Development and characterisation of an ultrasensitive monoclonal antibody for chloramphenicol

In this study, a highly sensitive monoclonal antibody (McAb) against chloramphenicol (CAP) was successfully raised and characterised by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). Three antigens coupled with different proteins were synthesised and determined by the UV-vis method. After five rounds of immunisation, antisera were collected from six Balb/C mice and screened by ic-ELISA. The standard inhibition curve of one CAP McAb (labelled 6F11) showed the best sensitivity to CAP after optimisation of the principal working conditions. The half-maximal inhibition concentration (IC₅₀) was calculated to be 0.01 ng/mL and the linear working range was from 0.0028 ng/mL to 0.040 ng/mL with a correlation coefficient of 0.996. This McAb showed high specificity, with negligible cross-reactivities detected towards CAP analogues (florfinicol, florfinicol amine, thiamphenicol). The recovery rate was 98.9–106.8% while the coefficient of variation was 4.45–6.05%. The IC₅₀ value and other parameters obtained for this CAP McAb suggest that it is likely to have a promising future in further applications in combination with different analytical techniques.     

Development of an immunochromatographic assay for hexestrol and diethylstilbestrol residues in milk

Hexestrol (HES) and Diethylstilbestrol (DES) are synthetic hormones, which have been found abuse use in animal-origin food production. We developed an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and immune-chromatographic strip to detect these compounds based on monoclonal antibody. Under optimized conditions, the half-inhibition concentration (IC₅₀) values of ic-ELISA were 0.15 µgL^?1 and 0.23 µgL^?1 for HES and DES, respectively. Spiked milk samples were analyzed. The recovery of both synthetic hormones in the milk samples was 60.48–102.19% (HES) and 89.34–100.16% (DES). In immune-chromatographic assay, the visual cutoff values at 0.5 and 1.0?µgL^?1 respectively for HES and DES, which allow us to detect milk samples of a concentration low to 10 µgL^?1 for HES and 15 µgL^?1 for DES.     

Development of an immunochromatographic strip for the rapid detection of 10 β-agonists based on an ultrasensitive monoclonal antibody

A sensitive monoclonal antibody, 3G10, derived from a mouse immunized with an immunogen of salbutamol hapten conjugated to keyhole limpet hemocyanin was developed against β-agonists, with a view to establishing a rapid gold nanoparticle immunochromatographic strip. The antibody displayed a 50% inhibitory concentration (IC₅₀) of 1.04?ng/mL for salbutamol and simultaneously detected nine other β-agonists (clenbuterol, cimatero, brombuterol, mabuterol, terbutaline, clenpenterol, carbuterol, mapenterol, pirbuterol) with uniform cross-reactivity. The immunochromatographic strip had cut-off values of 5?ng/mL for salbutamol and clenbuterol, 20?ng/mL for cimaterol, brombuterol and mabuterol, and 50?ng/mL for terbutaline, clenpenterol, carbuterol, mapenterol and pirbuterol. The collective advantages of the newly developed strip, such as high affinity, sensitivity and rapidity, support its utility in detecting β-agonists from actual biological samples, such as urine.     

Synthesis of olaquindox metabolite,methyl-3-quinoxaline-2-carboxylic acid for development of an immunoassay

A novel synthesis method of methyl-3-quinoxaline-2-carboxylic acid (MQCA) and the preparation of its hapten were described. MQCA was synthesised involving in two steps with a high purity. For improving the sensitivity of detection, five different haptens were synthesised and corresponding immunogens and coating antigens were prepared. After comparing the sensitivity of immunoassay with different pairs of antibody and coating antigen, a specific immunoassay was obtained using an antibody raised against hapten (four-atom spacer arm) – BSA and a suitable coating antigen with a heterologous spacer arm group. The 50% inhibitory concentration of MQCA by indirect competitive enzyme-linked immunosorbent assay with optimised antibody and coating antigen was 3.84 ng/ml and the limit of detection was 0.25 ng/ml.     

Development of an ultrasensitive ic-ELISA and immunochromatographic strip assay for the simultaneous detection of florfenicol and thiamphenicol in eggs

An ultrasensitive monoclonal antibody-based gold nanoparticle immunochromatographic strip assay and indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) were developed to detect florfenicol (FF) and thiamphenicol (TAP) in egg samples. The ic-ELISA, with optimized pH, methanol content and sodium chloride content, exhibited an IC₅₀ value of 0.2?ng/mL for FF and 0.27?ng/mL for TAP, with the working range of 0.05–0.77 and 0.05–1.42?ng/mL, respectively. The optimized ic-ELISA showed negligible cross-reactivity with other phenols and broad-spectrum antibiotics. The recoveries in egg samples using the ic-ELISA ranged from 84% to 115% with a coefficient of variation of less than 5%. Based on this monoclonal antibody, a rapid and ultrasensitive immunochromatographic strip assay was developed with a cutoff value of 1?ng/mL for FF and TAP. Our results indicated that both developed methods were highly useful for screening FF and TAP in eggs.     

