Immunoelectrophoresis crossed

A variation of the above method is crossed immunoelectrophoresis with an intermediate gel. The first electrophoretic steps including plate preparations, running buffer and sample application are the same as with the conventional procedure of Laurell s. The modification with the intermediate gel has been worked out to find out which of the peaks appearing on the complex electrophoretic profile of serum 

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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.

Cross-coupling reactions, alkyl, 12 835 Cross-dyeing, 9 198 Crossed cylinder test, 9 714 Crossed-immunoelectrophoresis, 9 755 Crossed-polarized light (XPL),... 

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... 

FIG. 12.15 Crossed immunoelectrophoresis of human serum with rabbit antihuman serum. (Redrawn with permission from B. Weeke, in A Manual of Quantitative Immunoelectrophoresis Methods and Applications (N. H. Axelsen, J. Kroll, and B. Weeke, Eds.), Universitetsforlaget, Oslo, Norway, 1973.)... 

Nielson, C. S. and Bjerrum, 0. J. 1977. Crossed immunoelectrophoresis of bovine milk fat globule membrane protein solubilized with non-ionic detergent. Biochim. Biophys. Acta 466, 496-509. 

Langeland, T. 1982a. A clinical and immunological study of allergy to hen s egg white. II. Antigens in hen s egg white studied by crossed immunoelectrophoresis (CIE). Allergy 37 323-333. 

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 6-7. 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.

The rocket technique or electroimmunodiffusion (EID), which allows a precise quantitation of antigen, is described in Chapter 21, the crossed immunoelectrophoresis (CIE), which is a qualitative and quantitative method, is dealt with in Chapter 22, and the crossed immunoaffinoelectrophoresis (CIAE), which is more specially designed for the detection of biospecific... 

Razor blades To cut out the gel and to transfer the gel from plate to plate (in crossed immunoelectrophoresis technique), a blade at least 2 X 11 cm is used. 

B0g-Hansen, T. C., Bjerrum, O. J. and, Ramlau,J (1975) Detection of biospecific interaction dunng the first dimension electrophoresis in crossed Immunoelectrophoresis Scand. J. Immunol 4(Suppl. 2), 141-147... 

Fig. 1. Templates for crossed immunoelectrophoresis. (a) First dimension After the first dimension, 5 mm of gel along the edges is discarded. O or CD sample well or slit, (b) Second dimension The first dimensicm strip is transferred to the bottom of the plate and the remaining area is filled with 12 mL of antiserum-containing agarose. bromophenol blue marker spot MKpaper wick positions t agarose containing antiserum.

Fig. 2. Templates fw the three steps for trap ge crossed immunoelectrophoresis. (a) preelectrophoresis (b) first dimension (c) second dimension. . strip to be removed and replaced by 1.2 mL of agarose containing monospecific antiserum /// agarose containing polyspecific antiserum m paper wick positimis bromophenol blue marker spot sample well.

Emmett, M. and Crowle, A. J. (1982) Crossed Immunoelectrophoresis Qualitative and quantitative considerations./. ImmunoL Methods 50, R65-R83. 

Laine, A., Ducourouble, M P., and Hannothiaux, M. H. (1987) Identification of proteins by crossed Immunoelectrophoresis with a trap-gel. AnaL Bxochem. 161, 39- 4. 

Kunidd TJ, Pidard D, Rosa JP, et al. Uie formation of a calcium dependent complex of platelet membrane GPIIb and Ilia in solution as detected by crossed immunoelectrophoresis. Blood 1981 58 268-278. 

Activation of C3 has been determined in the past by either immunofixation or two-dimensional, or crossed, Immunoelectrophoresis of plasma, with determination of the relative amounts of C3 and C3c. Currently, assays of fragments using antisera to neoaintigens (such as those on C3a) arfe preferred for this determination. For either type of assay, plasma samples must be obtained with precautions to prevent in vitro activation by plasmin, Cls, and leukocyte proteases The first few milliliters are discarded (or used for other purposes), then whole blood is collected through the same needle into a tube containing EDTA, Centrifugation should be performed as soon as possible and the separated plasma frozen below -40 C. 

D3. Delforge, D. G., and Aronson, L. D., Crossed Immunoelectrophoresis of alpha-1-antitrypsin using thin layer isoelectrofocusing in polyacrylamide as the first dimension. In Electrophoresis 78 (N. Catsimpoolas, ed.), pp. 253-260. Elsevier, Amsterdam, 1979. 

Fig. 4. Crossed Immunoelectrophoresis and crossed affinity immunoelectrophoresis patterns of human ceruloplasmin, c, Crossed immunoelectrophoresis with anti-ceruloplasmin c-wga, crossed affinity immunoelectrophoresis with WGA (T. vulgaris agglutinin) c-Ica, crossed affinity immunoelectrophoresis with LCA (L. culinaris agglutinin) y-wga, crossed affinity immunoelectrophoresis with WGA of component y from c y-Ica, crossed affinity immunoelectrophoresis with LCA of component y from c z-wga, crossed affinity immunoelectrophoresis with WGA of component z from c z-lca, crossed affinity immunoelectrophoresis with LCA of component z from c. All crossed affinity immunoelectrophoreses were performed with the lectin in the first dimension and anti-ceruloplasmin and appropriate lectin saccharide inhibitor incorporated into the second dimension gel. (Taken from ref. 311.)...

Crossed immunoelectrophoresis with an intermediate gel is a technique to identify a specific component in a mixture, such as that shown in Figure 28-10, p. 343. The technique (Svendsen, P.J., and Axelsen, N.H., J. Immunol. Methods, 1,169, 1972) is to add an intermediate gel that contains an antibody specific for the compoimd of interest and remove it from the pattern. The example described below is to identify human transferrin. Figure 28-10 shows the same second dimension spotting template as in Figure 28-9, with the addition of the intermediate gel application. 

Figure 28-10. Spotting template for intermediate gel crossed Immunoelectrophoresis and the results for one set of samples.

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