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

Abstract

A microsphere coagulation enzyme-linked immunosorbent assay (MC-ELISA) using Russell's viper venom factor X activator (RVV-XA) is described for the detection of Escherichia coli O157:H7. This microtitre plate assay comprises a standard sandwich immunoassay incorporating RVV-XA as the enzyme label. Coagulation substrate together with polystyrene microspheres are added to the wells of the microtitre plate. RVV-XA initiates the coagulation cascade causing formation of an artificial clot of polystyrene microspheres bound together with fibrin. As few as 10 2 E. coli O157 in a well (10 3 per ml) can be detected within 3 h. The assay is two orders of magnitude more sensitive than a standard ELISA and is a generic technique with the potential for widespread use in sandwich immunoassays. ß

Allosteric Aptamers Controlling a Signal Amplification Cascade Allow Visual Detection of Molecules at Picomolar Concentrations

“…Homogeneous BCC-MS Amplification Cascade. A biochemical signal amplification cascade, utilizing components of the BCC cascade in the presence of MS was described as a part of a heterogeneous ELISA assay (26). The BCC portion of the amplification cascade consisted of Factors X, Va, II (Prothrombin), and I (Fibrinogen).…”

“…The disadvantages of the above assay include its basis on a standard solid-support ELISA format that requires multiple washes and reagent transfers. Furthermore, the above assay relies on several specific, not readily available reagents, one of which is a monoclonal antibody conjugated to RVV-X (26).…”

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.…”