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Enzyme Immunoassay

Enzyme Immunoassay. Enzyme Immunoassay. The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used as labels

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Enzyme Immunoassay

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  1. Enzyme Immunoassay

  2. Enzyme Immunoassay • The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used as labels • The major prerequisite is that the antigen or antibody must be linked to an enzyme without destroying the immunologic or enzymatic activity of the antigen-antibody complex

  3. ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) • valuable tools for use in clinical labs • can measure biological materials (antibodies or antigens) • inexpensive, rapid, quantitative, specific • sensitive (pg/ml) • expensive equipment not required (but helps) • can be automated

  4. Major labeled immunoassays • RIA • EIA • Chemlum • IFA

  5. Key terms • Analyte • Immunoassay • Lable • Ligand • Reactant • seperation step • Solid phase • substrate

  6. BASIC FORMAT Solid phase = 96 / 384-well microplate

  7. Enzyme labels • Horseradish peroxidase (Horseradish ) • alkaline phosphatase (calf intestine) • Glucose oxidase (Aspergillus niger) • B-galactosidase (Ecoli): - Are not naturally in the patient's sample • - High specific activity • - Stability

  8. Enzymes used in immunoassay systems • must be stable • available in a highly purified state • have a high turnover rate • undergo minimal interference by substances likely to be in the test solution • and be specific for the substrate

  9. Types of protocols in labeled immunoassay • Competitive binding • Non-competitive binding • Sandwich technique • One-step assay • Homogeneous reaction • Heterogeneous reaction

  10. 1. Coat solid phase with antigen when analysing antibody antibody when analysing antigen Analyte = antibody Analyte = antigen Incubate, wash

  11. 2. Block free binding sites. Incubate. Wash. Analyte = antibody Analyte = antigen

  12. 3. Add sample. Incubate. Wash Analyte = antibody Analyte = antigen

  13. E E Enzyme labelled (rabbit) anti-(human) Ig (Second antibody) anti-(rabbit) Ig-enzyme Horse radish peroxidase (HRP) Alkaline phosphatase

  14. SUBSTRATES See Sigma catalogue for list of conjugates and substrates Orthophenylene diamine Tetramethyl hydrochloride (OPD) benzidine (TMP) Horse radish peroxidase (HRP) Orange, 490 nm Yellow, 450 nm Spectrophotometer

  15. The most widely used enzyme in EIA is horseradish peroxidase (HRP). • The substrate of HRP is hydrogen peroxide (H2O2) and the product is oxygen • This oxygen produced during the reaction is used to oxidize a reduced, colorless chromagen (usually reduced orthophenylenediamine) • The final product, oxidized orthophenylenediame, has a brown color

  16. Paranitrophenyl phosphate (PNP) Methyl umbelliferol phosphate Alkaline phosphatase Yellow, 405 nm Methyl umbelliferone Spectrophotometer 365 nm 445 nm Fluorimeter

  17. 5. Add substrate 6. Incubate, stop, measure colour change ENZYME Colourless OD CONCENTRATION

  18. AMPLIFICATION E Directly conjugated developing antibody may give weak signal

  19. amplify with E E unlabelled (rabbit) anti-(human) Ig followed by anti-(rabbit) Ig-enzyme a anti-rabbit labeled antibody

  20. or E S E-SBS-E streptavidin-enzyme streptavidin-enzyme Streptavidin-Enzyme Biotin-labelled anti-Ig followed by streptavidin-enzyme streptavidin-enzymesteptavidin-nzyme

  21. INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES • 3. Anti-(human) Ig-enzyme • 2. Sample (human) antibody • Antigen • ToxoplasmaIgG E E E

  22. INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES 4. Substrate 3. Anti-(human) Ig-enzyme 2. Sample (human) antibody 1. Antigen E E E

  23. ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES 2. Impure antigen eg tissue homogenate 1. Specific antibody

  24. ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES 3. Wash  pure antigen 2. Impure antigen 1. Specific antibody

