SEROLOGICAL/IMMUNOLOGICAL METHODS

SEROLOGICAL/IMMUNOLOGICAL METHODS

As the discipline of virology was emerging, the techniques of immunology were also developing, and, as with molecular biology more recently, the two disciplines have always been very closely linked. Understanding mechanisms of immunity to virus infections has, of course, been very important. Recently, the role that the immune system itself plays in pathogenesis has become known (see Chapter 7). Immunology as a discipline in its own right has contributed many of the classical techniques to virology (Figure 1.2). George Hirst, in 1941, observed haemagglutination of red blood cells by influenza virus (see Chapter 4). This proved to be an important tool in the study of not only influenza but also several other groups of viruses—for example, rubella virus. In addition to measuring the titre (i.e., relative amount) of virus present in any preparation, this technique can also be used to determine the antigenic type of the virus. Haemagglutination will not occur in the presence of antibodies that bind to and block the virus haemagglutinin. If an antiserum is titrated against a given number of haemagglutinating units, the haemagglutination inhibition titre and specificity of the antiserum can be determined.Also, if antisera of known specificity are used to inhibit haemagglutination, the antigenic type of an unknown virus can be determined. In the 1960s and subsequent years, many improved detection methods for viruses were developed, such as:

■ Complement fixation tests

■ Radioimmunoassays

■ Immunofluorescence (direct detection of virus antigens in infected cells or tissue)

■ Enzyme-linked immunosorbent assays (ELISAs)

■ Radioimmune precipitation

■ Western blot assays

These techniques are sensitive, quick, and quantitative.

In 1975, George Kohler and Cesar Milstein isolated the first monoclonal antibodies from clones of cells selected in vitro to produce an antibody of a single specificity directed against a particular antigenic target. This enabled virologists to look not only at the whole virus, but at specific regions—epitopes—of individual virus antigens (Figure 1.3). This ability has greatly increased our understanding of the function of individual virus proteins. Monoclonal antibodies are also finding increasingly widespread application in other types of serological assays (e.g., ELISAs) to increase their reproducibility, sensitivity, and specificity.

It would be inappropriate here to devote too much discussion to the technical details of what is also a very rapidly expanding field of knowledge; however, I strongly recommend that readers who are not familiar with the techniques mentioned above should familiarize themselves thoroughly with this subject by reading one or more of the texts given in the Further Reading for this chapter.

Figure 1.2 It is difficult to overestimate the importance of serological techniques in virology.The four assays illustrated by the diagrams in this figure have been used for many years and are of widespread value. (a) The complement fixation test works on the basis that complement is sequestered by antigen–antibody complexes. ‘Sensitized’ antibody-coated red blood cells, known amounts of complement, a virus antigen, and the serum to be tested are added to the wells of a multiwell plate. In the absence of antibodies to the virus antigen, free complement is present which causes lysis of the sensitized red blood cells (haemolysis). If, however, the test serum contains a sufficiently high titre of antivirus antibodies, then no free complement remains and haemolysis does not occur.Titrating the test serum by means of serial dilutions allows a quantitative measurement of the amount of antivirus antibody present to be made. (b) Immunofluorescence is performed using derivatized antibodies containing a covalently linked fluorescent molecule that emits a characteristically coloured light when illuminated by light of a different wavelength, such as rhodamine (red) or fluorescein (green). In direct immunofluorescence, the antivirus antibody itself is conjugated to the fluorescent marker, whereas in indirect immunofluorescence a second antibody reactive to the antivirus antibody carries the marker. Immunofluorescence can be used not only to identify virus-infected cells in populations of cells or in tissue sections but also to determine the subcellular localization of particular virus proteins (e.g., in the nucleus or in the cytoplasm). (c) Enzyme-linked immunosorbent assays (ELISAs) are a rapid and sensitive means of identifying or quantifying small amounts of virus antigens or antivirus antibodies. Either an antigen (in the case of an ELISA to detect antibodies) or antibody (in the case of an antigen ELISA) is bound to the surface of a multiwell plate. An antibody specific for the test antigen, which has been conjugated with an enzyme molecule (such as alkaline phosphatase or horseradish peroxidase), is then added. As with immunofluorescence, ELISA assays may rely on direct or indirect detection of the test antigen. During a short incubation, a colourless substrate for the enzyme is converted to a coloured product, thus amplifying the signal produced by a very small amount of antigen. The intensity of the product can easily be measured in a specialized spectrophotometer (‘plate reader’). ELISA assays can be mechanized and are therefore suitable for routine tests on large numbers of clinical samples. (d) Western blotting is used to analyse a specific virus protein from a complex mixture of antigens.Virus antigen-containing preparations (particles, infected cells, or clinical materials) are subjected to electrophoresis on a polyacrylamide gel. Proteins from the gel are then transferred to a nitrocellulose or nylon membrane and immobilized in their relative positions from the gel. Specific antigens are detected by allowing the membrane to react with antibodies directed against the antigen of interest. By using samples containing proteins of known sizes in known amounts, the apparent molecular weight and relative amounts of antigen in the test samples can be determined.

Figure 1.3 Monoclonal antibodies are produced by immunization of an animal with an antigen that usually contains a complex mixture of epitopes. Immature B-cells are later prepared from the spleen of the animal, and these are fused with a myeloma cell line, resulting in the formation of transformed cells continuously secreting antibodies. A small proportion of these will make a single type of antibody (a monoclonal antibody) against the desired epitope. Recently, in vitro molecular techniques have been developed to speed up the selection of monoclonal antibodies, although these have not yet replaced the original approach shown here.