Surfactant proteins in bronchoalveolar lavage fluid of horses: assay technique and changes following road transport.
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/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.
Publication Date: 2002-12-31 PubMed ID: 12503595DOI: 10.1136/vr.148.3.74Google Scholar: Lookup
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- Journal Article
Summary
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The researchers created a test to measure two types of surfactant proteins in the lung fluid of horses and then studied how these levels changed after the horses experienced long road trips.
Development of ELISA for Equine Surfactant Proteins
- The researchers began by developing an enzyme-linked immunosorbent assay (ELISA), a diagnostic tool that can measure specific substances in biological samples.
- This ELISA assay was customized to detect surfactant proteins SP-A and SP-D in bronchoalveolar lavage fluid (BALF), a type of fluid found in the lungs, specifically in horses.
- The researchers produced targeted antibodies against these proteins using hybridoma technology, which fuses normal cells with cancer cells to create a cell line that produces antibodies.
- These produced antibodies were purified and analyzed through Western blotting, a technique that detects specific protein molecules from among a mixture of proteins.
Detailed ELISA Procedure and Precision
- The researchers used a two-site sandwich ELISA, a method that involves coated antibodies on a solid surface which capture the target protein (SP-A or SP-D), and then a secondary antibody linked to an enzyme that creates a color change when it reacts with a substrate.
- The reactivity, recovery, and precision of the ELISA assay were tested using different combinations of antibodies, with the aim of measuring very low to moderate amounts of the surfactant proteins.
- They identified the best combinations: solid-phase TA08 with HRP-conjugated WA28 for SP-A, and solid-phase TD13 with HRP-conjugated WD19 for SP-D.
Effect of Road Transport on Equine Surfactant Proteins
- Finally, the researchers used their newly developed ELISA assay to investigate changes in surfactant protein levels after 41 hours of road transport.
- They gathered BALF samples from 30 horses before and after the transport, and found that SP-A decreased by 55% while SP-D decreased by 36%.
- This is comparable to their earlier findings where phosphatidylglycerol, a lung surfactant, also decreased in concentration after transportation stress.
Cite This Article
APA
Hobo S, Yoshihara T, Oikawa M, Jones JH.
(2002).
Surfactant proteins in bronchoalveolar lavage fluid of horses: assay technique and changes following road transport.
Vet Rec, 148(3), 74-80.
https://doi.org/10.1136/vr.148.3.74 Publication
Researcher Affiliations
- Clinical Science and Pathobiology Division, Equine Research Institute, Japan Racing Association, 321-4 Tokami-cho, Utsunomiya-shi, Tochigi 320-0856, Japan.
MeSH Terms
- Animals
- Antibodies, Monoclonal
- Blotting, Western
- Bronchoalveolar Lavage
- Bronchoalveolar Lavage Fluid / chemistry
- Dose-Response Relationship, Immunologic
- Enzyme-Linked Immunosorbent Assay / methods
- Enzyme-Linked Immunosorbent Assay / veterinary
- Female
- Horses / physiology
- Male
- Pulmonary Surfactant-Associated Protein A / analysis
- Pulmonary Surfactant-Associated Protein A / immunology
- Pulmonary Surfactant-Associated Protein A / isolation & purification
- Pulmonary Surfactant-Associated Protein D / analysis
- Pulmonary Surfactant-Associated Protein D / immunology
- Pulmonary Surfactant-Associated Protein D / isolation & purification
- Pulmonary Surfactants / analysis
- Pulmonary Surfactants / immunology
- Pulmonary Surfactants / isolation & purification
- Sensitivity and Specificity
- Transportation
Citations
This article has been cited 2 times.- Araki M, Ohtaki T, Kimura J, Hobo S, Taya K, Tsunoda N, Taniyama H, Tsumagari S, Nambo Y. Presence of surfactant proteins in the uteri and placentae of pregnant mares. J Vet Med Sci 2021 Jul 28;83(7):1167-1172.
- Yamaya Y, Suzuki K, Watari T, Asano R. Bronchoalveolar lavage fluid and serum canine surfactant protein A concentrations in dogs with chronic cough by bronchial and interstitial lung diseases. J Vet Med Sci 2014 Apr;76(4):593-6.
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