Page Banner

United States Department of Agriculture

Agricultural Research Service

Title: Production and Characterization of Monoclonal Antibodies Specific for the Lipopolysaccharide of Escherichia Coli O157

Authors
item Westerman, Ralph
item He, Yongsheng
item Keen, James
item Littledike, Ernest - OMAHA COL HLTH CAREERS
item Kwang, Hwei Sing

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 2, 1996
Publication Date: N/A

Interpretive Summary: E. coli (O157:H7) is particularly pathogenic to man but can exist in healthy or asymptomatic cattle. Consumption of undercooked ground beef contaminated with E. coli O157:H7 is a major cause of hemorrhagic colitis which is characterized by bloody diarrhea with occasional fatal complications. Therefore, the accurate and rapid identification of E. coli O157:H7 is essential for the prevention and treatment of the disease. E. coli are classified by serotype based on the composition of their O-antigens and H-antigens. The O-antigens are only a part of the very large molecules called lipopolysaccharides (LPS). The polyclonal antibodies present in the serum from animals injected with the LPS from E. coli O157:H7 react with all parts (epitopes) of these huge molecules. Many of these epitopes are identical to epitopes present on other types of bacteria besides E. coli, which could lead to false-positive identification of E. coli O157:H7. Monoclonal antibodies (MAbs) are produced by the fusion of a single antibody-producing cell with an immortalized myeloma (tumor) cell which has lost the ability to produce antibodies. The resulting hybrid (hybridoma) cell line has the desired attributes of rapid, continuous cell multiplication and the production of identical antibodies with a single specificity. This manuscript describes the production and characterization of eight different MAbs that are specific to the E. coli O157 serotype. These MAbs can rapidly detect and identify E. coli O157 strains, which is an essential part in the identification of E. coli O157:H7. However, other testing methods are required to identify the H-antigen, before an E. coli strain can be classified as serotype O157:H7.

Technical Abstract: Identification of the O157 antigen is an essential part of the detection of Escherichia coli O157:H7, which is recognized as a major etiologic agent of hemorrhagic colitis. However, polyclonal antibodies produced against E. coli O157:H7 lipopolysaccharide (LPS) are known to react with several other bacteria including: Brucella abortus, Brucella melitensis,(Pseudomonas) maltophilia. Eight monoclonal antibodies (MAbs) were produced which are specific for the LPS of E. coli O157. Western blots of both the phenol phase (smooth) and aqueous phase (rough) of hot phenol-water-purified LPS indicated that three of the MAbs were specific for the O-antigen and five were reactive with the LPS core. The eight MAbs could be further differentiated by their reactivity to Salmonella O30 LPS (Group N), which is reported to be identical to the E. coli O157 antigen. All eight MAbs reacted strongly to all of the 64 strains of E. coli O157, which included 47 strains of O157:H7 and 17 other O157 strains. None of the eight MAbs cross-reacted with any of the 38 other E. coli serotypes tested, which consisted of 29 different O-antigen serotypes, or with 38 strains (22 genera) of non-E. coli Gram-negative enteric bacteria.

Last Modified: 9/10/2014
Footer Content Back to Top of Page