Development and Use of Two-Site Sandwich ELISA for Determination of Surfactant Protein A in Serum of Horses

Abstract

In the previous study we reported that decreases in concentrations of phosphatidylglycerol, pulmonary surfactant protein A (SP-A), and pulmonary surfactant protein D (SP-D) in BALF of horses after transportation indicated a reduction in the quantity of surfactant in the lungs. However, it is unknown whether SP-A concentrations in sera change during transportation. The purpose of the study reported here was to develop a sandwich Enzyme-Linked Immunosorbent Assay (ELISA) for detection of SP-A in serum of horses and determine whether serum SP-A concentrations change during transportation of horses. The optimal concentration of Triton X-100 was determined by testing various concentrations of Triton X-100 with a sandwich ELISA. Analytical recovery and precision were assayed. Twenty Thoroughbreds were transported by road for 41 hr and five control horses were not transported to determine the effect of transportation on serum SP-A concentration. A combination of solid-phase TA08 and HRP-conjugated WA28 was found to be more sensitive than the other combinations. Maximum values for SP-A in the serum samples were obtained when 3% (v/v) Triton X-100 incubated at a reaction temperature of 37°C were used. This concentration of Triton X-100 and 37°C were used in subsequent assays. The changes of SP-A in sera during transportation had relationships to the condition of horses. Determination of SP-A concentrations by use of the ELISA developed in this study may be useful for the identification of lung conditions or to monitor the progression of lung disease in horses.