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

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Presentation on theme: "Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used."— Presentation transcript:

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4 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

5 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

6 Major labeled immunoassays RIA EIA Chemlum IFA

7 Key terms Analyte Immunoassay Lable Ligand Reactant seperation step Solid phase substrate

8 BASIC FORMAT Solid phase = 96 / 384-well microplate

9 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

10 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

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

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

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

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

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

16 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

17 The most widely used enzyme in EIA is horseradish peroxidase (HRP). The substrate of HRP is hydrogen peroxide (H 2 O 2 ) 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

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

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

20 AMPLIFICATION E Directly conjugated developing antibody may give weak signal

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

22 or Biotin-labelled anti-Ig followed by streptavidin-enzyme E-S B S-E ESES streptavidin-enzymesteptavidin-nzyme streptavidin-enzyme Streptavidin- Enzyme

23 INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES E E E 3. Anti-(human) Ig-enzyme 2. Sample (human) antibody 1.Antigen Toxoplasma IgG

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

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

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

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

28 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

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

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

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

32 1 2 2 HEALTH CANCER VIRUSES MYCOBACTERIA HELMINTHS ASTHMA, ALLERGY LUPUS RHEUMATOID ARTHRITIS MULTIPLE SCLEROSIS UVEITIS DIABETES IL2 IL12 IFN TNF IL4 IL5 IL6 IL10 CMI (AB ) Type 1Type 2 AB (CMI ) CYTOKINES

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

34 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

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36 HETEROPHILE ANTIBODIES

37 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

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

39 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

40 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

41 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

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

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44 How to deal with Hook effect 1. Run all samples in duplicate dilution 2. Ensure for adequate washings 3. Know the level of Hook phenomenon according to manufacturer suggestion 4. Good communication with clinicians

45 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

46 How to evade RF interference 1. Using isolated IgM preparation 2. Addition of precipitating anti-IgG 3. Removing the RF using an aggregated IgG 4. Using IgM capture assay problem can still occur: all IgM-specific antibody should be evaluated cautiously.

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