Obtaining a Monoclonal Antibody against a Novel Prometryn-Like Hapten and Characterization of Its Selectivity for Triazine Herbicides

In this study, a previously unreported 3-((4-(isopropylamino)-6-(methylthio)-1,3,5-triazin-2-yl) amino) butyric acid hapten was designed and synthesized. This maximized the exposure of the antigen-determinant isopropyl of prometryn to the immune system in animals to induce the production of anticipated highly specific anti-prometryn antibodies. The hapten has a molecular weight of 285.37 Da. The compound was confirmed by nuclear magnetic resonance hydrogen spectroscopy (1H NMR), nuclear magnetic resonance carbon spectroscopy (13C NMR), and high-resolution mass spectrometry (HRMS). By using the active ester approach, immunogens and coated antigens were created. Bovine serum albumin (BSA) was used as an immunogen, along with the successfully produced hapten, to immunize mice. The IC50 value of mouse monoclonal anti-prometryn antibody (mAb) 7D4 (the quantity of analyte that generated 50% prometryn inhibition) was 3.9 ng/mL. The anti-prometryn mAb was of the IgG1 subclass. The IC20 (80% binding level (B/B0) of prometryn)-IC80 (20% binding level (B/B0) of prometryn) range of the anti-prometryn monoclonal antibody standard curve working range was 0.9–18.1 ng/mL. The prepared mAb has good characteristics because it can specifically recognize prometryn, and the cross-reaction rates for ametryn, desmetryn, and terbumeton were 34.77%, 18.09%, and 7.64%, respectively. The cross-reaction rate with the other seven triazines was less than 1%. The hapten structure proposed can serve as an additional tool for modulating selectivity in detecting triazines.


Introduction
Prometryn (2-methylthio-4,6-di(isopropylamino)-1,3,5-triazobenzene) is a triazine herbicide that has been widely or selectively used to control a variety of weeds in agricultural and nonagricultural fields worldwide [1]. It is stable in slightly acidic or alkaline ergometrine media and hydrolyzed in strong acid or base conditions [2]. Prometryn inhibits the interception of electrons transferred to oxidized coenzyme II (NADP + ) by photosynthetic electrons, thus achieving the goal of weeding [3]. It is widely and excessively used in the environment, with a half-life of approximately 13 months [4]. These residues accumulate in soil and water, posing a serious threat to biological diversity and human health [5]. For example, using prometryn in cotton fields often negatively impacts cotton provide additional effective core elements for the establishment of immunoassay methods for triazines.

Synthesis of Prometryn Hapten
The reaction of reaction solution 1 and reaction solution 2 was carried out in a 250 mL flask. 2,4,6-Trichloro-1,3,5-triazine (C 3 Cl 3 N 3, 7.37 g, 40 mmol) was dissolved in tetrahydrofuran (120 mL), and the solution was cooled to 0 • C as reaction solution 1. Isopropylamine (C 3 H 9 N, 2.36 g, 40 mmol) and N,N-diisopropylethylamine (DIPEA, 7.74 g, 60 mmol) were dissolved in tetrahydrofuran (50 mL) and used as reaction solution 2. Next, dropping solution 2 into solution 1 took approximately 15 min to complete. Then, the mixture was reacted at 0 • C for 3 h, and thin-layer chromatography (TLC) was used to detect whether the reaction was complete. After the reaction was completed, the mixture was spun dry, and ethyl acetate (100 mL) was added to the spin-dried beaker. The obtained mixture was washed twice with deionized water (100 mL) and once with saturated sodium chloride aqueous solution (100 mL). The mixture was dried with anhydrous sodium sulfate. Finally, it was dried to obtain a yellow oil mixture (SM1, 7.85 g) with a 94.8% yield. 4,6-Dichloro-N-isopropyl-1,3,5-triazin-2-amine (SM1, 10.8 g, 52.2 mmol) and sodium methyl mercaptan (3.6 g, 52 mmol) were dissolved in N,N-dimethylformamide (DMF, 100 mL) in a 100 mL flask. The reaction was carried out at room temperature (25 • C) for 16 h. TLC was used to monitor whether the SM1 reaction was completed. After the reaction, the reaction mixture was poured into water (600 mL). The reaction mixture was extracted twice with ethyl acetate (150 mL), and the organic phase was combined after extraction. The organic phase was washed three times with deionized water (150 mL) and once with saturated sodium chloride solution (100 mL). The organic phase solution obtained was dried with anhydrous sodium sulfate. The dried solution was spun dry to obtain 13.5 g of a yellow oil. The yellow oil was passed through a silica gel column (petroleum ether (PE): ethyl acetate (EtOAc) = 25:1) to obtain yellow oil (SM2, 9 g) with a yield of 79.6%. The purity of the obtained compound was determined to be 60% using liquid chromatography-mass spectrometry (LC-MS), which can be tested in the following experiment.
In a 250 mL flask, homemade 4-chloro-N-isopropyl-6-(methylthio)-1,3,5-triazin-2amine (SM2, 9 g, 40 mmol), 3-aminobutanoic acid (SM3, 4 g, 40 mmol) and DIPEA (10.4 g, 80 mmol) were dissolved in ethanol (120 mL). The reaction mixture was heated to 90 • C and carried out for 8 h. TLC was used to monitor that the reaction stopped when approximately half of the raw material remained. The reaction mixture was cooled to room temperature (25 • C) and spun dry. A saturated sodium bicarbonate aqueous solution (50 mL) was added to the spin-dried reactant, and then the mixed solution was stirred for 20 min. The mixture after the reaction was extracted twice with ethyl acetate (100 mL). After extraction, the aqueous solution of the reaction liquid was collected, and the pH of the aqueous phase was adjusted to 4 with 3 M HCl. After pH adjustment, the solution was extracted three times with 100 mL dichloromethane. The extracted mixture was dried with anhydrous sodium sulfate. The dried mixture was spun dry to obtain 6.6 g of a yellow oil. Ethanol (50 mL) was added to the dried yellow oil, and the mixture was heated and refluxed for 1 h. After the reaction, the mixture was cooled to room temperature (25 • C). The white precipitate in the mixture was filtered and dried to 2.35 g.

