Mechanisms and application of enzyme immunoassays

Non-competitive EIA for antibody detection: components

Unknown: antibody

Solid phase: antigen

Non-competitive EIA for antibody detection: Results

Positive = color change

Negative = no color change

Non-competitive EIA for antibody detection: steps

1. add patient serum to solid phase and incubate

2. rinse and add conjugate

3. incubate and rinse

4. add substrate

5. results

Competitive EIA for antibody detection: Components

Unknown: antibody

Solid phase: antigen

Competitive EIA for antibody detection: Results

Positive = no color change

Negative = color change

Competitive EIA for antibody detection: Steps

1. add patient serum and conjugate to solid phase

2. incubate and rinse

3. add substrate

4. results

Non-competitive EIA for antigen detection: components

Unknown: antigen

Solid phase: antibody

Non-competitive EIA for antigen detection: results

Positive = color change

Negative = no color change

Non-competitive EIA for antigen detection: steps

1. add patient serum to solid phase

2. incubate and rinse

3. add enzyme labeled antiviral antibody

4. incubate and rinse

5. add substrate

6. results

Advantages and disadvantages of EIA testing

Advantages:

• standardized

• large volume testing

• cost effective

• less technical skill

• Qualitative and Quantitative

Disadvantages:

• unable to evaluate quality

• unable to distinguish specific vs non-specific

• may require repeat and/or confirmatory testing

IgM antibody capture methods

• Anti-human IgM bound to solid phase

• patient serum added and incubated

• IgM captured

• Rinse away IgG

• continue test assay

Serological assay types able to be modified for IgM specific testing

• Non-competitive EIA but with enzyme labeled anti-human IgM

• indirect immunofluorescence but with fluorescein labeled anti-human IgM

False positive and negative interference of IgM specific tests by IgG or rheumatoid factor IgM

• persistent IgM production

• Cross reactions or polyclonal activation of IgM

Solid phase of: BioMerieux vidas

Solid phase receptacle: coated pipette tips

Solid phase of: Microwell-based EIA instruments

Micowell wall

Testing steps common to BioMerieux Vidas

• Add patient serum to antigen well

• wash

• add conjugate

• add substrate

General format of the Multiplex Micro-bead system

Unit of measurement for chemiluminescence immunoassay (CLIA)

Emitted light

Reporting for Qualitative format by ELA testing

• Positive or negative for antibody detected

• no numerical value reported

Reporting for Quantitative format by ELA testing

• Numerical value reported

• Absorbance vs cutoff

Index value and how to calculate it

• Quantitative testing

• Index value: patient absorbance / cutoff absorbance

  • Greater than 1 is positive

  • Less then 1 is negative

Immunochromatography/lateral flow testing: mechanisms

Immunochromatography/lateral flow testing: components

Labeled monoclonal antibodies

• Sample line/area

Monoclonal antibodies to analyte being detected

• Test line

Monoclonal antibodies to IgG

• control line

Immunochromatography/lateral flow testing: results

Positive = both test and control line has color change

Negative = only control line has color change

Immunochromatography/lateral flow testing: Advantages and Disadvantages

Advantages:

• No technical expertise required

• Single step

• Turn around time is fast

• no special equipment needed

Disadvantages:

• limited antigen available

• cost per test may be high

• specimen quality is unable to evaluate

• sensitivity is low

Sensitivity in regard to evaluation

• ability of an assay to detect positive results

• use known POSITIVE samples

• # of true positives/ (# true positive + # of false negative) X 100

Specificity in regard to evaluation

• Ability of an assay to detect a negative result

• use known NEGATIVE samples

• # of true negatives (# of true negative + # of false positives) X 100