of agarose is required. Resolution is superior to that of filter paper. Large quantities of proteins can be separated and recovered. It adsorbs proteins relatively less when compared to other medium. Sharp zones are obtained due to less adsorption. Disadvantages Electro osmosis is high. Resolution is less compared to polyacrylamide gels. Applications I. Molecular Cloning This technique used in production of insulin in pharmaceutical manufacturing. Other applications of molecular cloning include adding fluorescent protein fusions to existing cellular proteins to study their location in cells and creating new genetic circuits to carry out specific functions, such as breaking down toxins. II. Genetic Fingerprinting This technique, known as DNA fingerprinting, can be used in areas such as forensics for criminal investigations, genealogy and parentage testing. III. Diagnostics Electrophoresis can be used in a range of diagnostic tests, primarily in the screening of genetic disorders but also to identify abnormal proteins. DNA can be extracted from patients, or even from embryos for pre-implantation screening, and subject to PCR and agarose gel electrophoresis to confirm the presence of certain genes or genetic abnormalities. Polyacrylamide gel electrophoresis (PAGE) It is prepared by polymerizing acrylamide monomers in the presence of methylene-bis-acrylamide to cross link the monomers. Structure of acrylamide (CH2=CH-CO-NH2)n Polyacrylamide gel structure held together by covalent cross-links. Polyacrylamide gels are tougher than agarose gels. 5  It is thermostable, transparent, strong and relatively chemically inert. Gels are uncharged and are prepared in a variety of pore sizes. Proteins are separated on the basis of charge to mass ratio and molecular size, a phenomenon called Molecular sieving. PAGE-procedure The gel of different pore sizes is cast into a column inside a vertical tube, often with large pore gel at the top and small pore gel at the bottom. Microgram quantity of the sample is placed over the top of the gel column and covered by a buffer solution having such pH so as to change sample components into anions. The foot of the gel column is made to dip in the same buffer in the bottom reservoir. Cathode and anode are kept above and below the column to impose an electric field through the column. Macromolecular anions move towards the anode down the gel column. There is no external solvent space, all the migratory particles have to pass through the gel pores. Rate of migration depends on the charge to mass ratio. Different sample components get separated into discrete migratory bands along the gel column on the basis of electrophoretic mobility and gel filtration effect. Advantage Gels are stable over wide range of pH and temperature. Gels of different pore size can be formed. Simple and separation speed is good comparatively. Types of PAGE A. Native-PAGE Native gels are run in non-denaturing conditions, so that the analyte natural structure is maintained. Separation is based upon charge, size, and shape of macromolecules. Useful for separation or purification of mixture of proteins. This was the original mode of electrophoresis. B. Denatured-PAGE or SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) When a detergent SDS added to PAGE the combined procedure is termed as SDS-PAGE. SDS coats protein molecules giving all proteins a constant charge-mass ratio. Due to masking of charges of proteins by the large negative charge on SDS binding with them, the proteins migrate along the gel in order of increasing sizes or molecular weights. 6  SDS is an anionic detergent which denatures secondary and non–disulfide– linked tertiary structures by wrapping around the polypeptide backbone. In so doing, SDS confers a net negative charge to the polypeptide in proportion to its length. Molecules in solution with SDS have a net negative charge within a wide pH range. The negative charges on SDS destroy most of the complex structure of proteins, and are strongly attracted toward an anode in an electric field. Native protein is unfolded by heating in the presence of mercaptoethanol and SDS. SDS binds to the protein so that it stays in solution and denatures. Large polypeptides bind more SDS than small polypeptides, so proteins end up with negative charge in relation to their size. Starch A suspension of granular starch should be boiled in a buffer to give a clear colloidal suspension. The suspension on cooling sets as a semisolid gel due to intertwining of the branched chains of amylopectin. In order to avoid swelling and shrinking petroleum jelly is used. Advantages High resolving power and sharp zones are obtained. The components resolved can be recovered in reasonable yield especially proteins. Disadvantages Electro osmotic effect. Variation in pore size from batch to batch Thin layer electrophoresis Studies can be carried out in thin layer of silica or alumina. Advantages Less time consuming and good resolution. Application Widely used in combined electrophoretic-chromatography studies in two dimensional study of proteins and nucleic acid hydrolysates. 