DETERMINATION OF ALPHA-1-ANTITRYPSIN PHEN OTYPES IN PLASMA USING ISOELECTRIC-FOCUSING ON THIN AGAROSE-GEL

Plasma alpha1 antitrypsin (alpha 1AT) is the major serine protease inh ibitor (Pi) in plasma. It is a glycoprotein, which presents many molec ular variants. Allelic phenotypes are classified alphabetically accord ing to their electrophoretic mobility in the Pi (Proteases inhibitor) system. More than 75 distinct protease inhibitor subtypes have been id entified using isoelectric focusing (IEF). The major interest for dete cting its microheterogeneity is the rare possibility of deficient alle les, which are responsible of low amounts in the alpha 1AT production. The clinical use of the alpha 1AT phenotyping is the diagnosis of her editary alpha 1AT deficiencies. The most commun normal phenotype is MM ; the major deficient phenotypes are MS, MZ, SS, SZ and ZZ. Hereditary deficiencies of the Pi, the most common inborn error in European peop le, lead to pulmonary emphysema in young adults or liver cirrhosis in children. IEF on polyacrylamide gels is the reference method for alpha 1AT phenotyping, but is very difficult to standardize. In the present study, we have developed IEF on agarose gels for Pi subtyping within a number of technical improvements. A 0,5 mm thin agarose gel (1,6%) i s cast on polyester film; focusing is performed using carrier ampholin es (pH = 4.2-4.9), using a very high voltage. Staining is done with a simplified silver nitrate method. The patterns of the different Pi phe notypes obtained with our technique are very attractive. The common su btypes corresponding to the alleles M1, M2, M3, S, Z are univocally de monstrated. Agarose gel allows the advantage of using a non. toxic sub stance. Further the gels are easy to produce and the method is accessi ble to all clinical laboratories.