Variability in a commercially available enzyme-linked immunosorbent assay system. I. Assay variability.
Three experiments were conducted to characterize the variation in enzyme-linked immunosorbent assay (ELISA) kits for infectious bronchitis virus (IBV) and infectious bursal disease virus (IBDV). Expt. 1 was carried out to determine the variation in assay results when the same pools of low-, medium-, and high-titered serum were assayed. Significant variation occurred among separate lots and among test plates within the same lots for the IBV and IBDV assays. In most cases, variability between days and among technicians was not significant. Coefficients of variation were larger than is acceptable for immune-type assays. In the IBDV assay with high-titered serum, most of the wells in the plates reached maximum absorbance and were not capable of detecting titers above 1:8000-1:9000. Expt. 2 was conducted to determine the effects of varying the length of the ortho-phenylene-diamine (OPD) incubation time upon assay results. Either 7-, 12-, or 15-minute OPD incubation times were used. Incubation time significantly affected mean titer at all combinations of assay types and times, except determinations on the low-titered IBV samples. Expt. 3 was conducted to determine the effects of three different dilution methods on observed IBDV titer. The use of non-standard dilutions had significant effects on observed titer. In the medium- and high-titered samples, the use of two different dilution methods at 1:5000 rather than 1:500 resulted in titers that were three to four times those observed at the 1:500 dilution.
Kreider
D L
DL
Department of Animal and Poultry Sciences, University of Arkansas, Fayetteville 72701.
Skeeles
J K
JK
Parsley
M
M
Newberry
L A
LA
Story
J D
JD
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards*; D001351:Infectious bronchitis virus / Q000276:immunology* / Q000302:isolation & purification; D007243:Infectious bursal disease virus / Q000276:immunology* / Q000302:isolation & purification; D011933:Reagent Kits, Diagnostic / Q000662:veterinary; D015203:Reproducibility of Results
D000914:Antibodies, Viral; D011933:Reagent Kits, Diagnostic