Immunoaffinity column

Sharman, M. and Gilbert, J., Automated aflatoxin analysis of foods and animal feeds using immunoaffinity column clean-up and high-performance liquid chromatographic determination, /. Chromatogr., 543, 220, 1991. 

Purification entails use of an immunoaffinity column containing immobilized murine antifactor VII antibody. It is initially produced as an unactivated, single-chain 406 amino acid polypeptide, which is subsequently proteolytically converted into the two-chain active factor Vila complex. After sterilization by filtration, the final product is aseptically filled into its final product containers, and freeze-dried. 

Fazekas, B. and Tar, A., Determination of zearalenone content in cereals and feedstuff s by immunoaffinity column coupled with liquid chromatography, J. AO AC Int., 84, 1453, 2001. 

Hodgson, R. J., Brook, M. A., and Brennan, J. D., CapiUary-scale monolithic immunoaffinity columns for immunoextraction with in-line laser-induced fluorescence detection. Analytical Chemistry 77(14), 4404-4412, 2005. 

Purification entails use of an immunoaffinity column containing immobilized murine antifactor VII antibody. It is initially produced as an unactivated, single chain 406 amino acid polypeptide, which is subsequently proteolytically converted into the two-chain active factor Vila complex. After sterilization by filtration, the final product is aseptically filled into its final product containers and freeze-dried. The excipients present in the product include sodium chloride, calcium chloride, polysorbate 80, mannitol and glycylglycine. When freeze-dried in the presence of these stabilizing substances and stored under refrigerated conditions, the product displays a shelf-life of at least 2 years. It has proved effective in the treatment of serious bleeding events in patients displaying anti-factor VIII or IX antibodies. 

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]. 

A continuing source of concern is the operating pressure of an immunoaffinity column. Excessively pressures will generate shear-type forces that could cause destruction of the antibody-support bond and lower the efficiency of the column. In general, pressures should not exceed the value of 0.34. IO Pa to prevent loss of immobilized antibody (167). This condition meets the major restriction of all immunoaffinity cleanup procedures, which is the need to use aqueous extracts. Analytes extracted from the original sample with organic solvents cannot be... 

The extent to which the immunoaffinity column can be reused depends mainly on the nature of the analyzed samples as well as the stability of the antibody and the support. The most important step is to remove any of the material physically adsorbed to the antibody so that the column may be reused with reproducibility. All that it is required for a column to be reequilibrated is the passage of several volumes of the starting buffer. Table 20.6 presents an outline of commercial column protocols used during analysis of steroid and -agonist residues in urine. Most columns have been shown to last at least 100 runs, provided that the sample is properly defatted and does not contain solid particles (169). 

Table 2O 6 Immunoaffinity-Column Protocols Recommended for Steroid and -Adrenergic Agonist Residue Analysis in Urine by Column Manufacturers... 

Steroids immunoaffinity columns -Agonists immunoaffinity columns ... 

Immunoaffinity chromatography cleanup has also been applied as an ideal and reliable strategy for residue analysis. Immunoaffinity columns prepared by coupling the antibodies to a cyanogen bromide-activated support were used to analyze avermectin BI residues in cattle tissues (359) and ivermectin in sheep serum (376). An immunoaffinity column prepared by an alternative activation/ coupling procedure with carbonyl diimidazole was also employed to analyze ivermectin residues in swine liver (361) since the earlier-reported methods did not work well in the analysis of this matrix. This recent work demonstrated the high specificity of tire antibody-mediated cleanup, but also showed that the immunoaffinity procedures could not always or completely eliminate matrix interference of samples. Therefore, application of additional cleanup steps before or after these procedures is often inevitable. 

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... 

Addition of appropriate amounts of salts, such as ammonium or sodium sulfate (2), or other chemicals, such as PEG, cause precipitation of IgG from serum. Caprylic acid can also be used to fractionate proteins from serum Although such IgG is usually contaminated with other proteins, the ease of these precipitation procedures coupled with the high yield of IgG produced has led to them being very widely used to produce enriched IgG preparations suitable for many immunochemical procedures, e.g., production of immunoaffinity columns, and as a starting point for further purification. The precipitated IgG is usually very stable and such preparations are ideally suited for long-term storage or distribution and exchange between laboratories. 

Purification by the immunoaffinity column can be carried out manually or by using a commercially available automated sample-preparation system. After the conditioning of the im-... 

LC Liquid chromatography CB Contamination Bureau SPE solid-phase extraction TFA trifluoroacetic acid MS mass spectrometry OPA o-phthaldialdehyde IA immunoaffinity column FD fluorescence detector BF best food UV ultraviolet. 

PM Scott, MW Trucksess. Application of immunoaffinity columns to mycotoxin analysis. J AOAC Int 80(5) 941-949, 1997. 

A Patey, M Sharman, J Gilbert. Liquid chromatographic determination of aflatoxins level in peanut butter using an immunoaffinity columns clean-up. method international collaborative trials. J Ass Off Anal Chem 74(1) 76-81, 1991. 

B Zimmerli, R Dick. Determination of ochratoxin A at the ppt level in human blood, serum, milk and some foodstuffs by high-performance liquid chromatography with enhanced fluorescence detection and immunoaffinity column cleanup methodology and Swiss data. J Chrom B 666 85-99,1995. 

MW Trucksess, ME Stack, S Nesheim, SW Page, RH Albert, TJ Hansen, KF Donahue. Immunoaffinity column coupled with solution fluorometry or liquid chromatography post-column derivati-... 

M Sharman, S Mac Donald, J Gilbert. Automated liquid chromatographic determination of ochratoxin A in cereals and animal products using immunoaffinity column clean-up. J Chiom 603 285-289, 1992. 

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