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 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/ml. 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.
Hobo
S
S
Clinical Science and Pathobiology Division, Equine Research Institute, Japan Racing Association, 321-4 Tokami-cho, Utsunomiya-shi, Tochigi 320-0856, Japan.
Yoshihara
T
T
Oikawa
M
M
Jones
J H
JH
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D018893:Bronchoalveolar Lavage; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry*; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D005260:Female; D006736:Horses / Q000502:physiology*; D008297:Male; D037662:Pulmonary Surfactant-Associated Protein A / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification; D037663:Pulmonary Surfactant-Associated Protein D / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification; D011663:Pulmonary Surfactants / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D014186:Transportation
D000911:Antibodies, Monoclonal; D037662:Pulmonary Surfactant-Associated Protein A; D037663:Pulmonary Surfactant-Associated Protein D; D011663:Pulmonary Surfactants