Minimizing ELISA background in the diagnosis of swine trichinellosis.
After confirming that long-term serum storage (frozen at -20 C for greater than 3 mo) causes optical density to drift upward, several modifications of an enzyme-linked immunosorbent assay (ELISA) protocol were evaluated to identify a protocol that would reduce background in porcine sera tested for trichinellosis. Modifications evaluated included blocking the antigen-coated ELISA plate with sample diluent containing 10% bovine serum albumin (BSA) or 10% nonfat milk powder (bovine lacto transfer optimizer or BLOTTO), diluting sera in sample diluent containing 10% BSA or 10% BLOTTO, and preincubating samples in sample diluent containing 10% BSA or 10% BLOTTO. Overnight preincubation (approximately 12 hr at 2 C) of fresh sera diluted (1:10) in sample diluent containing 10% BLOTTO significantly reduced background and improved the detection of experimentally infected pigs by enhancing positive-negative discrimination. When testing stored sera, the modified protocol effectively reduced the effect of storage and the kit revealed specificity of 98.4%; there was no loss in sensitivity. The effect of long-term storage at -20 C must therefore be considered when testing swine sera for trichinellosis by ELISA and possibly also when conducting other immunoglobulin assays. The modification described here may prove useful if there is no alternative to using serum stored for greater than 3 mo at -20 C.
van der Leek
M L
ML
Department of Infectious Diseases, College of Veterinary Medicine, University of Florida, Gainesville 32611.
Dame
J B
JB
Littell
R C
RC
D000818:Animals; D000909:Antibodies, Helminth / Q000096:biosynthesis* / Q000097:blood; D001667:Binding, Competitive; D001793:Blood Preservation; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards*; D005069:Evaluation Studies as Topic; D005431:Florida / Q000453:epidemiology; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000097:blood / Q000378:metabolism; D007814:Larva / Q000276:immunology; D015995:Prevalence; D011933:Reagent Kits, Diagnostic / Q000662:veterinary; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000453:epidemiology; D017160:Trichinella spiralis / Q000276:immunology*; D014235:Trichinellosis / Q000175:diagnosis / Q000453:epidemiology / Q000662:veterinary*
D000909:Antibodies, Helminth; D007074:Immunoglobulin G; D011933:Reagent Kits, Diagnostic