Surfactant protein D (SP-D) is a pattern recognition molecule that has an important role in pulmonary host defense. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) for bovine SP-D and determined the concentration of SP-D in bronchoalveolar lavage fluid (BALF) from calves. Bovine SP-D was purified from BALF by using a mannose-Sepharose 6B column. The obtained 44-kDa protein was identified as bovine SP-D by N-terminal amino acid sequence analysis and SDS-PAGE analysis. The peptides corresponding to bovine SP-D amino acid residues SDTRKEGT, which have little homology across bovine serum collectins, were synthesized and used to raise an antibody in rabbits. The obtained antibody was specific for bovine SP-D and did not react with collectins in serum. The anti-bovine SP-D antibody was purified, and an ELISA system was developed. The detection range of this assay was 4-125 ng/ml, and the intra-assay and inter-assay coefficients of variation were 5.6% and 9.7%, respectively. The concentrations of SP-D in BALF collected from calves experimentally infected with bovine adenovirus type-3 or Mannheimia haemolytica were determined by the ELISA. Elevation of SP-D was found in BALF from inoculated lobes of infected calves compared with those of non-inoculated lobes and those from control animals. These data suggest that the ELISA developed in this study may be useful in investigating the physiological role of bovine SP-D in bovine lung.
(Research Team for Production Diseases)
Miyamoto T. et al. (2010) J. Vet. Med. Sci. 72 (10): 1337-1343