Pharmacy 310 Immunoassays & ELISAs Lecture Notes PDF

Pharmacy 310 Immunoassays & ELISAs Dr. Michael R. Doschak Professor Faculty of Pharmacy & Pharmaceutical Sciences University of Alberta Email: [email protected]  LECTURE OUTLINE What are Immunoassays and ELISAs?...

Pharmacy 310 Immunoassays & ELISAs Dr. Michael R. Doschak Professor Faculty of Pharmacy & Pharmaceutical Sciences University of Alberta Email: [email protected]  LECTURE OUTLINE What are Immunoassays and ELISAs?  Components  Assay Formats and Types Clinical Applications and Current Research 3 WHAT IS AN IMMUNOASSAY ? A sensitive analytical test that utilizes highly specific antibody-antigen (Aby-Ag) complexes to produce a signal that can be measured and related to concentration of a compound in solution 3  FEATURES : IMMUNOASSAY Immunoassays differ from biochemical techniques as they measure Aby-Ag interaction – not just a chemical reaction Affinity, Avidity, Specificity and Sensitivity are central to Immunoassays Immunoassays are considered qualititative or semi- quantitative Indicate the presence or absence of analyte, more so than a precise quantification 4 CONCEPTS: Aby-Ag INTERACTION AFFINITY: the degree of attraction between the epitope on an Ag and the Aby binding site (paratope) Close fit between epitope and paratope stabilized by weak, non-covalent forces:  Ionic bonds  Hydrogen bonds  Van der Waals attraction  Hydrophobic effect 5 CONCEPTS: Aby-Ag INTERACTION AVIDITY: Combined strength (or binding intensity) of multiple binding sites for Ag Most Aby isotypes have 2 Ag-binding sites IgM in pentameric form has 10 Ag-binding sites. Even if attraction for the antigen is weak (i.e., low affinity for Ag), pentameric IgM will still have strong Avidity (10 binding sites versus 2) 6 CONCEPTS: Aby-Ag INTERACTION SPECIFICITY: The unique recognition of an antigenic determinant over that of another. Also defined as the property of Aby which enables them to react with some antigenic determinants, but not with others CROSS-REACTIVITY: Aby can bind with low affinity to closely related epitopes. SENSITIVITY: The detection limit of an Aby for an Ag The RIA is capable of sensitivity as low as the femto-mole (10-15) 7  IMMUNE PRECIPITATION Precipitates are formed by cross-linking between Aby-Ag complexes  Ag with multiple epitopes Aby binding will cross-link (i.e., connect) molecules  Witnessed as a precipitate  Termed a “precipitin” line when seen in a gel Precipitin line forms when ratios of Ag and Aby reach equivalence 8 CONCEPTS: Aby-Ag INTERACTION Very similar in nature and kinetics to enzyme-substrate rxns k1 Ag + Aby Ag-Aby k2 [Ag-Aby] K = [Ag] x [Aby] K= association constant Where [Ag], [Aby] and [Ag-Aby] are the molar concentrations 9  DETECTION METHODS Immunodiffusion and Precipitin formation Concentration gradients of Aby and Ag proteins formed by radial diffusion in gel Eventually an Aby to Ag conc. suitable for precipitin formation is achieved at a certain distance from original source of Aby/Ag in gel Immune precipitins are seen as thin white bands http://www.dshs.state.tx.us/lab/serology_id.shtm  DETECTION METHODS Agglutination: Latex particles coated with Aby will be cross- linked by presence of specific Ag in sample IgM: Highly effective at cross- linking and agglutination Agglutination of latex particles is visible due to the increase in particle size Positive Negative Agglutination (No Agglutination)  ASSAY FORMAT TYPES  Competitive  Non-Competitive 14  ASSAY FORMAT TYPES  Homogeneous  Competitive  Heterogeneous  Homogeneous  Non-Competitive  Heterogeneous 15  COMPETITIVE ASSAY Un-labelled analyte (Ag of interest) in test sample is measured based on its ability to compete with labeled Ag in the immunoassay Unlabeled Ag blocks the ability of labeled Ag to bind the Aby. There is a Competition between the two Ag in the system. 