Two efficient methods for the conjugation of smooth-form lipopolysaccharides (LPS) with probes bearing hydrazine or amino groups. II. LPS activation with a cyanopyridinium agent (CDAP)

FERNANDO BATTAGLINI; DIEGO PALLAROLA

Microbial Toxins: Methods and Protocols (Methods in Molecular Biology)

Humana Press Inc.

Lugar: Totowa, NJ. ; Año: 2009;

This chapter presents a conjugation method for coupling probes bearing hydrazine or primary amino groups to a lipopolysaccharide (LPS). LPS is modified by activation of the hydroxyl groups present in its O-antigen moiety with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP). The method yields conjugates with good labeling ratios, preserving the endotoxic activity of the lipid A moiety. Conjugation of smooth-form LPS from Salmonella enterica sv. with dansyl hydrazine and horseradish peroxidase yields labeling ratios above 110 nmoles dansyl per mg LPS, with nearly no loss of the original endotoxic 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 and an enzymatic activity of 120 units/mg.