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Title: MONOCLONAL ANTIBODIES FOR DETECTION OF THE H7 ANTIGEN OF ESCHERICHIA COLI

Author
item He, Yongsheng
item Keen, James
item Westerman, Ralph
item LITTLEDIKE, ERNEST - OMAHA COL HLTH CAREERS
item Kwang, Hwei Sing

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/15/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary: E. coli O157:H7 is an important bacterium which has caused several recent foodborne disease outbreaks in the United States. Most outbreaks have caused severe intestinal and/or kidney disease and many have been linked to consumption of meat products. There are hundreds of strains of E. coli bacteria as well as hundreds of other non-E. coli bacteria. Many of these bacteria are difficult to distinguish from pathogenic E. coli O157:H7, based on laboratory testing. Therefore, new methods are needed to make detection of this dangerous bacteria easier in food products or in clinical samples. At the same time, false positive reactions must be minimized. Monoclonal antibodies (MAbs) which react only with this harmful bacteria and not with other bacteria are one tool which can be used to either detect E. coli O157:H7 or to help understand how it causes disease. In this study, four MAbs which reacted only with the flagella (H antigen) of this bacteria were produced and characterized. (The flagellum is the whiplike motor of the bacteria which enables it to move about.) Out of 170 different bacteria tested, these antibodies reacted with all 48 E. coli O157:H7 strains and the E. coli (non-O157):H7- positive strains. They did not react, with very few exceptions, with any non-H7 E. coli nor with any non-E. coli bacteria. These MAbs also detected flagella from E. coli O157:H7 with much greater accuracy than either of two commercial sources of anti-H7 antibody (called polyclonal antibody) currently in use to classify and detect E. coli O157:H7. These unique antibodies could be used to detect and classify E. coli O157:H7 in applications ranging from meat or food inspection to medical specimen examination and research.

Technical Abstract: Four murine monoclonal antibodies (MAbs) (2B7, 46E9-9, 16D3, and 49B7) specific for the H7 antigen of Escherichia coli O157:H7 were produced and characterized. A total of 170 E. coli strains (48 O157:H7, 4 O157:H-, 23 O157:non-H7, 22 H7:non-O157, and 73 non-O157:H7), 17 Salmonella serovars, and 29 other Gram-negative bacterial strains were used to evaluate the specificity of the MAbs. Monoclonal antibodies 2B7 and 46E9-9 reacted strongly with H7, H23, and H24 positive E. coli strains. In addition to these strains, MAb 16D3 also reacted with O157:H19 and O157:H30, while 49B7 reacted with H52-positive and several other E. coli strains. The specificity of the MAbs to the H7 antigens was determined by enzyme-linked immunosorbent assay (ELISA) and confirmed by Western immunoblotting, using partially purified flagellins from O157:H7 and other non-O157:H7 strains. In the Western immunoblotting, MAb 46E9-9 appeared specific only for the H7 flagellin of O157:H7. Inhibition and competition ELISA analyses showed that 2B7, 46E9-9, and 16D3 shared, or partially shared, antigenic epitopes, but that 49B7 had a unique antigenic determinant. The specificities of these MAbs were also compared with two commercially available polyclonal anti-H7 sera. The polyclonal antisera showed similar reactivity to the H7-positive E. coli bacteria as these MAbs, but also showed significant cross-reactivity to many non-H7 bacteria by ELISA The polyclonal anti-H7 sera, like the MAbs, also strongly reacted with H23 and H24 positive E. coli strains. The H7 specific MAbs described in this report would be used alone or in combination with O157-specific MAbs to identify or detect E. coli O157:H7 in various applications.