Essential Guides for Isolation/purification of Immunoglobulins

The immunoglobulins (Igs), proteins produced by B lymphocytes, have been extensively studied both at molecular and genetic levels. They consist of two identical heavy chains and two identical light chains having therefore the same isotype and the same type, respectively. Igs are puriRed for three main purposes. (i) as therapeutic injections to patients; (ii) for use as a tool in research or clinical diagnosis; and (iii) for their biochemical analysis (speciRcity, isotype or clonal diversity). Most of these applications require that the binding activity of Igs be retained throughout all the puriRcation procedures. PuriRcation of Igs can be performed according to their physicochemical properties, their biological activities or a combination of both. The technique used will depend on the desired degree of purity and the amount and nature of the starting material. The methods that have been described are generally directly applicable to crude materials such as serum, ascitic Suid or cell culture supernatant. Twodimensional polyacrylamide gel electrophoresis (2DPAGE) affords an efRcient way of evaluating the degree of purity reached in afRnity puriRcations. Several aspects of 2D-PAGE analysis are described in detail in two other articles ‘Electrophoresis/Two-dimensional PAGE’ and &Clinical Applications of Electrophoresis/Electrophoresis’ in this Encyclopedia. As a general rule, and independently of the technique used, the starting material should always be devoid of any insoluble substances and the puriRcation be preceded by centrifugation or Rltration. Viscous Suids, such as serum, may be diluted before use, especially for chromatographic procedures. The solutions should contain a bacteriostatic agent, such as 0.02% sodium azide (NaN ), and be kept on ice. Ig solutions should be handled gently, avoiding bubbling or frothing, because such manipulations may be accompanied by denaturing effects, and may lead to protein precipitation.