Effect of gamma-irradiation on serum samples on the diagnostic performance of ELISA methods for the detection of trypanosomal antibodies.

The study investigated the effect of gamma-irradiation on bovine serum samples on the ability of enzyme-linked immunosorbent assay (ELISA) methods to detect trypanosomal antibodies. The serum samples were analysed using two standardised indirect ELISA systems. Higher measurement values were observed for most gamma-irradiated antibody positive and negative test samples. Using cut-off points, determined from the analysis of a non-irradiated trypanosomal antibody-negative population, the gamma-irradiated sera data showed that there was an increased risk of misclassifying samples as false positive or cross-reactive due to increased analytical sensitivity and decreased analytical specificity. The intraplate precision and agreement between tested and expected values of measurements were not altered throughout. The impact on the assays' diagnostic performance was estimated by analysing diagnostic sensitivity, diagnostic specificity and related parameters. The data demonstrated that although there was a bias of higher measurement values after gamma-irradiation, this could be compensated after readjustment of the cut-off points to obtain best separation of antibody-positive and -negative samples. Thus, for each assay, no significant difference of the diagnostic proficiency was found before and after gamma-irradiation. The practical implications are discussed of a serum sterilisation procedure using (60)Co gamma-rays for routine sample testing, assay validation and trypanosomosis monitoring and tsetse-fly control and eradication programmes.

Study of the effect of gamma-irradiation on bovine serum samples on the ability of monoclonal antibodies to detect invariant antigens of Trypanosoma congolense, T. vivax and T. brucei in enzyme-linked immunosorbent assays.

Samples of bovine serum from uninfected and African trypanosomes-infected animals were tested before and after gamma-irradiation, using three sandwich enzyme-linked immunosorbent assays (ELISA). Each test system utilized a different monoclonal antibody, reputedly allowing the specific detection of conserved-invariant cytoplasmic antigens of trypanonosomes, T. congolense, T. vivax, and T. brucei, respectively. Results have identified two groups of samples. The first contained samples where there were unequivocal ELISA results indicating positivity and negativity, for non-irradiated samples. In this group, irradiation had no effect on the diagnostic sensitivity of the assays. All samples shown to be positive before irradiation remained positive and those shown to be negative, remained negative. There was, however, a statistically significant reduction in signal in each of the ELISAs following irradiation. The second group contained samples identified before irradiation as flanking the diagnostic negative/positive threshold of OD > or =0.05. These showed a negative bias after irradiation of the order of OD -0.01, which was shown to be statistically significant by paired t-statistics. Without correction of the given diagnostic negative/positive threshold, bovine sera with OD values around the threshold were expected to deliver more false negative test results upon irradiation. This was confirmed when serological data were compared with parasitological findings; where three times more false negative test results were found from irradiated serum samples. Consequently, for this group of irradiated bovine samples tested by ELISA, the re-adjustment of the diagnostic negative/positive threshold of the ELISAs using defined irradiated serum samples is recommended; otherwise, the frequency of false negative results might be increased.