[Determination of alpha 1 antitrypsin phenotypes in plasma using isoelectric focusing on this agarose gel]. / Phénotypage de l'alpha 1 antitrypsine du plasma par isoélectrofocalisation sur gel fin d'agarose.

Plasma alpha 1 antitrypsin (alpha 1 AT) is the major serine protease inhibitor (Pi) in plasma. It is a glycoprotein, which presents many molecular variants. Allelic phenotypes are classified alphabetically according to their electrophoretic mobility in the Pi (Proteases inhibitor) system. More than 75 distinct protease inhibitor subtypes have been identified using isoelectric focusing (IEF). The major interest for detecting its microheterogeneity is the rare possibility of deficient alleles, which are responsible of low amounts in the alpha IAT production. The clinical use of the alpha 1AT phenotyping is the diagnosis of hereditary alpha 1AT deficiencies. The most common 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 people, 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%) is cast on polyester film; focusing is performed using carrier ampholines (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 phenotypes obtained with our technique are very attractive. The common subtypes corresponding to the alleles M1, M2, M3, S, Z are univocally demonstrated. Agarose gel allows the advantage of using a non toxic substance. Further the gels are easy to produce and the method is accessible to all clinical laboratories.

[Agarose gel isoelectric focusing: application to the study of abnormalities of immunoglobulin clonality in CSF and serum]. / L'isoélectrofocalisation sur gel d'agarose: application a l'étude des anomalies de clonalité des immunoglobulines dans le LCR et le sérum.

Since it is quite difficult to commonly use isoelectric focusing (IEF) of proteins in polyacrylamide gel for biological diagnosis, we have developed a method based on IEF in agarose gel, to split proteins from sera and cerebrospinal fluid (CSF). A prefocalisation at low voltage (250 V) is made on a custom thin gel of agarose (0.5 mm) containing some carrier ampholytes (pH 5-9). After deposition of biological samples, the gel is run at 500 V, thereafter at 1200 V. After focusing, the gel is fixed before being coloured by a simplified silver staining technique. In order to demonstrate the good resolution of the immunoglobulines (Ig) in the pH gradient, a transfer on a nitrocellulose membrane followed by an immunofixation was carried out from unstained gels after IEF. This separation on agarose gel shows several advantages, ie its speed (3H total), its lack of toxicity, its sensibility and its reproductibility. It is specially well suited for the diagnosis of diseases characterised by oligoclonal or monoclonal Ig, particularly those found in the CSF during neurologic diseases like multiple sclerosis. Several examples of focused sera and CSF are reviewed in the paper.