Immunization specific antibody separation

About 10% of serum proteins are immunoglobulins (Igs). After immunization, the specific antibodies produced are about 1"C5% of this fraction, so the required Ig (in ELISA) may be from 0.1 to 2.5% of the total protein in a serum. Some assays are favored by the relatively crude fractionation of serum to obtain Igs, e.g., for use in binding to plates in trapping (sandwich assays) to avoid competition for plastic binding sites by other serum proteins. Several methods for separation of Igs are available for use in ELISA. These procedures are suitable for polyclonal antibodies but not necessarily for mAbs. The isolation of total Igs as compared to the purification of specific Igs, is relatively simple. 

The primary alternative to the instrumental methods summarized above is immunodetection of EA with specific antibodies. Antiserum raised in response to immunization with lysergol (6a) coupled to human serum albumin recognizes several EA (100). Used for competitive enzyme-linked immunosorbent assay (ELISA), this broad-spectrum antiserum allows rapid detection of total EA in many samples concurrently. However, it does not separately quantify the different EA that contribute to the profile, and different EA show different reactivities to the antiserum. Monoclonal antibodies specific for individual EA species, such as ergotamine, also have been raised and provide an effective means for detection and quantification of the particular EA, as well as a valuable tool for physiological studies (101). 

Techniques for the detection or assay of various substances based on the reaction of those substances with specific antibodies (or vice versa, i.e. the detection and assay of antibodies using antigens). Such techniques include agglutination reactions, automated immune precipitation, complement fixation tests, crossed electrophoresis, counter electrophoresis, double diffusion, enzyme immunoassay, fluoroimmunoassay, haem-agglutination, immunoelectrophoresis, immunofluoresence, radial immunodiffusion, spin immunoassay, immunofixation, immunoradiometric assay and radioimmunoassay. See separate entries for these subjects. 

A technique that is used for the identification of proteins. It consists initially of separation of the proteins of the serum, or other fluid, by electrophoresis. This is usually carried out in agarose, although other support materials can be used. After electrophoresis, antiserum is placed in a trough adjacent and parallel to the direction of electrophoresis and the proteins and antibodies are allowed to diffuse towards each other. Interaction between the antibodies and the protein antigen results in the precipitation of immune complexes within the gel, which takes the form of an arc or bow for each protein. Thus if human serum has been separated and polyvalent antiserum has been placed in the trough, a series of arcs is obtained, each arc resulting from the precipitation of a serum protein with its own specific antibody. Polyvalent antiserum or monospecific antisera (anti-IgG, anti-IgA etc.) can be placed in the troughs. In the case of mono-specific antiserum, usually only one precipitin bow results. 

Fig. 4. Classification of reported noncompetitive immunoassays for haptens based on the assay principle. (A) Assays that include a chemical modification of hapten to allow sandwich-type detection. (B1) Improved single-antibody immunometric assays that separate immune complex and excess labeled antibody, either by using a hapten-immobilized affinity column or based on differences in their physical properties. (B2) A variation of single-antibody immunometric assays based on masking of unoccupied antibody by an immunoreactive macromolecule followed by selective capture and detection of the hapten-occupied antibody. (C) Assays employing a probe molecule specific to a hapten-antibody complex.

Figure 8-1. Electrophoretic separation of serum taken from a rabbit immunized with ovalbumin. The solid line indicates the result obtained before addition of ovalbumin to the serum and the broken line indicates the result obtained when ovalbumin antibodies are specifically precipitated prior to electrophoresis by addition of ovalbumin. [From A. Tiselius and E. A. Kabat, J. Exp. Med., 69 119 (1939) as redrawn by B. D. Davis, R. Dulbecco, H. N. Eisen, H. S. Ginsberg, and W. B. Wood, Jr., Microbiology, 2nd ed.. Harper and Row, New York, 1973.]...

Antibodies can be separated into different classes (IgM, IgG, IgA, and IgE) and subclasses (IgGi, IgCg, IgCg. and so on) on the basis of the structure of their heavy chains. This separation into different isotypes can be achieved using antisera directed against antigens on the heavy chains that are specific for the particular isotype. Members of an isotype share important properties, such as the ability to bind components of the comple-ment system, or to Fc-receptors present on certain effector cells of the immune system. 

Individual animals immunized with the same substance can produce antibodies that may differ in affinities, titer, and specificities. " Such differences are appeu-ent with antibodies studied by the more classical physical chemical procedures. However, even among anti-enzyme sera harvested from individual rabbits that had been immunized with -lactamase, some were found to neutralize the activity of the enzyme, others to stimulate its activity, and still others were stimulatory and then inhibitory at higher concentrations." In radioimmunoassay, each antiserum from an individual animal must be characterized separately to select those that have the proper affinities and specificities. The production of monoclonal antibodies by hybridoma technology can yield molecules with defined specificities and affinities. As this technology becomes affordable for the average research laboratory, problems associated with antibody heterogeneity are due to diminish. 

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