Immunoaffinity selective elution

Owing to the strength of the avidin-biotin complex, the immunochemical bond can be preferentially severed, permitting selective elution of the antigen. Variations on this same theme allow the immunoaffinity isolation of other components of the immune system, such as tmtibodies, complements, blood cell receptors, and the like. 

Analogous to two-dimensional LC, the on-line coupling of LC and CE has been carried out both in the heart-cut and the comprehensive mode. In a heart-cut LC-CE study, a protein G immunoaffinity LC column was used to selectively preconcentrate insulin from serum (171). A 1 p.1 elution plug comprising the insulin was switched on-line to the CE system where a part was injected into the capillary for final separation. With CE, efficient separations can be obtained in a... 

For small molecule analytes (see Note 6) for which a radiotracer form is available, sequentially load a known quantity of tracer dissolved in buffer and determine the amount of analyte in the eluant. When the radioactivity not retained by the immunoaffinity column plateaus, the column binding sites are saturated. Wash the column, and elute the retained radioactivity. The mass of analyte in the eluted volume is the apparent column capacity. In many instances a radio-labeled analyte may not be available. In such cases, high-performance liquid chromatography, UV spectroscopy, or any other analytical tool capable of selectively quantifying the analyte may be used to determine column capacity. 

An elegant study was presented by Thomas et al. who employed selective solid-phase extraction by immunoaffinity CEC (IACEC) to enhance detection limits. A model compound, fluorescein isothiocyanate biotin, was electrokinetically applied to a capillary column packed with an immunoaffinity stationary phase. The analyte was first selectively bound to the stationary phase, then eluted, migrated by zone electrophoresis, and detected by LIF [79],... 

The technique has been named immunoaffinity electrochromatography (1ACEC) [180]. In principle, the solute diluted in a large volume is applied to a capillary column packed with a support containing anti-solute IgG. The solute is selectively extracted by the immunoaffinity column, and the bound analyte is eluted and separated further (if desired) by capillary electrophoresis. 

Highly specific antibodies have been used for a similar purpose as above in immunoextraction recycling as shown in Fig. 9.13 [66]. This technique is based on the passage of a fiuorochrome-labelled sample through a battery of small immunoaffinity columns each column extracting a single specific analyte. Detection is realized by sequential elution of bound analytes via a valve system and laser-induced fluorescence. This system was applied for selective detection of different cytokines [66]. 

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