Immunoglobulin fraction

Va.ria.tions in Methods. The various immunochemical methods can differ in a number of ways. For example, the analytical reagent may be cmde antisemm, monoclonal antibodies, isolated immunoglobulin fractions, etc. The conditions under which the method is mn, detection of the antigen—antibody complex, and the techniques used to increase sensitivity or specificity of the reaction all maybe varied. 

The immunoglobulin fraction from each bleed was purified by use of a recirculating isoelectric focusing (RIEF) technique (Bier et al. 1979). The whole serum was diluted 1 3 with urea, to yield a final urea concentration of 3 M, and then desalted by electrodialysis. The urea was added to prevent precipitation under hypotonic conditions. Ampholine (1 percent w/v, pH 3.5 to 10, LKB... 

Electrophoretic Methods. Little use has been made of electrophoretic techniques for the fractionation of the whey proteins. Column isoelectric focusing has been used to fractionate further the crude immunoglobulin fraction obtained by Smith s procedure (Josephson et al. 1972). Two major peaks, a shoulder, and two minor peaks were obtained, but no attempt was made to identify the components in the peaks. 

The effectiveness of surface and interfacial tension depressants can be compared by plots of concentration versus tension. Various dilution studies of milk, skim milk, wheys, and solutions of milk proteins reveal that casein and the proteins of the lactalbumin fraction (/3-lactoglobu-lin, a-lactalbumin, and bovine serum albumin) are powerful depressants, while the proteins of the immunoglobulin fraction are somewhat... 

Dialyze the immunoglobulin fractions against a suitable buffer and store at 4°C (see Note 11). 

The total volume of the gradient should be 10-20-column volumes. In this method, albumin elutes immediately following the immunoglobulin fraction. The separation can be improved if necessary by applying a shallower gradient. 

The pivotal reagent common to all immunohistochemical techniques is the antibody. The availability of new antisera, their immunoglobulin fractions and monoclonal antibodies to an ever-increasing number of clinically useful tissue antigens has enormously expanded the quantity and quality of the immunohistologic repertoire. To better comprehend the potential of immunohistochemical staining methods as well as associated problems, it is necessary to have a basic knowledge of antibodies, their potentials and their limitations. 

Mild to moderate hemolysis in antiserum resulting from sub-optimal bleeding techniques probably does not interfere with most immunohistochemical staining procedures, but excessive hemolysis should be avoided. If excessive hemolysis or lipemia is encountered, isolation of the immunoglobulin fraction from the antiserum may be necessary. Such isolates will usually appear colorless and clear. Discard all immunochemicals, including antisera and normal non-immune sera contaminated with bacterial growth. Their use in... 

For polyclonal antibodies, negative reagent controls should be a dilution of immunoglobulin fractions or whole serum of normal/ non-immune serum of the same animal source. Again, the negative reagent control should be applied in the same concentration as the test antibody, and the same diluent should be used. 

The extraction and purification of serum-derived immunoglobulin fraction in hen egg yolk by the combined treatment of the raw egg yolk with caprylic acid and ammonium sulfate has been reported.64 This simple two-step method proved to be rapid, reproducible, and suitable for batch processing of pooled egg yolks. The extraction procedure had no adverse effects on antibody titer. Caprylic acid is, in fact, a natural substance the concentration of autologous caprylic acid in serum is in the range of 40 ppm.65 No adverse effect of caprylic acid should be expected because of traces present in a pharmaceutical solution. Caprylic acid has a low toxicity for vertebrates but acts as a bacteriostatic agent. 

McLaren, R. D., Prosser, C. G., Grieve, R. C., and Borissenko, M. (1994). The use of caprylic acid for the extraction of the immunoglobulin fraction from egg yolk of chickens immunized with ovine alpha-lactalbumin. /. Immunol. Methods 177, 175-184. 

The immunoglobulin fraction derived from the polyclonal hyperimmune serum of humans and animals has been used clinically in combating conditions such as bacterial infections and maternal immunization by Rh-positive cells from the fetus. Polyclonal sera raised against proteins have also been valuable diagnostic and research tools. However, the undefined composition of polyclonal antisera results in wide and unpredictable cross-reactivities. Furthermore, the quantities of antibody available are limited to the amount of serum an immunized animal can yield. Monoclonal antibodies described below circumvent these problems of definition and quantity associated with polyclonal sera. 

Untreated serum contains a set of 11 proteins called complement. These proteins are not immunoglobulins and do not increase quantitatively after immunization. However, they do bind to antigen-antibody complexes and can cause problems for certain immunochemical applications. These proteins may be inactivated by heating the serum to 56°C for 10 to 20 minutes. This treatment will not harm the immunoglobulin fraction if the temperature is carefully regulated. 

Preparation of Immunoglobulin Fractions from Whole Serum... 

Labeling should be carried out on immunoglobulin preparations derived from the selected antisera, so as to maximize the proportion of specific antibody to total protein and hence reduce non-specific activity in the final reagent. Immunoglobulin can be prepared by any of the methods described above, but only in the most demanding systems will it be necessary to prepare immunospecific antibody rather than a crude immunoglobulin fraction. 

Polyclonal Antibodies After an antigen is injected into an animal by a regimen designed to induce an optimal immune response, serum can be collected from the animal and the immunoglobulin fraction isolated. This antisera is enriched with antibodies specific for the original antigen. Because a large number of lymphocytes are involved in the production of the antisera, antibodies produced by this classical method are called polyclonal. 

Polyclonal antibodies can be used as whole serrim or in purified form, the latter either as the total immunoglobulin fraction or aflinity purified. Due to the heterogeneity of the antibody-binding sites of polyclonals, different selectivity for different antigens will appear and this problem is simply avoided by the use of monoclonal antibodies. However, even though it is often... 

Tishchenko G. Neosepta microporous ion-exchange membranes in dialysis desalination of immunoglobulin fraction of mouse ascitic fluids. J Membr Sci 1996 113 237-248. 

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