Evaluation of a simple method for improving the precision of an ELISA detecting antibody in serum.
A method of correcting OD values to improve between-assay precision (PCF4 method) was evaluated using an ELISA detecting anti-Dichelobacter nodosus antibody in ovine serum. Four control sera with pre-defined target OD were included on each plate and used to calculate a correction factor. The mean between-plate CV for eight reference sera tested over a period of 6 months was reduced from 15.2% to 9.4% in a polyclonal conjugate assay (114 plates) and from 13.3% to 9.8% in a monoclonal conjugate assay (128 plates) using this method. The PCF4 method was compared with an existing method that uses only one control serum and was found to be superior. Time course plots of PCF4 values were useful for laboratory quality control. The mean within-plate coefficients of variation for four reference sera tested in paired wells were 8.2% and 5.1% in the polyclonal and monoclonal conjugate assays respectively. Marked changes in OD due to experimental manipulation of antigen dilution, serum dilution and incubation time, conjugate dilution and chromogen-substrate incubation time were corrected by the method.
Whittington
R J
RJ
Microbiology Section, Elizabeth Macarthur Agricultural Institute, New South Wales Agriculture, Camden.
D000818:Animals; D000906:Antibodies / Q000097:blood*; D000907:Antibodies, Bacterial / Q000032:analysis; D001439:Bacteroides / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005069:Evaluation Studies as Topic; D011786:Quality Control; D015203:Reproducibility of Results; D012756:Sheep
D000906:Antibodies; D000907:Antibodies, Bacterial