Detection of genetic variants of α1-antitrypsin with site-specific monoclonal antibodies

Abstract

The serum protein α1-antitrypsin (α1-AT) serves as the major inhibitor of neutrophil elastase. The most common allele of the α1-AT gene is designated as PiM. The Z mutation is a single-base substitution of the normal M allele, causing a Glu → Lys change at position 342 in the molecule. The ZZ phenotype is associated with a severe deficiency of α1-AT, serum concentrations of the protein being 10% of normal. Individuals with an α1-AT deficiency are at an increased risk of developing emphysema. To generate antibodies that specifically detect the 342 position in the context of the flanking sequences, we synthesized several peptides that included the 342 position for both the M and the Z variant. Immunization with variantspecific peptide-carrier conjugates elicited α1-AT variantspecific responses, as determined in a direct enzymelinked immunoassay. Monoclonal antibodies (MAbs) were selected with different specificity for the 342 region: MAbs F43 recognize only the α1-AT sequence with 342Glu, i.e., all variant proteins that are non-Z, either from hetero- or homozygous individuals; MAbs F50 recognize only the sequence with 342LyS, i.e., all Z-variant proteins in ZZ or heterozygous individuals; MAbs F46 recognize α1-AT with either 342LyS or 342Glu, all variant proteins with sequences as in the peptides used. Z homo- and heterozygotes were detected with our MAbs in a rapid and simple immunoblot assay. Other variants (M, S, and F) can also be assigned on the basis of the electrophoretic pattern. This sensitive detection method is very easy, rapid, and straightforward and provides a powerful tool for diagnosis of the α1-AT deficiencies, allowing early treatment (augmentation of α1-AT) and proper advice on lifestyle practices.