7 Cellular acetate electrophoresis It contains 2-3 acetyl groups per glucose unit and its adsorption capacity is less than that of paper. It gives sharper bands. Provides a good background for staining glycoproteins. Advantages No tailing of proteins or hydrophilic materials. Available in wide range of particle size and layer thickness. Give sharp bands and offer good resolution. High voltage can be applied which will enhance the resolution. Disadvantages Expensive. Presence of sulphonic and carboxylic residue causes induced electro osmosis during electrophoresis. Moving boundary electrophoresis The moving boundary method allows the charged species to migrate in a free moving solution without the supporting medium. Capillary electrophoresis The principle behind electrophoresis is that charged molecules will migrate toward the opposite pole and separate from each other based on physical characteristics. Capillary electrophoresis has grown to become a collection of a range of separation techniques which involve the application of high voltages across buffer filled capillaries to achieve separations. Due to electro osmotic flow, all sample components migrate towards the negative electrode. The capillary can also be filled with a gel, which eliminates the electro osmotic flow. Separation is accomplished as in conventional gel electrophoresis but the capillary allows higher resolution, greater sensitivity, and on-line detection. The capillary is filled with electrolyte solution which conducts current through the inside of the capillary. The ends of the capillary are dipped into reservoirs filled with the electrolyte. Electrodes (platinum) are inserted into the electrolyte reservoirs to complete the electrical circuit. 8 Isotachophoresis The technique of isotachophoresis depends on the development of potential gradient. Based on principle of moving boundary electrophoresis. A leading electrolyte (e.g. chloride) with a higher mobility than the analytes, and a trailing electrolyte (e.g. glycinate) with a lower mobility are used. Solution in which the separation takes place is normally an aqueous medium, which contains sucrose to provide a higher density to the solution. Separation of the ionic components of the sample is achieved through stacking them into discrete zones in order of their mobility, producing very high resolution. The analyte are positioned between the electrolytes and, when the voltage is applied, they migrate in order of decreasing mobility. Isoelectric focusing All proteins have an isoelectric point pH. When electrophoresis is run in a solution buffered at constant pH, proteins having a net charge will migrate towards the opposite electrode so long as the current flows. The use of pH gradient across the supporting medium causes each protein to migrate to an area of specific pH. The pH of the protein equals the pH of the gradient, thus resulting in sharp well defined protein bands. 9  A procedure to determine the isoelectric point (PI) of proteins thus, a mixture of proteins can be electrophoresed through a solution having a stable pH gradient from the anode to the cathode and each protein will migrate to the position in the pH gradient according to its isoelectric point. This is called isoelectric focusing. Protein migrate into the point where its net charge is zero – isoelectric pH. Protein is positively charged in solutions at pH below its PI and will migrate towards the cathode. Protein is negatively charged in solution at pH above its PI will migrate towards the anode. They will be in the Zwitterion form with no net charge so the further movement will cease. 10  HPLC (High Performance Liquid Chromatography) General definitions of chromatography A technique for separating mixtures of unreacted contents into their components in order to analyze, identify, purify, and/or quantify the mixture or components. A technique that separates the components of a mixture by their distinctive attraction or by using the differential affinities for a mobile medium and for a stationary adsorbing medium through which they pass. Explanation Compound is placed on stationary phase. Mobile phase passes through the stationary phase Mobile phase solubilizes the components. Mobile phase carries the individual components a certain distance through the stationary phase, depending on their attraction to both of the phases. Terms ❖ Chromatograph: