Immunoaffinity techniques

Alternative methods to MS for the analysis of protein phosphorylation would involve immunoaffinity techniques using antibodies specific for phosphorylated residues. Surface plasmon resonance technology has been recently used for the determination of active concentration of phosphotyrosine-containing proteins at picomolar levels.117... 

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). 

During recent years, however, the distinction between these two types of methods has become less clear because of the improved methods of sample cleanup that allow selective isolation of groups of compounds. Mixed-mechanism solid-phase extraction procedures and multi-immunoaffinity techniques are clear examples of liquid chromatographic developments that have contributed greatly to the current state of the art within residue analysis. 

Immunoaffinity techniques were widely employed for the analyses and purification of proteins [191-193]. Immobilized antibodies were used, e.g., for industrial scale production of human interferon-a2a, interleukin-2, and interleukin-2 receptor, while protein A and protein G were successfully used in therapeutic applications including purification of human immunoglobulin G from plasma and semm [194—196]. 

T.M. Phillips and B.F. Dickens, Affinity and Immunoaffinity Purification Techniques, Eaton Publishing, Natick MA, 2000. ISBN 1881299228. 

Another subset of SPE is immunoaffinity extraction, in which an antibody specific to the analyte is incorporated into the SPE sorbent. This technique is very selective to the analyte and would be very effective in separating the marker residue from tissue-related matrix components. Disadvantages of immunoaffinity extraction are the need to develop a specific antibody-based SPE for each analyte. This approach holds promise for the future as the development of antibody-based methods becomes more commonplace. 

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. 

The literature contains numerous references to the use of MS/MS in the determination of new neuropeptides in identified cells of invertebrates (Bulau et al., 2004, for a recent example) and this technique is now being applied to in situ analysis of vertebrate tissues (Fournier et al., 2003). MS/MS is also used for studies of neuropeptide processing (Nilsson et al., 2001), pharmacokinetics of synthetic peptides (Mock et al., 2002), nonpeptide drug metabolism (Kamel et al., 2003), identification of peptides purified by immunoaffinity (Suresh Babu et al., 2004), and MALDI/MS/MS techniques adaptable to brain dialysis (Bogan and Agnes, 2004). 

A technique called on-line immunoaffinity CE has been presented (45) that was also coupled to MS. However, in this setup the affinity principle is used to extract the analyte from a complex matrix in a microchamber affinity device prior to CE separation. Therefore, it cannot be considered ACE. 

The type of ligand can be used to divide affinity techniques into various subcategories such as lectin, immunoaffinity, dye ligand etc. These techniques are placed as below [1]. 

Immunoaffinity chromatography is one of the most popular techniques of affinity derivatived method and it enables to produce ligands in case the ligand required is not available [7]. In this technique, stationary phase comprises of an antibody or antibody-related agent [1]. It is possible to isolate variable subtances using this technique due to high specifity of antibodies [1]. It is reported that immunoaffinity chromatography may be used for natural food contaminants such as aflatoxins, fumonisins and ochratoxins [11]. 

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 chromatography is probably the most highly specific of all forms of bioaffinity chromatography. However this technique has some disadvantages such as this technique relatively high cost, leakage of ligands may accur from the column and sometimes the desorption procedure results in partial denaturation of the bound protein [24]. 

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