Immunogen and Coating Antigen Preparation
When small molecule haptens of pesticides combine with large molecules (usually proteins), they have immunogenicity. This study used the active ester method to prepare coating antigens and immunogens. BSA and OVA were combined with proton hapten to make coating and immunogen antigens. Figure 1b depicts the immunogen and coating antigen synthesis pathways. The molar ratio of hapten to a carrier protein (BSA) used in the preparation of the immunogen was 60:1. The molar ratio of hapten to a carrier protein (OVA) used in the preparation of the coating antigen was 40:1. A total of 7.78 mg (0.027 mmol) of hapten, 6.28 mg (0.054 mmol) of N-hydroxysuccinimide (NHS), and 10.46 mg (0.054 mmol) of carbodiimide hydrochloride (EDC) were fully dissolved in 1 mL of anhydrous N,Ndimethylformamide (DMF) and reacted with magnetic stirring at 4 • C overnight (10 h). After the reaction, a reactant containing prometryn hapten activation was obtained, and the reaction product can be directly used for subsequent carrier protein coupling. The supernatant of 0.333 mL of hapten activation solution was added to the carrier protein OVA solution (the solution was obtained by dissolving 10 mg of OVA in 1 mL of 0.01 M phosphate buffer saline solution (PBS, pH = 7.4). The supernatant of 0.664 mL of hapten activation solution was added to the carrier protein BSA solution (the solution was obtained by dissolving 20 mg of BSA in 2 mL of 0.01 M PBS (pH = 7.4). The coupling reaction was stirred at 25 • C for 4 h. The reaction solution was dialyzed at 4 • C with a PBS solution with a pH value of 7.4 and a concentration of 0.01 mol/L. The dialysate was replaced every 4 h 6 times. After dialysis, the solution obtained from the reaction was quickly frozen in liquid nitrogen and stored at −20 • C. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) was used to characterize the synthesized coating antigen and immunogen and calculate the coupling ratio. The coupling ratio was calculated as follows: where the antigen conjugates are denoted by "M conjugate ," the BSA/OVA standard by "M carrier ," and the hapten by "M hapten ."

Immunization
The Institute of Quality Standards and Testing Technology for Agro-Product perimental Animal Welfare and Ethical Committee (IQSTAP-2021-05) approved th of animals in experiments. The buffer solution (0.01 M PBS, pH = 7.4) was used to d the immunogen (2 mg/mL) to 1 mg/mL. After that, Freund's adjuvant was added identical volume of 0.1 mL in a sterile glass vial. The mixture was placed on a mag stirrer and stirred and mixed rapidly in an ice bath to fully emulsify. Six 7-week-o male BALB/c mice were selected for immunization. At the beginning of the thir munization, 3-5 days after each immunization, blood was taken from the orbit, bated at 37 °C for 0.5 h and 4 °C for 2 h, and centrifuged at 1000 r/min for 10 min. S was collected, and the serum titer was determined. The immune strategy of mice the same as that previously reported [18]. Freund's incomplete adjuvant was utilize the first vaccination, and for the subsequent booster vaccination, Freund's incom adjuvant was utilized. Freund's adjuvant and immunogen during immunization sh be 1:1 and thoroughly mixed until dripping into the water to prevent diffusion. T terval of booster immunization was 2 weeks, and the immune dose was 0.1 mg. days before fusion, the mice with the best performance were immunized with a do 0.05 mg. The mice were intraperitoneally injected without adjuvant. Table S1 dis the serum titer and inhibition rate of the mice with the highest performance. Tab displays the serum titer and inhibition rate of the selected fusion mice.

