Preparation of anti-2,4-dichlorophenol and 2,4-dichlorophenoxyacetic acid monoclonal antibodies

Abstract

The ratios of hapten and bovine serum albumin (BSA) in an antigen conjugate were determined by matrixassisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Hybridomas secreting monoclonal antibodies against 2,4-dichlorophenoxyacetic acid (2,4-D) were produced by fusing 2,4-D-BSA conjugate-immunized splenocytes with a HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. A substantial cross-reaction was observed for 2,4-dichlorophenol (2,4-DP) when compared with that observed for 2,4-D. The full measurement range for this assay is 0.2-3 g ml À1 for 2,4-DP. On the other hand, the range for 2,4-D is between 1 and 20 g ml À1 .Abbreviations: ABTS -2,2 0 -azino-bis(3-etylbenzothiazoline-6-sulfonic acid) diammonium salt; BSAbovine serum albumin; 2,4-D -2,4-dichlorophenoxyacetic acid; 2,4-DP -2,4-dichlorophenol; ELISAenzyme-linked immunosorbent assay; MAb -monoclonal antibody; MALDI -matrix-assisted laser desorption/ionization; PBS -phosphate-buffered saline; TPBS -PBS containing 0.05% of Tween 20; SPBS -PBS containing 5% of skim milk

Novel surface plasmon resonance (SPR) immunosensor based on monomolecular layer of physically-adsorbed ovalbumin conjugate for detection of 2,4-dichlorophenoxyacetic acid and atomic force microscopy study

“…The protein conjugates of 2,4-D (2,4-D-OVA and 2,4-D-BSA) were synthesized similar to our procedure reported elsewhere [39]. Briefly, isobutylchloroformate (10 l) and Ntriethylamine (10 l) were added to a solution of 2,4-D (5 mg in 1 ml dioxane), and the mixture was stirred at room temperature for 2 h. It was added drop-by-drop to an aqueous solution of OVA or BSA (5 mg in 2 ml) and was kept stirring for 16 h. The reaction mixture was then dialyzed at 4 • C and lyophilized to get the conjugates.…”

“…A monoclonal antibody against 2,4-D (abbreviated as 2,4-DAb) was developed from a hybrid cell resulting from the fusion of an aminopterin-sensitive myeloma cell line and the spleen cell of mice immunized with 2,4-D-BSA [39]. Molecular weight (MW) of the conjugates was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using JMS time-of-flight mass monitor.…”

Construction, Expression, and Characterization of a Single-Chain Variable Fragment Antibody Against 2,4-Dichlorophenoxyacetic Acid in the Hemolymph of Silkworm Larvae

“…Moreover, it was revealed that 2,4-D-scFv could be applied to the determination/detection of the phenoxy family of herbicides, which include 2,4,5-T, 2,4-DP, and 2,4-DB, because high CR were obtained for these compounds. The CR of 2,4-D-scFv seems to be wider than that of MAb 2C4, which was determined in our previous study [13]. It is assumed that the differences in specificity between scFv and its parental monoclonal antibody mainly depend on the molecular weight of the target hapten and its tertiary structure.…”

“…In our previous study, we produced a monoclonal antibody against 2,4-D (MAb 2C4) that was secreted from hybridoma cells prepared by fusing splenocytes with the mouse myeloma cell line, P3-X63-Ag8-653 [13]. Although monoclonal antibodies (MAb) possess more specific activity than polyclonal immunoglobulins and are suitable for large-scale production, hybridoma cell lines are not stable, especially after cryopreservation [14].…”

Enzyme-Linked Immunosorbent Assay for Hyodeoxycholic Acid in Pharmaceutical Products Using a Monoclonal Antibody

“…Hyodeoxycholic acid-carrier protein conjugates were synthesized by a mixed anhydride method as previously reported (Tanaka et al 2003) with some minor modifications. Briefly, isobutylchloroformate (36 microliters) and tri-n-butylamine (36 microliters) were added dropwise to a dimethylformamide solution (0.5 milliliter) of hyodeoxycholic acid (19.0 milligrams) and stirred at room temperature for two hours.…”