A Rapid Subtractive Immunization Method to Prepare Discriminatory Monoclonal Antibodies for Food E. coli O157:H7 Contamination

Abstract

To detect food E. coli O157:H7 contamination rapidly and accurately, it is essential to prepare high specific monoclonal antibodies (mAbs) against the pathogen. Cyclophosphamide (Cy)-mediated subtractive immunization strategy was performed in mice to generate mAbs that react with E. coli O157:H7, but not with other affiliated bacteria. Specificity of 19 mAbs was evaluated by ELISA and/or dot-immunogold filtration assay (DIGFA). Immunogloubin typing, affinity and binding antigens of 5 selected mAbs were also analysed. MAbs 1D8, 4A7, 5A2 were found to have high reactivity with E. coli O157:H7 and no cross-reactivity with 80 other strains of bacteria including Salmonella sp., Shigella sp., Proteus sp., Yersinia enterocolitica, Staphylococcus aureus, Klebsiella pneumoniae, Citrobacter freundii and other non-E. coli O157:H7 enteric bacteria. Their ascetic titers reached 1∶106 with E. coli O157:H7 and affinity constants ranged from 1.57×1010 to 2.79×1010 L/mol. The antigens recognized by them were different localized proteins. Furthermore, immune-colloidal gold probe coated with mAb 5A2 could specifically distinguish minced beef contaminated by E. coli O157:H7 from 84 other bacterial contaminations. The Cy-mediated subtractive immunization procedure coupled with hybridoma technology is a rapid and efficient approach to prepare discriminatory mAbs for detection of E. coli O157:H7 contamination in food.

Multiple Tolerization Reduces Antibody Binding Against Tolerogen Cells: Implications for the Production of Monoclonal Antibodies

“…While investigating why antibodies reacted with nontumoral cells, (probably through immunodominant shared epitopes), we found a report by Jin et al, (9) in which the authors employed subtractive immunization and produced antibodies that were specific to a limited strain of enterohemorrhagic bacteria, the Escherichia coli O157:H7, (a common contaminant of food, which causes subsequent diarrhea). These mAbs were highly specific, and no crossreactivity with other enterobacteria was observed.…”

“…These mAbs were highly specific, and no crossreactivity with other enterobacteria was observed. (9) However, what immediately caught our attention was the fact that these researchers, unlike others, performed additional tolerization steps sequentially before introducing the actual immunogen in the host. Searching for similar reports, we could not find any that used multiple tolerization to produce antitumor antibodies.…”

Enhanced immunization techniques to obtain highly specific monoclonal antibodies

“…The identification of food contamination may be possible using mAbs capable of recognizing specific species of bacteria, as shown by Jin et al. , 7 who produced 3 mAbs that have high affinity for E. coli O157:H7 and do not cross-react with 80 other strains of bacteria. In this study, the authors used E. coli O157:H19 and E. coli O157:H7 as the tolerogen and immunogen strains, respectively.…”

Cryptic epitope for antibodies should not be forgotten in vaccine design

“…Cryptic epitopes are [14]). This approach was also used to produce diagnostic monoclonal antibodies that recognize cryptic epitopes on pathogenic E. coli [15].…”