SBIR-STTR Award

This project is designed to examine the technical feasibility of a new type of identification system for mycoplasmas. Mycoplasmas, bacteria-like microbes which cause human and animal disease and contaminate cell cultures, are ubiquitous. They musl bc identified to the genus and species level in order to identify their source and to aid in developing effective therapy and prevention measures. Bionique's immunoenzyme identification system will use colonies grown on artificial medium as a source of antigenic material. Colonies will be absorbed to small synthetic disks to facilitate immunoenzyme reactions. They will be placed in on a specially designed holder of porous material ("microfilter") for ease in handling and vacuum-mediated rinsing. Samples from a battery of specific antisera will be added to the disks and incubated. The antigenantibody reaction which identifies the agent will be detected through biochemical interactions involving protein A, biotin, avidin, and an enzyme marker. This type of sequence creates a multitude of marker molecules per antigen molecule. This type of amplification makes such a test several logs more sensitive than older assays in use (e.g., fluorescent antibody). Addition of the appropriate substrate (e.g., diaminobenzidine in the case of peroxidase) will cause grossly visible formation of chromogenic end product on and in the colonies adherent to the disk. The degree of response will be quantitated with a videodensitometer of Bionique's own design. Hard copy printout will serve to substantiate the identification. The entire system (protocol, disks, microfilter, electronic densitometer) represents a new and distinctive approach to the identification of antigenic particulates. This technique incorporates speed, simplicity, and sensitivity. Data will be objective, quantitative and compatible with automated methods and computer analysis and retrieval. This concept has direct and immediate application to the widespread problem of mycoplasma infection and contamination. It also has the potential for use in other biomedical areas such as the rapid speciation of bacteria, yeasts and fungi.National Institute of Allergy and Infectious Diseases

Current methods for identification of microbial isolates from clinical human and animal specimens are fairly slow and tedious. Fast and simplified assays for pathogenic isolates would allow more rapid initiation of appropriate therapy and epidemiological control Phase I demonstrated with mycoplasmas the feasibility of using an antigenantibody reaction that was unique in the manner in which microbial antigen was immobilized (on cellulose acetate strips) and tagged (with an enzyme marker, amplified by a biotin-avidin high avidity lattice reaction). Chromogenic product was quantitated with a novel videodensitometer.Phase 11 expands the study to include other common bacterial pathogens (Streptococcus, Hemophilus, and Salmonella). Specificity and sensitivity will be increased by using monoclonal antibodies and by developing blocking steps to reduce the number of naturally occurring active biotin sites on the microbial cells. The blocking agents evaluated will be bovine serum albumin (0.1 percent and 1.0 percent), horse serum (0.1 percent and 1.0 percent), and each of these serums with an additional 100 microgram per milliliter purified avidin. The densitometer will be modified to include both ultraviolet and white light (plus colored filters) used in a transmitted mode and a reflected mode. Significant effort will be made to facilitate automated data analysis by processing the densitornetric signal with an integrator/printer, an analog/digital converter, and a microcomputer. Prototypes of the improved protocol and densitometer (which also can be used for a wide variety of scanning functions such as electrophoresis gels and enzyme linked immunosorbent assay plates) will be ready for commercialization within 2 years, and will contribute directly to improved laboratory operations and patient care.National Institute Of Environmental Health Sciences