Simplified procedure for preparation of sensitized latex particles to detect capsular polysaccharides: application to typing and diagnosis of Actinobacillus pleuropneumoniae.

摘要

A novel, inexpensive method for obtaining immunoglobulin G (IgG) specific for capsular antigen is described for use in latex agglutination tests. Hyperimmune rabbit serum against encapsulated Actinobacillus pleuropneumoniae was thoroughly adsorbed with a nonencapsulated mutant. The capsule titer of the absorbed serum was unaffected, whereas reactivity to nonencapsulated cells was reduced to background levels, as determined by enzyme immunoassay. The IgG component of the adsorbed serum was recovered by protein A chromatography and was covalently coupled through a water-soluble carbodiimide to carboxylate latex beads. The sensitized latex particles (SLP) were agglutinated by 10 ng of homologous capsule or more per ml, were not agglutinated by heterologous capsules at concentrations of < 10 micrograms/ml, and were stable for over 1 year at 4 degrees C without loss of sensitivity. There was no difference in the sensitivity or specificity of latex particles coupled with IgG purified by capsule affinity chromatography. The SLP were agglutinated by all strains of bacteria of the homologous serotype but not by heterologous serotypes or strains of Pasteurella multocida, Actinobacillus suis, or Haemophilus parasuis tested at a density equivalent to a 0.5 McFarland standard. The SLP detected homologous capsule in lung tissue, nasal swabs, and concentrated urine samples from all pigs culture positive for A. pleuropneumoniae but one. Precoating of carboxylate latex particles with avidin followed by conjugation of biotin-hydrazide-labelled IgG to capsule increased the sensitivity of the assay approximately 10-fold. Adsorption of serum with nonencapsulated mutants may be used to prepare SLP with optimum sensitivity and specificity without the need to purify capsule or couple capsule to affinity columns.