PURPOSE: Human normal M-type A1PI occurs in plasma in several isoforms that are molecules with different numbers of sialic acids (isoforms M1, M2, M4, M6) or missing 5 N-terminal amino acids (M7, M8). In addition A1PI concentrates lack the C-terminal amino acid lysine. Less is known about the A1PI isoform pattern in broncheoalveolar lavage solutions. This is mainly because A1PI is found in low concentrations in the range of few μg/ml impeding a direct analysis with IEF. Here we describe a convenient method, relying on commercially available reagents and IEF gels, that enables the A1PI isoform pattern to be revealed in dilute solutions.
METHODS: Two commercially available A1PI concentrates were used for a feasibility study. IEF was done on Immobiline IPG 4.2-4.9 (GE Healthcare Bio-Sciences) using 1% Pharmalyte 4.2-4.9 and 20% glycerol for re-hydrating the gels. 0.2 M orthophosphoric acid and 0.2 M NaOH were used as anodic and cathodic solution. Dilute samples were concentrated/desalted by precipitation with 50% PEG 5000 in the presence of rabbit serum. The concentrated samples were applied cathodically, focused for 6 hours and transferred to nitrocelluose by Western blotting. For the immunodetection the antibodies rabbit anti-human α1-antitrypsin and goat anti-rabbit IgG-peroxidase were used. Peroxidase was detected using the Opti 4 CN kit.
RESULTS: The method could visualize very complex A1PI isoform patterns, even at the low total A1PI concentration of 0.5 μg/ml. The step introduced for concentration and/or desalting the samples had no influence on the specific pattern. The blotting procedure did not affect the high resolution of the IEF method as shown by the direct comparison with the IEF pattern obtained after Coomassie staining.
CONCLUSION: We describe a high resolution IEF method useful for visualizing the A1PI isoform pattern in dilute solution.
CLINICAL IMPLICATIONS: Due to the good sensitivity obtained the direct IEF immunoblotting method can be used to analyze BAL solutions.
DISCLOSURE: Alfred Weber, Employee All authors are employees of Baxter BioScience.