Antigen detection ELISAs: pretreatment of serum to reduce interference by specific host antibodies.
The pretreatment of serum to reduce interference by specific host antibodies was investigated as a means of improving the sensitivity of antigen detection ELISAs whilst screening serum samples. Four antigen detection assays based on monoclonal antibodies directed against antigens of the bovine filariid Onchocerca gibsoni were used in this study and, of these, three assays suffered a dramatic drop in sensitivity when detecting male O. gibsoni antigen in the presence of bovine serum as compared with antigen in buffer. A number of methods for pretreating serum to eliminate the problem of antibody interference with antigen detection were attempted, including heat and alkali treatments, detergent treatment of heat treated samples and the use of a reducing agent. The pretreatment of serum by boiling for 5 minutes in the presence of an equal volume of 0.1 M Na2EDTA pH 4.0 and recovery of the supernatant fluid following centrifugation at 16000 g was the most effective method of restoring the sensitivity of each of these three assays whilst screening bovine serum. Pretreatment of serum using this method produced up to a 512-fold increase in sensitivity compared with results obtained in assays with non-treated serum.
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S J
SJ
Graduate School of Tropical Veterinary Science and Agriculture, James Cook University of North Queensland, Townsville, Australia.
Copeman
D B
DB
D000818:Animals; D000909:Antibodies, Helminth / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000947:Antigens, Helminth / Q000032:analysis / Q000097:blood*; D002021:Buffers; D002417:Cattle; D002498:Centrifugation; D004492:Edetic Acid; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards*; D000939:Epitopes / Q000032:analysis; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008297:Male; D009854:Onchocerca / Q000276:immunology* / Q000302:isolation & purification; D011237:Predictive Value of Tests
D000909:Antibodies, Helminth; D000911:Antibodies, Monoclonal; D000947:Antigens, Helminth; D002021:Buffers; D000939:Epitopes; D004492:Edetic Acid