Ic-ELISA Procedure
The ic-ELISA method was used to screen the best-performing fusion mouse They were used to establish the standard curve of antibodies by detecting the sup tant of positive cells. The buffer required for ic-ELISA was the same as previous ported [18]. The specific steps of the operation are as follows: (1) The coated antigen added to the microplate by diluting it with the required coating solutions (CBS, pH 0.05 mol/L). Each well contained 100 µL, which was incubated at 37 °C for three h b

Production and Characteristics of Prometryn mAb 3.3.1. Immunization
The Institute of Quality Standards and Testing Technology for Agro-Products' Experimental Animal Welfare and Ethical Committee (IQSTAP-2021-05) approved the use of animals in experiments. The buffer solution (0.01 M PBS, pH = 7.4) was used to dilute the immunogen (2 mg/mL) to 1 mg/mL. After that, Freund's adjuvant was added to an identical volume of 0.1 mL in a sterile glass vial. The mixture was placed on a magnetic stirrer and stirred and mixed rapidly in an ice bath to fully emulsify. Six 7-week-old female BALB/c mice were selected for immunization. At the beginning of the third immunization, 3-5 days after each immunization, blood was taken from the orbit, incubated at 37 • C for 0.5 h and 4 • C for 2 h, and centrifuged at 1000 r/min for 10 min. Serum was collected, and the serum titer was determined. The immune strategy of mice was the same as that previously reported [18]. Freund's incomplete adjuvant was utilized for the first vaccination, and for the subsequent booster vaccination, Freund's incomplete adjuvant was utilized. Freund's adjuvant and immunogen during immunization should be 1:1 and thoroughly mixed until dripping into the water to prevent diffusion. The interval of booster immunization was 2 weeks, and the immune dose was 0.1 mg. Three days before fusion, the mice with the best performance were immunized with a dose of 0.05 mg. The mice were intraperitoneally injected without adjuvant. Table S1 displays the serum titer and inhibition rate of the mice with the highest performance. Table S2 displays the serum titer and inhibition rate of the selected fusion mice.

Ic-ELISA Procedure
The ic-ELISA method was used to screen the best-performing fusion mouse sera. They were used to establish the standard curve of antibodies by detecting the supernatant of positive cells. The buffer required for ic-ELISA was the same as previously reported [18]. The specific steps of the operation are as follows: (1) The coated antigen was added to the microplate by diluting it with the required coating solutions (CBS, pH 9.6, 0.05 mol/L). Each well contained 100 µL, which was incubated at 37 • C for three h before being washed three times with washing buffer (PBST, PBS containing 0.1% Tween-20). (2) With sample dilution buffer (PBSTG, PBST containing 0.1% gelatin), the standard prometryn sample and the antiserum/antibody from mice were diluted to the required concentration. The microplate was incubated for 30 min at 37 • C with 50 µL of diluted antiserum or antibody and standard samples of various concentrations. PBST was used three times to wash the dishes. (3) Then, 100 µL of PBSTG-diluent IgG HRP (labeled goat anti-mouse IgG with HRP) was added to each well. After 30 min at 37 • C, the plate was cleaned three times with PBST. (4) Each well was incubated for 15 min in the dark at 25 • C with 100 µL of TMB color developing solution. To stop the reaction, 50 µL of the hydrochloric acid solution was added to each well. The absorbance at OD450 nm was measured with a TECAN Infinite M200 PRO microplate reader from Männedorf, Switzerland.

