Generation and selection of anti-flagellin monoclonal antibodies useful for serotyping Salmonella enterica

Abstract

In developing countries, bacterial acute gastroenteritis continues to be an important cause of morbidity and mortality among young children. Salmonellosis constitutes a major cause of infectious enteritis worldwide, most of them associated to the consumption of contaminated food products. Traditionally, Salmonella has been classified in serovars based on varieties of O and H surface antigens. In the present work we generated and characterized a panel of anti-flagellin monoclonal antibodies (MAbs) in order to select antibodies useful for detecting the H surface antigen. Four different MAbs were obtained by somatic hybridization of splenocytes. We found two MAbs that recognised regions of flagellin conserved among different Salmonella serovars. Other two MAbs recognised structures restricted to Salmonella enterica sv. Typhimurium, being one of them suitable for agglutination tests. Using a diverse panel of S. enterica serovars with different H antigen varieties we confirmed that this MAb agglutinates specifically S. Typhimurium (antigenic formula: 4,12:i:1,2) or other serovars expressing flagellar factor i. In conclusion, we generated a valuable immunochemical tool to be used in simple assays for serotyping of epidemiologically relevant strains. The capacity to characterize specific strains and determine the primary sources of Salmonella contamination generate valuable information of the epidemiology of this microorganism, contributing to the improvement of public health.

A repertoire of high-affinity monoclonal antibodies specific to S. typhi: as potential candidate for improved typhoid diagnostic

“…Conceivably, this cross-reactivity of the pan mAb (P4D9) was due to the contribution of N or C terminus conserved epitopes of flagellin protein, since this mAb was unable to bind to the variable middle region of flagellin from both S. typhi and S. typhimurium. This phenomenon has also been reported earlier [47]. Additionally, enhanced specificity in antibody detection, by middle region, has been exploited against S. enterica serotype Enteriditis, in poultry [50,51], and in case of S. enterica serovar Brandenburg (S. brandenburg) infection, in sheep [52].…”

“…Isolated native flagellin from bacteria was immunogenic and elicited generation of four highly specific monoclonal antibodies, against S. typhi flagellin protein. Earlier reports showed isolation of native flagellin of *52 kDa from Salmonella culture [41][42][43][44][45] and production of anti-flagellin monoclonal antibodies [42,46,47]. We have also isolated genomic DNA, from the clinical isolate, to perform PCR amplification of fliC gene encoding phase 1-d flagellin.…”

Characterization of structural and immunological properties of a fusion protein between flagellin from Salmonella and lumazine synthase from Brucella

“…SDS-PAGE analysis along expression and purification process of the chimeric protein BLS-FliC131 indicated that the monomeric protein has the expected molecular weight of 52 kDa. The BLS-FliC131 chimeric protein is recognized by previously described anti-flagellin and anti-BLS monoclonal antibodies, 14,28 as shown by Western blot (Fig. 1).…”

“…Afterward, urea was gently removed by stepwise dialysis against Tris–HCl–EDTA buffer containing 4 M urea and then against PBS buffer. The solubilized protein was purified by affinity chromatography using monoclonal anti‐flagellin antibodies . Briefly, the 2B3C5 antibody was conjugated to an activated resin (NHS‐Activated Sepharose 4 Fast Flow G & E) according to the manufacturer's instructions.…”

Typhoid Fever as a Challenge for Developing Countries and Elusive Diagnostic Approaches Available for the Enteric Fever

“…The work of Fitts et al (1983) confirms at the gene level that the salmonellae constitute a distinct group. Using one strain of S. typhimurium, 10 unique sequences of DNA were isolated that appeared to specifically hybridize with all the Salmonellae examined by the DNA-DNA hybridization technique [20].…”