Immuno complex

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. 

Alternative protocols for covalent coupling were described by Wu et al. [48], Sims et al. [49], and McKie et al. [50]. In the latter approach, single-stranded DNA was linked in a multistep procedure with a short primer covalently coupled to the antibody. The single-stranded DNA-marker also included a Hind III restriction site. By adding the restriction enzyme previous to the PCR-step, the DNA-marker sequence was released from the immobilized immuno-complex to the supernatant liquid phase and subsequently transferred to capillary vessels (see Section 2.2.3). 

Fig. 6. Modes of interaction of actin with CCT. (A-H) Two-dimensional average images of different classes of CCT/a-actin (A, B, E, F) or a-actin subdomain 4 immuno-complexes with an anti-CCT-5 antibody (A-D) or an anti-CCT-a antibody (E-Fl). (I) Structure of actin showing the large and small domains and their division into four sub-domains (subs 1-4). (J) Two possible modes of interaction of actin with CCT. Reprinted from Llorca et al. (1999a), with permission.

Homogeneous assays can be performed without the need for separation. This can be advantageous with respect to convenience, time and cost of the assay and also facilitates automation. The detection method must be able to differentiate between bound and free antibodies without physically separating them from each other. An example of such a detection method is the turbidity measurement of the reaction mixture by laser light scattering or absorption. The more immuno-complexes are present in the reaction mixture, the higher its turbidity. Often, latex or gold particles are attached to the antibodies to enhance this turbidity effect. 

Separation of antibody-antigen complex from unreacted Ab and Ag has been achieved with a micellar electrokinetic capillary chromatography (MECC) method using cholate micelles and the appropriate buffer pH, temperature, voltage, and electrolyte and modifier concentrations [76], Mixing of antibody and antigen before their injection into the capillary showed that the immuno-complex has a longer retention time than antibody alone. 

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