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Antibody Detection CHAPTER 5 Antibody Detection KENNETH L. HERRMANN Subject: Serodiagnosis (use of known antigens as reagents to detect antibodies in patient sera for diagnosing recent or past viral infection). Serodiagnostic Principles: Mechanism of antibody production, response patterns to pri­ mary and recurrent infections, and significance of antibody to immunity in viral dis­ eases. Laboratory Methods: Neutralization, complement fixation, hemagglutination inhibition, immune adherence hemagglutination, passive agglutination, hemolysis in gel, radioim­ munoassay, enzyme immunoassay, indirect immunoftuorescencelftuoroimmunoassay, and Western immunoblotting. Interpretation of Test Results: Significance of presence of antibody or change in antibody level. Value of immunoglobulin type-specific assays in viral serodiagnosis. Problems and Pitfalls: Insensitive and nonspecific serodiagnostic tests resulting from heter­ ologous cross-reactions, immunologic interference, substandard reagents, or improper test performance. Introduction must have the proper antigen to detect antibody to this agent. Antibody detection plays an important role in the investigation of viruses as causes of human illness. Viral serology provides both clinicians and epidemi­ Immunoglobulin Specificity ologists with a powerful tool for diagnosing infection in an individual patient, for determining the suscepti­ The most basic feature of the immune response of bility of a person or population to a specific virus, the host to a foreign antigen is its specificity for that and for assessing the prevalence of a particular virus particular antigen. Our understanding of antibody in a community. Technologic advances during the specificity at the structural and the genetic level has past several years have led to more rapid, sensitive, been enhanced greatly during the past few years with and accurate tests for detecting and measuring anti­ the application of monoclonal antibodies to the anti­ viral antibodies. The development of automated genic and structural characterization of viral proteins technology has made these tests easier to perform and with immunochemical or genetic mapping of the and has encouraged more clinical laboratories to use antibody-combining sites on the immunoglobulin (Ig) them. molecule. The term serodiagnosis denotes diagnosis based The mechanism by which antibodies are formed on procedures that include known antigens as re­ has been debated for years. It is now generally ac­ agents to detect and measure antibodies in patient cepted that each person is endowed with a large pool serum. To use such tests to diagnose an infectious of B lymphocytes that possess surface receptors ca­ disease, one must suspect a specific causative agent pable of responding to or being triggered by an anti­ basedE. H. Lennetteon clinical et al., or Laboratory epidemiologic Diagnosis data. of Infectious One Diseasesalso genPrinciples (Mims and and Practice White, 1984). The initial step in anti- © Springer-Verlag New York Inc. 1988 5. Antibody Detection 77 body formation is phagocytosis of the invading efficient for attaching the antigen. Over time, the pathogen, the viral particle, by macrophages and the cells producing IgM switch to producing IgG. The presentation of specific viral antigens in some form mechanism for this switch involves genes controlling to B cells. When a B cell encounters the antigen, it the variable regions of the heavy chain of the IgM binds the antigens with receptors complementary to molecule; these genes recombine with genes control­ any of the several antigenic determinants (epitopes) ling the constant regions of IgG so that the IgG pro­ on that antigen. After receiving the appropriate anti­ duced is of the same specificity as the IgM. The IgG gen-specific and nonspecific signals from helper T antibody often has greater avidity for the antigen and cells, the B cell responds by dividing and differentiat­ is able to bind complement. ing into antibody-secreting plasma cells. Antibodies are either secreted into the blood or lymphatic fluid by plasma cells or remain attached to the surface of Antibody Responses to lymphocytes or other effector cells. Each clone of Viral Infections plasma cells secretes antibody of only a single Ig class corresponding to the particular receptors it ex­ Traditional serodiagnosis is based on the detection of presses and specific to the epitope that triggered it. specific antibody in the serum of the infected person. The binding strength of a specific antibody receptor Circulating antiviral antibodies, particularly neutral­ with its corresponding epitope is referred to as the izing antibodies, are widely accepted as prima facie affinity of the reaction. evidence of past infection with the particular virus. A