Cross reactivity analysis antibody specificity

Ehrenberg, J.P. and Gebre, N. 1987, Analysis of the antigenic profile of Mycobacterium leprae cross-reactive and unique specificities of human and rabbit antibodies. Scand J Immunol 26 673-681. 

The principal limitation of these data is the lack of definition of the individual forms for the CYP2C subfamily. Analysis of this subfamily has remained problematic due to high cross-reactivities of all of the distinct forms with most antibody preparations. In addition, Western blot analysis does not distinguish between active and inactive forms of the protein. Furthermore, distinct enzymes may have different affinities for coenzymes necessary for catalytic activity, which will serve to unlink abundance of the protein and its catalytic activity. Therefore the assumptions must be made that the ratios of active to inactive protein are similar for all forms and that all forms have similar affinities for coenzymes. These assumptions may not be justified. However, even with these limitations, the study of Shimada et al. (1994) contributes greatly to our understanding of relative enzyme abundance in human liver. In addition, the relative abundance data, coupled with the absolute P450 content (per unit protein) and the turnover numbers for enzyme-specific substrates (per unit protein), can provide an estimate of the turnover number for individual enzymes in the human liver membrane environment. This provides an important benchmark for evaluation of turnover number data from cDNA-expressed enzymes. 

Despite the fact that the preparation of chloramphenicol-specific antibodies was reported as early as in 1966 (36), it was 1984 before the first immunoassay was published for the determination of chloramphenicol residues in swine muscle, eggs, and milk (37). This first-published method was a radioimmunoassay that required an extraction procedure and special laboratory facilities to attain a quantification limit of 1 ppb. Employed polyclonal antibodies showed insignificant crossreactivity with structurally related compounds, except that thiamphenicol that did not interfere with the analysis. However, cross-reactivity was significant for metabolites deviating from the parent compound in the acyl side chain. 

Nevertheless, the potential for autoimmune disease would clearly exist if one considers the polyspecificity of immune recognition molecules such as B cell receptors, antibodies, T cell receptors and MHC molecules. Further, the polyspecificity of immune receptors is also able to transcend the biochemically defined classes of biomolecules. For instance, the monoclonal antibody SYA/J6 has been demonstrated to have a dual specificity, binding a specific carbohydrate or a specific peptide with comparable affinity.68 Detailed analysis of the interatomic interactions between the antibody combining site and either the carbohydrate or the peptide antigens demonstrated that functional mimicry is possible without exact structural mimicry. This example underlines the case that it is not possible to predict with certainty whether the molecular surfaces of all potentially cross-reactive epitopes, whether of foreign or self molecules, will, or will not, be able to bind to a specific antibody. 

Beta-endorphin is a frequently measured opioid peptide. Immunoassay is the method of choice for the analysis of plasma (3-endorphin. Both RIAs and direct IRMAs have been developed for this purpose. Commercial reagent kits are widely available, and many commercial reference laboratories offer P-endorphin assays. The concentrations of P-endorphin are usually very low to undetectable in normal subjects, and it may be necessary to use extraction procedures to detect meaningful concentrations in plasma. The specificity of commercial antibodies for p-endorphin relative to p-LPH varies widely in some immunoassays, 50% cross-reactivity is seen with p-LPH. With polyclonal antibodies, results may be spuriously high owing to crossreactivity with serum immunoglobulin G (e.g., in patients with immunoglobulin G myeloma). 

Immunofluorescent detection of TS protein was done with the use of monoclonal antibodies, developed by in vivo immunization of Balb/c mice with homogeneous recombinant rat hepatoma TS protein as an antigen. The specific anti-rat TS antibodies recognized also T. spiralis TS, as indicated by cross-reactivity on Western blot. Localization of the enzyme was based on analysis of pictures collected by confocal microscopy. Two types of T spiralis muscle larvae preparations were studied muscle larvae isolated from mouse muscles by a procedure destroying nurse cells and muscle larvae remaining in nurse cells, isolated as an intact nurse cell preparation. 

Antibody Production. Immunogens were prepared by covalently linking l,N -ethenoadenosine and 3,N -ethenocytidine to bovine albumin (76). The use of the ribose forms of the adducts did not create a problem of specificity as the analytical samples were purified free of RNA. Ultimately, HPLC purification of EdA and EdC prior to analysis obviated all potential cross-reactivity problems. Rabbits were immunised by the multisite intradermal method (17) and high-titre antisera were generated. 

Western blotting. Both FNR and cyt 553 from A. variabilis were detected immunologically, either in purified preparations or in cell-free extracts, by Western blot analysis of SDS-PAGE (15% acrylamide) gels. Cross-reactivity of the antibody against Anabaena sp. 7119 cyt 553 with the same protein from A. variabilis and specificity of both antisera were demonstrated. 

The commonly used methods for the determination of these drugs are immunoassays and chromatography. Most immunoassays for immunosuppressants are semiautomated since extraction of drugs from the whole blood is needed before analysis. Immunoassays are convenient due to automation, but have problems with cross-reactivity with drug metabolites (3, 6). Both polyclonal and monoclonal antibody-based assays are available. Monoclonal antibody-based immunoassays are more specific. HPLC with ultraviolet detection and tandem mass spectrometry are commonly used chromatographic methods for the assay of immunosuppressants. Due to their specificity and sensitivity, tandem mass spectrometry assays are preferred and are now in wide use (7, 8). The other major advantage of tandem mass spectrometry assays is their ability to simultaneously measure several immunosuppressants (7-10). Pharmacokinetic properties of CSA, sirolimus, and tacrolimus are shown in Table 1 (3, 6, 11). 

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