It is equipment that enables an advanced separation. ❖ Chromatogram: It is the visual output of the chromatograph. ❖ Stationary phase (fixed): The chromatographic packing material needed to effect the separation, it is held inside the column hardware. ❖ Mobile phase (Movable): It is the phase which moves in a definite direction. ❖ Analyte (Sample): It is the substance to be separated during chromatography. ❖ Retention time: It is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions. It is taken as the elapsed time between the time of injection of a solute and the time of elution of the peak maximum of that solute. ❖ Eluent: The eluent is the "carrier" portion of the mobile phase. It moves the analytes through the chromatograph ❖ Eluate: Analyte + eluent that emerges from the column to the detector. ❖ Elution: Involves analyte migration through the entire system and analyte detection as it emerges from the column (isocratic elution and gradient elution) ▪ Isocratic elution: mobile phase is constant over the complete testing time. ▪ Gradient elution: the eluent mixture is changed during measurement. 11 HPLC principle ▪ Stationary phase: Column ▪ Mobile phase: liquid ▪ Commonly used to analyze, separate, identify, and quantify the active compounds. ▪ HPLC is really the automation of traditional liquid chromatography under conditions which provide for enhanced separations during shorter periods of time, utilizing very small particles, small column diameters, and very high fluid pressures. ▪ It is a technique by which a mixture sample is separated into components for identification, quantification and purification ofmixtures. Instrumentation Solvent Reservoirs The mobile phase contents are contained in a glass reservoir that carry sample into the column. The mobile phase, or solvent, in HPLC is usually a mixture of polar and non-polar liquid components whose respective concentrations are varied depending on the composition of the sample. (Filtration and degassing of solvent should be performed before use to eliminate air bubble). Isocratic elution: single solvent separation technique Gradient elution: two or more solvents, varied during separation Pump Pump, also called the solvent delivery system. Produce an appropriate pressure to push solvent into the sample. The role of the pump is to force a liquid (mobile phase) through the liquid chromatograph at a specific flow rate. A pump capable of pumping solvent up to a pressure of 4000 psi and at flows of up to 10 ml/min A pump can deliver an isocratic or a gradient mobile phase Sample Injector The injector serves to introduce the liquid sample into the flow stream of the mobile phase. May be auto-sampler or manual A fixed-volume loop of between 1 – 200 µl (20 µl is often used as standard). 12 Columns ❖ Columns are usually made of polished stainless steel, 50 -300 mm long and with internal diameter 2 -5 mm. ❖ They are commonly filled with a stationary phase with a particle size of 3–10 µm. Columns with internal diameters of less than 2 mm are often referred to as micro bore columns. ❖ Ideally the temperature of the mobile phase and the column should be kept constant during an analysis. (Column should be continuously wet with mobile phase to avoid cracks allow mobile phase to run only about 20 min before and between runs). ❖ For different compounds the internal diameter, particle size, pore size parameters can be changed according to their nature and chemical properties. ▪ Internal diameter: The internal diameter (ID) of an HPLC column is a critical aspect that determines quantity of analyte that can be loaded onto the column and also influences sensitivity. Larger columns are usually seen in industrial applications such as the purification of a drug product for later use. Low ID columns have improved sensitivity and lower solvent consumption. ▪ Particle size: Most traditional HPLC is performed with the stationaryphase attached to the outside of small spherical silica particles (verysmall beads). Smaller particles generally provide more surface area and better separations. ▪ Pore size: Many stationary phases are porous to provide greater surface area. Small pores provide greater surface area while larger pore size has better kinetics especially for larger analytes. Pore sizedefines an ability of the analyte molecules to penetrate inside the particle and interact with its inner surface Types of HPLC according to column type Types of HPLC generally depends on phase system used in the process i. Normal phase chromatography or NPHPLC In this method a polar stationary phase (silica (-Si-OH), and A non-polar mobile phase (hexane or heptane mixed with a slightly more polar solvent such as isopropanol, ethyl acetate or chloroform) are used. Separates