Washington Research Foundation

Other

The United States of America As Represented by the Department of Health

Kirk C Chen

Thomas M Buchanan

William Pearce

Florence J Knapp

King K Holmes

A novel colorimetric method for beta-lactamase assay and its applications are disclosed. The novel assay method for beta-lactamase is based on the discovery described in this invention. The chromophore formed by oxidation of either the N-alkyl derivative of p-phenylenediamine or the 3,3',5,5'-tetraalkyl derivative of benzidine can be decolorized by the open beta-lactam ring end product resulting from the hydrolysis of a penicillin in the presence of a mercury-containing compound. The same chromophore can be decolorized by the open beta-lactam ring end product resulting from the hydrolysis of a cephalosporin in either the presence or the absence of the mercury-containing compound. None of the intact beta-lactam antibiotics can decolorize the chromophore in either the presence or the absence of the mercury-containing compound. The final concentration of the mercury-containing compound in the decolorization mixture ranges from approximately 0.005 mM to approximately 3 mM, and the pH of the decolorization mixture ranges from 3.5 to 8.0. Additional applications derived from coupling this novel assay method to methods in the art include: a method for specific detection of microbial beta-lactamases; a method for simultaneous detection of microbial cytochrome c oxidase systems and penicillinases, and its application to simultaneous presumptive identification of Neisseria gonorrhoeae in urethral exudates, and differentiation of the infecting gonococci into penicillinase-producing N. gonorrhoeae and non-penicillinase-producing N. gonorrhoeae; and a simultaneous assay method for peroxidase and penicillinase.

Colorimetric method for beta-lactamase assay and its applications

An improved developer for a method for the specific detection of the presence of beta-lactamase from microbial sources is disclosed. The method utilizes a beta-lactam ring-containing substrate whose amide bond is hydrolyzed in the presence of beta-lactamase. A substrate which includes a beta-lactam antibiotic with an acyl side chain containing an alpha-amino group and an alpha-phenyl group or its derivatives is first contacted with an organism thought to produce beta-lactamase or a cell-free beta-lactamase preparation, and, subsequently, it is determined whether the reaction product between the unhydrolyzed substrate, the end products and the organism or the preparation fluoresces. Fluorescence is the indication of .beta.-lactamase activity. The developer comprises the addition of an oxidizing agent (preferably also containing formaldehyde) to the reaction product to enhance the fluorescence. The oxidizing agent is selected from the group consisting of Ag.sup.+, Hg.sup.++, H.sub.2 O.sub.2, I.sub.3.sup.-, IO.sub.4.sup.-, persulfate, Pd.sup.++, p-hydroxymercuribenzoate, and mixtures thereof.

Detection of microbial beta-lactamase

A rapid method for the specific detection of the presence of Beta-lactamase from microbial sources is disclosed. The method utilizes a Beta-lactam ring containing substrate whose amide bond is hydrolyzed in the presence of Beta-Lactamase. A substrate, which includes a Beta-Lactam antibiotic with an acyl side chain containing an .alpha.-amino group and a .alpha.-phenyl group and/or its derivatives is first contacted with an organism thought to produce Beta-lactamase or a cell-free Beta-lactamase preparation, and, subsequently, it is determined whether the reaction product between the substrate and the organism or the preparation fluoresces.

Fluorescence assay for microbial beta-lactamase

A vaccine affording protection against attachment of Neisseria gonorrhoeae (N.g.) to human cells, and consequent infection, is prepared from a fragment of pili protein isolated from gonococci. This fragment contains the shared antigens between pili from different N.g., in an exposed form.

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Seattle, WA, United States of America

Kirk C Chen