In-vitro Production of Anti LPS Antibody by Peritoneal and Spleen B-Lymphocyte of Balb/c mice

CD5B1 lymphocytes are the major cell subpopulation for defense found in many organs including peritoneum and splenic follicles. They can produce natural antibodies with poly specific reaction with an important Ligand: Lipopolysaccaride (LPS). The aim of this study was isolation and purification of this unique population from cellular content of peritoneum, spleen and blood and determining their functional activity in producing IgM antibody in response to stimulants in cell culture under experimental conditions.

With direct heart puncture, splenic puncture, and peritoneal lavage from inbred Balb/C mice, cells were collected and purified through Ficole density gradient and nylon wool column for purification of B lymphocytes. In complete tissue culture medium (Bwool), cells were harvested and divided into 2 Groups, experimental and control. LPS stimulation was performed on the experimental group for different durations: 24, 48 and 72 hours. Finally culture supernatants were assayed for IgM concentration with ELISA Technique. The proliferation rate was defined by M.T.T assessment. Immunophenotyping studies for confirmation of cellular purity were carried out by CD3 and CD5 markers.

Lymphocytes from spleen and peritoneum organs had significantly higher levels of IgM secretory activity in 24 hours, as compared to the control groups. In Immuno-phenotyping studies, purified B lymphocytes from peritoneum showed highest levels of CD5 marker.

The findings of this research indicate that Cells collected from splenic puncture and peritoneal fluid are excellent source for IgM antibody with polyspecific properties against LPS in the laboratory and extremely useful for research purposes.