Surfactant protein A (SP-A) is one of the four known surfactant-associated proteins found in human lungs. It plays a major role in determining regulation of surfactant uptake and resecretion. Qualitative and quantitative deficiencies of SP-A may contribute to neonatal respiratory distress syndrome. The measurement of its level in amniotic fluid or neonatal tracheal aspirate may be useful in the assessment of replacement therapy using natural or synthetic surfactants. In order to develop an in-house immunoassay to detect the level of SP-A, we used a discontinuous sucrose density gradient to isolate SP-A from amniotic fluid. Polyacrylamide gel electrophoresis was carried out on the isolates with low molecular weight markers. We successfully isolated SP-A from 12 out of 31 samples of amniotic fluid. The isolates were found to be relatively pure and have a molecular weight of about 35 kD. The isolated SP-A were used as immunogens to raise antibodies in rabbits for the immunoassay.
An in-house enzyme-linked immunoabsorbant assay (ELISA) for SP-A was successfully developed using in-house polyclonal anti SP-A and a commercial polyclonal anti-rabbit immunoglobulin horseradish peroxidase conjugate system. The standard curve, generated by using 50 ng of SP-A to coat the plate and 1:500 dilution of polyclonal anti SP-A as a primary antibody, was linear for concentrations of SP-A ranging from 4 micrograms/l to 4000 micrograms/l and reproducible. Results of recovery study of SP-A from a known sample of tracheal aspirate ranged from 94%-114%. Intra- and inter-assay coefficients of variations were 2.7% and 5.6% respectively for a known sample of tracheal aspirate. Interference study showed that tracheal aspirate did not interfere with the assay. The assay developed was intended to be used for SP-A measurement in tracheal aspirates obtained from neonates with and without respiratory distress syndrome.