Crossreactivity, antibodies

Analytical sensitivity can be assessed by end point dilution analysis, which measures the dilution of serum at which antibodies are no longer detectable. Analytical specificity is best assessed by examining test performance using a panel of sera derived from animals that have experienced related infections that may stimulate crossreactive antibody. If, e.g., the assay does not detect antibody in limiting dilutions of serum with the same efficiency as other assays, or crossreactivity is common when sera from animals with closely related infections are tested, the reagents need to be recalibrated or replaced, or the assay abandoned. 

The animal species chosen can be important. The animal species most often used in laboratories are rabbits, goats, guinea pigs, pigs, sheep, and rats. Commercial companies may favor horses and donkeys for large-scale preparations. Many animals contain crossreactive antibodies in their serum before immunization this could complicate their use in ELISA, and some simple absorption technique may be required (or may have been performed in commercial preparations) to block such reactions. Another point to remember is that many smaller animals can be immunized as compared to only a few larger animals. 

Schneider CH, Kasper MF, de Week AL, Rolh H, Angst BD Diagnosis of antibody-mediated drug allergy. Pyrazohnone and pyrazohdinedione crossreactivity relationships. Allergy 1987 42 597-603. 

The limitations of ELISA methods include the specificity of antibodies, the concentrations of primary antibody and antigen, and the type of reaction solution. Nonspecific binding of either of the antibodies to related antigens, unrelated proteins of other bacteria, or even the microtiter plate may lead to false positive reactions.49,52 57 Use of a monoclonal antibody may decrease crossreactivity with other antigens. For detection of low numbers of bacteria, as in drinking water, the sample may be filtered to concentrate the cells or cultured in a selective broth until it reaches the minimum detection limit for ELISA.49,58 Commercial test kits using dipsticks, immunoblots, and sandwich ELISA methods have been developed for the identification of pathogenic bacteria.58,59... 

El-Kasmi, K. C., Deroo, S., Theisen, D. M., Brons, N. H. C. and Muller, C. P. (1999), Crossreactivity of mimotopes and peptide homologues of a sequential epitope with a monoclonal antibody does not predict crossreactive immunogenicity , Vaccine, 18, 284-290. 

H., Gottesman, M. M., Pastan, I., Willingham, M. C., Immunohisto-chemical localization in normal tissues of different epitopes in the multidrug transport protein PI70 evidence for localization in brain capillaries and crossreactivity of one antibody with a muscle protein, J. Histochem. Cytochem. 1989, 37, 159-164. 

Fig. 2. (Continued). (C) Control protein tissue prints with antibody omitted. (D) Tissue-prints incubated with Phaseolus vulgaris IAA peptide antibody. Crossreaction visualized by chemiluminescence. Arrows indicate strong localization of the IAA-peptide in seeds.

The S6 document also mentions monoclonal antibody products. Indeed, many of the considerations for rDNA products are also applicable to monoclonal antibodies (including hybridized antibodies). With monoclonal antibodies, there is the additional concern of crossreactivity with nontarget molecules. 

Cells exist in an electrically charged environment. To prevent antibodies from binding as a result of excess charges on the tissue surface, a proteinaceous solution is used to bind to these sites in advance of the antibody incubations. A 10% normal animal serum from the same species as that providing the secondary or detecting system antibody in PBS is used. The use of other species semm may cause the antibodies to adhere nonspecifically or crossreact. The... 

The specificity of fluorescence localization is dependent on the specificity of the primary antibody, a property that must be tested and controlled by other methods, such as immunoprecipitation or immunoblotting. Controls for the labeling procedure described include deletion of the primary antibody step, which controls for the second-step reagent, or inclnsion of a similar, but nonreactive antibody as a first step. In the case of the availability of purified primary antigen, competition controls can be used, but they only control for the reactivity of the antibody with one antigen, and do not mle ont the possibility of a crossreactive, but unrelated antigen. 

As a negative control, the crossreactivities of the secondary antibodies may be checked by exchanging them in single-staining methods. 

The experiment must be designed without possibility of crossreaction between the two parallel assays. For example, avoid using a goat primary antibody that needs detection with antisheep antibody since this may cross-react with the sheep anti-FAM antibody. Also, the anti-FAM antibody should not be used after applying TSA-FITC reagent. 

Calkohven, P. G., Aalbers, M., Koshte, V. L., Pos, O., Oei, H. D., Aalberse, R. C. (1987). Cross-reaetivity among birch pollen, vegetables and fruits as detected by IgE antibodies is due to at least three different crossreactive structures. Allergy, 42, 382-390. 

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. 

Strong crossreactivity to chlortetracycline has been also observed when a commercialized kit (70) was applied to analyze tetracycline, chlortetracycline, and oxytetracycline residues in honey (71). The detection limit for both tetracycline and chlortetracycline was at 15 ppb, but for oxytetracycline at 250 ppb due to the low crossreactivity of the used antibodies to this analyte. Experiments using honey free of tetracyclines showed that dilution of honey with buffer at a ratio of a 1 50 was sufficient to eliminate matrix interferences. 

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