Comparison of techniques of detecting immunoglobulin-binding protein reactivity to immunoglobulin produced by different avian and mammalian species.

The rationale of this study was to use several immunological assays to investigate the reactivity of immunoglobulin binding protein (IBP) to immunoglobulins from various avian and mammalian species. The IBP studied were Staphylococcal protein A (SpA), Streptococcal protein G (SpG), Peptostreptococcal protein L (SpL) and recombinant protein LA (SpLA). The various immunological techniques used were double immunodiffusion (Ouchterlony technique) that tested positive high protein reactivities, direct and competitive enzyme-linked immunosorbent assays (ELISAs) that tested moderate and low positive protein binding capacities, respectively. In addition to sandwich ELISAs, immunoblot analyses and Ig-purification by SpA-affinity chromatography, which were sensitive tests and helpful in the screening and confirmatory tests were also used. The Ouchterlony technique showed that compared to the other proteins, SpLA had the highest range of reactivity with animal sera and purified immunoglobulins while SpL was least reactive. With the direct ELISA, SpL reacted with the raccoon sera, rabbit IgG and with IgY from bantam hens and pigeons. While with the direct ELISA, SpA reacted with sera from skunk, coyote, raccoon, mule, donkey and human. The sandwich ELISA revealed high reactivity of both SpG and SpLA with mammalian sera titres ranging from 1:32 (raccoon serum) to 1:1024 (mule and donkey sera). These results suggest that IBP can be used for the detection of immunoglobulin using various immunological assays and this is important for the diagnosis of infectious diseases in animal and bird populations studied and in the purification of immunoglobulins.

Detection of red blood cell antibodies using bacterial antiglobulins: a new assay [abstract]

OBJECTIVE: To develop an agglutination technique using Staphylococcal protein A (SpA) and Steptococcalprotein G (SpG) to detect human red blood cell antibodies. METHODS: Blood samples were obtained from the National Transfusion Service of Jamaica. SpA, SpG, and anti-IgG, -C3d were commercial preparations. SpA and ApG were incubated with sensitized human red blood cells (RBC) to assess their RBC agglutinating capacity. The Ouchterlony technique was used to determine binding between the bacterial antigens and the IgG in human serum. Polyethyleneglycol (PEG) was used as an agglutination enhancer. Agglutination techniques for a large number of samples were developed, using 96 percent well polystyrene microplates and a microscope for visualizing the agglutination techniques to detect human anti-RBC IgG was compared with the traditional Coombs' test. Sensitivity and specificity were determined. RESULTS: SpA and SpG did not appear to cause agglutination of the red cells sensitized in vivo and in vitro. However, no precipitation bands were formed between human serum and the supernatant obtained after reaction of the sensitized RBC with SpA and SpG (Ouchterlony technique). These results indicated that indeed there was binding of SpA and SpG with the sensitized cells since they were not available for binding with human serum. In additon, SpA and SpG agglutinated the sensitized red blood cells in the presence of the PEG. When compared to the Coombs' test, the following results were obtained with new techniques. For the direct method, sensitivity was 93.8 percent and 95.1 percent for SpA and SpG respectively (n= 81), and specificity was 91.4 percent and 93.5 percent for SpA and SpG, respectivley (n= 93). For the indirect method, sensitivity was 96.3 percent and 97.5 percent for SPA and SpG, respectively (n= 81) and its specificity was 100 percent for both proteins (n= 85). CONCLUSION: Agglutination techniques using SpA and SpG constitute alternative and feasible tests for the detection of human red blood cell antibodies. (AU)