Antigens for Monoclonal Antibodies

Although the hybridoma technique does not require pure antigen for the production of specific monoclonal antibodies, it still re- 

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Chen, M. S., Huber, A. B., Van Der Haar, M. E. etal. Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature 403 434-439, 2000. 

Several amperometric immunosensors have been developed for monoclonal antibodies (IgG), a-interferon and the pesticide 2,4-dichlorophenoxy-acetic acid (2,4-D) by using a flow-cell with the catching antibody covalently bound to a cellulose acetate or activated nylon membrane as shown in Fig. 3.31. B. With multiepitope antigens (e.g. a protein), after the antigen is bound and washed, a second enzyme-labelled antibody is used to form a sandwich... 

De Waele, M., De Mey, K., Moeremans, M., De Brabender, M., and Van Camp, B (1983) Immunogold staining for the light microscopic detection of leukocyte cell-surface antigens with monoclonal antibodies. J Histochem. Cytochem 31, 938-944. 

Otsuki, Y., Maxwell, L. E, Magan, S., and Kubo, H. (1990) Immunogold-silver staining method for light and electron microscopic detection of lymphocyte cell-surface antigens with monoclonal antibodies. J Histochem Cytochem. 38, 1215—1221. 

A direct detection method was recently developed for these adducts in stratum comeum of human skin based on immunofluorescence microscopy (30). Three partial sequences of keratins containing glutamine or asparagine, adducted with a 2-hydroxyethyl-thioethyl group at the omega-amide function, were synthesized and used as antigens for raising antibodies. After immunization, monoclonal antibodies were obtained with affinity for keratin isolated from human callus exposed to 50 xM sulfur mustard (see Plate 1). In contrast to the immunochemical... 

This class includes Carnoy s, Methacarn and others. They have been used for IHC purposes primarily in order to try to avoid the loss of antigenicity caused by excessive formalin fixation, or for monoclonal antibodies that reacted against an epitope destroyed by formalin. These fixatives typically found most of the application in looking at lymphocytes using CD-specific markers, and in looking for immunoglobulins such as IgG, A, and M. 

The repeated pulse injection method was applied to determine the association rate constant of the antigen-immobilized monoclonal antibody reaction 124.25). These monoclonal antibodies differ by their specificity and affinity for HSA. The epitope recognized by the HA6 antibody is located between residues I and 128. The epitope of the commercial antibody (m-anti-HSA) is located between residues 124 and 298 of the HSA molecule. They differ by their affinity for HSA. with equilibrium binding constants of respectively 6.7 x 108 mol-dm and 1.6 x 10K dm3 mol-1 for the HSA/HA6 and the HSA/m-anti-HSA association in solution 25). Close binding rate constants are obtained on both immobilized monoclonal antibodies with Aa = 2.5 x I0 i dm -moC -s-1. 

Wt ener, C, Kruger, U., and Shively, J. E. (1990) Selective precipitation of biotin-labeled antigens or monoclonal antibodies by avidin for determining epitope specificities and affinities in solution-phase assays. MethodsEnzymoL 184, 518-529. 

Explain why Scatchard plots for antigen binding are linear for monoclonal antibodies, and curved for polyclonal antibodies. 

Another interesting application is the analysis of the specificity of monoclonal antibodies, i.e., whether they recognize different epitopes on the antigen (Friguet et al., 1983). After coating the antigen, two monoclonal antibodies are added, either to separate or to the same well and the amount of bound antibody is quantitated with enzyme-labeled anti-mouse IgG. Additivity of enzyme activity is observed when the monoclonal antibodies bind to distinct epitopes. The additivity index (A/) for a pair of antibodies is then defined as ... 

FIGURE 1.8 (A) Direct biotin-avidin method. The primary antibody is linked to biotin (B) an avidin-peroxidase-conjugate (A-Px) is then added. (B) Indirect biotin-avidin method. Used for monoclonal antibodies, the primary antibody is not conjugated its localization is detected by a biotinylated secondary antibody. Boxed asterisk represents antigen determinant on primary antibody. Px, peroxidase label A, avidin B, biotin. From Taylor CR, Cote RJ, eds. Immunomicroscopy A Diagnostic Tool for the Surgical Pathologist. 3rd ed. Philadelphia Elsevier 2005 21. 

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