Enzyme immunoassays (EIA) unpacked

There are many types of EIA used in microbiology and biochemistry.  Depending on the format of the test, some have alternative names such as ELISA and CLIA (chemoluminescent immunoassay).

There is usually a solid phase, a surface or particle with an attached specific antibody or antigen. A patient sample is added; after time for binding, unbound patient antigen or antibody with a wash step. Then a ‘conjugate’, a non-human antibody with required specificity that has an enzyme attached, is added. A further wash step and then a colourless substrate for the enzyme is added to enable a colour change in the reaction mix proportionate to the amount of bound conjugate. A stop reagent is added to halt the reaction after a precise period. An absorbance value is then obtained with a spectrophotometer.  Automation to achieve higher reproducibility of the result is the norm for modern assays.

SnipImage

Image credit: K. James.

EIAs are used for microbial antigen or antibody detection from a range of fluids and samples. Immunochromatographs (ICTs), also called lateral flow assays, are a special sort of EIA.

  1. How is the absorbance value interpreted?

In general, the absorbance value for the patient sample is compared to a reference standard – this varies according to the assay. Some uncalibrated assays (usually manual assays) require a known low positive control to be run with every assay run. An index value is calculated (index = patient sample absorbance/low positive control).  A ‘reactive’ or positive result equates to an index of greater than 1.1 (this varies with the assay).

A calibrated assay provides an absolute measure of the quantity of a certain antibody –e.g. HBsAb, Rubella IgG.  In these assays, commonly a reference ‘standard’ (generally either WHO or European standards) is run across a dilution series of 5-6 points.  Using the run data, a calibration curve is constructed by the machine to enable the patient result to be expressed as an absolute value – e.g. 20 IU/mL.  These assays are almost always on automated platforms.

  1. How is antibody seroconversion documented using an EIA?

A non-reactive going to a reactive result in the convalescent sample (IgG total or IgG) represents an EIA seroconversion.  Four-fold seroconversion only applies to titred tests like IF assays (see below).  Problems obviously arise if the acute sample is taken too late in the illness or the convalescent sample is taken too early.

  1. What is the significance of a positive IgM EIA result and what are common causes of false positives and false negative IgM results?

Detection of IgM from a single serum sample is less reliable than demonstrating a seroconversion given various considerations below.  However, if the patients symptoms are compatable (i.e. good pretest probability ),  and sufficient time has elapsed since start of the illness – e.g. 7 days for an arbovirus infection, measles, rubella etc.,  then the result is reliable. Earlier detection or confirmation of an IgM often requires direct detection of a microbial antigen or gene sequence- e.g. use of an NS1 antigen test for acute dengue  with illness < 7 days.

False negative IgM results may arise from :

  • Laboratory error
  • Sample taken too early in the illness
  • Immune suppression
  • Competition from pathogen-specific IgG
  • Other interfering substances

False positive IgMs:

  • Rheumatoid factor (a rogue human IgM with IgG Fc specificity)
  • Cross-reacting antibody (e.g. dengue vs chikungunya)
  • Polyclonal B cell stimulation (e.g acute EBV, HIV or Q fever and others)
  • Persistence of IgM out to many months after the illness  – may confuse diagnosis of a second infection
  • Herpesviridae – asymptomatic reactivation may throw up positive IgMs of no significance
  • Immunoglobulin or blood transfusion – exogenous antibody
  1. How do titred serological test results differ from EIA results? 

EIA assays are not usually repeated at different patient serum dilutions. There is one-off result, positive (reactive) or negative (unreactive).  The value of the result (whether an absorbance index or an estimated absolute antibody level) is NOT therefore a titre as such and it is not appropriate to refer to it in that way.

Compare this with complement fixation tests, indirect immunofluorescence tests and similar which are repeated at half step dilutions above a screening dilution to determine the highest titre that exhibits a positive result. We can associate these titres with seroconversion ( > 4 fold rise) but that requires testing of the acute and convalescent serum in parallel.

Whatever the type of test, care required for microbial agents (e.g. Herpesviridae, Toxoplasma, polyoma viruses etc) where persistent latent infection is the norm.  Immunocompetent people will remain seropositive for life and the value fluctuates in a meaningless way.

Reference

James-K. The Immunoserology of Infectious Diseases. Clinical Micro Reviews, 1990. 

Free text link- this is an exhaustive old review. I am not aware of anything more recent that hits the spot.  Please let me know if you know of something!

About mdjkf

Microbiologist and Infectious Diseases Physician
This entry was posted in Module-virology, Part 2 M Med Micro topics and tagged . Bookmark the permalink.

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