Novel haptens synthesis and development of a monoclonal antibody-based enzyme-linked immunosorbent assay for leuco-malachite green in fish

Abstract

To produce specific antibodies against leuco-malachite green (LMG), 15 haptens were synthesized and characterized, and conjugated to carrier protein for immunization. One antigen with excellent reactogenicity and immunogenicity was discovered. Specific monoclonal antibody with high sensitivity for LMG in competitive indirect enzyme-linked immunosorbent assay (ciELISA) was screened and selected. After the screening of a series of heterologous coating antigen and optimization of working conditions, the proposed ciELISA showed the 50% inhibition value (IC 50) of 1.16 ng mL −1 and the limit of detection of 0.06 ng mL −1 for LMG. The average recoveries of LMG from spiked fish samples ranged from 78.0% to 101.0%, with coefficients of variation below 15%. Good correlation (R 2 = 0.9977) was obtained between the results of ciELISA analysis and those of standard liquid chromatography-tandem mass spectrometry analysis. The proposed ciELISA is ideal for the rapid and sensitive detection of LMG with a low cost and high throughput.

Generation of functional single-chain fragment variable from hybridoma and development of chemiluminescence enzyme immunoassay for determination of total malachite green in tilapia fish

“…The specificity of competitive CLEIA using anti-LMG scFv-AP was determined by testing the cross-reactivity of other structurally related compounds (Table S9). The anti-LMG scFv-AP fusion protein was highly selective for LMG, which is in line with its parent monoclonal antibody (Wang, et al, 2017). Only slight cross-reactivity was observed for LCV and MG (4.65% and 1.36%, respectively), and low cross-reactivity (<0.01%) for other tested analogs.…”

“…The plasmid named pLIP6/GN was a kind gift of Dr. Frédéric Ducancel (CEA-Saclay, France). The hybridoma cell line secreting Mab against LMG had previously been generated in our laboratory (Wang, et al, 2017). E.coli.…”

“…ELISA with heterologous coating antigen (Table S2) was performed to validate the binding activity of scFv−AP fusion protein compared with parent Mab (Wang, et al, 2017). The coating antigen (LMG hapten H14-OVA) was diluted to 1.2 μg/mL and 100 μLper well was pipetted into the transparent microplates.…”

“…However, broad-specific assay is not suitable for quantitative analysis of neither LMG nor MG. Recently, a monoclonal antibody (Mab) against LMG was successfully produced in our laboratory, with no cross-reactivity toward other triphenylmethane dyes (Wang, Yang, Shen, Sun, Xiao, Lei, et al, 2017). Though homemade ELISA kit based on this Mab showed excellent specificity and sensitivity for detection of LMG, it requires an additional step of secondary antibody incubation.…”

Hapten synthesis and production of specific antibody against 3-amino-5-morpholinomethyl-2-oxazolidone for immunoassay without derivatisation

“…Five haptens (H1-H5) were coupled to OVA for coating antigens. The active ester method was adopted to conjugate H1, H2, H3 and H5 to the carrier protein according to Wang et al (2017). H4 and H1-SPAMAM 4 were connected with BSA via glutaraldehydes according to Romestand et al (2010).…”