16  COMPETITIVE ASSAY The greater the amount of Ag in the test sample, the lesser the signal generated Inverse correlation of signal intensity to the amount of analyte Ag present in sample 17  NON-COMPETITIVE ASSAY Most common format referred to as “Sandwich” Immunoassay Antigen usually “sandwiched” between antibodies specific to two different epitopes The measurement of signal is directly proportional to the amount of target Ag present in the sample 18  HETEROGENEOUS IMMUNOASSAYS Require separation of immuno-complex from the free/unbound forms Separation accomplished by:  Immobilization of Ab on a matrix such as plastic beads, plastic dish with circular wells, or on a test tube surface, such that unbound antigen can be washed away  For small molecular haptens in samples, free hapten can be removed by using absorbent materials such as charcoal and membranes Hapten: a small molecule (not antigenic by itself), that can elicit an immune response only when attached to a larger carrier, such as a protein (notably albumin) 19  HOMOGENEOUS IMMUNOASSAYS Do not require separation of immuno-complex from reaction mix More desirable format for Point-of-Care testing (e.g., immunoassay dip-strips, pregnancy tests) 20  IMMUNOASSAY TYPES Types of Detection Labels and Assay Designs:  Radiolabel: RIA (Radioimmunoassay)  Enzyme: EIA (Enzyme Immunoassay)  ELISA (Enzyme Linked ImmunoSorbent Assay 21  RADIOIMMUNOASSAY (RIA) RIA is a competitive protein binding assay that needs separation techniques (Note: Heterogeneous)  involves competition of un-labeled Ag in sample with radio- labeled Ag as tracer 22  RADIOIMMUNOASSAY (RIA) RIA is a competitive protein binding assay that needs separation techniques (Note: Heterogeneous)  involves competition of un-labeled Ag in sample with radio- labeled Ag as tracer Exam Q: Does the assay signal intensity increase or decrease with greater analyte in the sample? Inverse or proportional? 22  RADIOIMMUNOASSAY (RIA) RIA is a competitive protein binding assay that needs separation techniques (Note: Heterogeneous)  involves competition of un-labeled Ag in sample with radio- labeled Ag as tracer Exam Answer: The assay signal intensity DECREASES with greater analyte in the sample. It is an INVERSE relationship 22 RADIOIMMUNOASSAY (RIA) Non-competitive RIA format also developed Uses labeled secondary antibody (Note: Heterogeneous) 23  RADIOIMMUNOASSAY (RIA) Non-competitive RIA format also developed Uses labeled secondary antibody (Note: Heterogeneous) Exam Q: Does the assay signal intensity increase or decrease with greater analyte in the sample? Inverse or proportional? 23  RADIOIMMUNOASSAY (RIA) Non-competitive RIA format also developed Uses labeled secondary antibody (Note: Heterogeneous) Exam Answer: The assay signal intensity INCREASES with greater analyte in the sample. It is a PROPORTIONAL relationship 23  ENZYME IMMUNOASSAY Most conventional format of immunoassay Ab is immobilized on surface of matrix such as polystyrene/polyacrylamide Mostly involves antibody conjugated to an enzyme that converts substrate to yield a measurable read out (i.e., an ELISA) Usually heterogeneous: involves washing and separation steps Formats used:  Direct ELISA  Indirect ELISA 24  DIRECT ELISA Antigen is coated onto the surface and a corresponding enzyme labelled or fluorescent tagged antibody is used to obtain a read-out ADVANTAGES:  Quick: since only one Ab is used and fewer steps  Cross reactivity of secondary Ab is eliminated DISADVANTAGES:  Labelling by chemical conjugation of the primary Ab with enzymes/fluorescent tags may compromise its immuno-reactivity  Labelling of several primary Abs for experiments can be time consuming and expensive  Limited flexibility between choice of labelled primary antibodies from one experiment to the other  Less scope for signal amplification 25  INDIRECT ELISA INDIRECT ELISA Antigen is coated onto the surface and a specific primary antibody used to bind the antigen. Thereafter, a secondary enzyme-labelled (or fluorescent tagged) antibody specific for the primary antibody is used to obtain a read- out 26  INDIRECT ELISA ADVANTAGES:  Immuno-reactivity of primary Aby is not compromised due to labelling/tagging  Offers potential for signal amplification as primary Aby contains multiple sites for labelled secondary Abs. Thus, assay sensitivity is improved 27  INDIRECT ELISA DISADVANTAGES  Non-specific signal may result from cross reactivity (or non-specific binding) of the labelled secondary antibody  Additional “blocking” incubation step using albumin often required to reduce non-specific adsorption of labelled secondary antibody 28  Test Tube or Multi-Well plate types Mostly used in lab tests Requires spectrophotometry equipment Non-competitive & Heterogeneous format DESIGN AND WORKING:  Capture Aby coated on test tube  Sample containing Ag is added to tube and allowed to bind with Aby  Washed with water/buffer  Then, signaling Aby is added  Incubation of this mixture  Enzyme assay solution is added  Wait for color to develop Intensity of color (absorbance reading) determines concentration of Ag 29  ELISA Using 96 well plate and micropipetter 32  Test Tube or Multi-Well