Immunoglobulin separation

Class 1 and class II MHC molecules bind peptide antigens and present them at the cell surface for interaction with receptors on T cells. The extracellular portion of these molecules consists of a peptide-binding domain formed by two helical regions on top of an eight-stranded antiparallel p sheet, separated from the membrane by two lower domains with immunoglobulin folds. These domains are differently disposed between the two protein subunits in class I and class II molecules. 

FIGURE 7.17 Separation of a complex mixture on Fractogel EMD BioSEC (S) with a column dimension of 1000 X 50 mm (Superformance glass column). The sample contained ferritin (I), immunoglobulin G (2), transferrin (3), ovalbumin (4), myoglobin (5), aprotinin (6), and vitamin B, (7). Five milliliters of the mixture was injected onto the column at a flow rate of 3 ml/min (eluent 20 mAI sodium phosphate buffer, 0.1 M NaCI, pH 7.2). 

Another application shows the preparative purification and polishing of a therapeutic fusion protein with a humanized recombinant IgG protein. The fusion protein was expressed by the fermentation of baby hamster kidney cells. The filtered culture supernatant (155 liters) contained 2.2 g of IgG and 75.5 g of total protein. After the immunoglobulins were isolated by expanded bed adsorption and rebuffering, the IgG fraction was bound to Fractogel EMD SOj (M). This column achieved baseline separation of complete antibodies (fusion protein) from small amounts of antibodies lacking the fusion part. The resulting highly purified IgG fraction (110 ml) was diluted to 150 ml and... 

The L chains and H chains are synthesized as separate molecules and are subsequently assembled within the B cell or plasma cell into mature immunoglobulin molecules, all of which are glycoproteins. 

Each immunoglobulin light chain is the product of at least three separate strucmral genes a variable region... 

The first is glycosaminoglycan, a compound produced by the body that coats the epithelial cells of the bladder. This compound essentially separates the bladder from the urine by forming a protective layer against bacterial adhesion.14 A second compound known as Tamm-Horsfall protein is secreted into the urine, and prevents E. coli from binding to receptors present on the surface of the bladder. Other factors implicated in contributing to host defense mechanisms include immunoglobulins, specifically IgA, and lactobacilli, bacteria that are part of the normal vaginal flora.13,15... 

S. Aoyagi, R. Imai, K. Sakai, and M. Kudo, Reagentless and regenerable immunosensor for monitoring of immunoglobulin G based on non-separation immunoassay. Biosens. Bioelectron. 18, 791-795 (2003). 

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.

The only cases that were omitted from Fig. 48 are the disulfides that span the /3 barrels in immunoglobulins (Epp et ah, 1975 Saul et ah, 1978). They are unusually long, with Ca separations of 6.6 to 7.4 A, which is achieved by having both x and x close to 180°. These long disulfides are trans-gauche-trans (180°, 90°, 180°) in x Xs> X >... 

Immunoglobulins in the form of monoclonal antibodies are manufactured commercially for therapeutic and diagnostic uses. Major areas of consideration in these applications are quality control and bioactivity. Separation of immunoglobulins has proven to be a challenge even for well-established techniques such as HPLC. Difficulties arise due to the large size of antibodies and their surface properties, which increase their tendency to interact with proteins and matrix. 

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