The affinity of the 2C1 antibody for Z α1-antitrypsin polymers was
entirely abolished by the Gly117Phe mutation when the double variant (Gly117Phe/Z) was transiently expressed in COS-7 cells (data not shown). This was despite the expression of the Gly117Phe/Z mutant resulting in comparable levels Luminespib clinical trial of polymeric material as Z α1-antitrypsin in cell lysates and supernatants of transfected cells.21 When used in western blot analysis of nondenaturing PAGE, the 2C1 antibody detected polymers of M and Z α1-antitrypsin formed by heating in vitro and, most importantly, polymers of Z α1-antitrypsin isolated from the liver of an individual with α1-antitrypsin
deficiency (Fig. 2B, left panel). It did not detect the monomeric form of either variant. Another mAb generated at the same time, this website 2D1, detected all the species present on the same membrane with similar intensities (Fig. 2B, right panel). The 2C1 mAb did not recognize the stable monomeric latent conformer of α1-antitrypsin22 when assessed by western blot analysis (results not shown). The mAb 2C1 was also assessed in immunocytochemistry. COS-7 cells were transfected with cDNA for M or Z α1-antitrypsin and costained with a rabbit polyclonal antibody that detects all α1-antitrypsin (Fig. 3A, red) and the 2C1 mAb (Fig. 3A, green, overlap in yellow). Although all cells showed strong staining with the polyclonal antibody, only cells expressing polymerogenic Z α1-antitrypsin showed a strong signal with mAb 2C1, indicating that it also recognized polymers in this technique. Moreover, the 2C1 mAb was able to detect pathological polymers in paraffin-embedded liver sections from PI*Z homozygotes (Fig. 3B, middle panel). Our new mAb 2C1 is thus a powerful tool for the study of polymerization selleck chemicals llc of α1-antitrypsin both in vivo and in experimental models of α1-antitrypsin
deficiency. Inclusions of α1-antitrypsin can also form in the liver as a result of mutations in the shutter region, which is distinct from the Z variant (Fig. 1). The 2C1 antibody was used to investigate a novel severe shutter region mutant that we have termed α1-antitrypsin King’s. This mutation came to clinical attention in a 6-week-old Caucasian boy presented with prolonged neonatal jaundice. He was born after an uneventful dichorionic and diamniotic twin pregnancy and elective caesarean section at 34 weeks gestation. Physical examination revealed mild jaundice but was otherwise normal. His paternal grandfather was recently diagnosed with unresectable hepatocellular carcinoma.