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In this work, a quantitative sandwich ELISA was optimized, through a full factorial design of experiments (DOE) in successive steps of a preliminary protocol obtained by the method of one factor at a time (OFAT). The specificity of the optimized ELISA, the lower limit of quantification, the quantification range and the analytical sensitivity of the antigen quantification curve were evaluated, in comparison with the curve obtained from the preliminary protocol. The full factorial DOE was linked to a simple statistical processing, which facilitates the interpretation of the results in those laboratories where there is no trained statistician. The step-by-step optimization of the ELISA and the successive incorporation into the protocol of the best combination of factors and levels, allowed obtaining a specific immunoassay, with an analytical sensitivity 20 times greater and with a lower limit of antigen quantification that decreased from 156.25 at 9.766 ng/mL. As far as we know, there are no reports of optimization of an ELISA following the step-by-step scheme used in this work. The optimized ELISA will be used for the quantification of the TT-P0 protein, the active principle of a vaccine candidate against sea lice. [Display omitted] • ELISA optimization followed a step-by-step scheme not described previously in scientific papers. • The full factorial design of experiments was linked to a simple statistical processing. • The optimized ELISA has a 20-fold higher analytical sensitivity. • The lower limit of quantification of antigen decreased from 156.25 to 9.766 ng/mL. [ABSTRACT FROM AUTHOR]

Lambda-cyhalothrin is a pyrethroid widely used in crop, fruit and vegetable production, but has potential health threats to human. Immunoassay is a cheap, rapid and facile method to detect lambda-cyhalothrin, yet wide application of this method still requires improvement in the construction of antigen. In this study, we developed a one-step lambda-cyhalothrin hapten synthesis that transformed the cyanide group in lambda-cyhalothrin to amide. Complete antigen was assembled by coupling the amide with succinic-anhydride-activated carrier proteins, and corresponding polyclonal antibodies were generated using Balb/c mice. Using antibody generated by the method in this paper, the competitive ELISA demonstrated the lowest detection limit of 3.772 μg/L for lambda-cyhalothrin, and no significant cross-reactivity for other pyrethroid pesticides was observed. All the results suggested we have established a more efficient technique of generating lambda-cyhalothrin antibody. Furthermore, since the activated proteins used in this study are highly controllable, we believe these proteins could potentially be the prototype of a series of standardized carrier proteins for the synthesis of complete antigens. Image 1 • A safe and high-yielding one-step lambda-cyhalothrin hapten synthesis strategy was developed. • A novel succinic anhydride activating technology was developed which provided the carrier proteins with more reactive sites. • The carrier protein activation method has provided a new solution to the poor-conjugation problems. [ABSTRACT FROM AUTHOR]