  25. ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES 5. Anti-human Ig-enzyme 4. Sample (human antibody) 3. Wash  pure antigen 2. Impure antigen 1. Specific antibody E E

  26. ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES 6. Substrate 5. Anti-human Ig-enzyme 4. Sample (human antibody) 3. Wash  pure antigen 2. Impure antigen 1. Specific antibody

  27. ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS eg. hormones drugs tumour antigens cytokines 1. Anti-analyte

  28. ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS E S 3. anti-analyte- biotin followed by streptavidin-enzyme E S E-SBS-E E-SBS-E 2. Sample 1. Anti-analyte

  29. ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS 4. Substrate 3. anti-analyte-biotin followed by streptavidin-enzyme 2. Sample 1. Anti-analyte

  30. CYTOKINES Type 1 Type 2 HEALTH AB (CMI) CMI (AB) IL2 IL12 IFN TNF IL4 IL5 IL6 IL10 CANCER VIRUSES MYCOBACTERIA HELMINTHS ASTHMA, ALLERGY LUPUS RHEUMATOID ARTHRITIS MULTIPLE SCLEROSIS UVEITIS DIABETES 2 1 2

  31. Quadruplicate capture anti-cytokine antibody spots Overlay with standards, samples. Incubate, wash Add fluorophore-detection antibody. Incubate, wash Scan MICROARRAY Eg. Novagen ProteoPlex Std 1 Std 2 IL1 IL4 IL6 IL8 IL12 IL18 IFN TGF IL1 IL2 IL5 IL7 IL10 IL16 gmCSF TNF Std 3 Std 4 Std 5 Std 6 #1 #2 #3 #4 #5 #6 #7 #8 #9 #10

  32. Erroneous Results Caused by antibodies in immunoassay • The application of Abs as an analytical reagent • MoAb novel detection signals • In spite of technologic evolution during past 30 years, still : • False False • Positive Negative

  33. HETEROPHILE ANTIBODIES

  34. Heterophil antibodies • Endogenous antibodies produced against poorly defined antigens • Can react with several animal species (mouse is most common) • Usually react with Fc • IgG or IgM but can be IgA, IgE • Frequency : up to 40% (0.05% may present clinical significance) • Produced by enviromental contact, transplacental passage or viral infection

  35. Human anti-animal antibodies (HAAA) • Response to parenteral administration of animal MoAb • Following radioimaging, cancer therapy, transplant immunotherapy • High concentration, high avidity, epitope specific

  36. The steps to minimize interferences 1. The use of chimeric Abs (animal Fab + human Fc) 2. Using other methods based upon different MoAb 3. Removal of unusual Ab by PEG 4. Making dilutions usually produce non linear results 5. Commercial mixture of animal proteins for pre-analysis incubation

  37. Antibody cross reactivity • Although MoAb enhances specificity , still remains a source of error • Drug assay vs active, non active metabolites • Cross-reactants 1- positive interferent bias 2- Unexpected negative interferent

  38. Hook effect • Unexpected fall in the amount of analyte resulting in the gross under-estimation of the analyte • Particularly in sandwich immunoassays when sample contain extremely high level • Upon further dilution, the result will be out of range • Most commonly occurred in measurement of IgE, hCG, Ferittin, tumor marker, infectious Ag-Ab

  39. Choices of antibodies • Polyclonal • IgG fraction or F (ab') 2 fragments • Affinity purified polyclonal • Monoclonal

  40. How to deal with Hook effect • Run all samples in duplicate dilution • Ensure for adequate washings • Know the level of Hook phenomenon according to manufacturer suggestion • Good communication with clinicians

  41. Rheumatoid Factor, RF • An IgM that act to IgG Fc • 70% RA patients • May bind to rabbit, sheep, goat and mouse Ig • Special technical problem for IgM quantitation • False positivity is common, but competitive inhibitor may cause false negative

  42. How to evade RF interference • Using isolated IgM preparation • Addition of precipitating anti-IgG • Removing the RF using an aggregated IgG • Using IgM capture assay problem can still occur: all IgM-specific antibody should be evaluated cautiously.

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