Serology — Questions

Both are functional assays, detecting what antibody does rather than that it binds, and both are read as titres.

• Haemagglutination inhibition detects antibody by its ability to block virus-induced agglutination of red cells. It is quick and needs no cell culture, used for agglutinating viruses such as influenza.
• Plaque (neutralisation) assay measures antibody that blocks viral infectivity in cultured cells, counting the reduction in plaques. It correlates with protection and is a reference standard, but it is slow, labour-intensive and uses live virus.

Both have been largely replaced by enzyme immunoassay for routine work, surviving mainly in reference and vaccine-evaluation settings.

A complete answer defines the two performance measures, shows why sensitivity alone is insufficient, and links this to how serological testing is structured.

Sensitivity is the proportion of truly infected people a test calls positive, and a screening assay is deliberately built for it so that few true cases are missed. But sensitivity says nothing about the false positives a test produces. Specificity, the proportion of truly uninfected people called negative, governs that, and a test can be very sensitive yet poorly specific.

The practical consequence is that predictive value depends on prevalence. In a low-prevalence setting, even a specific test yields a low positive predictive value, because most positives are false. A single sensitive result is therefore not a diagnosis.

This is why serology is built as screen-then-confirm: a sensitive first assay to avoid missing infection, then a specific confirmatory assay to remove false positives, with every result read against the patient’s pre-test probability.

A complete answer tracks each generation by its antigen, what it detects, and how each step narrowed the window between infection and a positive result.

First-generation assays coated the plate with whole viral lysate and detected IgG only, so they were relatively insensitive early and prone to cross-reactivity.

Second-generation assays replaced lysate with recombinant proteins or synthetic peptides, improving specificity and sensitivity.

Third-generation assays used an antigen-sandwich (double-antigen) format that detects IgM as well as IgG, becoming positive earlier and so shortening the window.

Fourth-generation assays are combined tests that add p24 antigen detection to antibody detection. Because p24 appears before antibody, this shortens the window further, narrowing the interval in which a recently infected, infectious person tests negative.

The patient’s antibody is sandwiched between antigen on both sides, so the format detects virus-specific antibody, not antigen. Needing specific binding on both arms also makes it more specific than an indirect assay.

It is not an antigen assay here, and it will not register rheumatoid factor, non-specific immunoglobulin or complement, because only a specific antibody can bridge the two antigens.

A fourfold or greater rise in IgG titre across paired sera shows an active, evolving antibody response and so marks recent infection. No change would indicate past exposure.

A stable titre is past immunity or carriage, a genuine rise is not an error, and passive antibody would decline rather than rise.

p24 antigen appears before antibody, so a combined assay that detects it alongside antibody turns positive earlier and narrows the window. It remains a serological assay, not a molecular one.

Fourth-generation assays do not detect viral RNA (that is a nucleic acid test), and neutralising antibody, avidity and rheumatoid factor play no part in shortening the window.

Antibody is absent for some weeks after infection, so a sample taken this early falls in the diagnostic window and reads negative even though infection is present. A convalescent sample is needed.

The virus does induce antibody in time; rheumatoid factor and the hook effect cause false results in other settings; and passive maternal antibody is irrelevant in an adult.

At an extremely high analyte level the capture and detector reagents are saturated separately, so few complete sandwiches form and the result reads falsely low or negative: the high-dose hook effect. Diluting the sample restores a positive.

The window and passive antibody concern antibody assays, rheumatoid factor causes false positives, and an assay defeated by excess antigen is not one of low sensitivity.

The difference is the label: ECLIA uses a ruthenium label that emits light when triggered electrically at an electrode, rather than the enzyme-and-substrate colour reaction of an ELISA. The immunoassay format is otherwise unchanged.

Both can detect antigen or antibody, both use a solid phase, and ECLIA is in fact the basis of fully automated high-throughput analysers, not a functional cell-culture assay.

In the indirect format, patient antibody binds the fixed antigen, and the enzyme-labelled anti-human immunoglobulin then binds that patient antibody, generating signal.

The detector does not bind the antigen or free antigen directly, there is no capture antibody in an indirect assay, and it binds human immunoglobulin generally, not only rheumatoid factor.

Antibody avidity matures with time, so low-avidity IgG marks recent primary infection while high-avidity IgG marks past infection. This is why avidity testing helps date an infection, notably in pregnancy.

Past infection gives high avidity, passive antibody reflects the donor’s mature response, and avidity is a property of genuine antibody rather than an artefact or a reactivation marker.

IgG rises later than IgM and persists, so IgG without IgM signifies past infection and often immunity. This is the pattern used to define immune status.

Susceptibility would show neither antibody; acute infection would show IgM; and the result is a normal mature response, not an artefact.

Antibody from blood crosses a damaged blood-brain barrier, so CSF antibody is diagnostic only when intrathecal synthesis is shown, by comparing the CSF-to-serum ratio of specific antibody against that of total IgG or albumin.

Isotype, assay method, titre and fever do not by themselves distinguish local production from passive transfer.

By capturing the patient’s IgM first, the format removes IgG before antigen is added, so it avoids both the rheumatoid-factor false positive and the false negative from competing specific IgG.

It is not chosen for cost or speed, it targets IgM specifically rather than IgG, it still requires viral antigen, and it does not measure avidity.

a. The discordance most likely represents a false-positive screen. The screening assay is built for sensitivity, so it produces some false positives, and in a person with low pre-test probability a true positive is unlikely; the specific confirmatory assay is the more trustworthy result here.

b. The usual explanation is cross-reactivity or non-specific binding in the sensitive screen. The alternative that must be excluded is very early infection within the window, when antibody is only beginning to appear and the confirmatory assay has not yet turned positive.

c. Repeat testing on a fresh sample and a later follow-up sample, and resolve with a more specific confirmatory assay or a molecular test; interpret throughout against the clinical picture rather than acting on the screen alone.