Electrophoresis immuno

See crossed electrophoresis, counter electrophoresis, immuno-electrophoresis... 

Anderson, N. G., and Caton, J. E. (1973), High resolution electrophoresis Immuno-subtraction. In preparation. 

Four of the entries shown in the comment column of Table 3 for macromolecules also indicate that problems exist. The subjects of the first, tenth and thirteenth entries in Table 3, namely sample pre-treatment procedures, choice of detector and collection of separated fractions, are all connected in that they arise from the complexity of the analyte mixtures, a subject discussed previously. The subject of the first entry constitutes the major, at present unsolved, problem in the separation of macromolecules. As a result it may be confidently predicted that much of the instmment manufacturers research and development efforts at the present time lies in this area of automated sample pretreatment devices suitable for mixtures of macromolecules because this area must be automated if the whole HPLC process is to be automated. The problem indicated in the tenth entry of Table 3, concerning the choice of detector for macromolecules was also discussed in the previous part. Therefore it is sufficient to note here that because of the lack of a universal, sensitive detector for macromolecules two or more of the available detector molecules, arranged in tandem, may need to be employed. Alternatively if a single detector module of the type already discussed in the previous section is used, then discrete fractions of the eluate must be collected for subsequent off-line analysis by say, gel electrophoresis, immuno- or bio-assay procedures. This alternative practice accounts for the optional entry number 13 in Table 3 regarding the provision of a fraction collector. 

Two variations of the basic technique are isoelectric focusing and immuno-electrophoresis. The former offers improved resolution and sharper bands in the separation of weak acids, weak bases and ampholytes through the use of pH and density gradients superimposed along the potential gradient. The latter employs specific antigen-antibody interactions (Chapter 10) to visualize the separated components of serum samples. 

NHH Heegaard. Determination of antigen—antibody affinity by immuno-cap-illary electrophoresis. J Chromatogr A 680 405-412, 1994. 

NHH Heegaard, DT Olsen, KLP Larsen. Immuno-capillary electrophoresis for the characterization of a monoclonal antibody against DNA. J Chromatogr A 744 285-294, 1996. 

CAE employing antibodies or antibody-related substances is currently referred to as immunoaf-hnity capillary electrophoresis (lACE), and is emerging as a powerful tool for the identification and characterization of biomolecules found in low abundance in complex matrices that can be used as biomarkers, which are essential for pharmaceutical and clinical research [166]. Besides the heterogeneous mode utilizing immobilized antibodies as described above, lACE can be performed in homogeneous format where both the analyte and the antibody are in a liquid phase. Two different approaches are available competitive and noncompetitive immunoassay. The noncompetitive immunoassay is performed by incubating the sample with a known excess of a labeled antibody prior to the separation by CE. The labeled antibodies that are bound to the analyte (the immuno-complex) are then separated from the nonbound labeled antibody on the basis of their different electrophoretic mobility. The quantification of the analyte is then performed on the basis of the peak area of the nonbonded antibody. 

PLASMA. The portion of the blood remaining after removal of the white and red cells and the platelets it differs from serum in that it contains fibrinogen, which induces clotting by conversion into fibrin by activity of the enzyme thrombin. Plasma is made up of more than 40 proteins and also contains acids, lipids, and metal ions. It is an amber, opalescent solution in which the proteins are in colloidal suspension and the solutes (electrolytes and nonelectrolytes) are either emulsified or in true solution. The proteins can be separated from each other and from the other solutes by nltrafiltration, nltracentrifugation, electrophoresis, and immuno-chemical techniques. See also Blood. 

Eig. 5. Several endpoint detection methods were compared for the detection of immuno-polymerase chain reaction (IPCR) amplificate from a direct IPCR (Fig. 3A) of mouse-IgG. Although all IPCR/DNA-detection combinations were able to improve the detection limit of a comparable enzyme-linked immunosorbent assays (ELISA) of approximately 10 amol IgG in a 30-fL sample volume, several differences were observed in actual detection limit, and the linearity of the concentration/signal ratio dependent on the DNA quantification was applied. Best results were obtained for PCR-ELISA (see also Fig. 6) in combination with fluorescence- or chemiluminescence-generating substrates (b, c). With photometric substrates (d) or gel electrophoresis and subsequent spot densitometry (a), a 10-fold decrease in sensitivity was observed. In addition to the more sigmoid curve in gel electrophoresis, an enhanced overall error of 20% compared to 13% in PCR-ELISA was observed for two independent assays. The simple addition of a double-strand sensitive intercalation marker to the PCR-amplificate and measurement in a fluorescence spectrometer further decreased sensitivity (e) and appears therefore to be unsuited for IPCR amplificate quantification. (Figure modified according to references 37 and 65.)... 

K2. Kohn, J., Small-scale and Micro membrane filter electrophoresis and immuno-electrophoresis. Protides of the Biological Fluids, Proceedings of the Sixth Colloquium, Bruges, pp. 74-78. Elsevier, Amsterdam, 1958. 

Although the cellular and humoral response in experimental animals tends to be relatively uniform, it must be remembered that in man (and domestic animals) the immune responses can vary enormously. This is undoubtedly related to human genetic diversity - unlike the uniform genetic background of most experimental animals. These responses have been much studied in hydatid disease and (T. solium) cysticercosis. In the latter case, the frequency of different precipitation bands in serum immuno-electrophoresis (Fig. 11.8) and of the immunoglobulin classes (Table 11.4) show great variation between patients (226). Moreover, some patients show no humoral or cellular response whatsoever (226). Similarly, there is much variation in the immune responses to hydatid disease and, again, some patients show no detectable antibody (734). 

The purified hemagglutinin was homogeneous by gel filtration and immuno-electrophoresis, and in the analytical ultracentrifuge.63,560,561 A molecular weight of 79,000 was determined by the sedimentation equilibrium method561,569 (100,000 by sedimentation and velocity measurements,83 and 53,000 by gel filtration563). 

Hirsch-Marie and Burtin (H7a) determined proteolytic activity of human gastric juice after histamine by electrophoresis on agar, using, as a substrate, human or bovine serum albumin at 0.2% concentration in a glycine-HGl buffer of 0.2 molarity and pH 2. Acid gastric juices with pH below 5.8 showed 4 proteolytic constituents on agar gel electrophoresis. These corresponded in their pH optimum of activity and in immuno-... 

Transferrin is commonly assayed by immunochemical methods, including immunoturbidimetry and immuno-nephelometry. It migrates in the Pi region on routine serum electrophoresis as noted previously, genetic variants may cause problems in interpretation of these patterns. 

Paper electrophoresis/paper Tryptic digests, e.g., immuno- W4... 

PI. Peltre, G., and Brogren, C. H., Immuno-isoelectric focusing analysis of antibodies fractionated by isotachophoresis. In Electrofocusing and Isotachophoresis (B. ]. Radola and D. Craesslin, eds.), pp. 577-585. Walter de Gruyter, Berlin, 1977. Poehling, H. M., and Neuhoff, V., One- and two-dimensional electrophoresis in micro-slab gels. Electrophoresis 1, 90-102 (1980). 

Phillips, T.M. (1998) Determination of in situ tissue neuropeptides by capillary immuno electrophoresis. Analytica Chimica Acta, 372,209 218. 

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