Surfactant proteins in bronchoalveolar lavage fluid of horses: assay technique and changes following road transport

An enzyme-linked immunosorbent assay (ELISA) was developed for equine surfa ctant proteins SP-A and SP-D in bronchoalveolar lavage fluid (BALF). Anti-e quine SP-A or SP-D monoclonal antibodies (mAb) were produced by hybridoma t echnology, purified by the antibody purification reagent, and analysed by W estern blotting analysis. The immunoreaction (two-site sandwich ELISA) with a mAb. peroxidase-labelled mAb and BALF sample was carried out simultaneou sly and analytical recovery and precision were assayed. Six mAb for SP-A an d four mAb for SP-D were successfully cloned in limiting dilution to monocl onality. These mAb were reacted with equine SP-A or SP-D on Western blottin g analysis. For SP-A, a combination of solid-phase TAO8 and horseradish per oxidase (HRP)-conjugated WA28 was found to be more sensitive than other com binations, gave a good dose response and was capable of measuring 0.78 to 1 00 ng of protein/ml. For SP-D, a combination of solid-phase TD13 and HRP-co njugated 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 transpor t on the concentrations of SP-A and SP-D in the BALF of 30 horses. The conc entrations of SP-A and SP-D decreased by 55 per cent and 36 per cent, respe ctively, decreases similar to the decrease in phosphatidylglycerol concentr ation previously reported by the authors.