Theoretical Calculation and Experimental Verification Demonstrated the Impossibility of Finding Haptens Identifying Triphenylmethane Dyes and Their Leuco Metabolites Simultaneously.

Detection of triphenylmethane dyes (TDs), especially the widely used malachite green (MG) and crystal violet (CV), plays an important role in safety control of aquatic products. There are two chromatic forms of TDs: oxidized or reduced. Usually, only one form can be detected by reported ELISA antibodies. In this article, molecular shape superimposing and quantum mechanics calculation were employed to elucidate the differences between MG, CV, and their reduced chromatic forms (leucomalachite green, LMG and leucocrystal violet, LCV). A potential hapten was rationally designed and synthesized. Polyclonal antibodies were raised through immunizing New Zealand white rabbits and BALB/C mice. We tested the cross-reactivity ratios between the hapten and TDs. The cross-reactivity ratios were correlated with the difference in surface electrostatic potential. The determination coefficients (r²) of the correlations are 0.901 and 0.813 for the rabbit and mouse antibody, respectively. According to this linear model, the significant difference in the atomic charge seemed to make it impossible to find a hapten that can produce antibodies with good cross-reactivities with both reduced and oxidized TDs.