Immunoaffinity chromatography, small

Both small and large analytes can be determined using direct detection in lAC. Additionally it is possible to use this technique either separately or in combination with other chromatographic techniques [1]. If this technique is performed as part of HPLC system the method can be referred as high performance immunoaffinity chromatography. 

Immunoaffinity columns are extremely versatile and have been used for the isolation and concentration of a diverse number of analytes from a wide array of matrices (2). Analytes may include macromolecules such as proteins and receptors or small molecules such as environmental toxins, antibiotics, or pesticides. Matrices may include animal tissues or excreta, plant extracts, cell culture medium, or virtually any milieu encountered in biological work. Because of its value as a research tool, immunoaffinity chromatography has found extensive use by the pharmaceutical industry to purify therapeutic proteins, the food safety community to purify small amounts of toxins from food and as a general tool for analytical chemists to purify analytes for subsequent instrumental analysis. 

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. 

The majority of reports have used electrospray ionization mass spectroscopy (ESI-MS) as an analytical detection method because of its sensitivity and the soft namre of its ionization procedure, which generally only leads to the detection of the molecular ions of the positive library members. Many separation techniques have been coupled to ESI-MS, including affinity chromatography (49), size exclusion chromatography (50, 51), gel filtration (52), affinity capillary electrophoresis (53-58), capillary isoelectric focusing (59), immunoaffinity ultrafiltration (60), and immunoaffinity extraction (61). ESI-MS has also been used alone (62) to screen a small carbohydrate library. Other examples reported alternative analytical techniques such as MALDI MS, either alone (63, 64) or in conjunction with size exclusion methods (65), or HPLC coupled with immunoaffinity deletion (66). 

Number of antibodies that can be produced is practically unlimited, hence wide development of immunoassays not only for determination of macromolecules, which induce immunoreactions, but also for small molecule analytes inducing such reaction after binding into suitable conjugates. Besides immunoassays carried out in various formats and immunoaffinity liquid chromatography, since many years very broad studies are carried out on design of integrated immunosensors.127129 In principle their construction should simplify the immunochemical determination by elimination various steps necessary in conventional immunoassays based on the use of labels to monitor the immunochemical reaction. In immunochemical process, the... 

Albumin, a major constituent in serum (60-80 mg/L), is known to act as a transport carrier for small proteins. Also, many antineoplastic drugs bind to albumin, often at the 80-95% level. Removal or depletion of albumin is a major problem there are problems with ultrafiltration (e.g., membrane binding of small proteins and drugs) and other approaches, e.g., Cibacron dye columns and immunoaffinity-based protein subtraction chromatography. Albumin removal using acetonitrile may be a simple alternative [54] however, there is no truly satisfying method at this time. 

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