Production of mAb
Ic-ELISA was used to measure the inhibition rate and serum titer of six immunized mice. Three days before cell fusion, Balb/c mice with the highest performance were selected and immunized. SP2/0 myeloma cells and mouse spleen cells were combined for fusion. After fusion, the supernatant of hybridoma cells was determined by indirect competitive enzyme-linked immunosorbent assay, the positive wells were screened, and the hybridoma cells were cloned to obtain hybridoma cell lines that can stably secrete monoclonal antibodies against prometryn. After the positive cell lines were expanded and cultured, mice treated with paraffin 7 days earlier were injected intraperitoneally. Ascites were collected after abdominal swelling and centrifuged at 4 • C at 1000 rpm, and the potency and inhibition rate of ascites were determined and frozen at −20 • C for purification. The 7D4 mAb against prometryn was obtained by purifying the culture medium with the octanoic acid-saturated ammonium sulfate method. The steps of antibody purification were as follows: (1) The pH was adjusted to 4.5 by diluting the antiserum or ascites (produced by hybridoma cell lines in the abdominal cavity of mice) with 60 mmol/pH 4 acetate buffer solution in a 1:4 ratio. Dropwise, 75 µL/mL serum was added, stirred for 30 min at room temperature, incubated for 1 h at 4 • C, and then centrifuged for 30 min at 10,000 rpm.
(2) Qualitative filter paper was used to filter the sediment, and 0.1 mol/L PBS was added to the supernatant. A 1:10 (PBS: pH was adjusted to 7.4 with 1 mol/L NaOH, the supernatant) dilution ratio was added, and the sample was precooled for 15 min at 4 • C. (3) (NH 4 ) 2 SO 4 was added to a mixed solution containing 0.277 g/mL, stirred for 30 min, chilled for 2 h at 4 • C, centrifuged for 20 min at 12,000 rpm, and then discarded. (4) The precipitate was dialyzed four times with 0.01 mol/L PBS at 4 • C, pH 7.4, and twice with 0.01 M PB (0.2 g KH 2 PO 4 , 2.96 g Na 2 HPO 4 • 12H 2 O, 1 L) after being dissolved in a small amount of 0.01 mol/L PBS. (5) Dry freeze and store at −20 • C until needed. (6) Safety Tips: If the time is short, it is best to extract immunoglobulin at 2-4 • C. This can also be done at room temperature, but all materials should be cooled before use. Ic-ELISA was used to measure the inhibition rate and titer of freeze-dried antibody powder. The procedure was identical to that of the antiserum. The obtained dried product was impure. High-performance liquid chromatography (HPLC) was used to purify the product, and 800 mg of oil was obtained in a 6% yield. Finally, the molecular weight and structure of the hapten were characterized by HRMS, 1 H NMR, and 13 C NMR. Hapten was confirmed by liquid chromatography, 13   The determination results of the chemical structural formula of prometryn hapten are shown in Figure 2d. The theoretically accurate molecular weight [M + + H] of C11H19N5O2S was 285.37, which was 286.1398. The results showed that the expected hapten structure of prometryn was synthesized. 1

Identification of the Prometryn Antigen and Immunogen
The molar ratio of prometryn used in the coating synthesis was 40:1, and the molar ratio of prometryn used in the immunogen synthesis was 60:1. The single charge ion peaks of the OVA standard and coated antigen detected by MALDI-TOF-MS were 44,586.160 and 46,144.036, respectively. The BSA standard and immune antigen had single charge ion peaks of 72,079.531 and 67,308.849, respectively. It was calculated from the coupling ratio formula of the carrier protein and hapten. The determined OVA standard and coating antigen, BSA standard, and immune antigen are shown by MALDI-TOF-MS in Figure 3. The determination results of the chemical structural formula of prometryn hapten are shown in Figure 2d.

Identification of the Prometryn Antigen and Immunogen
The molar ratio of prometryn used in the coating synthesis was 40:1, and the molar ratio of prometryn used in the immunogen synthesis was 60:1. The single charge ion peaks of the OVA standard and coated antigen detected by MALDI-TOF-MS were 44,586.160 and 46,144.036, respectively. The BSA standard and immune antigen had single charge ion peaks of 72,079.531 and 67,308.849, respectively. It was calculated from the coupling ratio formula of the carrier protein and hapten. The determined OVA standard and coating antigen, BSA standard, and immune antigen are shown by MALDI-TOF-MS in Figure 3.

Characterization of mAb
The ic-ELISA method was used to determine the ideal antibody and antigen m The coating antigen and antibody concentration used in the experiment was 1 mg/m The dilution multiples of the coating were 1 × 10 3 , 2 × 10 3 , 4 × 10 3 , and 8 × 10 3 . The pro etryn standard was 1000 ng/mL. The best concentration combination of antigen and tibody was selected to establish the standard curve of the antibody. The determinat results of the optimal combination are shown in Table S3. Prometryn standards w diluted to 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625, and 0 ng/mL, w PBSTG for subsequent ic-ELISA experiments. The standard curve of the prometryn m 7D4 is shown in Figure 4. The standard curve was determined by ic-ELISA. The IC50 w the concentration of analyte that generated 50% prometryn inhibition. Each measur OD value in the curve is the average of three independent repeats. The 7D4 mAb's in bition rate was well linearly correlated with the prometryn concentration. The IC50 of 7D4 mAb was 3.9 ng/mL, and the working range of the standard curve (IC20-IC80) w 0.9-18.1 ng/mL. The 7D4 mAb type was identified as IgG1 using a mouse antibody i typing kit from Sigma ® , St. Louis, MO, USA.

