An enzyme-linked immunosorbent assay (ELISA) for the determination of mucin levels in bronchoalveolar lavage fluid.

ABSTRACT

INTRODUCTION: A method to measure the mucin concentration in bronchoalveolar lavage (BAL) fluid was developed to aid efforts to identify pharmacologically the mechanisms that modulate pathophysiological mucin secretion. Mucins are the major macromolecular components of mucus. In the airways, mucus is the first line of defense against inhaled microorganisms (infection) and particulates (irritation). METHODS: An enzyme-linked immunosorbent assay (ELISA) was developed, comparing two monoclonal anti-mucin antibodies (A10G5 and 45M1) raised to human mucin, to quantify the mucin in BAL fluid from animal models of pulmonary inflammation. To validate the ELISA method, rats were exposed to ovalbumin (OVA, in sensitized rats), lipopolysaccharide (LPS), vanadium pentoxide (V(2)O(5)), or saline. One hundred microliters of BAL fluid was analyzed for mucin concentration. Pooled BAL fluid from untreated rats was used as an internal "plate standard", as a standard mucin that cross-reacts with A10G5 was unavailable. RESULTS: We found both antibodies reacted with rat, human, and guinea-pig mucin; where the 45M1 antibody also reacted with the mucin in porcine BAL, while A10G5 did not. We determined the mucin concentration in each BAL fluid sample relative to the standard, defined as a mucin concentration of 100 plate units. BAL fluid from LPS (218+/-25 plate units, n=5), OVA (386+/-31, n=3), V(2)O(5) (1208+/-450, n=6) challenged rats displayed significantly elevated mucin concentration over their saline controls (126+/-22, n=12). Subsequently, the 45M1 antibody displayed immunoreactivity with a commercially available crude preparation of porcine stomach mucin, allowing us to calculate the concentration of mucin directly compared to the known concentration of the porcine stomach mucin standard. Both the 45M1 and A10G5 based ELISA assays detected higher mucin content in the saline challenged rat than the saline challenged guinea pig BAL. DISCUSSION: The recent availability of the 45M1 antibody and the use of the crude purification of porcine stomach mucin as a reference standard should allow for direct comparison of mucin concentration in BAL (and other fluids).