Immunoaffinity column application

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. 

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

DRAGACCI s and FREMY J M (1996), Application of immunoaffinity column cleanup to aflatoxin Ml , J Food Protect, 59, 1011-1013. 

For mycotoxin analyses radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISAs) and affinity chromatography are the principal immunochemical methods in commercial application. Immunoaffinity columns or cartridges for specific mycotoxins are now being increasingly used in preliminary clean-up of extracts prior to final analysis by HPLC or GLC methods. 

Forskolin, a labdane diterpenoid, was isolated from the tuberous roots of Coleus forskohlii Briq. (Lami-aceae) [1], C. forskohlii has been used as an important folk medicine in India. Forskolin was found to be an activator of adenylate cyclase [2], leading to an increase of c-AMP, and now a medicine in India, Germany, and Japan. The production of forskolin is completely dependent on the commercial collection of wild and cultivated plants in India. We have already set up the production of monoclonal antibodies (MAbs) against forskolin [3]. The practical application of enzyme-linked immunosorbent assay (ELISA) for the distribution of forskolin contained in clonally propagated plant organs and the quantitative fluctuation of forsko-lin depend on the age of C. forskohlii [4,5]. As an extension of this approach, we present the production of the immunoaffinity column using anti-forskolin MAb and its application [6]. 

In some cases a cleanup step is introduced, in which the analyte of interested is separated from the matrix. This can be carried out by Cis-columns or immunoaffinity columns. ) A very interesting approach is the application of supercritical fluid extraction (SFE) prior to immunoanalysis. These methods generally employ CO2 or CO2 containing various modifiers. 

The usefulness of immunochemical techniques for the fluorimetric determination of mycotoxins has been widely demonstrated. In addition to the use of immunoaffinity column cleanup followed by LC with fluorescent detection, several FIAs have been described for the determination of aflatoxins, deoxynivalenol, and fumonisins using fluorescence polarization immunoassay or time-resolved fluor-oimmunoassay. Also, although the application of flu-oroimmunosensors to food analysis has been relatively limited, there are some examples of the use of this approach to the determination of some mycotoxins such as aflatoxin Bi and fumonisin B2. 

Yanagihara, H. Minami, H. Tanaka, H. Shoyama, Y. Murakami, H. Immunoaffinity column chromatography against forskolin using an anti-forskolin monoclonal antibody and its application. Anal. Chim. Acta 1996, 335, 63-70. 

The most frequently used CSPs for biological applications in the reversed-phase mode are based on macrocyclic antibiotics, proteins, or oligosaccharides, but some of the applications utilize phases based on polysaccharides, low-molecular-weight selectors, crown ethers, or columns based on immunoaffinity techniques (Table 17.5). 

Columns, dialysis membranes, capillaries or beads may be used in immunoaffinity application which is a non-covalent, irreversible purification process based on highly specific interactions between analyte and antibody [11]. 

Depending on the application, immunoaffinity matrices can be packed into open columns, such as screening columns (Fisher Scientific, Pittsburgh, PA), and a variety of glass columns, such as those provided by Amersham Pharmacia Biotechnology, Bio-Rad, or other vendors. The column material must be compatible with your analysis because either plastic or glass can cause problems with low concentrations of some analytes. Column dimensions are dependent... 

---