Characterization and Cross-Reactivity of the 7D 4 mAb 4.2.1. Characterization of mAb
The ic-ELISA method was used to determine the ideal antibody and antigen mix. The coating antigen and antibody concentration used in the experiment was 1 mg/mL. The dilution multiples of the coating were 1 × 10 3 , 2 × 10 3 , 4 × 10 3 , and 8 × 10 3 . The prometryn standard was 1000 ng/mL. The best concentration combination of antigen and antibody was selected to establish the standard curve of the antibody. The determination results of the optimal combination are shown in Table S3. Prometryn standards were diluted to 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625, and 0 ng/mL, with PBSTG for subsequent ic-ELISA experiments. The standard curve of the prometryn mAb 7D4 is shown in Figure 4. The standard curve was determined by ic-ELISA. The IC 50 was the concentration of analyte that generated 50% prometryn inhibition. Each measured OD value in the curve is the average of three independent repeats. The 7D4 mAb's inhibition rate was well linearly correlated with the prometryn concentration. The IC 50 of the 7D4 mAb was 3.9 ng/mL, and the working range of the standard curve (IC 20 -IC 80 ) was 0.9-18.1 ng/mL. The 7D4 mAb type was identified as IgG1 using a mouse antibody isotyping kit from Sigma ® , St. Louis, MO, USA.

Cross-Reactivities of the 7D4 mAb
Eleven triazine herbicides were selected to determine their cross-reactivity with 7D mAb. The determination and comparison results of the cross-reaction rate of 7D4 mA are shown in Table 1. The cross-reaction rate of mAb prepared by the hybridoma ce line 7D4 screened in this study with prometryn was 100%, which indicated that the pr pared 7D4 mAb could specifically recognize prometryn. The cross-reaction rates wit ametryn, desmetryn, and terbumeton were 34.77%, 18.09%, and 7.64%, respectively. Th cross-reaction rate with the other seven triazines was less than 1%. The hybridoma ce line 7D4 screened in this study can specifically recognize prometryn. However, this doe not mean that the antibodies prepared by the positive hybridoma cell strains screene after immunizing host animals with hapten proposed in this study can specifically re ognize prometryn. The cross-reaction rate may vary depending on the immunoassa format and the competitive hapten preparation used.

Cross-Reactivities of the 7D 4 mAb
Eleven triazine herbicides were selected to determine their cross-reactivity with 7D4 mAb. The determination and comparison results of the cross-reaction rate of 7D4 mAb are shown in Table 1. The cross-reaction rate of mAb prepared by the hybridoma cell line 7D4 screened in this study with prometryn was 100%, which indicated that the prepared 7D4 mAb could specifically recognize prometryn. The cross-reaction rates with ametryn, desmetryn, and terbumeton were 34.77%, 18.09%, and 7.64%, respectively. The crossreaction rate with the other seven triazines was less than 1%. The hybridoma cell line 7D4 screened in this study can specifically recognize prometryn. However, this does not mean that the antibodies prepared by the positive hybridoma cell strains screened after immunizing host animals with hapten proposed in this study can specifically recognize prometryn. The cross-reaction rate may vary depending on the immunoassay format and the competitive hapten preparation used.

Cross-Reactivities of the 7D4 mAb
Eleven triazine herbicides were selected to determine their cross-reactivity with 7D4 mAb. The determination and comparison results of the cross-reaction rate of 7D4 mAb are shown in Table 1. The cross-reaction rate of mAb prepared by the hybridoma cell line 7D4 screened in this study with prometryn was 100%, which indicated that the prepared 7D4 mAb could specifically recognize prometryn. The cross-reaction rates with ametryn, desmetryn, and terbumeton were 34.77%, 18.09%, and 7.64%, respectively. The cross-reaction rate with the other seven triazines was less than 1%. The hybridoma cell line 7D4 screened in this study can specifically recognize prometryn. However, this does not mean that the antibodies prepared by the positive hybridoma cell strains screened after immunizing host animals with hapten proposed in this study can specifically recognize prometryn. The cross-reaction rate may vary depending on the immunoassay format and the competitive hapten preparation used.

Cross-Reactivities of the 7D4 mAb
Eleven triazine herbicides were selected to determine their cross-reactivity with 7D4 mAb. The determination and comparison results of the cross-reaction rate of 7D4 mAb are shown in Table 1. The cross-reaction rate of mAb prepared by the hybridoma cell line 7D4 screened in this study with prometryn was 100%, which indicated that the prepared 7D4 mAb could specifically recognize prometryn. The cross-reaction rates with ametryn, desmetryn, and terbumeton were 34.77%, 18.09%, and 7.64%, respectively. The cross-reaction rate with the other seven triazines was less than 1%. The hybridoma cell line 7D4 screened in this study can specifically recognize prometryn. However, this does not mean that the antibodies prepared by the positive hybridoma cell strains screened after immunizing host animals with hapten proposed in this study can specifically recognize prometryn. The cross-reaction rate may vary depending on the immunoassay format and the competitive hapten preparation used.