analytes based on polarity; the polar analyte interacted with and is retained by the polar stationary phase. Adsorption strengths increase with increased analyte polarity, and the interaction between the polar analyte and the polar stationary phase increases the elution time. the least polar compounds elute first and the most polar compounds elute last This method used for the analysis of solutes readily soluble in organic solvents, based on their polar differences such as amines, acids, metal complexes also steroid hormones and phospholipids. ii. Reversed phase HPLC ▪ the most popular method In which a non-polar stationary phase (alkyl hydrocarbons are the preferred stationary phase; C18 is the most common stationary phase, but C8 and C4 are also used in some applications) andan aqueous, moderately polar mobile phase (The mobile phase is generally a binary mixture of water and a miscible polar organic solvent like methanol, acetonitrile or tetrahydrofuran (THF)). ▪ The most polar compounds elute first and least polar compounds elute last 13  ▪ It operates on the principle of hydrophobic interactions, which result from repulsive forces between a polar eluent, the relatively non-polar analyte, and the non-polar stationary phase. ▪ Organic compounds less to mid polar can be separated. Detector The HPLC detector, located at the end of the column, detect the analytesas they elute from the chromatographic column. Commonly used detectors are UV-spectroscopy, fluorescence, mass- spectrometric and electrochemical detectors. Data Collection Devices Using specific software that is connected to HPLC machine. Receive the information from HPLC machine and present it as a graph. The graph describes about qualitative data (Retention time) and quantitative data (height and area of peak) HPLC chromatogram Quantitative Analysis Measurement of the amount of compound in a sample (concentration). Determination of the peak height or area. Determination the concentration of a compound, the peak area or peak height is plotted versus the concentration of the substance. For peaks thatare well resolved, peak area and peak height are proportional to the concentration. Several dilutions of standards should be prepared, starting from 5 mg/mL and going down to well calculate the calibration curve and find the linearityrange. Later, the content of a certain compound in the sample should be within this range. 14 Applications HPLC has wide applications in different fields in term of quantitative and qualitative estimation of active molecules. ❖ Pharmaceutical applications Identification of active ingredients of dosage forms Pharmaceutical quality control ❖ Environmental applications Detection of phenolic compounds in Drinking Water Bio-monitoring of pollutant ❖ Forensics Quantification of the drug in biological samples. Determination of cocaine and metabolites in blood ❖ Clinical Estimation of bilirubin and bilivirdin in blood plasma in case ofhepatic disorders. Detection of endogenous neuropeptides in extracellular fluidsof brain. ❖ Food and Flavor Ensuring the quality of soft drink and drinking water. Sugar analysis in fruit juices Advantages Needs a small sample with a high accuracy Non-destructed sample during operation compared to GC. Disadvantages Need a skill to run the instruments Solvents consuming 15  Spectrophotometer Spectrophotometer is a device that measures the intensity of electromagnetic energy at each wavelength of light in a specified region. A UV-visible-NIR spectrophotometer operates in the ultraviolet, visible, and near-infrared regions. The spectrophotometer consists of a light source, a monochromator that separates the light, and a detector. The spectrophotometer allows the researcher to acquire spectra by illuminating a sample and measuring the intensity of light returned from the sample relative to its wavelength. It is a non-destructive technique. Principle of spectrophotometer A spectrophotometer is based on the Beer-Lambert law, which states that the absorbance (amount of light absorbed) of the solution has a linear relationship with the length of light and the concentration of a sample. That is; A= εLC Where; (A) is the absorbance, (ε) is the molar extinction coefficient; the value of which is constant for a specific molecule. (L) is the optical path length, (C) is the concentration of the sample. We know that a standard spectrophotometer uses a cuvette of a length of 1 cm, which is the path length. Since path length (L), absorbance (A), and molar extinction coefficient (ε) are known, the concentration of the solution (C) can now be calculated. The fraction of the incident light that is absorbed by a solution depends on three factors: 1- The thickness of the sample or path length. 