Nanobodies are one-tenth the size of conventional antibodies and are naturally obtained from the atypical heavy-chain-only antibodies present in camelids. Their small size, high solubility, high stability, and strong resilience to organic solvents facilitate their use as novel analytical reagents in immunochemistry. In this study, specific nanobodies against pesticide carbofuran were isolated and characterized from an immunized library via phage display platform. We further established an indirect competitive enzyme-linked immunosorbent assay (ELISA) using nanobody Nb316 to detect carbofuran in vegetable and fruit samples. The results showed a half-maximal inhibitory concentration (IC₅₀) of 7.27 ng/mL and a detection limit of 0.65 ng/mL. A simplified sample pretreatment procedure omitting the evaporation of organic solvent was used. The averaged recovery rate of spiked samples ranged between 82.3% and 103.9%, which correlated with that of standard UPLC–MS/MS method. In conclusion, a nanobody with high specificity for carbofuran was characterized, and a nanobody-based sensitive immunoassay for simple and rapid detection of carbofuran in real samples was validated.

The haptens 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)carbonyl]-amino]butanoic acid (BFNB) and 6-[((2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)-carbonylamino]hexanoic acid (BFNH) were synthesized and then used to develop a rapid,specific and sensitive ELISA method to determine residues of the pesticide carbofuran in avariety of matrices. A hybridoma cell line (5D3) producing anti-carbofuran monoclonalantibodies (MAbs) was also established. Based on the MAbs in combination with theheterologous hapten BFNH coupled to either horseradish peroxidase (HRP) or ovalbumin(OVA), four ELISAs (formats I-IV) for the quantification of carbofuran were developedand compared. Among them, the optimized format II (the conjugate-coated directcompetitive ELISA) showed the best characteristics, with an IC50 value of 18.49 ng/mL, alimit of detection of 0.11 ng/mL and the shortest assay time (1 h). This ELISA method wasthen applied to the determinations of carbofuran in environmental water, soil and foodsamples. The relative standard deviations (R.S.D.s) ranged from 1.8% to 21.3% and themean recoveries were 104.6%, 108.3%, 106.3% and 100.1% for water, soil, lettuce andcabbage, respectively. Thus, the ELISA method of format II exhibited the potential todevelop commercial ELISA kits for a rapid detection of carbofuran for human health andenvironmental safety.

Paclobutrazol (PBZ) is a plant growth inhibitor and fungicide, but it is also carcinogenic and teratogenic, and has potential harm to human health. In this study, two PBZ haptens (PBZ-1, PBZ-2) were synthesized and conjugated with carrier proteins to get artificial antigens. A highly specific monoclonal antibody (mAb) against PBZ was prepared. The antibody subtype was IgG₁ and the concentration was 11.03 mg/mL. A sensitive and rapid time-resolved fluorescence microsphere lateral flow immunoassay (TRFMs-LFIA) was established based on the mAb. The activated pH, the mAbs diluents, the mAb reacting concentration and the probe amount were optimized. The visual limit of detection (vLOD) and quantitative limit of detection (qLOD) of the TRFMs-LFIA for PBZ were 50 and 1.72 ng/mL respectively, and the 50% inhibiting concentration (IC₅₀) was 9.38 ng/mL. The pretreatment procedures are simple and rapid, and the detection time of TRFMs-LFIA strip is 6 min. Qualitative and quantitative analysis of PBZ could be achieved under a UV light or with a portable fluorescence immunoassay analyzer. The average recovery rates ranged from 96.2% to 111.9% and the corresponding coefficients of variation (CV) were 4.0%–11.2% in spiked wheat and rice samples. Twenty real wheat and rice samples were measured by the TRFMs-LFIA and compared with Ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS). The measured values showed a good accordance. These results indicated that the proposed assay will provide a novel effective strategy for on-site detection of PBZ.

A novel and efficient immunoaffinity column (IAC) based on bispecific monoclonal antibody (BsMAb) recognizing aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) was prepared and applied in simultaneous extraction of AFB₁ and OTA from food samples and detection of AFB₁/OTA combined with ic-ELISA (indirect competitive ELISA). Two deficient cell lines, hypoxanthine guanine phosphoribosyl-transferase (HGPRT) deficient anti-AFB₁ hybridoma cell line and thymidine kinase (TK) deficient anti-OTA hybridoma cell line, were fused to generate a hybrid-hybridoma producing BsMAb against AFB₁ and OTA. The subtype of the BsMAb was IgG₁ via mouse antibody isotyping kit test. The purity and molecular weight of BsMAb were confirmed by SDS-PAGE method. The cross-reaction rate with AFB₂ was 37%, with AFG₁ 15%, with AFM₁ 48%, with AFM₂ 10%, and with OTB 36%. Negligible cross-reaction was observed with other tested compounds. The affinity constant (Ka) was determined by ELISA. The Ka (AFB₁) and Ka (OTA) was 2.43 × 10⁸ L/mol and 1.57 × 10⁸ L/mol, respectively. Then the anti-AFB₁/OTA BsMAb was coupled with CNBr-Sepharose, and an AFB₁/OTA IAC was prepared. The coupling time and elution conditions of IAC were optimized. The coupling time was 1 h with 90% coupling rate, the eluent was methanol–water (60:40, v:v, pH 2.3) containing 1 mol/L NaCl, and the eluent volume was 4 mL. The column capacities of AFB₁ and OTA were 165.0 ng and 171.3 ng, respectively. After seven times of repeated use, the preservation rates of column capacity for AFB₁ and OTA were 69.3% and 68.0%, respectively. The ic-ELISA for AFB₁ and OTA were applied combined with IAC. The IC₅₀ (50% inhibiting concentration) of AFB₁ was 0.027 ng/mL, the limit of detection (LOD) was 0.004 ng/mL (0.032 µg/kg), and the linear range was 0.006 ng/mL~0.119 ng/mL. The IC₅₀ of OTA was 0.878 ng/mL, the LOD was 0.126 ng/mL (1.008 µg/kg), and the linear range was 0.259 ng/mL~6.178 ng/mL. Under optimum conditions, corn and wheat samples were pretreated with AFB₁-OTA IAC. The recovery rates of AFB₁ and OTA were 95.4%~105.0% with ic-ELISA, and the correlations between the detection results and LC-MS were above 0.9. The developed IAC combined with ic-ELISA is reliable and could be applied to the detection of AFB₁ and OTA in grains.