Cross-Reactivities of the 7D4 mAb
Eleven triazine herbicides were selected to determine their cross-reactivity with 7D4 mAb. The determination and comparison results of the cross-reaction rate of 7D4 mAb are shown in Table 1. The cross-reaction rate of mAb prepared by the hybridoma cell line 7D4 screened in this study with prometryn was 100%, which indicated that the prepared 7D4 mAb could specifically recognize prometryn. The cross-reaction rates with ametryn, desmetryn, and terbumeton were 34.77%, 18.09%, and 7.64%, respectively. The cross-reaction rate with the other seven triazines was less than 1%. The hybridoma cell line 7D4 screened in this study can specifically recognize prometryn. However, this does not mean that the antibodies prepared by the positive hybridoma cell strains screened after immunizing host animals with hapten proposed in this study can specifically recognize prometryn. The cross-reaction rate may vary depending on the immunoassay format and the competitive hapten preparation used.

Cross-Reactivities of the 7D4 mAb
Eleven triazine herbicides were selected to determine their cross-reactivity with 7D4 mAb. The determination and comparison results of the cross-reaction rate of 7D4 mAb are shown in Table 1. The cross-reaction rate of mAb prepared by the hybridoma cell line 7D4 screened in this study with prometryn was 100%, which indicated that the prepared 7D4 mAb could specifically recognize prometryn. The cross-reaction rates with ametryn, desmetryn, and terbumeton were 34.77%, 18.09%, and 7.64%, respectively. The cross-reaction rate with the other seven triazines was less than 1%. The hybridoma cell line 7D4 screened in this study can specifically recognize prometryn. However, this does not mean that the antibodies prepared by the positive hybridoma cell strains screened after immunizing host animals with hapten proposed in this study can specifically recognize prometryn. The cross-reaction rate may vary depending on the immunoassay format and the competitive hapten preparation used.

Conclusions
This study proposed a new structure design and synthesis route, which provides an additional tool for adjusting the selectivity in the detection of triazines. It is maximally exposed to the isopropyl group of the prometryn epitope to produce specific monoclonal antibodies against prometryn. The molecular weight of the prometryn hapten is 285.37 Da. The mAb 7D4 for the specific detection of prometryn was obtained, and its IC50 value was 3.9 ng/mL. The antibody type of 7D4 mAb was determined to be IgG1. The working range of the standard curve (IC20-IC80) was 0.9-18.1 ng/mL. The 7D4 mAb can be used as the core material for establishing prometryn immunoassays. The cross-reactivity values are not invariable parameters of a given antibody and may vary depending on the immunoassay format and the competing hapten preparation used, which provides additional tools for modulating selectivity in the detection of triazines.
This study once again confirmed that the isopropyl group of the structural formula of triazine pesticides is also a crucial antigenic determinant. We previously verified the hapten design of atrazine and concluded that the isopropyl group of triazine pesticides could be the epitope [18]. The hapten structure of triazine pesticides previously reported is shown in Table 2. Compared with previous reports on the hapten structure of prometryn [18][19][20][21][22][23][24], the current hapten design has better performance in cross-reaction rate and specificity with other triazines. Although the hapten designed and synthesized in this study has only one connecting arm (-CH2), it can still stimulate host animals to produce monoclonal antibodies against prometryn with high specificity. This study and the previous hapten design and synthesis of atrazine [18] have confirmed that the isopropyl group of triazine pesticides is also a critical antigenic determinant. Even when there is

Conclusions
This study proposed a new structure design and synthesis route, which provides an additional tool for adjusting the selectivity in the detection of triazines. It is maximally exposed to the isopropyl group of the prometryn epitope to produce specific monoclonal antibodies against prometryn. The molecular weight of the prometryn hapten is 285.37 Da. The mAb 7D4 for the specific detection of prometryn was obtained, and its IC50 value was 3.9 ng/mL. The antibody type of 7D4 mAb was determined to be IgG1. The working range of the standard curve (IC20-IC80) was 0.9-18.1 ng/mL. The 7D4 mAb can be used as the core material for establishing prometryn immunoassays. The cross-reactivity values are not invariable parameters of a given antibody and may vary depending on the immunoassay format and the competing hapten preparation used, which provides additional tools for modulating selectivity in the detection of triazines.
This study once again confirmed that the isopropyl group of the structural formula of triazine pesticides is also a crucial antigenic determinant. We previously verified the hapten design of atrazine and concluded that the isopropyl group of triazine pesticides could be the epitope [18]. The hapten structure of triazine pesticides previously reported is shown in Table 2. Compared with previous reports on the hapten structure of prometryn [18][19][20][21][22][23][24], the current hapten design has better performance in cross-reaction rate and specificity with other triazines. Although the hapten designed and synthesized in this study has only one connecting arm (-CH2), it can still stimulate host animals to produce monoclonal antibodies against prometryn with high specificity. This study and the previous hapten design and synthesis of atrazine [18] have confirmed that the isopropyl group of triazine pesticides is also a critical antigenic determinant. Even when there is

