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Immunological techniques for the serum determination of specific-IgE levels among workers exposed to seafood allergens
Elliott, Alicia Rochelle
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Allergic conditions among workers processing seafood are most often related to inhalation of the seafood antigens or via direct unprotected handling of the seafood and its products. This can cause sensitised individuals to suffer from asthma, rhino-conjunctivitis, urticaria and protein contact dermatitis, which are IgE mediated. Food intolerance may also occur which is a non-IgE mediated reaction, however the exact mechanism is yet to be determined. There is therefore a need to develop reliable tests to identify sensitised workers processing seafood. The objective of this study was to prepare specific seafood extracts from raw and cooked lobster; raw and cooked saltwater bony fish species (mackerel, red eye, maasbanker, pilchard and anchovy) and fishmeal dust obtained from a fish-processing factory. These extracts were tested by SDS-Polyacrylamide Gel Electrophoresis to characterise the seafood proteins, and the allergenicity was confirmed by the Western blot technique. Polyclonal IgG antibodies were also successfully generated in rabbits, using the specific seafood extracts isolated from the various species. The second objective was to optimise and standardize an Enzyme Allergosorbent Test (EAST) method to quantify specific IgE antibodies in the sera of factory workers. This EAST was optimised and validated to detect allergen-specific IgE to each of the different fish species and also one crustacean species (rock lobster). Sera from a group of workers were selected and analysed for specific IgE antibodies by the optimised EAST (S) (South African laboratory), and commercial RAST techniques. Analysis was performed for the three most important extracts, pilchard (canned), anchovy, and lobster. The same samples were analysed by EAST (R) in the reference laboratory (Dr Gerald Reese; Paul-Ehrlich-Institute, Germany). The different techniques, and the EAST (R) and the EAST (S) results were compared by using a statistical software package. An EAST method was successfully developed, however, compared to the results obtained by the reference laboratory the sensitivity and specificity was below 80%. The main reason for the low agreement between the two laboratories was the fact that the South African laboratory used a modified EAST method, and different data calculation methods, for categorising positive results. The South African laboratory did not use a kit-based assay and a serum dilution of 1:4 and not 1:2 were used when compared to the reference laboratory. When the EAST results were compared to the RAST results, poor agreement was found due to the fact that canned pilchard was used in the EAST while raw pilchard in the commercial RAST assay. For pilchard, anchovy and lobster EAST, different species were utilized compared to the RAST, and this can also explain the poor level of agreement. Future directions would be to further standardise the EAST method and to introduce reference sera and a standard curve to determine positive results, thereby ensuring more reproducible results between laboratories.