plate types Advantages:  Larger number of samples can be tested at the same time  A colorimeter (e.g., spectrophotometer) will enable a semi- quantitative readout of Antigen amount  Most kits come with +ve and -ve controls for validation Disadvantages:  Several handling and rinsing steps required (may be automated)  Have to consider enzyme kinetics and thus terminate all the test rxns at the same time  Shelf life: Often storage is at 4°C, some formats can be frozen  No good after expiry date (protein Aby denaturation issues) 30  COATED DIP STICK TYPE DESIGN AND WORKING:  Capture Aby are coated on surface of plastic at one end  Stick is dipped into sample  Rinsed  The stick is then dipped into solution of enzyme-Aby conjugate (signaling Aby)  Extensive rinsing to remove unbound signaling Aby  Stick is dipped into solution for enzymatic assay  Appearance of color at Aby- coated end of stick indicates a +ve result 31  LECTURE OUTLINE What are Immunoassays and ELISAs?  Components  Assay Formats and Types Clinical Applications and Current Research 33  IDEAL HOME TESTING KITS Desirable traits:  Should be easy to use, with simple and reliable results for easy interpretation  High specificity  High sensitivity  Immunoassays usually considered qualitative (or semi-quantitative at best) – more sensitive lab- based blood or tissue sample analysis often required to confirm diagnosis 34  LATERAL FLOW TYPE WORKING PRINCIPLE:  Antigen in sample migrates  it encounters signaling Aby  Ag-Aby complexes are formed The soluble complex migrates further until captured by immobilized anti-Antigen polyclonal Aby on strip A line of immobilized anti- signaling Aby is used after the capturing Aby line: this line used to confirm negative result Locations of Aby on +ve -ve Test strip Result Result 35 RAPID COVID-19 IMMUNOASSAY Government of Alberta Rapid Testing Program https://www.alberta.ca/rapid-testing-program.aspx Youtube Video of Rapid COVID-19 antigen test https://youtu.be/LEDib8tjhrI  RAPID COVID-19 IMMUNOASSAY Note: A positive result would show another red line at the T (Test) location, indicating Add 5 COVID-19 drops Antigen in the of test sample sample Negative Result Sample fluid Sample and Aby emulsifies pass 2 immobilized Only one line of Colour-labeled Capture Aby lines Colour-labeledAby Aby which at T (Test) and Immobilized at C starts the C (Control) (Control), proving capillary flow the assay worked  OTC PREGNANCY KITS Based on ELISA principle applied to hCG (human chorionic gonadotropin)  glycoprotein hormone produced by placenta during pregnancy  present in significant amounts in urine of pregnant women  detectable 4-5 days post conception/implantation of embryo hCG molecule:  2 sub-units: α and β  α sub-unit : common to many other hormones  β sub-unit is unique to hCG  Abs against β sub-unit are used in developing immunodiagnostic kits 36  LIMITATIONS  Increase in Ag concentration can result from unrelated physiological or pathological conditions (e.g., trophoblastic disease)  Infectious disease Immunoassay kits mostly measure specific bacterial/viral antigen; may not be able to detect variants/mutants  Dead organisms can give false positives  Complex biological samples can have inhibitors/activators of enzymes used in the assay  Improper Storage conditions can impact stability of Aby/enzymes in test kit  Human error 37  CLINICAL APPLICATIONS Rapid detection of: – Pathogen Antigen (bacterial, viral, fungal) – Drug substance (in patient plasma – for PK) – Hormone / cytokine levels in test body fluid sample – Cancer antigen theranostics – Serological Aby response in patient 38  CLINICAL APPLICATIONS Serological (blood Aby) Tests – Do not detect the pathogen (e.g., virus) itself – They detect the Aby produced in response to previous infection, or in response to vaccination – IgM indicates recent exposure, IgG suggests specific Aby to pathogen – Have been used to inform public health responses https://www.canada.ca/en/health-canada/services/drugs-health- 38 products/covid19-industry/medical-devices/testing/serological.html  REFERENCES Immunodiagnostics and Immunoassay  Chapter 10: Immunology for Pharmacy Students, Shen WC, Louie SG; Harwood Academic Publishers, Netherlands, 1999 The Immune System, Peter Parham; Garland Science Publishing The Immunoassay Handbook (Third Edition), David Wild Ghaffari, A.; Meurant, R.; Ardakani, A. COVID-19 Serological Tests: How Well Do They Actually Perform? Diagnostics 2020, 10, 453. https://doi.org/10.3390/diagnostics10070453 40