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Surfactant proteins in bronchoalveolar lavage fluid of horses: assay technique and changes following road transport
  1. S. Hobo, DVM, PhD1,1,
  2. T. Yoshihara, DVM, PhD1,
  3. M. Oikawa, DVM, PhD1 and
  4. J. H. Jones, DVM, PhD2
  1. 1 Clinical Science and Pathobiology Division, Equine Research Institute, Japan Racing Association, 32 1-4 Tokami-cho, Utsunomiya-shi, Tochigi 320-0856, Japan
  2. 2 Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, CA 95616, USA

Abstract

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.

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      Footnotes

      • Dr Hobo's present address is Racehorse Clinic, Miho Training Center, Japan Racing Association, 2500-2 Mikoma, Miho-mura, Inashiki-gun, Ibaraki 300- 0439, Japan

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