Immunoassay antibody-protein conjugates

Figure 1 Schematic of the quasi-equihbria using heterologous haptens in coating antigen immunoassay formats. Ka represents the equilibrium constant for binding of antibody (Y) to target analyte (A). Kh is the equilibrium constant for the binding of antibody to hapten-protein conjugate (H-) immobihzed on a solid phase...

Alternatively, competitive ELISA can be used to estimate the hapten density if an antibody that specitically recognizes the hapten is available. At first observation this approach seems circular because the immunoassay developed is used to determine hapten density on proteins used for immunization. However, if a small molecule mimic of the protein conjugate is used as a standard, the method can be accurate. For example, a hapten containing a carboxylic acid can be coupled to phenethylamine or tyramine, its structure confirmed and the material used to generate a calibratron curve to estimate hapten density. 

Grammer LC, Harris KE, Malo JL, et al. 1990. The use of an immunoassay index for antibodies against isocyanate human protein conjugates and application to human isocyanate disease. J Allergy Clin Immunol 86(1)94-98. 

The utihzation of fluorescence dyes for analytical measurements enhances the sensitivity for the detection of the molecules of interest. First, Cronick and Little made use of evanescent wave excitation for a fluorescence immunoassay, in 1975. By using totally internally reflected light, they excited the fluorescence of a fluorescein-labeled antibody which has become bound to a hapten-protein conjugate adsorbed on a quartz-plate in an antibody solution [41]. Contrary to the label-free high-refractive-index sensors where the mass of the molecule of interest is... 

Monoclonal antibodies were obtained against atrazine and its metabolite hydroxyatrazine by immunizing mice with atrazine or hydroxyatrazine protein conjugates. By competitive ELISA, we observed that the antibodies raised against hydroxyatrazine cross-reacted mainly with hydroxypropazine. The antibodies raised against atrazine cross-reacted with propa-zine, prometone, prometryne, and to a much lower extent with a few other s-triazines and hydroxy-s-triazines. Atrazine could be detected in water samples down to 50 ppt. Average recoveries measured by ELISA from soil samples fortified with atrazine or hydroxyatrazine were comparable to those measured by GLC or HPLC. Soil samples of unknown atrazine content were analyzed by GLC, GC-MS, and by ELISA. The results show that the ELISA immunoassay represents a valuable detection method for trace amounts of atrazine and hydroxyatrazine in soil. 

The cross-reactivity of polyclonal antibodies raised to an atrazine-protein conjugate (linked at the chlorine group) was evaluated, using a peroxidase-atrazine labeled species linked in the same manner, in order to determine the reliability of a newly available competitive immunoassay kit for environmental atrazine monitoring.28 A wide variety of similar compounds (Fig. 6.14) were screened, and the concentrations given beside the compounds indicate the apparent concentration of atrazine that would be erroneously recorded from the responses obtained to 1-pg/ mL concentrations of each of these interferents. 

In recent years, immunoassays for pesticides, phytopharmaceuticals, and industrial pollutants (polychlorinated biphenyls, dioxins, etc.) have become accepted as methods that complement traditional analytical procedures also in the field of food analysis (including drinking water). In the case of pesticides, both quantitative and semiquantitative kits are available. Crossreactivity may occur - although with different efficiency - between an antibody raised against a protein conjugate of a given compound, and structurally... 

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

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