3. 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

4. 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

5. Major labeled immunoassays
RIA
EIA
Chemlum
IFA

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

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

8. 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

9. 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

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

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

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

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

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

15. 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

16. 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

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

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

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

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

21. Biotin-labelled anti-Igor E S
E-S B S-E
streptavidin-enzyme
streptavidin-enzyme
Streptavidin-Enzyme
Biotin-labelled anti-Ig
followed by
streptavidin-enzyme
streptavidin-enzymesteptavidin-nzyme

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

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

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

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

26. 4. Sample (human antibody) 3. Wash  pure antigen 2. Impure antigen
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

27. 4. Sample (human antibody)
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

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

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

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

31. 1 2 CYTOKINES Type 1 Type 2 AB (CMI) CMI (AB) HEALTH IL2 IL12 IFN
TNF
IL4
IL5
IL6 IL10
CANCER
VIRUSES
MYCOBACTERIA
HELMINTHS
ASTHMA, ALLERGY
LUPUS
RHEUMATOID ARTHRITIS
MULTIPLE SCLEROSIS
UVEITIS
DIABETES 2 1 2

32. MICROARRAY #1 #2 #3 #4 #5 #6 #7 #8 #9 #10
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

33. 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

35. HETEROPHILE ANTIBODIES

36. 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

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

38. 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

39. 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

40. 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

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

43. 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

44. 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

45. 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.