Viral Serology :General Principles
المؤلف:
Patricia M. Tille, PhD, MLS(ASCP)
المصدر:
Bailey & Scotts Diagnostic Microbiology
الجزء والصفحة:
13th Edition , p816-818
2025-12-21
39
Serology was the primary means of laboratory diagnosis of viral infections until the mid-1970s. At that time, culture and detection of viral antigen became more widely available because of commercially available reagents, such as cell cultures, and a broad range of immunodiagnostic test kits and the production of virus specific monoclonal antibodies to detect viral antigen in patient specimens. Viral serology is now used primarily to determine immune status and to confirm the diagnosis of infection when the virus cannot be cultivated in cell culture or detected readily by immunoassay or molecular assays.
In most viral infections, IgM is undetectable 1 to 4 months after the acute infection resolves, but detectable levels of IgG remain for the life of the patient. If a patient is infected with an antigenically similar virus or the original strain has remained latent and reactivates at a later time, these virus-specific IgG and IgM antibody levels may again rise. The secondary IgM response may be difficult to detect; however, a significant (fourfold) IgG titer rise is readily apparent in immunocompetent patients.
An immune status check measures whether a particular virus has previously infected a patient. A positive result with a sensitive, virus-specific IgG test indicates past infection. Some immune status tests include methods that can detect both IgG and IgM; these are used to identify recent or active infections.
To diagnose active disease, two approaches are helpful. Detection of virus-specific IgM in an acute-phase specimen collected at least 7 to 14 days after the onset of infection indicates current or very recent disease. Detection of a fourfold (or equivalent increase if twofold dilutions are not tested) antibody titer rise between acute and convalescent sera also indicates current or recent disease. Acute-phase serum should be collected as soon as possible after the onset of symptoms. The convalescent specimen should be collected 2 to 3 weeks after the acute-phase specimen. If a single postacute serum, collected between acute and convalescent times, or a convalescent specimen is all that is available for testing, an extremely high, virus-specific IgG titer may suggest infection. The exact titer specific for active disease, if known at all, varies with each testing method and virus. In general, titers high enough to be diagnostic are unusual, and single specimens should not be tested. A reasonable policy would involve using IgM tests, where available, and performing IgG tests only on paired acute and convalescent specimens. IgG tests are not needed on the first, acute specimen until receipt of the convalescent specimen. This eliminates useless testing of single specimens when a second sample is never submitted for analysis.
Many serologic methods are or have been routinely used to detect antiviral antibody. Prominent among these are complement fixation (CF), ELISA, indirect immunofluorescence, anticomplement immunofluorescence (ACIF), and Western immunoblotting. CF is a labor intensive, technically demanding method best fitted to batch testing. As less demanding, easily automated techniques for batch testing are developed (e.g., ELISA), the need for CF testing will disappear. Additional advantages of ELISA are that it can be used to detect IgM-specific antibodies free of common interfering factors, particularly through use of an antibody-capture technique. Indirect immunofluorescence is best used for individual specimens or small-batch testing.
Immunofluorescence also can be used to detect virus specific IgM; however, it requires prior separation and elimination of the IgG fraction, which if present can result in both false-positive and false-negative results. IgM and IgG can be separated by ion exchange chromatography (Figure 1), by immune precipitation, or with an IgG inactivation reagent, such as Gullsorb, (Meridian Bioscience) a reagent containing an anti-human IgG reagent (caprine) capable of neutralizing up to 15 mg/ mL of IgG antibody in human serum.
Fig1. IgM is separated from human serum by passing the serum through an ion exchange column.
IgG indirect FA testing is subject to false-positive results because of antibody-Fc receptors that occur in cells infected with virus. Indirect immunofluorescence antibody (IFA) testing is performed using virus-infected substrate cells fixed to a microscope slide. When the substrate cells are overlaid with patient serum, the Fc portion of the antibody molecule binds to these receptors. Fluorescent-labeled antiglobulin attaches to both homologous antibody (bound to viral antigen) and to Fc-bound antibody. Subsequent fluorescence of Fc-bound antibody results in a false-positive or falsely elevated reading. To avoid this complication, the ACIF test can be used. Because fluorescent-labeled complement binds only to antigen-antibody complexes, the nonspecific anti body attached by Fc receptors, which is complement free, does not fluoresce. Western immunoblotting is also used for viral antibody detection. Because complex antigens are separated into individual components during the Western blot procedure, and positive or negative reactions are observed with each of these components, the Western blot provides a more specific result than other serologic tests, such as EIA.
False-positive and false-negative results can occur when testing for virus-specific IgM antibodies. False-positive results occur when rheumatoid factor, an anti-IgG/IgM type globulin, combines with homologous or virus-specific IgG present in the patient specimen. Labeled anti-IgM combines with either bound virus-specific IgM or rheumatoid factor, causing falsely positive fluorescence. False negative IgM test results occur when high levels of strongly binding homologous IgG antibodies prevent binding of IgM molecules, decreasing or eliminating IgM-specific fluorescence. Both problems can be eliminated by testing the IgG-free serum fraction.
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