Immunoaffinity sample preparation

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. 

Starting with a description of the analytical challenge in Chapter 19, the third part, which is devoted to analytical attitudes, proceeds with a detailed description in Chapter 20 of modern sample preparation procedures including solid-phase extraction, matrix solid-phase dispersion, use of restricted-access media, supercritical fluid extraction, and immunoaffinity cleanup. Flexible derivatization techniques including fluorescence, ultraviolet-visible, enzymatic, and photochemical derivatization procedures are presented in Chapter 21. 

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-... 

Key Words Immunoaffinity chromatography IAC isolation purification sample preparation immunoaffinity columns. 

The analyte binding efficiency is matrix dependent. Some matrices, such as urine and tissue extracts, can be directly loaded onto the column, other matrices such as milk may need sample processing prior to loading onto an immunoaffinity column. The simplest sample preparation method is dilution this method has been applied to serum, liver, and kidney extracts after removal of particulates. Sometimes dilution alone is not sufficient to eliminate the matrix effect and classical sample preparation techniques (solvent/solvent extraction, solid phase extraction, etc.) will be necessary prior to immunoaffinity chromatography. We found milk often needs this type of treatment. 

With the exception of immunoaffinity extraction, which is a specialized and elaborate sample-preparation approach [27,28], solid-phase extraction generally provides the cleanest extract of all sample-preparation techniques in terms of selectivity. The price paid for this performance is that method development is generally the most complex and time consuming [29—31]. Generic conditions for automated 96-well solid-phase greatly reduced the need for extraction method development and work for about 85% of the small organic molecule analytes typically encountered in drug discovery [32]. These approaches are described later. 

The first publication that reported the use of LC—MS for quantification of IsoPs in urine used reversed-phase LC coupled with ESI/MS. The method used only 1 mL urine and the clean-up procedure using solid phase extraction (SPE) columns gave quantitative recovery of the IsoP. The chromatographic runs and SPE purification methods are short, and this results in a very easy and user-friendly procedure (Li et al., 1999). A comprehensive review by Tsikas et al. describes sample preparation techniques and compares GC—MS methods with the most recent LC—MS/MS methods (Tsikas et al., 2003). The focus is primarily on 8-f5o-PGF2ci and highlights the difficulty to detect only one IsoP isomer without immunoaffinity chromatography preparation. The large concentration differences for the various IsoP classes in urine are also addressed. 

Zolotaijova, N. Boyes, B. MartoseUa, J. Yang, L.-S. Nicol, G. Zhang, K. Szafranski, C. Bailey, J. Immunoaffinity depletion of high-abundant proteins for proteomic sample preparation. In Separation Methods in Proteomics CRC Press Boca Raton, 2006 63-79. 

Immunoaffinity (lA) methods are based on the principle of molecular recognition via very selective antigen-antibody interactions. Their use for sample preparation prior to TLC has been mostly for determination of toxins. The following are selected examples the steroid animal drug trenbolone and its metabolite in bovine urine " fumonisin B1 in corn with methanol-water (80 20) extraction followed by lA column cleanup " aflatoxins Bl, B2, Gl, and G2 in foods regulated within... 

The use of immunoaffinity purification as a sample preparation method for downstream proteomics applications is emerging for applications involving identification of proteins in functional complexes and global identification of modified proteins. This approach allows investigators to use proteomics approaches to address hypothesis-driven research questions. Although a detailed protocol was presented here, it should be noted that each sample type and antibody will require optimization of the protocol—different buffers may work better for lysis, antibodies may bind more or less... 

Proper sampling procedures have to be applied to obtain representative samples from analyzed biological material since mycotoxins are not homogenously distributed in contaminated grain stocks. Sample preparation usually involves a few cleanup steps to eliminate compounds present in the matrix that might coelute with the mycotoxins. The cleanup methods may involve either liquid-liquid extraction (LLE), solid-phase extraction (SPE) on columns that contain various types of solid phases, or immunoaffinity columns [39,42,43]. The recovery achieved with a cleanup method is critical for the final results. 

High-performance affinity chromatography has recently been reported with trypsin-modified avidin supported on 5 pm silica. While the separations were successful and a wide range of foods were studied, elution times were 80 minutes and ADAM post-column reactions were still required (Hayakawa et al. 2009). However, such affinity columns within a solid-phase extraction (SPE) platform make realistic choices for sample preparation, whereby the biotin can be purified and concentrated prior to reversed-phase HPLC. R-Biopharm has recently developed a commercially available antibody-based immunoaffinity column to bind biotin from aqueous extracts, providing an excellent technique to clean up complex samples. 

I n Fig. 3, two chromatograms are compared, which show the difference between sample preparation with classical Cjg SPE and immunoaffinity extraction (afEnity SPE). The main metabolite, 1-hydroxypyrene, can be examined with both methods all other metabolites, which occur in lower concentrations, carmot be quantified without affinity purification. The detection limit with affinity extraction can be improved even further ifa concentrating Cjg-SPE is subsequently applied. Eigure 3 clearly shows the improvement that can be achieved with affinity purification. Furthermore, it is much easier to identify new metabolites that have not been identified before because the chromatogram contains only very few peaks. Hyphenation with mass spectrometry would be particularly suitable to achieve a structural analysis. 

Zolotarjova N, Boyes B, Martosella J. Immunoaffinity depletion of high-abundant proteins for proteomic sample preparation. In Smqkal GB, Lazarev A, editors. Separation methods in proteomics. Boca Raton, FL CRC Press 2006. p. 63—79. Moser AC, AC, Hage DS, DS. Chromatographic immunoassays. In Hage DS, editor. Handbook of affinity chromatography. 2nd ed. Boca Raton, FL CRC Press 2005. p. 789-836. 

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). 

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). 

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