An efficient method for the conjugation of a lipopolysaccharide from Salmonella enterica sv. Minnesota with probes bearing hydrazine or amino functional groups

DIEGO PALLAROLA; FERNANDO BATTAGLINI

ANALYTICAL BIOCHEMISTRY

Elsevier

Lugar: Amsterdam; Año: 2008 vol. 381 p. 53 - 58

0003-2697

A conjugation method for coupling probes bearing hydrazine or primary amino groups to a lipopolysaccharide (LPS) is described. LPS is modified through the hydroxyl groups present in its O-antigen moiety by activation with cyanogen bromide in aqueous acetone using triethylamine to enhance the electrophylicity of CNBr. The method yields conjugates with good labeling ratios, preserving the endotoxic activity of the lipid A moiety, which in blood exerts pleiotropic effects on many tissues and organs, resulting in multiple organ damage, circulatory collapse, and death. Conjugation of smooth-form LPS from Salmonella enterica sv. Minnesota with dansyl hydrazine and horseradish peroxidase was carried out yielding a labeling ratio of 330 nmoles dansyl per mg LPS, with nearly no loss of the original endotoxin activity. In the case of horseradish peroxidase, introducing a spacer, a ratio of 28 nmoles HRP per mg LPS were obtained, preserving 65% of the original endotoxic activity. This work shows that under these conditions of CNBr activation, the labeling process has practically no effect on the endotoxic behavior of LPS. The method can be used effectively for the conjugation of LPS with probes bearing primary amino, hydrazine, or hydrazide functional groups.