Production and Selection of Antibody-Antigen Pairs for the Development of Immunoenzyme Assay and Lateral Flow Immunoassay Methods for Carbofuran and Its Analogues

Abstract

To produce a sensitive monoclonal antibody (mAb) for the simultaneous detection of carbofuran, benfuracarb, carbosulfan and 3-hydroxy-carbofuran, 2,3-dihydro-2,2-dimethyl-7-benzofuranmethanamine (DDB) was conjugated to bovine serum albumin (BSA) to prepare the immunogen DDB-BSA and mice were immunized. Coating antigens were prepared by conjugating DDB and 5-methoxy-2,3-dihydrobenzofuran-3-acetic acid (MDA) to BSA and ovalbumin (OVA), respectively. Furthermore, the effect of different antibody-antigen pairs on the sensitivity of ELISA and LFIA methods for the detection of carbofuran was investigated. After the immunization, a high-affinity mAb 13C8 was obtained. The ability of the coating antigen to compete with carbofuran for binding antibodies was found to be significantly different between ELISA and LFIA methods. With the antibody-antigen pair 13C8-MDA-OVA, the IC₅₀ values of the ELISA and QD-LFIA methods for carbofuran were 0.18 ng/mL and 0.67 ng/mL, respectively. The cross-reactivity (CR) values of the two methods for benfuracarb, carbosulfan and 3-hydroxy-carbofuran ranged from 72.0% to 83.7%, while, for other carbamate pesticides, the CR values were less than 1%. The spiked recoveries of carbofuran in vegetables by the QD-LFIA method were 83-111%, with a coefficient of variation below 10%, and the test results of the actual samples were consistent with the HPLC-MS method. Overall, this study provides key materials for the development of immunoassays for carbofuran and its analogues, and the antibody-antigen pair selection strategy established in this study provides useful insights for the development of sensitive immunoassays for other compounds.

Keywords: benfuracarb; carbofuran; carbosulfan; immunoassay; monoclonal antibody.

Figure 1

Chemical structures of carbofuran and its analogues. (a) Carbofuran. (b) Benfuracarb. (c) Carbosulfan. (d) 3-Hydroxy-Carbofuran. (e) BFNB. (f) BFNH. (g) 3-Succinyl-Carbofuran. (h) BFOA. (i) DDB. (j) MDA.

Figure 2

The synthetic routes for the immunogen and coating antigen. (a) Conjugation progress of hapten MDA to carrier protein (b) Conjugation progress of hapten DDB to carrier protein.

Figure 3

The sensitivity of colloidal gold-based lateral flow immunoassay for carbofuran with 13C8 and different coating antigens. 1  =  0 ng/mL, 2  =  1 ng/mL, 3  =  3 ng/mL, 4  =  5 ng/mL, 5  =  100 ng/mL, 6  =  1000 ng/mL.

Figure 4

Standard curve for the determination of carbofuran by ELISA; the abscissa is the logarithm of the carbofuran concentration (pg/mL) and the ordinate is the inhibition rate.

Figure 5

Standard curve for the determination of carbofuran by QD-LFIA; the abscissa is the logarithm of the carbofuran concentration (pg/mL) and the ordinate is the inhibition rate.