Fig. 4

Detection of SP-A: (A) The complexity of BAL; (B) polyclonal anti-human SP-A detects two bands of reduced SP-A; (C) detection of SP-A from gel filtration peaks. (A) Reduced partially purified SP-A (5 µg), BAL (30 µl), and human serum (1 µl) were run on an SDS-PAGE gel (10% w/v acrylamide). Proteins on the gel were detected by silver stain (Bio-Rad). Partially purified SP-A was a gift from Mr. P. Strong (MRC Immunochemistry Unit). (B) IgG was purified from polyclonal rabbit anti-human SP-A by sodium sulfate precipitation and preabsorbed against “aged” serum, and was shown to be specific for SP-A. Reduced SP-A, BAL, and serum were run on an SDS-PAGE gel (10% w/v acrylamide) before blotting to PVDF membrane. The blot was blocked with PBS-EDTA, 0.05% Tween-20 (PBS-Tween), and probed with the anti-human SP-A antibodies (30 µg/ml in PBS-Tween). Bound antibody was detected with goat anti-rabbit IgG alkaline phosphatase conjugate (Sigma; diluted to recommended concentration in PBS-Tween). (C) Representative fractions from within the four peaks of SP-A seen on gel filtration (hex-amer, fraction 10; tetramer, 13; dimer, 17; and polypeptide, 21) were run reduced on an SDS-PAGE gel (10% w/v acrylamide) before blotting to Immobilon P membrane (Millipore). A representative sample of BAL was compared on the same blot. The blot was blocked and analyzed as above. Background staining of IgG heavy chain is seen where IgG is present (BAL and dimer fraction). The 30 and 60 kD bands of SP-A are visible in the hexamer-polypep-tide fractions, showing that there is no apparent proteolytic degradation. The protein standards used for all the above gels were Sigma high-molecular weight (Cat. No. MW-SDS-BLUE) and Gibco BRL low molecular weight (Cat. No. 16040-016).