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

Abstract

An enzyme-linked immunosorbent sandwich assay (ELISA) has been developed to detect and quantify both the K88 ®mbrial antigen and the concentration of bacterial cells in fecal and other samples using anti-®mbrial chicken egg-yolk and anti-®mbrial rabbit serum antibodies. The assay has a sensitivity of 40 ng ml À1 for K88 ®mbrial antigen and 10 7 CFU ml À1 for intact cells and cell homogenate. The inter-and intra-assay coef®cients of variation (CV) for intact cells, homogenates, and ®mbrial antigens were similar, suggesting that reproducible results can be obtained with any of the three preparations. In all cases the CV increased with decreasing concentration of the antigen, especially when very low concentrations of antigen were used. Fimbrial antigens had lower CV (values ranged from 2 to 37%, depending on its concentration) than standards prepared from either whole cells or homogenized cells (values ranged from 1 to 67%). The correlation between the actual number of cells as counted and the number as estimated from the ELISA using the amount of ®mbrial antigen was r = 0.98, P`0.001. The anti-K88 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 speci®c and provides a good estimate of the amount of ®mbrial protein or the number of K88 ETEC in the sample.

Isolation, Affinity Purification, and Identification of Piglet Small Intestine Mucosa Receptor for Enterotoxigenic Escherichia coli K88ac+ Fimbriae

“…The local strain of hemolytic ETEC K88ϩ bacteria was identified as being K88ac (11). The K88ϩ fimbrial antigen was purified by using a modification of the method of Erickson et al (6).…”

“…This binding prevents the removal of the bacteria by intestinal peristalsis and is a prerequisite for virulence (6,10,18,19,20). Neonatal piglets are extremely sensitive to infection by ETEC but can be protected by specific anti-K88 egg yolk antibodies (11,14,17). Piglets are commonly infected by E. coli strains expressing several serological types of the K88 fimbrial antigens.…”

A net fishing enrichment strategy for colorimetric detection of E. coli O157:H7

“…With a short time enrichment, excellent sensitivity was demonstrated. With a sandwich-like pattern comprising of antibody modified on the nitrocellulose membrane-target bacteria-antibody and HRP modified GNP probes, as low as 100 CFU/ml of E. coli O157:H7 could be recognized with the generated colorimetric signal, exhibiting an improved sensitive compared to the methods which use similar sandwich-like pattern such as the conventional ELISA [35,36], nanoparticle enhanced ELISA [3] and magnetic enrichment-assisted ELISA methods [37,38]. Validity of the method to detect pathogens in pineapple juice showed the feasibility of the proposed method in real food samples.…”

A sensitive microsphere coagulation ELISA for Escherichia coli O157:H7 using Russell's viper venom

“…By comparison, standard immunological tests (including latex agglutination, immunomagnetic separation, lateral £ow immunoassays and ELISA) for the detection and con¢rmation of bacterial pathogens are used frequently. However, for the direct detection of bacterial pathogens, methods such as ELISA typically have sensitivities of 10 51 0 7 bacteria ml 31 [12,13] and hence require overnight enrichment of the sample prior to analysis [14]. More sensitive immunological methods have been reported using electro-chemiluminescence [15] and rapid £ow through systems [16].…”