Hemagglutination assay procedure

Cost Effectiveness. As with the other advantages of immunochemical analysis, cost may be quite variable. Reagent costs for several automated systems have been estimated at under 1.25 per sample. The cost is obviously much lower for less sophisticated assay systems, especially if some reagents are prepared in house. A major consideration is the expense of new instrumentation. For dedicated or automated instrumentation for either RIA or ELISA procedures, the cost may be 50-100,000. However, most analytical laboratories already have the basic instrumentation needed for immunoassays. Moderate sensitivity can be obtained through the use of numerous procedures such as radial immunodiffusion and hemagglutination. These procedures require no expensive equipment or reagents and they may be very useful in areas where equipment acquisition or maintenance is a problem. 

Diagnostic procedures include dark-field microscopy12, non-treponemal exams10 (i.e., the Venereal Disease Laboratory and the rapid plasma reagin test), and treponemal exams (i.e., enzyme immunoassay, the T. pallidum hemagglutination test, the fluorescent treponemal antibody test, and the enzyme-linked immunosorbent assay). 

This procedure is of great potential utility for the identification and enumeration of cells that secrete an antigenic product. Patterned after the assay just described, it employs similar techniques with two major differences The coating for the indicator red cells consists of purified antibody, as in the reverse passive hemagglutination previously described, and an antiserum against the secretion product is used as the developing agent. 

The antibody-coated red cells are prepared as previously described. It is particularly important for this procedure that the indicator red cells are absolutely free of clumps. The sensitivity of coated cells can be assayed by reverse passive hemagglutination if, as in the model under consideration, the antigen is available in soluble form. The cells under study are washed in suitable tissue culture medium or other buffered solution and suspended at a concentration of 10 per milliliter in the same diluent to which serum has been added (usually 1% fetal calf serum). A small volume (50-100 /U.1) of the cell suspension is placed in a 10 x 75 mm disposable tube. The addition of an equal volume of 1% coated red cells results in a mixture that contains about 25 red cells per lymphocyte. Linkage of antibody on the red cells to the corresponding antigen determinant on the surface of the lymphocyte results in the formation of a rosette or lymphocyte surrounded by red cells. The mixing of cells and incubation for at least 1 hr are done in an ice bath. The tubes are then centrifuged very briefly (1 min at 1000 g), and a drop of dye is added to tint the lymphocytes (e.g., crystal violet or brilliant cresyl blue). The mixture is then aspirated four or five times with a Pasteur pipette and examined in a hemacytometer chamber at about 400 x. A cell is scored as a rosette if it is surrounded by three or more adherent erythrocytes, and usually 300 cells are counted. 

Practicability. Both hemagglutination-inhibition and latex inhibition tests are easy to perform and are inexpensive. In addition, the results of the tests can be read in a few hours. As mentioned previously, their main disadvantage is the need to use large quantities of purified hormones in order to coat the red cells. Stockell Hartree (S25) has shown that these assays are extremely useful in order to monitor hormone extraction procedures, but in the absence of a supply of purified hormones their sphere of applicability to clinical problems is limited. When crude hormones and nonspecific antisera are used the quantitative significance of assays based on hemagglutination-inhibition reactions is doubtful. 

The evaluation of anti-BPO antibodies of the IgG class, by a similar procedure to the one used in the RAST assay, has proved of interest in the evaluation of penicillin-induced hematological disorders, such as hemolytic anemia or leukopenia (Neftel et al. 1981), although anti-BPO IgG antibodies are also encountered in asymptomatic individuals treated with high doses of penicillins. Previously described methods, such as hemagglutination or bacteriophage inhibition, are cumbersome, less reproducible, and may be considered as obsolete. 

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