Antibodies immunoaffinity chromatography

Affinity Chromatography of Immunoglobulins on immobiiized Antibodies (Immunoaffinity Chromatography, iAC)... 

Zarba A, Wild CP, Hall AJ, Montesano R, Hudson GJ, Groopman JD (1992) Aflatoxin Mi in human breast milk from The Gambia, west Africa, quantified by combined monoclonal antibody immunoaffinity chromatography and HPLC. Carcinogenesis, 13(5) 891-894. 

Another example of vims clearance is for IgM human antibodies derived from human B lymphocyte cell lines where the steps are precipitation, size exclusion using nucleases, and anion-exchange chromatography (24). A second sequence consists of cation-exchange, hydroxylapatite, and immunoaffinity chromatographies. Each three-step sequence utilizes steps based on different properties. The first sequence employs solubiUty, size, and anion selectivity the second sequence is based on cation selectivity, adsorption, and selective recognition based on an anti-u chain IgG (24). 

Immunoaffinity chromatography utilizes the high specificity of antigen—antibody interactions to achieve a separation. The procedure typically involves the binding, to a soHd phase, of a mouse monoclonal antibody which reacts either directly with the protein to be purified or with a closely associated protein which itself binds the product protein. The former approach has been appHed in the preparation of Factor VIII (43) and Factor IX (61) concentrates. The latter method has been used in the preparation of Factor VIII (42) by immobilization of a monoclonal antibody to von WiHebrand factor [109319-16-6] (62), a protein to which Factor VIII binds noncovalenfly. Further purification is necessary downstream of the immunoaffinity step to remove... 

Figure 6.15 Principle of immunoaffinity chromatography. Only antigen that is specifically recognized by the immobilized antibody will be retained on the column...

Figure 12.7 Purification of factor VIII complex using immunoaffinity chromatography. The immobilized anti-factor VIII antibody is of mouse origin. Antibodies raised against specific epitopes on both the VIII C and vWF components have both been successfully used. Industrial-scale columns would often exhibit a bed volume of several litres. Note that the different elements in this diagram are not drawn to the correct scale relative to each other...

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... 

Affinity-purified antibodies are isolated from antisera by immunoaffinity chromatography using antigens coupled to agarose beads. 

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]. 

Three forms of purified FSH are available. Urofollitropin, also known as uFSH, is a purified preparation of human FSH that is extracted from the urine of postmenopausal women. Virtually all the LH activity has been removed through a form of immunoaffinity chromatography that uses anti-hCG antibodies. Two recombinant forms of FSH (rFSH) are also available follitropin alfa and follitropin beta. The amino acid sequences of these two products are identical to that of human FSH. They differ from each other and urofollitropin in the composition of the carbohydrate side chains. The rFSH preparations have a shorter half-life than preparations derived from human urine but stimulate estrogen secretion at least as efficiently and, in some studies, more efficiently. The rFSH preparations are considerably more expensive. 

A specific cleanup procedure based on immunoaffinity chromatography with polyclonal antibodies has been described by Gude et al. (50) for the gas chromatographic determination of chloramphenicol in swine tissues. In this method, tissue sample is extracted with acetonitrile/4% sodium chloride (1 1) Following centrifugation, the supernatant is purified with n-hexane, and chloram-... 

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. 

Also included in this group is the highly specific technique of immunoaffinity chromatography, in which an antibody directed against an epitope on the protein surface is used to pull out the desired protein from a mixture. 

Multi-immunoaffinity chromatography columns were prepared by the coupling of monoclonal antibodies against AMP to activated sepharose. Both sensitivity and specificity were tested for the most commonly used penicillins (AMP, AMO, PenG, OXA, CLO, DICL). Recoveries ranging from 67% to 100% were obtained from phosphate buffer solutions, and it was assumed... 

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). 

R Dietrich, E Usleber, E Martlbauer. The potential of monoclonal antibodies against ampicillin for the preparation of a multi-immunoaffinity chromatography for penicillins. Analyst 123 2749-2754, 1998. 

Immunoaffinity chromatography makes use of immobilized antigen molecules to bind and separate specific antibody from a complex mixture. After the preparation of an... 

Hyde, G.E., Wilberding, J.A., Meyer, A.L., Campbell, E.R. Campbell, W.H. (1989). Monoclonal antibody-based immunoaffinity chromatography for purifying corn and squash NADH nitrate reductases. Evidence for an interchain disulfide bond in nitrate reductase. Plant Molecular Biology 13, 233-46. 

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