Immunoglobulin separation from serum

Bueno SMA, Flaupt K, and Vijayalakshmi MA. Separation of immunoglobulin G from human serum by pseudobioaffinity chromatography using immobilized L-histidine in hollow fibre membranes. J. Chromatogr. B. 1995 667 51-61. 

The whole serum contains other proteins that block the immunoglobulins involved in capture. The IgG is separated from such proteins and can attach to plastic. The whole serum thus has a propone of poor capture activity. When the... 

Collected blood should be allowed to clot and centrifuged as described previously to separate the serum (Harlow and Lane 1988). Polyclonal antisera should be stable at 4°C for several years if sodium azide (0.02%) is added to inhibit bacterial growth. However, it is usually best to aliquot precious reagents and store them at -80 C. Working stocks should be kept at 4°C, and repeated freeze-thaw cycles avoided. Some investigators prefer to partially purify immunoglobulins from serum using 50% ammonium sulfate (Harlow and Lane 1988). 

Figure 11.15 Immunoelectrophoresis of human serum proteins. The proteins are separated electrophoretically from wells cut in a suitable gel. After electrophoresis, a trough is cut in the gel parallel to the direction of migration and filled with an antiserum. The components are allowed to diffuse for 24-48 hours for precipitation lines to develop. Human serum contains many proteins, among which the immunoglobulins can be identified.

Protein samples used to conduct a protein transfer were bovine serum albumin (BSA, 67,000 daltons), chicken egg ovalbumin (Ovalb, 45,000 daltons), and human serum cryo-precipitate. These agents were obtained from plasma containing a mixture of proteins including Fibrinogen (340,000 daltons), human serum albumin (HSA, 67,000 daltons), and immunoglobulin G (IgG, 47,000-56,000 daltons). The cryo-precipitate was diluted with 20 ml of buffer solution prior to separation. 

Liquid-liquid partition chromatography techniques based on aqueous-aqueous systems have successfully been employed in the fractionation of crude human serum, purification of steroid hormone-binding proteins from human serum, isolation of basic proteins from crude bacterial extracts, purification of immunoglobulins and monoclonal antibodies, DNA fractionations by size, topology and base sequence, as well as the isolation of soluble and ribosomal RNAs in preparative amounts from bulky mixtures [10]. Highspeed CCC using PEG-dextran system has also been employed in the separation of proteins [6]. 

This method is based on the accessibility of the pores in the stationary phase for 99mrpc-iabeled molecules of different molecular sizes. The sample is eluted from a vertical column packed with porous beads of the gel by gravity or low pressure. Smaller Tc species penetrate the pores and are retained on the column, while larger molecules are excluded and are therefore rapidly eluted from the column. This separation technique has particular application for macromolecules, proteins (serum albumin, immunoglobulins [e.g., monoclonal antibodies and their fragments]), but has also been used for separation of small-molecular-weight Tc-diphosphonate complexes. 

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