2- The concentration of the absorbing sample. 3- The chemical nature of the compound. The relationship between the concentration, path length, and the amount of light absorbed or transmitted can be exposed mathematically in the Beer and Lambert law. Components of spectrophotometer 16  1) Light source In spectrophotometer three different sources of light are commonly used to produce light of different wavelengths. a. The most common source of light used in the spectrophotometerfor the visible spectrum is a tungsten lamp. b. For Ultraviolet radiation sources are the hydrogen lamp and the deuterium lamp. c. Nernst filament is the most satisfactory source for IR (Infrared) radiation. 2) Monochromator It is used to break the polychromatic radiation into component wavelength or bands of wavelengths. There are two types of monochromators: a. Prisms: A prism disperses polychromatic light from the source into its constituent wavelengths by its ability to reflect different wavelengths to a different extents. b. Grating: Gratings are often used in the monochromators of spectrophotometers operating in all ultraviolet, visible and infrared regions. 3) Transport vessels (cuvettes) Samples to be studies are put in containers known as “CUVETTES”. Cuvettes meant for the visible region are made up of either ordinary glass or disposable plastic cuvettes. For the IR region, glass and quartz cuvettes can be used. For the ultraviolet region, quartz cuvettes are used. 4) Photodetector system Most detectors depend on the photoelectric effect. The current is then proportional to the light intensity and therefore a measure of it. Radiation detectors generate electronic signals which are proportional to the transmitter light. 5) Measuring device The current from the detector is fed to the measuring device – the galvanometer. The meter reading is directly proportional to the intensity of light. Types of spectrophotometer Visible light spectrophotometer: This type of spectrophotometer uses visible light from a tungsten lamp. It is typically used for routine laboratory work. UV/Visible spectrophotometer: A visible light spectrophotometer is turned into a UV-visible unit with the aid of a second lamp.Used for scanning function. Near-infrared spectrophotometer: It measures the response of a sample when exposed to infrared light. It helps in monitoring highly absorbing solids. It also provides essential information like fat, protein, fibre, and starch content. Nuclear Magnetic Resonance spectroscopy: Used to determine the structure of organic compounds. 17  Atomic absorption spectrophotometer: A flame evaporates water from the sample causing it to dissociate into ions. The dissociation leads to changes in the intensity of light as seen by the detector. It helps in finding out the concentration of the sample. It useful in toxicology, environmental testing, and quality control laboratories. Mercury spectrophotometer/analyzer: It measures the trace amount of mercury in water. Fluorometers: These measures the fluorescence release once the object being studied is exposed to a single wavelength of light. It is commonly used in the chemical dosing control systems of cooling towers and boilers. Applications of Spectrophotometer Qualitative analysis, like identifying classes of compounds. Quantitative analysis such as; to determine the unknown concentration of a given specimen through absorption spectrometry like nucleic acid (DNA or RNA). Identifying the molecular weight of a particular sample such as amines, ketone compounds, aldehyde, and sugar. Identifying impurities. Identifying the dissolved oxygen content in a body of water. Analysis of gases in hospitals Functional group detection. Determines the phase of reaction by measuring the formation and disappearance rate of the light- absorbing compounds. Organic compounds structure illustration. 18  Enzyme-linked immunosorbent assay (ELISA) It is a biochemical technique mainly used in immunology to detect the presence of an antibody or an antigen in a sample. It is used for the detection and quantification of peptides, proteins, antibodies, enzymes and hormones. ELISA is based on: The principle of ELISA is antigen-antibody interaction. Here, the specific antibodies associate or bind to its target antigen. Only when the interaction takes place, the substrate can bind to the enzyme, thereby substrate conversion can be observed, and hence a positive result is obtained. This technique is used to detect HIV. Components of ELISA 1. Antigen Any substance that stimulates an immune response is known as antigen. It is the target protein in the sample that binds to the antibody. 