Abstract
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.
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- Anderson P., Pitt J., Smith D. H. Synthesis and release of polyribophosphate by Haemophilus influenzae type b in vitro. Infect Immun. 1976 Feb;13(2):581–589. doi: 10.1128/iai.13.2.581-589.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gilbride K. A., Rosendal S. Evaluation of a selective medium for isolation of Haemophilus pleuropneumoniae. Can J Comp Med. 1983 Oct;47(4):445–450. [PMC free article] [PubMed] [Google Scholar]
- Inzana T. J., Clark G. F., Todd J. Detection of serotype-specific antibodies or capsular antigen of Actinobacillus pleuropneumoniae by a double-label radioimmunoassay. J Clin Microbiol. 1990 Feb;28(2):312–318. doi: 10.1128/jcm.28.2.312-318.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Inzana T. J., Ma J., Workman T., Gogolewski R. P., Anderson P. Virulence properties and protective efficacy of the capsular polymer of Haemophilus (Actinobacillus) pleuropneumoniae serotype 5. Infect Immun. 1988 Aug;56(8):1880–1889. doi: 10.1128/iai.56.8.1880-1889.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Inzana T. J., Mathison B. Serotype specificity and immunogenicity of the capsular polymer of Haemophilus pleuropneumoniae serotype 5. Infect Immun. 1987 Jul;55(7):1580–1587. doi: 10.1128/iai.55.7.1580-1587.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Inzana T. J. Purification and partial characterization of the capsular polymer of Haemophilus pleuropneumoniae serotype 5. Infect Immun. 1987 Jul;55(7):1573–1579. doi: 10.1128/iai.55.7.1573-1579.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Inzana T. J., Todd J., Koch C., Nicolet J. Serotype specificity of immunological assays for the capsular polymer of Actinobacillus pleuropneumoniae serotypes 1 and 9. Vet Microbiol. 1992 Jun 15;31(4):351–362. doi: 10.1016/0378-1135(92)90127-f. [DOI] [PubMed] [Google Scholar]
- Inzana T. J., Todd J., Veit H. P. Safety, stability, and efficacy of noncapsulated mutants of Actinobacillus pleuropneumoniae for use in live vaccines. Infect Immun. 1993 May;61(5):1682–1686. doi: 10.1128/iai.61.5.1682-1686.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- James K. Immunoserology of infectious diseases. Clin Microbiol Rev. 1990 Apr;3(2):132–152. doi: 10.1128/cmr.3.2.132. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lucas A. H. Expression of crossreactive idiotypes by human antibodies specific for the capsular polysaccharide of Hemophilus influenzae B. J Clin Invest. 1988 Feb;81(2):480–486. doi: 10.1172/JCI113345. [DOI] [PMC free article] [PubMed] [Google Scholar]
- O'Shannessy D. J., Dobersen M. J., Quarles R. H. A novel procedure for labeling immunoglobulins by conjugation to oligosaccharide moieties. Immunol Lett. 1984;8(5):273–277. doi: 10.1016/0165-2478(84)90008-7. [DOI] [PubMed] [Google Scholar]
- Staros J. V., Wright R. W., Swingle D. M. Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions. Anal Biochem. 1986 Jul;156(1):220–222. doi: 10.1016/0003-2697(86)90176-4. [DOI] [PubMed] [Google Scholar]