Validation of antibody reagents for mucin analysis in chronic inflammatory airway diseases

Abstract

In chronic inflammatory airway diseases, mucins display disease-related alterations in quantity, composition and glycosylation. This opens the possibility to diagnose and monitor inflammatory airway disorders and their exacerbation based on mucin properties. For such an approach to be reasonably versatile and diagnostically meaningful, the mucin of interest must be captured in a reliable, patient-independent way. To identify appropriate mucin-specific reagents, we tested anti-mucin antibodies on mucin-content-standardized, human bronchoalveolar lavage fluid samples in immunoblot assays. All commercially available monoclonal antibodies against the major airway mucin MUC5AC were screened, except for those with known specificity for carbohydrates, as glycosylation patterns are not mucin-specific. Our results indicated considerable inter-patient and inter-antibody variability in mucin recognition for all antibodies and samples tested. The best results in terms of signal strength and reproducibility were obtained with antibodies Mg-31, O.N.457 and 45M1. Additional epitope mapping experiments revealed that only one of the antibodies with superior binding to MUC5AC recognized linear peptide epitopes on the protein backbone.

Keywords: Asthma; COPD; MUC5AC; chronic inflammatory airway diseases; mucin capturing; mucin quantification; mucus.

Figure 1.

Quantification of MUC5AC in human BALF samples. MUC5AC content (dark bars) and relative MUC5AC amount in relation to total protein content (light bars) as determined by commercial MUC5AC quantitation kit (mean + SD from two independent experiments).

Figure 2.

Performance of 15 monoclonal anti-MUC5AC antibodies on MUC5AC-content-matched human BALF samples. 150 ng MUC5AC-containing BALF samples from six non-asthmatics and twelve asthmatics were dotted onto nitrocellulose membranes. Each immunoblot was incubated with a different primary anti-MUC5AC antibody and the same fluorophore-labeled secondary antibody. Fluorescence signals were quantified with the LI-COR Odyssey Classic system. Background fluorescence of the membrane was measured and subtracted from the values of the BALF sample dots. Depicted are the mean values of three independent experiments with standard deviation.

Figure 3.

Performance of the five most promising monoclonal anti-MUC5AC antibodies on human BALF samples. 150 ng MUC5AC-containing BALF samples from four non-asthmatics and eleven asthmatics were dotted onto nitrocellulose membranes. Each immunoblot was incubated with one of five anti-MUC5AC antibodies or one irrelevant antibody. Binding of the primary antibodies was detected with a fluorophore-labeled secondary antibody. Fluorescence signals were quantified with the LI-COR Odyssey Classic system. Background fluorescence of the membrane was measured and subtracted from the values of the BALF sample dots. Mean values of three independent experiments with standard deviation are shown.

Figure 4.

Epitope mapping of anti-MUC5AC antibody 2H7. 15mer peptides spanning the MUC5AC protein backbone were synthesized and spotted onto cellulose-coated glass slides. Slides were incubated with monoclonal anti-MUC5AC antibody 2H7 and binding to individual peptides was detected by incubation with fluorescent secondary antibody and quantitation of fluorescence signal with the LI-COR Odyssey Classic system. (A) Regions of the MUC5AC protein backbone covered by the slides 1 – 6 and read-outs for one representative experiment. (B) Detailed analysis of the fluorescence signals from slide #6, containing the sequence regions of MUC5AC recognized by the 2H7 antibody (N_experiments = 6). Peptide sequences of all spots displaying signals above the cut-off value (defined as the mean value of all spot signals on a slide plus three standard deviations; here: 0.22) are given.