Improved Methods for Detection and Serotyping of Coagulase from Staphylococcus aureus

Abstract

Improved methods for detection and serotyping of staphylocoagulase were concomitantly devised. We devised an improved method for detection of coagulase activity on agarose film in the same manner as single radial immunodiffusion. The amounts of reagents required for detection of coagulase on agarose film were successfully diminished by adding polyethylene glycol (PEG) to the original formula described by Boothby et al. Using microplates in another improved method for coagulase serotyping, the amount of reaction fluid required was considerably less compared with the conventional tube method. PEG was found to be also effective to increase the efficacy of coagulase serotyping. In the presence or absence of anti-coagulase antisera, culture supernatants of staphylococcal strain grown in brain heart infusion broth were incubated with the reaction fluid containing bovine fibrinogen, rabbit plasma, 6-amino caproic acid, polyethylene glycol 6,000. Coagulase activity was visualized as a turbid mass formed in the wells. Turbid mass formation due to coagulase activity was type-specifically inhibited in the presence of type-specific antisera. Detailed procedures of the methods are precisely described with some preliminary results obtained by the methods.

The von Willebrand factor-binding protein (vWbp) of Staphylococcus aureus is a coagulase

“…In the plate coagulation assay, adapted from Hwang et al [9], 5 ll of recombinant protein in concentrations ranging from 40 to 2.5 lg ml À1 , was added to small wells punched out in agarose plates (0.9%) containing 0.4% PEG 8000, 3 mg ml À1 bovine fibrinogen and 1% plasma (PFP-plates). Coagulation zones were measured after incubation at 37°C overnight.…”

Identification and functional activity of a staphylocoagulase type XI variant originating from staphylococcal food poisoning isolates

“…A plate coagulation assay was performed according to published methods with a slight modification (Hwang et al . ; Bjerketorp et al . ).…”

“…A plate coagulation assay was performed according to published methods with a slight modification (Hwang et al 1989;Bjerketorp et al 2004). Aliquots (20 ll) of rCoa310 or bovine serum albumin (Biorad) in concentrations ranging from 0Á25 to 1 mg ml À1 were added to small wells (3 mm in diameter) punched out of an agar plate (Bactoagar; Becton Dickinson) containing 20% rabbit plasma (Denka Seiken).…”

The von Willebrand factor-binding protein (vWbp) ofStaphylococcus aureusis a coagulase

“…In the plate coagulation assay, adapted from Hwang et al [9], 5 μl of recombinant protein in concentrations ranging from 40 to 2.5 μg ml −1 , was added to small wells punched out in agarose plates (0.9%) containing 0.4% PEG 8000, 3 mg ml −1 bovine fibrinogen and 1% plasma (PFP‐plates). Coagulation zones were measured after incubation at 37 °C overnight.…”