2. Antibody An antibody is a protein made in response to an antigen. Each antibody binds in the epitope region of its antigen. Two types of antibodies; monoclonal or polyclonal can be produced and used as the capture and detection antibodies. The monoclonal antibodies are used as detection antibody as they have mono specificity toward a single epitope and allows fine detection and quantitation of small differences in antigen. 3. Enzyme conjugate An enzyme conjugate is an antibody joined with an enzyme. The joining of the enzyme to antibody is called conjugation. The detection enzyme can be linked directly to the primary antibody or introduced through a secondary antibody that recognizes the primary antibody. The most commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (ALP). 4. Substrate A substrate is a compound that undergoes change. The substrate binds to active sites on the surface of enzyme and gets converted by the change of color. The choice of substrate depends upon the assay sensitivity and instrumentation available for signal-detection such as spectrophotometer, fluorimeter or illuminometer. 19 Types of ELISA ELISA tests can be classified into four types depending upon the different methods used for binding between antigen and antibodies, namely: Direct ELISA –Antigen-coated plate; screening antibody. Indirect ELISA – Antigen is coated to the microtiter well-screening antigen/antibody. Sandwich ELISA – Antibody is coated on the microtiter well; screening antigen Competitive ELISA – Microtiter well which is antigen-coated is filled with the antigen-antibody mixture; screening antibody. 1. Direct ELISA For direct detection, an antigen coated to a multi-well plate is detected by an antibody that has been directly conjugated to an enzyme. This detection method is a good option if there is no commercially available ELISA kits for your target protein. Advantages Quick because only one antibody and fewer steps are used. Cross-reactivity of secondary antibody is eliminated. 2. Indirect ELISA Antibody can be detected or quantitatively determined by indirect ELISA. In this technique, antigen is coated on the microtiter well. Serum or some other sample containing primary antibody is added to the microtiter well and allowed to react with the coated antigen. Any free primary antibody is washed away and the bound antibody to the antigen is detected by adding an enzyme conjugated secondary antibody that binds to the primary antibody. Unbound secondary antibody is then washed away and a specific substrate for the enzyme is added. Enzyme hydrolyzes the substrate to form colored products. The amount of colored end product is measured by spectrophotometric plate readers that can measure the absorbance of all the wells of 96-well plate. 20 Procedure of Indirect ELISA 1. Coat the micro titer plate wells with antigen. 2. Block all unbound sites to prevent false positive results. 3. Add sample containing antibody (e.g. rabbit monoclonal antibody) to the wells and incubate the plate at 37°c. 4. Wash the plate, so that unbound antibody is removed. 5. Add secondary antibody conjugated to an enzyme (e.g. anti- mouse IgG). 6. Wash the plate, so that unbound enzyme-linked antibodies are removed. 7. Add substrate which is converted by the enzyme to produce a colored product. 8. Reaction of a substrate with the enzyme to produce a colored product. Advantages Increased sensitivity, since more than one labeled antibody is bound per primary antibody. A wide variety of labeled secondary antibodies are available commercially. Maximum immunoreactivity of the primary antibody is retained because it is not labeled. Versatile because many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection. Flexibility, since different primary detection antibodies can be used with a single labeled secondary antibody. Cost savings, since fewer labeled antibodies are required. Different visualization markers can be used with the same primary antibody. 3. Sandwich ELISA Antigen can be detected by sandwich ELISA. In this technique, antibody is coated on the microtiter well. A sample containing antigen is added to the well and allowed to react with the antibody attached to the well, forming antigen-antibody complex. After the well is washed, a second enzyme-linked antibody specific for a different epitope on the antigen is added and allowed to react with the bound antigen. Then after unbound secondary antibody is removed by washing. Finally substrate is added to the plate which is hydrolyzed by enzyme to form colored products. 21 Procedure of sandwich ELISA 1. Prepare a surface to which a known quantity of antibody is bound. 2. Add the antigen-containing sample to the plate and incubate the plate at 37°c. 3. Wash the plate, so that unbound antigen is removed. 4. Add the enzyme-linked antibodies which are also specific to the antigen and then incubate at 37°c. 5. Wash the plate, so that unbound enzyme-linked antibodies are removed. 