Naifeng Xu

Development of an immunoassay for carbendazim based on a class-selective monoclonal antibody

In the present study, a class-selective monoclonal antibody (mAb) for carbamate fungicides was obtained using two haptens. One hapten was designed as immunogen and the other one for coating. Under the optimum assay conditions of coating antigens H2-ovalbumin, in the assay buffer of sodium chloride 1.6%, no methanol, and pH 6.5, the assay system was more sensitive. The linear range was from 0.06 to 7.47 ng/mL. The cross-reactivity test was carried out with IC50 values of 0.45 ng/mL for carbendazim, 0.57 ng/mL for benomyl, and 9.70 ng/mL for thiabendazole. Other related compounds thiophanate methyl, fenbendazole, mebendazole, methyl paraoxon, methyl parathion, chlorpyrifos, and chlorothalonil did not cross-react significantly in this assay. To evaluate the proposed immunoassay, real samples such as apples, tomatoes, and cucumbers were subjected to recovery test with carbendazim. The recovery and coefficient variation range from 73% to 125% and 0.36% to 11.80%, respectively. The developed method was also compared with the commercial kit for assaying carbendazim in the fruits and vegetables and the results were consistent. All results indicated that this developed enzyme-linked immunosorbent assay (ELISA) is useful for the detection of carbendazim residues in fruits and vegetables.

An ultrasensitive immunochromatographic assay for non-pretreatment monitoring of chloramphenicol in raw milk

A non-pretreatment monitoring method based on immunochromatographic test (ICT) strips was developed to test for chloramphenicol (CAP) in raw milk. This assay was designed to measure competitive binding affinities for CAP molecules in raw milk and CAP antigen immobilized on strips with colloidal gold nanoparticles (GNPs, average diameter of 30 nm) labeled with anti-CAP monoclonal antibody (CAP mAb). Several working conditions, including the CAP solvent, different types and concentrations of CAP mAb, coating antigen, and GNP, were optimized for performance. After optimization, the detection process was carried out in 8 min with a visual limit of detection (LOD) of 0.3 ng/mL for qualitative detection and a LOD of 0.064 ng/mL for semi-quantitative detection. No cross-reactivity was detected toward CAP analogs. Based on these results, this simple and ultrasensitive assay could be thoroughly applied in the on-site testing of CAP in raw milk.

Development of a lateral flow immunoassay for the detection of total malachite green residues in fish tissues

We developed a sensitive lateral flow immunoassay (LFI) for the detection of total malachite green (MG) residues in fish tissues. LFI was based on a colloidal gold nanoparticle-labeled monoclonal antibody against MG , which was generated by fast immunization of carboxymalachite green-bovine serum albumin. The half-maximum inhibition concentration (IC50) of MG monoclonal antibody (MG mAb) was 0.057 ng/ml with a linear range of 0.020–0.162 ng/ml. Following the optimization of LFI, a qualitatively visual limit of detection of 1 ng/ml and a semi-quantitatively limit of detection of 0.418 ng/ml (IC50 of the calibration curve) were obtained for total MG residues in 8 min. Cross-reactivity with most MG analogs was negligible, expect with crystal violet (CV; 78.6% cross-reactivity). LFI is a novel and sensitive assay based on MG mAb that has applications for the rapid detection of total MG and CV in fish products.

Development and application of one-step ELISA for the detection of neomycin in milk

A sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of neomycin (NEO) in milk was developed. Two conjugates were synthesized as the immunogen and coating antigen. Four BALB/c female mice were immunised and the antisera were screened by indirect competitive ELISA (icELISA). The icELISA was further optimised using different coating methods, pH values, ionic strengths of assay buffer and reaction times. After optimised, the indirect competitive ELISA (icELISA) could be finished in 85min with an IC50 value of 0.74±0.05 ng/mL. One-step indirect competitive ELISA (icELISA) exhibited similar sensitivity with an IC50 value of 0.73±0.03 ng/mL, which was sensitive enough for NEO detection and could be finished in 55 min. No cross-reactivity of the antibody was observed with other aminoglycosides based on icELISA, indicating that the antibody is highly specific for neomycin. Milk samples were further analyzed with recovery ranging from 85 to 110% at spiked level of 0.25–10 ng/mL, and the detection limit was 0.08 ng/mL.

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.