Monoclonal immunoaffinity purification

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. 

Recently, an immunoaffinity purification method using antimicrocystin-LR monoclonal antibodies (named M8H5) has been developed.This purification method was found to be remarkably effective in the removal of coexisting substances and in the enrichment of microcystins in samples.This work will focus on the immunoaffinity purification methods for microcystins in lake ° and tap water samples, and the analysis methods for microcystins and their metabolites in mouse and rat livers.It will also cover the reuse of the immunoaffinity column. 

Interferring compounds from the sample matrix can become a major problem in the analysis of cytokinins that normally occur at trace levels. Immunoaffinity purification methods that are based on polyclonal or monoclonal antibodies enable a selective single-step clean-up and concentration of a certain group of cytokinins. These methods are usually combined with various techniques of final analysis such as HPLC-UV, HPLC-ELTSA, HPLC-MS [277-279]. However, the antibodies prepared so far do not show suitable affinity to certain metabolites (O-glucosides, N -glucosides, nucleotides). 

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

Brizzard, B. L., Chubet, R. G., and Vizard, D. L. (1994). Immunoaffinity purification of FLAG epitope-tagged bacterial alkaline phosphatase using a novel monoclonal antibody and peptide elution. Biotechniques 16, 730-735. 

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... 

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... 

Forskolin Purification Using an Immunoaffinity Column Combined with c Monoclonal Antibody... 

Preparation of immunoaffinity adsorbents require reasonably pure antibodies and usually salt/solvent fractions of antisera [28,33] or affinity purified polyclo-nal/monoclonal antibodies [18,22] are employed. Antibody purification strategies have been reviewed extensively [53, 65] and do not fall with in the purview of this article. It is however of interest to point out of a novel strategy employing thermo-sensitive immunomicrospheres that appear highly effective in the large scale purification of antibody from the sera of immunized animals [66,67]. 

Immunoaffinity chromatography can provide extensive purification of endogenous hormones in plant extracts [60] (see Figs. 6 and 7 in Section 6.2). Both monoclonal and polyclonal antibodies have been used to produce immunoaffinity supports for lAA [60,61], GAs [62,63] and cytokinins [64,65]. Despite the enormous potential of the procedure, it has as yet not found widespread application in plant hormone purification protocols. The situation is unlikely to change until a range of immunoaffinity supports are available from commercial sources at affordable prices. The raising of antibodies against plant hormones, the preparation of a variety of immunoaffinity supports and their application in plant hormone analysis are discussed and evaluated in Chapter 3. 

Yoshimura, K., Ishikawa, T., Wada, K., et al. (2001) Characterization of Monoclonal Antibodies against Ascorbate Peroxidase Isoenzymes Purification and Epitope-Mapping Using Immunoaffinity Column Chromatography. Biochimica et Biophysica Acta (BBA)—General Subjects, 1526 (2), 168-174. 

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