6. Add substrate which is converted by the enzyme to produce a colored product. 7. Reaction of a substrate with the enzyme to produce a colored product. Advantages High specificity, since two antibodies are used the antigen is specifically captured and detected. Suitable for complex samples, since the antigen does not require purification prior to measurement. Flexibility and sensitivity, since both direct and indirect detection methods can be used. 4. Competitive ELISA This test is used to measure the concentration of an antigen in a sample. In this test, antibody is first incubated in solution with a sample containing antigen. The antigen-antibody mixture is then added to the microtitre well which is coated with antigen. The more the antigen present in the sample, the less free antibody will be available to bind to the antigen-coated well. After the well is washed, enzyme conjugated secondary antibody specific for isotype of the primary antibody is added to determine the amount of primary antibody bound to the well. The higher the concentration of antigen in the sample, the lower the absorbance. 22 Procedure of competitive ELISA 1. Antibody is incubated with sample containing antigen. 2. Antigen-antibody complex are added to the microtitre well which are pre-coated with the antigen. 3. Wash the plate to remove unbound antibody. 4. Enzyme linked secondary antibody which is specific to the primary antibody is added. 5. Wash the plate, so that unbound enzyme-linked antibodies are removed. 6. Add substrate which is converted by the enzyme into a fluorescent signal. Advantages High specificity, since two antibodies are used. High sensitivity, since both direct and indirect detection methods can be used. Suitable for complex samples, since the antigen does not require purification prior to measurement. Applications of ELISA The presence of antibodies and antigens in a sample can be determined. It is used in the food industry to detect any food allergens present. To determine the concentration of serum antibody in a virus test. During a disease outbreak, to evaluate the spread of the disease, e.g. during recent COVID-19 outbreak, rapid testing kits are being used to determine presence of antibodies in the blood sample. Detect and Measure the Presence of Antibodies in the Blood Autoantibodies (anti-dsDNA, anti-dsg1, ANA, etc.) Antibodies against infectious disease (antibacterial, antiviral, antifungal), Hepatitis A, B, C, HIV, etc. Detect and Estimate the Levels of Tumor Markers Prostate-specific antigen (PSA), Carcinoembryonic Antigen (CEA) Detect and Estimate Hormone Levels 23  Luteinizing hormone, Follicular stimulating hormone, Prolactin, Testosterone, and Human chorionic gonadotropin (hCG) Tracking Disease Outbreaks Cholera, HIV, and Influenza Detecting Past Exposures HIV, Lyme disease, and Hepatitis Screening Donated Blood for Possible Viral Contaminants anti-HIV-1/2, anti-HCV, and HBsAg Detecting Drug Abuse Amphetamine, Methamphetamine, and Cocaine Benzoylecgonine. Terms Definitions ❖ Solid phase: Usually a microtiter plate well. Specially prepared ELISA plates are commercially available. These have an 8 ¡Á 12 well format and can be used with a wide variety of specialized equipment designed for rapid manipulation of samples including multichannel pipets. ❖ Adsorption: The process of adding an antigen or antibody, diluted in buffer, so that it attaches passively to the solid phase on incubation. This is a simple way for immobilization of one of the reactants in the ELISA and one of the main reasons for its success. ❖ Washing: The simple flooding and emptying of the wells with a buffered solution to separate bound (reacted) from unbound (unreacted) reagents in the ELISA. Again, this is a key element to the successful exploitation of the ELISA. ❖ Antigens: A protein or carbohydrate that when injected into animals elicits the production of antibodies. Such antibodies can react specifically with the antigen used and therefore can be used to detect that antigen. ❖ Antibodies: Produced in response to antigenic stimuli. These are mainly protein ❖ in nature. In turn, antibodies are antigenic. ❖ Antispecies antibodies: Produced when proteins (including antibodies) from one species are injected into another species. Thus, guinea pig serum injected into a rabbit elicits the production of rabbit anti¨Cguinea pig antibodies. ❖ Enzyme: A substance that can react at low concentration as a catalyst to promote a specific reaction. Several specific enzymes are commonly used in ELISA with their specific substrates. ❖ Enzyme conjugate: An enzyme that is attached irreversibly to a protein, usually an antibody. Thus, an example of