Immunoblot method

Once transfectomas have been generated, they must be screened for the production of the desired fusion. The binding site provided by the expression of the VNP heavy chain in J558L cells and association of the heavy chain with the resident light chain means that the protein fusion can be captured on a solid phase by NP or NIP, and detected using commercially available antibody to mouse X chain and an appropriate enzyme conjugate, in a simple ELISA screening procedure. Similarly, purification can be achieved by affinity adsorption of the fusion onto an NP matrix. An immunoblot method is described here for characterization of selected transfectomas, which allows the mol-wt of the fusion product to be estimated. 

Kamps, M. P. (1991) Generation of anti-phosphotyrosine antibodies for immunoblotting. Methods Enzymol. 201,101-110. 

Kamboh MI, Ferrell RE. Genetic studies of human apolipoproteins. XV. An overview of IEF immunoblotting methods to screen apolipoprotein polymorphisms. Hum Hered. 1990, 40 193-207. 

Varjonen, E., Vainio, E., Kalimo, K., Juntunen-Backman, K., Savolainen, J. 1995. Skin-prick test and RAST responses to cereals in children with atopic dermatitis. Characterization of IgE-binding components in wheat and oats by an immunoblotting method. Clin Exp Allergy 25 1100-1107. 

Howe, J. G. and Hershey, J. W. B. (1981) A sensitive immunoblotting method for measuring protein synthesis initiadon factor levels in lysates of tcAmcAta colt. J. BtoL Chem. 256, 12836-12839. 

Overproduction of OP-detoxifying esterases in mosquitoes was also demonstrated by an immunoblot method. Esterase Bi of C. qulnquefasclatus was shown to be overproduced by a factor of at least 500x, approximating the level of resistance to the OP insecticide chlorpyrifos (ca. 800x) as determined by bioassay (.22). ... 

The previous discussion has illustrated the role of monooxygenase systems in fish for the metabolism of xenobiotics. Measurements of this activity have therefore been used as a measure of the extent to which fish have been exposed to xenobiotics at the same time, of course, increased levels enable the fish to metabolize xenobiotics effectively (Kleinow et al. 1987). Although specific assays of cytochrome P-450 activity may be made by immunoblot methods (Monosson and Stegeman 1991), it may be expedient to measure specific enzyme activity using defined substrates. Two assays have been widely used (1) aryl hydrocarbon hydroxylase activity that may be assayed using benzo[a]pyrene as substrate, although this substrate has been replaced recently by the less hazardous 2,5-diphenyloxazole, and (2) activity for O-deethylation of 7-ethoxyresorufin (EROD) has been extensively used and is a simple and convenient assay. 

Purity for a small molecule is a relatively simple concept. Normally, an HPLC method is sufficient to measure the content and impurity levels of a small molecule drug. A macromolecule, such as a protein, has a much more complex behavior. Determining protein concentration by UV absorption spectroscopy can give a measure of the total protein in the product, but it will not necessarily differentiate between active protein and inactive protein (i.e., denatured or otherwise degraded). A validated method or methods to determine the biological activity of the molecule is needed. So, whereas protein concentration is usually tested as part of the specifications, it is also normally accompanied by one or more methods that measure or correlate to biological activity. This is the bioassay. These methods can be animal-based or cell-based, protein interaction assays, binding methods such as surface plas-mon resonance or ELISA (enzyme-linked immunosorbent assay) and immunoblot methods. 

Immunochemical properties Binding assays, ELISA, western-blot (immunoblotting methods)... 

Laboratory confirmation is vital to effective treatment of HSV, especially in individuals in whom a clinical diagnosis cannot be obtained. There are several methods by which a definitive diagnosis may be acquired, and these include virologic typing, serologic diagnosis, rapid point-of-care antigen detection, enzyme-linked immunosorbent assay (ELISA), immunoblot, and DNA polymerase chain reaction.27... 

The practicing clinician has a number of different tests available to aid in the evaluation of patients with suspected hepatitis C. These include measurement of alanine aminotransferase (ALT) levels, liver biopsy, serological tests (ELISA and recombinant immunoblot assay), and molecular methods for detection and quantitation of HCV RNA. 

The limitations of ELISA methods include the specificity of antibodies, the concentrations of primary antibody and antigen, and the type of reaction solution. Nonspecific binding of either of the antibodies to related antigens, unrelated proteins of other bacteria, or even the microtiter plate may lead to false positive reactions.49,52 57 Use of a monoclonal antibody may decrease crossreactivity with other antigens. For detection of low numbers of bacteria, as in drinking water, the sample may be filtered to concentrate the cells or cultured in a selective broth until it reaches the minimum detection limit for ELISA.49,58 Commercial test kits using dipsticks, immunoblots, and sandwich ELISA methods have been developed for the identification of pathogenic bacteria.58,59... 

A significant advantage of this method is that is provides quantitative information since the IEF technique actually separates the phospho- and dephospho- forms of the protein, the ratio of these species as detected by IEF/immunoblotting is a direct readout of the protein s phosphorylation status within the cell (assuming no selective loss of one species during isolation and analysis). 

Immunoassay kits, 14 152 Immunoassay methods alternative, 14 151 comparison of, 14 151-153 Immunoassay technology, 12 97 Immunoblotting, 9 756 Immunochromatographic assay, 14 141-142... 

The specificity of fluorescence localization is dependent on the specificity of the primary antibody, a property that must be tested and controlled by other methods, such as immunoprecipitation or immunoblotting. Controls for the labeling procedure described include deletion of the primary antibody step, which controls for the second-step reagent, or inclnsion of a similar, but nonreactive antibody as a first step. In the case of the availability of purified primary antigen, competition controls can be used, but they only control for the reactivity of the antibody with one antigen, and do not mle ont the possibility of a crossreactive, but unrelated antigen. 

Figure 8. Increased chromosome instability in ubrlA S. cerevisiae (3). (a) Sectoring assay for the chromosome loss using the SUPJJ-marked YPH277 (UBRl) strain and its ubrlA derivative (see Methods), (b) Immunoblot analysis of...

Often the question arises, whether the total amount of connexin is altered. Because immunohistology is mostly a semiquantitative method, the following biochemical method for isolation of gap junctions may be a suitable alternative. After isolation of the gap junction pellet SDS-PAGE has to be carried out and the gels have to be stained. For control an immunoblot is recommended. 

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