Antibody-Based Assays

In order to avoid the described problems, antibody-based assays have been developed. As mentioned above, usually a primary antibody recognizes the modification, which in turn is then quantitated. One of these approaches is based on the technology of AlphaScreen (Amplified luminescent Proximity Homogeneous Assay) [51, 52], also known as luminescent oxygen channeling immunoassay (LOCI) [53], a method that can be used to study protein-protein interactions in general. In this case the enzymatic transfer of acetyl groups to a histone peptide is determined. 

Lysine methyltransferases catalyze the transfer of methyl groups from the cosubstrate SAM to certain lysine residues in histone proteins. To characterize modulators of these transferases, the above-mentioned antibody-based assay protocols are also applicable. 

In this chapter we will focus on the methods of immobilization of aptamers and the detection of aptamer-ligand interactions by various methods. The application of aptamers in therapy and in conventional analytical assay has been reviewed elsewhere [2,4,5,8]. A comparison of aptamer- and antibody-based assays was recently published in a review by Leca-Bouvier and Blum [14]. 

Protein Expression Specific protein expression Antibody based assays (e.g. western blots, immunohistochemistry, immunofluorescence, enzyme immune assays, protein microarrays)... 

The approach of implementing a biological assay as a postcolumn reaction detection system after liquid chromatography can not only be applied to antibody-based assays (immunoassays) but also to assays employing other affinity interactions with high association and low dissociation rate constants, such as receptors. Information obtained from such a detection system not only provides quantitative results but also indicates the biological activity of the detected compound. 

It is beyond the scope of this chapter to discuss all potential applications and all present technologies associated with biological drugs. We wish to concentrate here on the newer technologies that are actually used on either an investigational or approved basis in human beings, and can only provide an overview. Thus, vaccines, fermented antibiotics, blood products, diagnostic products (e.g. antibody-based assay systems) and devices... 

The MIPs have also been utilized as the recognition elements in pseudoimmunoassays. " In this approach, MIPs are substituted for antibodies to quantify the amount of analyte in a biological sample, such as blood plasma. Most MIP immunoassays are competitive binding studies in which a radio- or fluorescent-labeled analyte is added to a mixture of the MIP and imlabeled analyte. After equilibrium is reached, some fraction of the labeled species is bound to the polymer surface and thus can be separated from the supernatant. The supernatant is then analyzed via scintillation or fluorescence techniques to determine the concentration of the original unlabeled analyte. Mosbach et al. have demonstrated that MIP-based immunoassays can rival the selectivity of antibody-based assays. Imprinted polymers for the opioid receptor ligands enkephalin and morphine were prepared and showed submicromolar (pM) level selectivity in a radioligand competition assay in aqueous buffers. The analysis... 

Binding protein/antibody-based assay on a light addressable potentiometric transducer pH changes in whole cells... 

This technique may be useful as a supplement to antibody-based assays, because imprinted polymers can be stable under severe conditions where natural molecules cannot survive, such as in organic sol-... 

Although antibody-based assays have many attractive features, we must emphasize that classical analytical methods must be used to confirm and validate immunoassays which are used for quantitative and/or qualitative analysis of specific molecular structures. Since most immunization procedures generate antibodies... 

Figure 5. Crop product decisions influence the utilization of variable crop inputs. Crop management decisions occur through all stages of the production cycle. Antibody based assays can provide accurate, timely information to support such decisions.

Analyte specific monitors chronic toxicity and employs tests specific for a single toxin or for analysis of a class of toxins. This covers a wide spectrum of tests including antibody-based assays. 

---