Monoclonal immunoaffinity

Table 1 Design Table for Screening Studies on Affinity Purification of a Protein Using Monoclonal Immunoaffinity Column (2 1 Fractional Factorial Design)...

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... 

Factor IX may also be purified by immunoaffinity chromatography, using immobilized anti-IX murine monoclonals. Purification to homogeneity is particularly important in the case of... 

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... 

Fusions may also be designed against which antibodies may be raised that can be used for detection. An example is the tripeptide Glu-Glu-Phe motif for the immunoaffinity of HIV enzymes which is recognized by the YLl/2 monoclonal antibody to a-tubulin (Stammers et ah, 1991). 

Selectivity in lAC depends on the specificity of the immobilized antibody and, thus, monoclonal antibodies are preferentially used. In that case, a large amount of sample can be subjected to immunoaffinity cleanup without any retention of matrix components. This opens the possibility to determine very low concentrations of drug residues in edible animal products. For example, 20 ng chloramphenicol in 1 L milk can be determined with a recovery of 99% when 1 L of defatted milk is submitted to immunoaffinity cleanup. The chromatograms obtained after LC analysis were as clean as those obtained when 10 ml milk containing the same amount of chloramphenicol was also submitted to immunoaffinity cleanup (170). 

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). 

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. 

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. 

Yin B, Whyatt RM, Perera FP, Randall MC, Cooper TB, Santella RM (1995) Determination of 8-hydroxy-guanosine by an immunoaffinity chromatography-monoclonal antibody-base ELISA. Free Rad Biol Med 18 1023-1032... 

CR Harrington, DM O Hara, PE Reynolds. Characterization of a monoclonal antibody and its use in the immunoaffinity purification of penicillin-binding protein 2 of methicillin-resistant Staphylococcus aureus. FEMS Microbiol Lett 53 143-147, 1989. 

Affinity chromatography techniques have shown less utility in analytical testing than in preparative separations for a variety of reasons, including cost and the difficulty of validating consistent operation as the column changes over time. Protein A affinity has been commonly used to quantitate the total antibody content of either ascites or cell culture fluids. To provide guidance in the development of a purification process, specific immunoaffinity resins are either available or can be readily prepared to quantitate the levels of unrelated protein contaminants. To rapidly determine what the active species in a mixture is, a monoclonal antibody that... 

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. 

Partially purified receptor preparations have been used for production of polyclonal and monoclonal antibodies to receptors. If these antibodies are available they might be used for immunoaffinity chromatography of receptors. This procedure permits rapid isolation of highly purified receptors and has been used, e.g., for separation of different phosphorylated forms of progestin receptors [30], The right choice of a suitable reagent for elution of receptors from the immunoaffinity column in a form still able to bind the steroid was crucial in these studies. 

Immunoaffinity separation chromatography can also be performed using immobilized specific antibodies to separate immunoglobulins. Actually immunoglobulins are antigenic molecules and, as such, they can produce specific antibodies in animals. The use of these specific antibodies as immobilized affinity ligands is the second aspect of immunoaffinity chromatography and constitutes an effective specific way to purify monoclonal and polyclonal antibodies. 

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