Radiolabeled antigen

The rapid turnover rate of some enzymes allows ELISAs to be designed that surpass the sensitivity of radiolabeling techniques. In addition, substrates can be chosen to produce soluble products that can be accurately quantified by their absorbance or fluorescence. Alternatively, substrates are available which form insoluble, highly colored precipitates, excellent for localizing antigens in blots, cells, or tissue sections. The flexibility of enzyme-based assay systems makes the chemistry of enzyme conjugation one of the most important application areas in bioconjugate techniques. 

Immunoassays employ monoclonal or polyclonal antibody preparations (Chapter 13) to detect and quantify the product (Box 7.1). The specificity of antibody-antigen interaction ensures good assay precision. The use of conjugated radiolabels (RIA) or enzymes (EIA) to allow detection of antigen-antibody binding renders such assays very sensitive. Furthermore, when compared with... 

Preclinical studies should address the potential toxicity due to inappropriate release of the conjugated toxin. Preclinical toxicology of monoclonal antibodies may not require extensive animal studies but should be examined for cross-reactivity with antigenic epitopes present on normal cells in vitro and for the presence of human or rodent vimses. Early clinical trial should involve biodistribution studies with radiolabelled material. 

Immunoassay Methods. Radioimmunoassay (RIA) allows measurement of biologically active materials which are not detectable by traditional cold chemistry techniques. RIAs can be used to measure molecules that cannot be radiolabeled to detectable levels in vivo. They also are used for molecules unable to fix complement when bound to antibodies, or they can be used to identify cross-reacting antigens that compete and bind with the antibody. 

Immunoradiometric assays (IRMAs) are like RIAs in that a radiolabeled substance is used in an antibody-antigen reaction, except that the radioactive label is attached to the antibody instead of the hormone. Furthermore, excess of antibody, rather than limited quantity, is present in the assay. All the unknown antigen becomes bound in an IRMA rather than just a portion, as in a RIA IRMAs are more sensitive. In the one-site assay, the excess antibody that is not bound to the sample is removed by addition of a precipitating binder. In a two-site assay, a molecule with at least two antibody-binding sites is adsorbed onto a solid phase, to which one of the antibodies is attached. After binding to this antibody is completed, a second antibody labeled with 125I is added to the assay. This antibody reacts with the second antibody-binding site to form a sandwich, composed of antibody-hormone-labeled antibody. The amount of hormone present is proportional to the amount of radioactivity measured in the assay. 

Mix a fixed volume (fixed concentration ) of antiserum containing the specific antibody with a constant amount of radiolabelled antigen,... 

The antibody reacts with both the radioactive and unlabelled hapten forming an antibody-radiolabelled antigen and antibody-unlabelled antigen complexes,... 

Radioimmunoassay. Radioimmunoassay (RIA) was first described by Berson and Yalow (34) and Luft and Yalow (35). The assay is based upon the competition for an antibody between a radiolabelled antigen and its unlabelled counterpart. The greater the amount of unlabelled antigen in the test sample, the less radiolabelled antigen bound. The concentration of antigen in a test sample can be determined from comparisons with standard curves. 

Transfer 50-pL aliquots of the mixtures to the antibody or antigen-coated PVC multiwell plate so that each well contains 1-4 x 104 cpm of radiolabel. 

Detection of Antibody-Antigen Complexes Using Radiolabeled Secondary Antibody... 

Immunoprecipitation has been widely used to visualize the antigens recognized by various antibodies, both polyclonal and monoclonal. However, there are several problems inherent to immunoprecipitation, including the need to radiolabel the antigen, coprecipitation of tightly associated macromolecules, occasional... 

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