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Surfactant proteins in bronchoalveolar lavage fluid of horses: assay technique and changes following road transport
Author(s) -
Hobo S.,
Yoshihara T.,
Oikawa M.,
Jones J. H.
Publication year - 2001
Publication title -
veterinary record
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.261
H-Index - 99
eISSN - 2042-7670
pISSN - 0042-4900
DOI - 10.1136/vr.148.3.74
Subject(s) - horseradish peroxidase , bronchoalveolar lavage , microbiology and biotechnology , monoclonal antibody , chemistry , chromatography , blot , pulmonary surfactant , immunoassay , peroxidase , antibody , enzyme , biology , biochemistry , immunology , medicine , lung , gene
An enzyme‐linked immunosorbent assay (ELISA) was developed for equine surfactant proteins SP‐A and SP‐D in bronchoalveolar lavage fluid (BALF). Anti‐equine SP‐A or SP‐D monoclonal antibodies (mAb) were produced by hybridoma technology, purified by the antibody purification reagent, and analysed by Western blotting analysis. The immunoreaction (two‐site sandwich ELISA) with a mAb, peroxidase‐labelled mAb and BALF sample was carried out simultaneously and analytical recovery and precision were assayed. Six mAb for SP‐A and four mAb for SP‐D were successfully cloned in limiting dilution to monoclonality. These mAb were reacted with equine SP‐A or SP‐D on Western blotting analysis. For SP‐A, a combination of solid‐phase TA08 and horseradish peroxidase (HRP)‐conjugated WA28 was found to be more sensitive than other combinations, gave a good dose response and was capable of measuring 0.78 to 100 ng of protein/mi. For SP‐D, a combination of solid‐phase TD13 and HRP‐conjugated WD19 was found to be more sensitive than other combinations, had a good dose response and was capable of measuring 0.78 to 200 ng of protein/ml. The assay was used to determine the effect of 41 hours of road transport on the concentrations of SP‐A and SP‐D in the BALF of 30 horses. The concentrations of SP‐A and SP‐D decreased by 55 per cent and 36 per cent, respectively, decreases similar to the decrease in phosphatidylglycerol concentration previously reported by the authors.