Immunoaffinity assay

LC-microbial and immunoaffinity assays In Table 2 the different methods are summarized. 

The selectivity of immunosensors for steroid analytes is achieved with the use of appropriately selected monoclonal antibodies. The carbon working electrode provides a suitable surface for passive adsorption of proteins, and can therefore be tailored with an appropriate antibody, so that it will act as an immunoactive surface upon which an immunoaffinity assay can be performed an electrochemical signal can then be generated by monitoring the production of an electroactive species at the underlying electrode surface. We and other workers have found that to retain maximum monoclonal antibody activity, it is desirable to use a primary antibody (rabbit IgG), which serves both to capture (e.g., from a culture medium) and to orientate the mAb. Hence this approach... 

McConnell RJ, Fitzgerald SP, Lamont JV (1992) Trenbolone and 19-nortestosterone residue analysis by immunoaffinity chromatography and high-performance liquid chromatography and/or an enzyme-linked immunosorbent assay. In Morgan MRA, Smith CJ, Williams PA (eds) Food safety and quality assurance applications of immunoassay systems. Elsevier, Barking, p 245... 

Immunoaffinity chromatography (IAC), 6 400—402 12 137, 145 Immunoanalyzers, automated, 14 150 Immunoassay(s), 14 135-159. See also Immunoassay- DNA probe hybrid assays Immunoassay methods Immuno(bio)sensors antibody-antigen reaction, 14 136-138 basic technology in, 14 138-140 chemiluminescent, 14 150-151 classification of, 14 140-153 design of, 14 139-140 enzyme, 14 143-148 fluorescence, 14 148-150 highly specific, 14 153 historical perspective on, 14 136 microarrays and, 14 156—157 microfluidics in, 26 968—969 monoclonal versus polyclonal antibodies in, 14 152-153... 

Other common impurities, such as immunoglobulins and protein A, result from the immunoaffinity purification of recombinant proteins or MAbs.16 If affinity chromatography is used to purify an antigen, then an ELISA can be used to detect contaminating levels of MAbs leached from the column. An assay for the antibody needs to detect the antibody in the presence and absence of its specific antigen. 

Lucas, C., C. Nelson, M.L. Peterson, S. Frie, D. Vetterlein, T. Gregory, and A.B. Chen (1988). Enzyme-linked immunosorbent assays (ELISAs) for the determination of contaminants resulting from the immunoaffinity purification of recombinant proteins. J Immunol Methods 113(1) 113-122. 

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). 

All the antibody—toxin conjugates bind in their entirety to the appropriate immunoaffinity HPLC column and are completely separated from free antibody. There is no adverse affect on the integrity of the conjugates as judged by gel electrophoresis, size exclusion HPLC, and assays of cytotoxic potency (5)... 

For OTA detection, the optimized immunosensors and the protocol that was implemented on the electrochemical device allowed the detection of 0.4 pg/kg, with within- and between-assay variability less than 5% and 10%, respectively. The method was evaluated with respect to a reference instrumental method (HPLC/immunoaffinity clean-up method based on the AOAC Official Method 2000.03) obtaining good agreement (r — 0.9992). 

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. 

Kuhn E, Addona T, Keshishian H, Burgess M, Mani DR, Lee RT et al (2009) Developing multiplexed assays for troponin I and interleukin-33 in plasma by peptide immunoaffinity enrichment and targeted mass spectrometry. Clin Chem 55 1108-1117... 

The continuing refinement in the selection of reference materials and the production of antibodies to complex protein mixtures has resulted in immunoassay systems of remarkable sensitivity and specificity. In particular, the selection and enrichment of the antibody population by immunoaffinity purification against the reference impurities has afforded an additional level of control over the production and validation of these reagents and served to improve the assay range and sensitivity (6,17). This normalization of the antibody population to a stoichiometric relationship with the reference impurities has suggested the term Antigen Selected Immunoassay (ASIA) for these methods. 

Maurer, H.H., Schmidt, C.J., Weber, A.A., Kraemer, T. Validated electrospray LC MS assay for determination of the mushroom toxins alpha- and beta-amanitin in urine after immunoaffinity extraction. J. Chromatogr. B Biomed. Sci. Appl. 748, 125-135 (2000)... 

Immunoaffinity-based assays are routinely developed for new biologicals and products of the biotechnology industry as part of their characterization as new agents. In contrast, assays used for pharmacokinetic studies of new chemically synthesized entities are less likely to be immunoaffinity based because analysts are required to measure accurately the concentration of the administered parent drug. Metabolism of the parent drug can result in metabolites that are structurally very similar and that cross-react with antibodies to the parent drug, but exhibit different pharmacological activity. For this reason, determination of the structures of these metabolites and, commonly, the measurement of their concentrations... 

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