Conclusions
This study proposed a new structure design and synthesis route, which provides an additional tool for adjusting the selectivity in the detection of triazines. It is maximally exposed to the isopropyl group of the prometryn epitope to produce specific monoclonal antibodies against prometryn. The molecular weight of the prometryn hapten is 285.37 Da. The mAb 7D4 for the specific detection of prometryn was obtained, and its IC50 value was 3.9 ng/mL. The antibody type of 7D4 mAb was determined to be IgG1. The working range of the standard curve (IC20-IC80) was 0.9-18.1 ng/mL. The 7D4 mAb can be used as the core material for establishing prometryn immunoassays. The cross-reactivity values are not invariable parameters of a given antibody and may vary depending on the immunoassay format and the competing hapten preparation used, which provides additional tools for modulating selectivity in the detection of triazines.
This study once again confirmed that the isopropyl group of the structural formula of triazine pesticides is also a crucial antigenic determinant. We previously verified the hapten design of atrazine and concluded that the isopropyl group of triazine pesticides could be the epitope [18]. The hapten structure of triazine pesticides previously reported is shown in Table 2. Compared with previous reports on the hapten structure of prometryn [18][19][20][21][22][23][24], the current hapten design has better performance in cross-reaction rate and specificity with other triazines. Although the hapten designed and synthesized in this study has only one connecting arm (-CH2), it can still stimulate host animals to produce monoclonal antibodies against prometryn with high specificity. This study and the previous hapten design and synthesis of atrazine [18] have confirmed that the isopropyl group of triazine pesticides is also a critical antigenic determinant. Even when there is

Conclusions
This study proposed a new structure design and synthesis route, which provides an additional tool for adjusting the selectivity in the detection of triazines. It is maximally exposed to the isopropyl group of the prometryn epitope to produce specific monoclonal antibodies against prometryn. The molecular weight of the prometryn hapten is 285.37 Da. The mAb 7D4 for the specific detection of prometryn was obtained, and its IC50 value was 3.9 ng/mL. The antibody type of 7D4 mAb was determined to be IgG1. The working range of the standard curve (IC20-IC80) was 0.9-18.1 ng/mL. The 7D4 mAb can be used as the core material for establishing prometryn immunoassays. The cross-reactivity values are not invariable parameters of a given antibody and may vary depending on the immunoassay format and the competing hapten preparation used, which provides additional tools for modulating selectivity in the detection of triazines.
This study once again confirmed that the isopropyl group of the structural formula of triazine pesticides is also a crucial antigenic determinant. We previously verified the hapten design of atrazine and concluded that the isopropyl group of triazine pesticides could be the epitope [18]. The hapten structure of triazine pesticides previously reported is shown in Table 2. Compared with previous reports on the hapten structure of prometryn [18][19][20][21][22][23][24], the current hapten design has better performance in cross-reaction rate and specificity with other triazines. Although the hapten designed and synthesized in this study has only one connecting arm (-CH2), it can still stimulate host animals to produce monoclonal antibodies against prometryn with high specificity. This study and the previous hapten design and synthesis of atrazine [18] have confirmed that the isopropyl group of triazine pesticides is also a critical antigenic determinant. Even when there is

Conclusions
This study proposed a new structure design and synthesis route, which provides an additional tool for adjusting the selectivity in the detection of triazines. It is maximally exposed to the isopropyl group of the prometryn epitope to produce specific monoclonal antibodies against prometryn. The molecular weight of the prometryn hapten is 285.37 Da. The mAb 7D4 for the specific detection of prometryn was obtained, and its IC50 value was 3.9 ng/mL. The antibody type of 7D4 mAb was determined to be IgG1. The working range of the standard curve (IC20-IC80) was 0.9-18.1 ng/mL. The 7D4 mAb can be used as the core material for establishing prometryn immunoassays. The cross-reactivity values are not invariable parameters of a given antibody and may vary depending on the immunoassay format and the competing hapten preparation used, which provides additional tools for modulating selectivity in the detection of triazines.
This study once again confirmed that the isopropyl group of the structural formula of triazine pesticides is also a crucial antigenic determinant. We previously verified the hapten design of atrazine and concluded that the isopropyl group of triazine pesticides could be the epitope [18]. The hapten structure of triazine pesticides previously reported is shown in Table 2. Compared with previous reports on the hapten structure of prometryn [18][19][20][21][22][23][24], the current hapten design has better performance in cross-reaction rate and specificity with other triazines. Although the hapten designed and synthesized in this study has only one connecting arm (-CH2), it can still stimulate host animals to produce monoclonal antibodies against prometryn with high specificity. This study and the previous hapten design and synthesis of atrazine [18] have confirmed that the isopropyl group of triazine pesticides is also a critical antigenic determinant. Even when there is

