Immunoelectrophoresis antibodies

Fig. 3. Immunological reactions, where Ag is antigen and Ab is antibody, for detection in electrophoresis (a) Ouchtedony technique (b) single-radial diffusion (c) rocket immunoelectrophoresis and (d) crossed immunoelectrophoresis.

The Mancini and Ouchtedony techniques are the basis of the techniques employed in immunoelectrophoresis. A technique referred to as rockets (59) is named as such because of the appearance of a rocket-shaped antigen—antibody precipitin formed after an antigenic sample is electrophoresed through a gel-containing antibody (Fig. 3c). 

Separation of a mixture of proteins by electrophoretic techniques such as polyacrylamide gel, SDS polyacrylamide or iso-electric focusing usually results in a complex pattern of protein bands or zones. Interpretation of the results often involves a comparison of the patterns of test and reference mixtures and identification of an individual protein, even using immunoelectrophoresis (Figure 11.15), is very difficult. However, specific proteins can often be identified using an immunoblotting technique known as Western blotting. The prerequisite is the availability of an antibody, either polyclonal or monoclonal, against the test protein. 

Alternatively, electrophoretic separation in one direction may be followed by a second electrophoresis in a perpendicular direction, the latter into a gel containing antibodies. This technique is called crossed immunoelectrophoresis and combines high resolution with the possibility of quantification by measuring the area of the precipitate formed. Figure 12.15 is a... 

In immunoelectrophoresis two sequential procedures are applied to the analysis of complex protein mixtures (1) separation of the protein mixture by agarose gel electrophoresis, followed by (2) interaction with specific antibodies to examine the antigenic properties of the separated proteins. 

Immunoelectrophoresis A Antigen is placed in sample wells. B Electrophoresis c Antiserum containing antibody is placed in trough D Insoluble antigen-antibody complexes form precipitin arcs... 

Receptor mimics (anti-idiotypic antibodies) Immunoelectrophoresis (rocket electrophoresis)... 

Second, methods for the characterization of complex antisera are difficult. Antisera to E. coli protein mixtures have been developed with impressive spectra of reactivity using conventional immunization methods (6,22-23). An exact assessment of the spectrum of antibody reactivity is often limited, however, by the resolution of the analytical methods used. Counter immunoelectrophoresis is limited by the relatively low sensitivity of detection and resolution for complex mixtures of reacting species. One dimensional silver stained SDS-PAGE and immunoblotting provides sensitive detection limits but lacks resolution. Therefore, methods which have a high degree of resolution and sensitivity are required to best compare potential improvements in the production of antibodies to minor components in the mixture. 

Combination of electrophoresis in different formats with affinity reactions started in the early 1950s, when Tiselius cells were used for the first time to characterise antigen-antibody interactions. A real milestone report in the field was the determination of the equilibrium constants for the binding of Ca2+ and Zn2+ to serum albumin by gel electrophoresis in 1960 [1]. Finally, the term "affinity electrophoresis" was proposed [2,3]. Its modification or, better said, its particular case, named immunoelectrophoresis, has been used for many years in reports relying on agarose gel separation in conjunction with immunoprecipita-tion. Appearance of precipitated zones on a gel is indicative for the antigen-antibody reaction and can be used for the identification of an analyte and also for its quantification. 

In crossed immunoelectrophoresis, antigen and antibody solutions are loaded at opposite ends of a short agarose gel. With the correct choice of pH, the antigen and antibodies will move towards one another forming a precipitin band at the equivalence point (Figure 6-7)... 

Figure 6-6. Rocket immunoelectrophoresis. anode, and the antibodies to the cathode. Preci-...

Figure 6-7. Crossed Immunoelectrophoresis. Antigen and antibody are loaded into separate wells in an agar gel. On electrophoresis, the antigen and antibody migrate towards one another forming a single precipitin band.

Figure 6-8. Immunoelectrophoresis. Antigen mixtures are loaded into the two centrally located wells in the agarose gel, and then separated by electrophoresis. When the electrophoresis is complete, the antibody mix (antiserum)...

Fig. 2. Immunoelectrophoresis of undigested goat IgG (upper well), and of Fab and Fc from papain digestion of goat IgG (lower well), showing the electrophoretic difference and the reaction of nonidentity between the Fab and the Fc fragments. The slot contains antibody to goat IgG.

Fig. 3. Immunoelectrophoresis of separated Fab (upper three wells) and Fc (lower three wells), foHowing DEAE chromatography of a papain digest of goat IgG. The slots contain antibody to goat IgG.

Assays for insulin antibodies fall into three categories (1) quantitative radioimmunoelectrophoresis, which measures the binding of IgG antibody to radiolabeled insulin by rocket Immunoelectrophoresis into anti-IgG-containing agarose (2) RIAs with separation of bound and free insulin by precipitation with PEG or a second antibody and (3) solid phase immobilization of insulin to test tubes or Sepharose. These are discussed in more detail in Reeves. ... 

Immunological Properties. These should be examined by methods such as immunoassay, Immunoelectrophoresis, and antibody neutralization. 

Quantitative immunoelectrophoresis (Birkmeyer et al., 1981) and reverse quantitative immunoelectrophoresis (Birkmeyer et a)., 1982) allow the determination of average affinity of different antibody populations for an antigen. Plots of rocket area vs. the amount of antibody applied yield straight lines, the slope of which is indicative of affinity (steeper if the affinity is higher). Actual values of K are not obtained. Relative avidity indices can also be obtained with the test of Farr (1958) (Griswold and Nelson, 1984). 

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