antispecies enzyme conjugate is rabbit antiguinea linked to horseradish peroxidase. ❖ Substrate: A chemical compound with which an enzyme reacts specifically. This reaction is used, in some way, to produce a signal that is read as a color reaction (directly as a color change of the substrate or indirectly by its effect on another chemical). ❖ Chromophore: A chemical that alters color as a result of an enzyme interaction with substrate. ❖ Stopping: The process of stopping the action of an enzyme on a substrate. It has the effect of stopping any further change in color in the ELISA. ❖ Reading: Measurement of color produced in the ELISA. This is quantified using special spectrophotometers reading at specific wavelengths for the specific colors obtained with particular enzyme/chromophore systems. Tests can be assessed by eye. 24  Flow Cytometry ▪ The basic principle of flow cytometry is the passage of cells in single file in front of a laser so they can be detected, counted and sorted. ▪ Cell components are fluorescently labelled and then excited by the laser to emit light at varying wavelengths. Flow- Cells in motion Cyto- Cell Metry- Measure (Measuring properties of cell while in a fluid stream). Components of Flow Cytometry The basic building blocks of a Flow cytometer are Fluidics, Optics, and Electronics. 1. Fluidics The fluidics system is responsible for transporting sample from the sample tube to the flow cell. Once through the flow cell (and past the laser), the sample is either sorted (in the case of cell sorters) or transported to waste. 2. Optics The components of the optical system include excitation light sources, lenses, and filters used to collect and move light around the instrument and the detection system that generates the photocurrent. 3. Electronics The electronics are the brains of the flow cytometer. Here, the photocurrent from the detector is digitized and processed to be saved for subsequent analysis. 25 Work of a Flow Cytometer The fluorescence can be measured to determine the amount and the type of cells present in a sample. Up to thousands of particles per second can be analyzed as they pass through the liquid stream. A beam of laser light is directed at a hydro dynamically-focused stream of fluid that carries the cells. Several detectors are carefully placed around the stream, at the point where the fluid passes through the light beam. One of these detectors is in line with the light beam and is used to measure Forward Scatter or FSC. Another detector is placed perpendicular to the stream and is used to measure Side Scatter (SSC). Since fluorescent labels are used to detect the different cells or components, fluorescent detectors are also in place. The suspended particles or cells, which may range in size from 0.2 to 150μm, pass through the beam of light and scatter the light beams. The fluorescently labelled cell components are excited by the laser and emit light at a longer wavelength than the light source. This is then detected by the detectors. The detectors therefore pick up a combination of scattered and fluorescent light. This data is then analyzed by a computer that is attached to the flow cytometer using special software. The brightness of each detector (one for each fluorescent emission peak) is adjusted for this detection. Using the light measurements, different information can be gathered about the physical and chemical structure of the cells. Generally, FSC can detect the cell volume whereas the SSC reflects the inner complexity of the particle such as its cytoplasmic granule content or nuclear structure. Applications A few of the major applications used in the scope of modern clinical settings both therapeutic and research oriented include: Protein expression throughout the entire cell, even the nucleus. Protein post translational modifications; including cleaved and phosphorylated proteins. RNA; including both miRNA, and mRNA Transcripts. 26  Cell health status; detection of apoptotic cells or cell death. Identification and characterization of distinct subsets of cells within a heterogeneous sample; including distinguishing central effector memory cells from exhausted T cells or regulatory T cells. Conclusion ▪ First sample containing cells or particles is suspended in a fluid and injected into the flow cytometer, then pass through narrow tube causing cells form single line before pass through laser. ▪ Each cell pass through laser individual, when light hit the cells it scattered in forward and side. Light scattered is characteristic to the cells and their components it detects by doctor. Forward scattered detect the size of cell and side detect shape and complex of cell. The computer put the results in graph each cell has different scattered profile. 27