Conclusions
This study proposed a new structure design and synthesis route, which provides an additional tool for adjusting the selectivity in the detection of triazines. It is maximally exposed to the isopropyl group of the prometryn epitope to produce specific monoclonal antibodies against prometryn. The molecular weight of the prometryn hapten is 285.37 Da. The mAb 7D4 for the specific detection of prometryn was obtained, and its IC50 value was 3.9 ng/mL. The antibody type of 7D4 mAb was determined to be IgG1. The working range of the standard curve (IC20-IC80) was 0.9-18.1 ng/mL. The 7D4 mAb can be used as the core material for establishing prometryn immunoassays. The cross-reactivity values are not invariable parameters of a given antibody and may vary depending on the immunoassay format and the competing hapten preparation used, which provides additional tools for modulating selectivity in the detection of triazines.
This study once again confirmed that the isopropyl group of the structural formula of triazine pesticides is also a crucial antigenic determinant. We previously verified the hapten design of atrazine and concluded that the isopropyl group of triazine pesticides could be the epitope [18]. The hapten structure of triazine pesticides previously reported is shown in Table 2. Compared with previous reports on the hapten structure of prometryn [18][19][20][21][22][23][24], the current hapten design has better performance in cross-reaction rate and specificity with other triazines. Although the hapten designed and synthesized in this study has only one connecting arm (-CH2), it can still stimulate host animals to produce monoclonal antibodies against prometryn with high specificity. This study and the previous hapten design and synthesis of atrazine [18] have confirmed that the isopropyl group of triazine pesticides is also a critical antigenic determinant. Even when there is

Conclusions
This study proposed a new structure design and synthesis route, which provides an additional tool for adjusting the selectivity in the detection of triazines. It is maximally exposed to the isopropyl group of the prometryn epitope to produce specific monoclonal antibodies against prometryn. The molecular weight of the prometryn hapten is 285.37 Da. The mAb 7D4 for the specific detection of prometryn was obtained, and its IC50 value was 3.9 ng/mL. The antibody type of 7D4 mAb was determined to be IgG1. The working range of the standard curve (IC20-IC80) was 0.9-18.1 ng/mL. The 7D4 mAb can be used as the core material for establishing prometryn immunoassays. The cross-reactivity values are not invariable parameters of a given antibody and may vary depending on the immunoassay format and the competing hapten preparation used, which provides additional tools for modulating selectivity in the detection of triazines.
This study once again confirmed that the isopropyl group of the structural formula of triazine pesticides is also a crucial antigenic determinant. We previously verified the hapten design of atrazine and concluded that the isopropyl group of triazine pesticides could be the epitope [18]. The hapten structure of triazine pesticides previously reported is shown in Table 2. Compared with previous reports on the hapten structure of prometryn [18][19][20][21][22][23][24], the current hapten design has better performance in cross-reaction rate and specificity with other triazines. Although the hapten designed and synthesized in this study has only one connecting arm (-CH2), it can still stimulate host animals to produce monoclonal antibodies against prometryn with high specificity. This study and the previous hapten design and synthesis of atrazine [18] have confirmed that the isopropyl group of triazine pesticides is also a critical antigenic determinant. Even when there is

Conclusions
This study proposed a new structure design and synthesis route, which provides an additional tool for adjusting the selectivity in the detection of triazines. It is maximally exposed to the isopropyl group of the prometryn epitope to produce specific monoclonal antibodies against prometryn. The molecular weight of the prometryn hapten is 285.37 Da. The mAb 7D 4 for the specific detection of prometryn was obtained, and its IC 50 value was 3.9 ng/mL. The antibody type of 7D4 mAb was determined to be IgG1. The working range of the standard curve (IC 20 -IC 80 ) was 0.9-18.1 ng/mL. The 7D4 mAb can be used as the core material for establishing prometryn immunoassays. The cross-reactivity values are not invariable parameters of a given antibody and may vary depending on the immunoassay format and the competing hapten preparation used, which provides additional tools for modulating selectivity in the detection of triazines.

This work
Supplementary Materials: The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Table S1: Serum potency and inhibition rate in mice; Table S2: Serum titer and inhibition rate of mice for the fusion experiment; Table S3: Optimal ic-ELISA combination of coated antigen and antibody.  Table S1: Serum potency and inhibition rate in mice; Table S2: Serum titer and inhibition rate of mice for the fusion experiment; Table S3: Optimal ic-ELISA combination of coated antigen and antibody.