single virus may contain many different anti­ Current or very recent infection is usually detected gens that the host will recognize as foreign, so infec­ by an initial antibody response or an increase in the tion with one virus may cause many different anti­ level of specific antibody. Most viruses induce de­ bodies to appear. In addition, some antigenic tectable levels of specific antibodies after a primary determinants of a virus may not be available for rec­ infection and may boost the titer after reinfection or ognition by the host until the virus has been dis­ reactivation of latent infection. Serodiagnosis of a rupted by enzymatic action. Certain internal, or viral infection generally requires paired acute- and core, antigens of a virus may not be "visible" to the convalescent-phase serum samples. Confirmation of host immune system until the virion has been broken a suspected virus infection usually requires demon­ down to reveal these internal components. strating a significant rise of specific antibody, not When large amounts of antigen are present, as in merely the presence of antibody to the viral agent. the early stages of an acute infection, the antigen­ In some situations, however, an antibody rise reactive B cells may be triggered even if their recep­ may not be detected in conjunction with an acute tors fit the epitope with relatively poor binding viral infection. Such exceptions include 1) cases in strength. Antibody secreted by the resulting plasma which antibodies had reached peak levels before the cell clones are thus likely to have low affinity for the acute-phase ·serum sample was collected, 2) infec­ antigen. Later, when only small amounts of antigen tions in immunocompromised hosts, 3) superficial in­ are present, B cells with receptors of high antigen fections, such as respiratory infections, that fail to binding strength are selected; hence the affinity of induce a humoral antibody response despite signifi­ the antibody secreted increases correspondingly, cant illness, and 4) acute infections acquired in the sometimes loo-fold. presence of passively transferred antibody. Congeni­ Each antibody-producing cell makes only one tal and neonatal infections may be difficult to diag­ specificity of antibody. The spleen and lymph nodes nose by antibody rise because high levels of placen­ receive foreign antigens via the blood or lymphatic tally transferred maternal antibody are often present system and synthesize humoral antibodies, mainly of in the newborn's serum and may mask the develop­ the IgM and IgG classes. On the other hand, the ment of specific antibodies by the newborn infant. interstitial lymphoid follicles of the respiratory and As variables inherent in serologic procedures can alimentary tracts, such as the tonsils and Peyer's result in substantial differences in the results ob­ patches, receive antigens directly from the overlying tained from one sample tested at different times, epithelial cell and secrete local antibodies mainly of acute- and convalescent-phase samples must be the IgA class. Viremic infections lead to the produc­ tested together in the same run for differences in tion of both specific IgM and IgG antibodies in most antibody levels to be meaningfully interpreted. In persons. Localized virus infections, however, often tests that use doubling dilutions of the serum, a four­ stimulate little or no increase of humoral IgM or IgG fold or greater rise in antibody titer has traditionally antibody levels. The larger number of antigen bind­ been considered significant, whereas a twofold dif­ ing sites on the IgM molecules may help to clear the ference is considered within the technical error of the invading pathogen more quickly, even though each technique (Hall and Felker, 1970). In tests using individual antigen-binding site may not be the most other methods of quantitating antibody, significant 78 K. L. Herrmann differences in antibody levels must be established agents other than the one responsible for the current independently (Wood and Durham, 1980). Fre­ infection (Morgan-Capner et aI., 1983). Such true quently, a critical ratio of convalescent- to acute­ heterologous IgM responses are probably rare, but phase antibody levels is used to define a significant should be considered when the results of IgM tests antibody rise. are used in determining clinical management. As mentioned previously, each virus generally possesses several antigens, some of which are spe­ Exogenous Reinfection cific for the particular strain of virus and others that are shared by related viruses. Infection with a partic­ The antibody response to viral reinfection is often ular virus commonly stimulates antibody responses quite brisk and usually begins within a day or two to several viral antigens, and
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