Polyclonal antisera cross-reactivity

A competitive enzyme immunoassay for the quantification of ivermectin residues in bovine liver has also been reported recently (85). This method uses a polyclonal antiserum raised in rabbits against 5-O-succinoylivermectin-trans-ferrin conjugate. Cross-reactivity was demonstrated with doramectin, a member... 

In 1993, another immunoassay for the detection of monensin was developed but, unfortunately, was never applied to biological material (91). Quite recently a competitive ELISA and a compatible extraction procedure suitable for screening monensin in poultry liver samples was described (92). In this assay, a polyclonal antiserum raised against a monensin-transferrin conjugate and prepared via an acid anhydride intermediate (93) was used. Significant cross-reactivity with other polyethers commonly used by the broiler industry, such as maduramicin, lasalocid, salinomycin, and narasin, was not found. A detection limit of 3 ppb could be readily attained when liver samples were submitted to extraction with aqueous acetonitrile, partitioning between aqueous sodium hydroxide solution and a hexane-diethyl either mixture, evaporation of the organic phase, and reconstitution in ethanol/sodium acetate solution. 

Antibodies to one protein antigen will often react (cross-react) with a wide variety of similar proteins from other species. For example, 15% of the antibody in a polyclonal antiserum to bovine serum albumin will react with human serum albumin. The molecular basis for this cross-reaction is not clearly understood, but cross-reactivity is believed to be caused by the presence of a configuration of amino acid side chains on another protein molecule that has a sufficient overall similarity to the specific configuration on the original antigen that induced antibody formation. 

The specificity of these EIA is determined by the uniqueness of the interaction between antibody and antigen, as well as to environmental factors. Non-specificity of the immune reaction can be traced to two phenomena, shared reactivity and cross-reactivity, discussed in Section 8.6. Specificity of a polyclonal antiserum is generally higher than that of its component antibodies, due to the specificity bonus (Section 8.6). 

Fig. 8.4. Concepts of shared (I) and cross-reactivity (II). Two antigens (A and B) may have one or more identical epitopes and a subpopulation of antibodies from a polyclonal antiserum will react equally with both antigens (shared reactivity). If monoclonal antibodies against the common epitope were used, it would not be possible to distinguish A and B. In cross-reactivity, antibodies reactive with the homologous antigen are also reactive with different epitopes (generally less fit thus lower affinity).

A specific antibody is desirable because it enables an IA to be performed with limited or no sample clean-up. For reagent-excess methods, it is very important to choose specific antibodies. Cross-reactivity of polyclonal antiserum from each blood collection or each clone of monoclonal antibodies is tested against known metabolites, drug degradants, concomitant drugs, and the protein carrier... 

A number of enzyme-linked immunosorbent assay (ELISA) methods have also been reported. Koppelman et al. (2007) reported the development of ELISA methods to detect mustard protein contamination of mustard seed oil [6]. A rabbit polyclonal antiserum was raised to B.juncea and used to develop an inhibition ELISA assay with a reported detection limit of 1.5 ppm (mg/kg). Weak cross-reactivity with soy (0.016%) and milk (0.28%) was reported. 

ELISA Systems Mustard Seed Protein Residue kit was released in June 2007. A polyclonal rabbit antiserum was raised and used to develop a quantitative sandwich ELISA that has been demonstrated to detect mustard seed protein from aU three species of mustard plants S. alba, B. nigra, and B. juncea [5]. The detection limit of the kit has been shown to be less than 0.5 ppm (mg/kg) of soluble mustard protein, which corresponds to mustard seed concentrations below 3.4ppm S. alba, below 4.9 ppm B. nigra, and below 5.5 ppm B. juncea. An example of a calibration curve is presented in Figure 23.1. Cross-reactivity studies were conducted on full-strength extracts from 50 plants and other common foods, and cross-reactivity was observed only with rapeseed (Canola), Brassica napus. This cross-reactivity was approximately 50%, but purified canola oil did not cross react. 

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