Development of sensitive and fast immunoassays for amantadine detection

A sensitive indirect competitive enzyme-linked immunosorbent assay(ic-ELISA) method and a gold nanoparticle immunochromatographic strip were developed to detect amantadine (AM) in foods. A novel AM hapten was prepared, and a sensitive monoclonal antibody against AM was developed. The optimum ic-ELISA conditions included a pH of 7.4 and an ionic strength of 1.6% with no organic solvents. The half-inhibition concentration (IC₅₀) value of ic-ELISA was 1.92?ng/ml, with a limit of detection of 0.62?ng/ml. The immunochromatographic test strip method had a visual cut-off value at 5?μg/kg. Chicken samples were spiked with three concentrations of AM (1, 2, and 5?µg/kg) and analysed by ic-ELISA. Good recoveries were obtained (92.3% at 1?µg/kg AM, 116.1% at 2?µg/kg AM, and 91.8% at 5?µg/kg AM).     

Development of ic-ELISA and lateral-flow immunochromatographic assay strip for the detection of vancomycin in raw milk and animal feed

A highly sensitive monoclonal antibody (mAb) 3H4 against vancomycin (VAN) was prepared. Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and lateral-flow immunochromatographic assay (ICA) were developed based on the mAb. The 50% inhibition concentration (IC₅₀) value and limit of detection (LOD) value of ic-ELISA method for vancomycin were 0.59 and 0.06?ng/mL, and for norvancomycin were 1.51 and 0.13?ng/mL under optimized conditions as pH 7.4, 0.4% (m/v) NaCl, and 5% (v/v) acetonitrile. In lateral-flow ICA, the visual limit of detection (vLOD) value and cut-off values for vancomycin were 1 and 2.5?ng/mL, and for norvancomycin were 5 and 10?ng/mL under optimized conditions as pH 8.6 with 1?mg/mL coating antigen and 1?µg/mL gold nanoparticle-labeled mAb. In raw milk and animal feed samples, recovery rates from ic-ELISA ranged from 89.2% to 121.6%. The vLOD and cut-off value were 5–10?ng/g and 100–200?μg/kg, respectively. Therefore, both methods were sensitive, rapid, and effective for the on-site detection and rapid mass screening of samples.     

Development of ic-ELISA and lateral-flow immunochromatographic assay strip for the detection of citrinin in cereals

A sensitive and specific anti-citrinin (anti-CIT) monoclonal antibody 1F2 was obtained following immunization and cell fusion. An indirect competitive enzyme-linked immunosorbent assay was developed with a 50% inhibitory concentration of 0.761?ng/mL and a limit of detection of 0.089?ng/mL. The recovery rates for CIT-spiked cereals (maize, wheat, and rice) ranged from 112% to 123%. A lateral-flow immunochromatographic assay was developed for both semi-quantitative and quantitative detection. With CIT-spiked cereals, the visual limit of detection was 8?ng/g and the cut-off value was 40?ng/g (semi-quantitative analysis with naked-eye detection). Using a strip scan reader, the calculated limit of detection was 1.28–1.8?ng/g for different CIT-spiked cereals. The recovery rates ranged from 110% to 127%. Therefore, both methods were effective for CIT detection and suitable for on-site detection and rapid screening of samples.     

Development of ic-ELISA and an immunochromatographic strip assay for the detection of methylmercury

In this study, we developed an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and an immunochromatographic strip assay based on a monoclonal antibody (mAb) against methylmercury (MeHg) to detect the presence of MeHg in tap water. Under optimum conditions (pH 8.0, 0.8% NaCl, and 0.1% Tween 20), the 50% half maximal inhibitory concentration (IC₅₀) and limit of detection (LOD) were 16.64 and 2.03?ng/mL, respectively. The anti-MeHg mAb was speci?c to mercury with no cross-reactivity with other metal ions. The cut-off value of the immunochromatographic strip assay was 500?ng/mL for semi-quantitative detection, and the LOD was 11.3?ng/mL for quantitative detection. The average recovery rates of the ic-ELISA and immunochromatographic strip assay were 98.13% and 107.87%, respectively, in tap water. Therefore, ic-ELISA and the immunochromatographic strip assay can be used to detect MeHg in tap water.     

Rapid enzyme-linked immunosorbent assay and immunochromatographic strip for detecting ribavirin in chicken muscles

The use of ribavirin (RBV) as an antiviral drug for livestock has been prohibited in China, the USA, and many other countries. In this study, we developed a rapid and sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and a gold nanoparticle immunochromatographic (ICA) strip test for detecting RBV in chicken muscles. Under the optimum assay conditions, where the assay employed phosphate-buffered saline at pH 7.4, no acetonitrile, and an ionic strength of 0.8%, the quantitative working range was 1.43–26.47?ng/ml with an IC₅₀ of 6.15?ng/ml. The recovery rate for RBV in real samples ranged from 82.1% to 112.3%. The immunochromatographic test strip method had a visual cutoff value of 50?μg/kg. Given their high recovery rates and good sensitivity, the proposed ic-ELISA and ICA methods could be useful for the RBV analysis in chicken tissue samples.