Immunoaffinity elution

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... 

Elution of the bound antibody-enzyme conjugate occurs by only a slight shift in pH to acidic conditions or through the inclusion of a metal-chelating agent like EDTA or imidazole in the binding buffer. Either method of elution is mild compared to most immunoaffinity separation techniques (discussed in the previous section). Thus, purification of the antibody-enzyme complex can be done without damage to the activity of either component. 

Especially if samples with a high content of unspecific proteins, e.g., sera, are processed by immunoaffinity chromatography, it is recommended to use a sequential washing/elution protocol When all sample is applied, wash the column with PBS or TBS until O.D.280 < 0.1, then with 1 column volume 1 M sodium chloride, followed by 5 vol. PBS or TBS. Elute with 1 vol. elution buffer, e.g., glycine-HCl pH 2.5, then apply 1 vol. alkaline buffer, e.g., 0.1 M borate pH 10. Finally, regenerate the column with PBS or TBS. 

This is the most suitable buffer for elution in immunoaffinity chromatography. 

Initially, the antibodies should be purified prior to prepare the immunoaffinity column. Precipitation with ammonium sulfate, ion-exchange chromatography, gel filtration chraoma-tography or affinity chromatography may be employed with the aim of antibody purification. Activated beads which are coated with bacterial proteins A or G may be used as the support material. Some parameters may be changed for the elution of the sample solution for example the ionic conditions of mobile phase may be changed or chaotropic buffers may be used [11]. 

Immunoaffinity cleanup was first applied in drug residue analysis for the determination of chloramphenicol in swine muscle tissue by LC (113). The lAC column was prepared using monoclonal antibodies originally developed for an enzyme-linked immunosorbent assay (ELISA) method (171) specific for chloramphenicol. Meat samples were extracted with water, and a concentrated phosphate buffer was added to the filtered extracts before immunoaffinity cleanup. A phosphate buffer was used in the washing process, whereas chloramphenicol was eluted from the column with a glycine/sodium chloride solution of pH 2.8. For subsequent LC analysis, this eluate was extracted with ethyl acetate, evaporated, and reconstimted in the mobile phase. The same analytical scheme was later successfully applied for the determination of chloramphenicol in eggs and milk as well (170, 172). 

Solid-phase extraction for milk, urine, and feces samples is carried out by washing the loaded Cig cartridge successively with 5 ml water, 5 ml acetone/ water (20 80), 5 ml methanol/water (20 80), 5 ml dichloromethane/hexane (20 80), and 5 ml ethyl acetate/hexane (10 90). The corticosteroids are eluted with 3 ml ethyl acetate. The eluate is evaporated, and the residual is reconstituted in 0.5 ml ethanol and 5 ml phosphate-buffered saline, pending subsequent immunoaffinity column cleanup. The solid-phase extraction procedure differs for liver samples. In that case, washing of the cartridge is performed with 5 ml water, 5... 

Following solid-phase extraction, all extracts are adjusted in the pH range 7-7.5, and submitted to additional cleanup on an immunoaffinity column containing a mixture of dexamethasone- and prednisolone-specific gels. Column washing is performed with water, while elution of the analytes with 3 ml methanol/water (80 20). Aliquots of the eluates are submitted to oxidative reaction with pyridin-ium chlorochromate, and the oxidized corticosteroid derivatives are then analyzed by gas chromatography-mass spectrometry under the conditions shown in Table... 

Monoclonal antibodies against STR were used for the preparation of an immunoaffinity chromatography column. Milk samples were defatted by centrifugation and diluted with phosphate-buffered saline. After loading onto the column, this was washed with saline, and STR and DIHS were eluted with the glycine-HCl buffer. The column bounded 80.4% and 88.7% of milk samples containing 100 ppb STR and DIHS, respectively (117). 

The general principle of immunoaffinity chromatography is illustrated in Fig. 1. The analyte in the sample matrix is loaded onto the column, the column is washed to remove interfering substances, and the analyte is eluted from the column for subsequent use. The column is the heart of the purification system and must bind the analyte specifically enough to allow other substances to be rinsed off the column, allow the elution of the analyte under conditions that do not elute interferences, and permit the column to be regenerated multiple times for subsequent use. 

Important considerations when generating immunoaffinity columns include the conjugation of antibody to the supporting (column) material, packing the column, developing suitable washing and elution protocols, and constructing appropriate tests to evaluate column performance. Of primary importance to the utility of an... 

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. 

Immunoaffinity chromatography can be used to purify protein antigens by immobilizing the relevant antibodies on an inert matrix such as polysaccharide beads. When exposed to a protein mixture, only the protein recognized by that antibody will bind to the beads and can be eluted later in pure or almost pure form. Cells bearing the antigen on their surface can also be purified using a similar procedure. 

---