CHARACTERIZATION OF LACTOBACILLUS RHAMNOSUS GG LIPOTEICHOIC ACID PURIFICATION PROCESS AND DEVELOPMENT OF SPECIFIC QUANTIFICATION TECHNIQUES.

FRIED RICH, ADRIAN; WEILL, FEDERICO; LEONI, JULIANA; GONZÁLEZ MAGLIO, DANIEL

Cordoba

Congreso; LXI Reunión Anual de la Sociedad Argentina de Inmunología; 2013

Sociedad Argentina de Inmunologìa

18) CHARACTERIZATION OF LACTOBACILLUS RHAMNOSUS GG LIPOTEICHOIC ACID PURIFICATION PROCESS AND DEVELOPMENT OF SPECIFIC QUANTIFICATION TECHNIQUES.  Lipoteichoic acid (LTA) is a major component of Gram positive cell wall. Its role in inflammation and sepsis by pathogenic bacteria has been profusely described, as well as its immune -regulatory effect by probiotic bacteria. Within this group, Bifidobacterium and Lactobacillus are the most studied. Lactobacillus rhamnosus GG (LGG), whose properties have been extensively described, is used worldwide in the dairy industry. Despite the fact that extraction and purification of LTA is well known, there are few works that describe this process and no one that describe specific quantification. For this reason, we decided to develop immune and non -immune based strategies to analyze LTA along its purification process. The purity of LTA was checked in each purification fraction by two different techniques: a) SDS-PAGE with a subsequent periodic acid oxidation and silver staining procedure; and b) Western Blot using a self produced serum anti-LGG cell wall. Afterwards, a competitive ELISA was developed to quantify LTA in each fraction. At last, in order to check the integrity and functionality of LTA (pooled fractions) an in vitro bioassay stimulating peritoneal macrophages and a Mass Sprectrometry by MALDI-TOFF were performed. Purified LTA was used to produce monoclonal antibodies. High purity and identity in all fractions were proven by SDS-PAGE and Western Blot. The competitive ELISA developed showed a linear range between 0,25 -12,5 ug/ml with a R2 of 0,9241. The whole purification process yielded 10 mg of LTA from 3 liters of culture media. Mass spectrometry showed 3 major peeks (5378, 6946 and 8596 m/z); while peritoneal macrophages responded producing TNF-a in a dose ?dependent manner after LTA stimuli. Two adequate IgM producing hybridomas were obtained. The present work shows a variety of assays that allows the evaluation of LTA purification process and describes a specific quantification technique, which can be optimized by us ing the monoclonal antibody obtained