Two efficient methods for the conjugation of smooth-form lipopolysaccharides (LPS) with probes bearing hydrazine or amino groups. I. LPS activation with cyanogen bromide

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 smooth(S)-form lipopolysaccharide (LPS). LPS is modified by activation of the hydroxyl groups present in its O-antigen moiety with cyanogen bromide in aqueous acetone. 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. Minnesota with dansyl hydrazine and horseradish peroxidase yields labeling ratios above 300 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 per mg LPS is obtained, preserving 65% of the original endotoxic activity.