Production and characterization of monoclonal antibodies against S-layer proteins from Lactobacillus kefir

AMBROSIS, NICOLÁS; CARASI, PAULA; GERBINO, ESTEBAN; SERRADELL, MARÍA

Mar del Plata

Congreso; VIII Congreso Argentino de Microbiología General; 2012

Sociedad Argentina de Microbiología General (SAMIGE)

The S-layer constitutes a crystalline structure of (glyco)proteins which has been described in many different bacterial species, including the genus Lactobacillus. Our laboratory has several potentially probiotic strains of L. kefir which contain S-layer proteins. These proteins were partially characterized and a high structural heterogeneity was observed among them. Moreover, we have demonstrated their capacity to inhibit the invasion of Salmonella enteritidis to Caco-2 cells and the antagonism of Clostridium difficile toxins. The objective of the present work was to obtain monoclonal antibodies (Mabs) against S-layer proteins from L. kefir, in order to get immunochemical tools to advance into the study of the structure, function and properties of these S-layer proteins. Mabs-producing hybridomas were obtained by the method described by Galfré and Milstein. The immunization protocol consisted in 3 serial intraperitoneal injections of a mixture of the S-layer proteins from L. kefir CIDCA 8321 (aggregating strain) and JCM 5818 (non-aggregating strain) with Freund adjuvant in BALB/c mice. The follow-up of the immunization protocol was carried out by titration of sera by indirect ELISA. Splenic cells from the mouse presenting the major anti S-layer titer were fused with NSO cells (myeloma line). As a result of the hybridization, one Mab- producing hybridoma was obtained: 4G11H9. This antibody showed reactivity against 20 of 22 strains of L. kefir (both aggregating and non-aggregating ones), 1 of 3 strains of L. parakefir and L. brevis ATCC 8287 by dot-blot assay. The same profile of recognition was obtained by immunoblotting against S-layer proteins extracted from the different strains. These results suggest that Mab 4G11H9 recognizes a conserved epitope present in S-layer proteins from L. kefir and also in phylogenetically related species such as L. parakefir and L. brevis. The Mab 4G11H9 will be purified by isoelectric precipitation of gamma-globulin fraction from ascites and further affinity chromatography. Based on the characteristics of reactivity, purified Mab 4G11H9 could be employed as a tool for the detection of whole bacteria and to reveal the presence of surface proteins belonging to different strains of L. kefir. Additionally, 4G11H9 will be potentially useful to inhibit the S-layer functions in different in vitro assays.