Use of chicken egg-yolk antibodies against K88+fimbrial antigen for quantitative analysis of enterotoxigenic Escherichia coli (ETEC) K88+by a sandwich ELISA

An enzyme-linked immunosorbent sandwich assay (ELISA) has been developed to detect and quantify both the K88+ fimbrial antigen and the concentration o f bacterial cells in fecal and other samples using anti-fimbrial chicken eg g-yolk and anti-fimbrial rabbit serum antibodies. The assay has a sensitivi ty of 40 ng ml(-1) for K88+ fimbrial antigen and 10(7) CFU ml(-1) for intac t cells and cell homogenate, The inter- and intra-assay coefficients of var iation (CV) for intact cells, homogenates, and fimbrial antigens were simil ar, suggesting that reproducible results can be obtained with any of the th ree preparations, In all cases the CV increased with decreasing concentrati on of the antigen, especially when very low concentrations of antigen were used, Fimbrial antigens had lower CV (values ranged from 2 to 37%, dependin g on its concentration) than standards prepared from either whole cells or homogenized cells (values ranged from 1 to 67%), The correlation between th e actual number of cells as counted and the number as estimated from the EL ISA using the amount of fimbrial antigen was r = 0.98, P <0.001. The anti-K 88+ polyclonal antibodies from chicken serum and egg yolk had little or no cross-reactivity with K99, 987P ETEC, The correlation (r) between the fecal score for diarrhea and the number of E coli in the fecal swab was high, r = 0.80 (n = 64, P < 0.0001). The assay is sensitive and specific and provid es a good estimate of the amount of fimbrial protein or the number of K88ETEC in the sample, (C)) 1999 Society of Chemical Industry.