The Antibody

Species origin tests, used to determine whether the specimen is human or from another source, are immunological in nature. Host animals, usually rabbits, are injected with protein from another species. The animal creates antibodies to the unknown material. Semm from the host animal, containing species (human, bovine, equine, canine, etc) specific antibodies, is tested against a dilute solution of blood (antigens) collected as evidence. A positive reaction is determined by a visible band where the antibodies and antigens come into contact. 

If an antibody to the protein of interest is available, it is sometimes possible to use vector sequences, eg, the beta-galactosidase promoter sequence, to direct the transcription of the passenger DNA into messenger RNA and the translation of that mRNA into protein which can be recognized by the antibody. Although this method is somewhat less reHable than the use of nucleic acid probes, specialized vectors are available for this purpose. 

The definition of an association constant for an antibody—antigen reaction can become more complex if the antibody—antigen reaction iavolves a multivalent antigen, as is the case when a polyclonal antisemm is used for detection of an antigen. This type of multivalent binding is termed avidity and is defined by the equation ... 

Definition of the association (or avidity) constant for such multivalent antibody—antigen reactions must consider not only the heterogeneity of the antibodies and the antigen determinant site(s), but also an apparent additive effect of binding two antigen molecules to a single antibody. Such effects lead... 

Fig. 2. The basic approach for a competitive immunoassay. The analyte (A) and analyte-indicator ( 0) compete for sites on the antibody which...

Most modem RJAs utilize a competitive assay format (Fig. 2) in which radiolabled antigen, Ag, competes with unlabeled antigen, Ag, in a sample for binding to the antibody. Ah. The free antigens are then separated from the antigen—antibody complexes, and the amount of radioactivity in the... 

EIAs can be used per se or with a spectrophotometer. Traditionally, EIAs have been developed in 96-weU microtiter plates which provide the immobilization support for the assay, the reaction vessel, and, when linked to a spectrophotometer-based reader, a rapid means to detect and quantify the color resulting from interaction of a substrate with the antibody—antigen—enzyme complex. Automated immunoassay analyzers targeted primarily for use in the clinical laboratory have taken automation one step further, utilizing robotics to carry out all reagent additions, washings, and final quantification including report preparation. 

As shown in Table 2, free DAS, as expected, is its own best displacing agent, whereas only DAS—HMS showed any appreciable displacing capabiUty. This can be expected because the hemisuccinate linker is also immunogenic and leads to the production of antibodies specific for the linker in the polyclonal antibody population. AH the other toxins had at least lOOx less the avidity for the antibody, illustrating the specificity of the aDAS for DAS. 

Most immunoassay kits and many commercial immunoassay analyzers are based on heterogenous EIA or FIA. These include an immunoassay system that uses FIA linked to radial partition chromatography of the antibody—antigen complex (39) a system that uses antibody-coated tubes for enzyme immunoassay of a variety of hormones and dmgs (40) and a system that uses either a sandwich or competitive FIA format to measure a variety of analytes (41). 

In 1975, the first successful production of MAbs was reported (44). By fusing normal antibody-producing cells with a B-ceU tumor (myeloma), hybridoma cell lines resulted which produced antibodies having a specificity to only one deterrninant on an antigen ie, all the antibodies produced from the cell line are identical. These studies resulted in a standard approach to MAb production. In this approach, the hybridoma cells are produced in large quantities in culture and screened to select specific clones producing the desired MAb using an appropriate assay. The selected clones are then expanded in culture (or in animals), the cells are collected, and the MAbs are extracted and purified. 

As the result of high specificity and sensitivity, nucleic acid probes are in direct competition with immunoassay for the analytes of some types of clinical analytes, such as infectious disease testing. Assays are being developed, however, that combine both probe and immunoassay technology. In such hybrid probe—immunoassays, the immunoassay portion detects and amplifies the specific binding of the probe to a nucleic acid. Either the probe per se or probe labeled with a specific compound is detected by the antibody, which in turn is labeled with an enzyme or fluorophore that serves as the basis for detection. 

Fig. 8. Basic components of a biosensor. In the case of an immunosensor, the antibody (or antigen) would be immobilized onto the transducer.

Fig. 9. Immunosensor approaches where A is the analyte, is the labeled analyte, and Y is the antibody, (a) Direct immunosensors where the actual antigen—antibody interaction is measured (b) indirect immunosensors 1 and 2 which utilize formats similar to competitive and displacement...

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