Enzyme Immunoassay

Enzyme ImmunoassayThe 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

• 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

Major labeled immunoassays

RIAEIAChemlumIFA

Key terms

AnalyteImmunoassayLableLigandReactantseperation stepSolid phasesubstrate

BASIC FORMAT

Solid phase = 96 / 384-well microplate

Enzyme labelsHorseradish 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

Enzymes used in immunoassay systemsmust be stableavailable in a highly purified statehave a high turnover rateundergo minimal interference by substances

likely to be in the test solutionand be specific for the substrate

Types of protocols in labeled immunoassay

Competitive bindingNon-competitive bindingSandwich techniqueOne-step assayHomogeneous reactionHeterogeneous reaction

Analyte = antibody Analyte = antigen

Incubate, wash

1. Coat solid phase withantigen when analysing antibody

antibody when analysing antigen

2. Block free binding sites. Incubate. Wash.

Analyte = antibody Analyte = antigen

3. Add sample. Incubate. Wash

Analyte = antibody Analyte = antigen

Enzyme labelled (rabbit) anti-(human) Ig

(Second antibody)anti-(rabbit) Ig-enzyme

EE

Horse radish peroxidase (HRP)Alkaline phosphatase

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

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

Paranitrophenyl phosphate (PNP) Methyl umbelliferol phosphate

Alkaline phosphatase

Yellow, 405 nm Methyl umbelliferone Spectrophotometer

365 nm 445 nm Fluorimeter

5. Add substrate

6. Incubate, stop, measure colour change

Colourless

ENZYME

OD

CONCENTRATION

E

Directly conjugated developingantibody may give weak signal

unlabelled (rabbit) anti-(human) Ig followed by

anti-(rabbit) Ig-enzyme

EE

a anti-rabbit labeled antibody

Biotin-labelled anti-Ig followed by

streptavidin-enzyme

E-S B S-E

ES

streptavidin-enzymesteptavidin-nzyme

streptavidin-enzyme

streptavidin-enzyme

Streptavidin-Enzyme

INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES

E E E

3. Anti-(human) Ig-enzyme

2. Sample (human) antibody

1.Antigen

Toxoplasma IgG

INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES

E E E

4. Substrate

3. Anti-(human) Ig-enzyme

2. Sample (human) antibody

1. Antigen

ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES

2. Impure antigen eg tissue homogenate

1. Specific antibody

ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES

3. Wash pure antigen

2. Impure antigen

1. Specific antibody

ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES

E E5. Anti-human Ig-enzyme

4. Sample (human antibody)

3. Wash pure antigen

2. Impure antigen

1. Specific antibody

ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES

6. Substrate5. Anti-human Ig-enzyme

4. Sample (human antibody)

3. Wash pure antigen

2. Impure antigen

1. Specific antibody

ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS

eg. hormones drugs tumour antigens cytokines

1. Anti-analyte

ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS

1. Anti-analyte

2. Sample

3. anti-analyte- biotin followed by streptavidin-enzyme

E-S B S-E

ES

E S E-S B S-E

ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS

1. Anti-analyte

2. Sample

4. Substrate

3. anti-analyte-biotin followed by streptavidin-enzyme

1

2

2

HEALTH

CANCER VIRUSES

MYCOBACTERIAHELMINTHS

ASTHMA, ALLERGYLUPUS

RHEUMATOID ARTHRITISMULTIPLE SCLEROSIS UVEITISDIABETES

IL2IL12IFNTNF

IL4 IL5 IL6 IL10

CMI(AB)

Type 1 Type 2

AB(CMI)

CYTOKINES

MICROARRAYEg. Novagen ProteoPlex

Std 1

Std 3 Std 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 spotsOverlay with standards, samples. Incubate, washAdd fluorophore-detection antibody. Incubate, washScan

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

Heterophil antibodiesEndogenous 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

Human anti-animal antibodies (HAAA)Response to parenteral administration of animal MoAb

Following radioimaging, cancer therapy, transplant immunotherapy

High concentration, high avidity, epitope specific

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

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

Hook effectUnexpected 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

Choices of antibodiesPolyclonal

IgG fraction or F (ab') 2 fragments

Affinity purified polyclonal

Monoclonal

How to deal with Hook effect1. 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

Rheumatoid Factor, RFAn IgM that act to IgG Fc70% RA patientsMay bind to rabbit, sheep, goat and mouse IgSpecial technical problem for IgM quantitationFalse positivity is common, but competitive inhibitor may cause false negative

How to evade RF interference1. Using isolated IgM preparation

2. Addition of precipitating anti-IgG

3. Removing the RF using an aggregated IgG4. Using IgM capture assay problem can still occur: all IgM-specific antibody should be

evaluated cautiously.