INVESTIGADORES
GONZALEZ MAGLIO Daniel Horacio
congresos y reuniones científicas
Título:
Characterization of Lactobacillus rhamnosus GG lipoteichoic acid (LTA) purification process and development of specific quantification techniques
Autor/es:
FRIEDRICH, A.D.; WEILL, F.S.; LEONI, J.; GONZÁLEZ MAGLIO, D.H.
Lugar:
Los Cocos, Córdoba
Reunión:
Congreso; LXI Reunión Anual de la Sociedad Argentina de Inmunología; 2013
Resumen:
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 0,9218. 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 using
the monoclonal antibody obtained.