Antibody affinities, detection using

In a direct immunoassay the immobilized antibody binds to the corresponding antigen. The competitive immunoassay relies upon the competition of the analyte with a labelled analyte for antibody binding. These formats are widely used for high throughput affinity arrays. A sandwich immunoassay is based on the trapping or capture of the analyte by another antibody. In ELISA (enzyme linked immunosorbent assays) the second antibody is conjugated with an enzyme. The bound enzyme labelled antibody is detected by its ability to break down its substrate to a colored product. 

At the EM level, detection usually involves using a probe (oligonucleotide) in which a hapten has been incorporated. Incorporation of the hapten does not interfere with the hybridization of the complementary sequences. The next step is the binding of a reporter (may be an antibody) to the hapten. The reporter is then subjected to a binding molecule (may be a secondary antibody) that is coupled with an electron-dense material such as colloidal gold for visualization. Nonetheless, the many affinity-detection and immunodetection systems developed for immuno-cytochemistry may now with ingenuity be applied to molecular biology at the EM level. 

For many years, due to the availability and low cost of radioisotope-labeled secondary antibodies, radioactive detection was the method of choice in Western blotting. Newer methods that are less hazardous and easier to use, while maintaining comparable sensitivity, have been developed. Today, Western blotting detection methods can be light-based, (chemiluminescence, bioluminescence, chemifluorescence, and fluorescence), radioactivity-based, or color-based. It is important to note that the detection sensitivity depends on the affinity of the primary antibody for the antigen and on the affinity of the secondary antibody for the primary antibody and can therefore vary considerably from one protein sample to another and from one antibody batch to another. 

Fig. 4. Classification of reported noncompetitive immunoassays for haptens based on the assay principle. (A) Assays that include a chemical modification of hapten to allow sandwich-type detection. (B1) Improved single-antibody immunometric assays that separate immune complex and excess labeled antibody, either by using a hapten-immobilized affinity column or based on differences in their physical properties. (B2) A variation of single-antibody immunometric assays based on masking of unoccupied antibody by an immunoreactive macromolecule followed by selective capture and detection of the hapten-occupied antibody. (C) Assays employing a probe molecule specific to a hapten-antibody complex.

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. 

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