Development of enzyme-linked immunosorbent assay for bovine surfactant protein D in bronchoalveolar lavage fluid.
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 using a mannose-Shepharose 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 available to investigate the physiological role of bovine SP-D in bovine lung.
Production Disease Research Team, National Institute of Animal Health, Kannondai, Tsukuba, Ibaraki, Japan. miyomoto@affrc.go.jp
Toru
T
Miyamoto
Motoko
M
Takanashi
Rieko
R
Horino
Harumichi
H
Yamanaka
Minoru
M
Narita
D000257:Adenoviridae Infections / Q000662:veterinary; D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry* / Q000276:immunology; D002417:Cattle; D002418:Cattle Diseases / Q000821:virology; D037222:Collectins / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D010446:Peptide Fragments / Q000737:chemistry; D037663:Pulmonary Surfactant-Associated Protein D / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D011817:Rabbits / Q000276:immunology; D012712:Serum Globulins / Q000737:chemistry
D037222:Collectins; D010446:Peptide Fragments; D037663:Pulmonary Surfactant-Associated Protein D; D012712:Serum Globulins; C024014:conglutinin