Prevention of false-positive reactions in whooping cough ELISA
Abstract number: R2384
Dalby T., Seier-Petersen M., Krogfelt K.A.
Objectives: Since 1991 is has been known that heat inactivation of serum results in very high false-positive values when analysing for the presence of antibodies to pertussis toxin (PT) by ELISA [Tada, Dev. Biol. Stand. 1991, 73: 175184]. Since PT is widely agreed to be the antigen of choice for whooping cough ELISA, prevention of false-positive reactions is crucial when analysing sera with unknown treatment. No reports so far have addressed this issue. Furthermore, heat could present a major problem e.g. in tropic areas.
Methods: An indirect ELISA based on pertussis toxin for detection of antibodies in human serum was optimised for the removal of false-positive results occurring at analysis of heat treated sera. Heat treated sera (56°C, 30 minutes) with known low contents of PT-antibodies were used, as well as non-treated sera from patients with confirmed whooping cough.
Results: When analysing heat treated sera with known low contents of PT-antibodies in the non-optimised PT-ELISA, the resulting estimates for PT-antibody contents were similar to, or even higher than, the values obtained when analysing sera from patients with confirmed whooping cough. Dilution curves of the heat treated sera moreover produced markedly different slopes than those obtained with non-treated sera, as was also seen by Lopez in 1998 [Lopez, Diagn. Microbiol. Infect. Dis. 1998, 30: 2124].
An ELISA-method based on Maxisorp microtiter plates (Nunc®, Denmark), using 1% milk in blocking solution and 0.1% milk in sample dilution, was seen to be a stable assay with elimination of the false-positive results. PT-antibody values obtained with heat treated sera were identical to values obtained with the corresponding non-treated sera.
Conclusion: False-positive results for PT-antibodies arising from heat-inactivation of sera were eliminated by a blocking step with 1% milk. Studies involving measurements of PT-antibodies in historic sera should involve such a blocking step, since historic sera might have been subjected to routinely heat inactivation. Moreover, in settings where sera have been heat-inactivated for safety reasons, an accurate analysis of PT-antibodies is now feasible.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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