RAPID IDENTIFICATION OF MYCOPLASMAS WITH IMMUNOENZYMES

THE PROJECT WHICH WE PROPOSE IS DESIGNED TO EXAMINE THE TECHNICAL FEASIBILITY OF A NEW TYPE OF IDENTIFICATION SYSTEMFOR MYCOPLASMAS. MYCOPLASMAS, BACTERIA-LIKE MICROBES WHICH CAUSE HUMAN AND ANIMAL DISEASE AND CONTAMINATE CELL CULTURESARE UBIQUITOUS. THEY MUST BE IDENTIFIED TO THE GENUS AND SPECIES LEVEL IN ORDER TO IDENTIFY THEIR SOURCE AND TO AID IN DEVELOPING EFFECTIVE THERAPY AND PREVENTION MEASURES. OUR 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 ANTIGEN-ANTIBODY REACTION WHICH IDENTIFIES THE AGENT WILL BE DETECTED THROUGHBIOCHEMICAL INTERACTIONS INVOLVING PROTEIN A, BIOTIN, AVIDINAND 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 A ANTIBODY). ADDITION OF THE APPROPRIATE SUBSTRATE (E.G., DIAMINOBENZIDINE IN THE CASE OF PEROXIDASE) WILL CAUSE GROSSLY-VISIBLE FORMATION OF CHROMOGENIC ENDPRODUCT ON AND IN THE COLONIES ADHERENT TO THE DISK. THE DEGREE OF R RESPONSE WILL BE QUANTITATED WITH A VIDEODENSITOMETER OF OUROWN 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, SIMPLICITYAND SENSITIVITY. DATA WILL BE OBJECTIVE, QUANTITATIVE, AND COMPATIBLE WITH AUTOMATED METHODS AND COMPUTER ANALYSIS AND RETRIEVAL. THIS CONCEPT HAS DIRECT AND IMMEDIATE A 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. THIS PROJECT, DEVELOPED FOR THE SBIR PROGRAM, IS CONSISTENT WITH THE PROGRAM DESCRIPTION OF NIAID MICROBIOLOGY AND INFECTIOUS DISEASES SECTION UNDER "INNOVATIVE BIOMEDICAL TECHNOLOGIES".