Development and Characterization of Monoclonal Antibodies Specific for 3-(1-naphthoyl) Indole Derivatives

Chemicals and instruments

Horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody, bovine serum albumin (BSA; agarose gel electrophoresis grade, art. no. A3675), keyhole limpet hemocyanin (KLH), complete Freund's adjuvants (CFA), incomplete Freund's adjuvants (IFA), and RPMI1640 were purchased from Sigma-Aldrich (St. Louis, MO). Fetal calf serum (FBS) was purchased from (Hyclone, Thermo Scientific, Cramlington, United Kingdom).

1-methyl-3-(1-naphthoyl) indole, 1-ethyl-3-(1-naphthoyl) indole, 1-octyl-3-(1-naphthoyl) indole, 1-methyl-3-(4-methyl-1-naphthoyl), 1-ethyl-3-(4-methyl-1-naphthoyl), and 1-octyl-3-(4-methyl-1-naphthoyl) indole were purchased from Cayman Chemicals (Ann Arbor, MI). Naphtoic acid, 4-methyl-naphtoic acid, indole, n-hydroxysuccinimide (NHS), N, N-dicyclohexylcarbodiimide (DCC), sodium bicarbonate, sodium dodecylsulfate (SDS), dichloromethane, oxalyl chloride, 2,4,6-trinitrobenzene sulfonic acid (TNBSA), hydrochloric acid, ethylmagnesium bromide, ether, ammonium chloride, methanol, sodium hydroxide, N,N-dimethylformamide, dimethyl sulfoxide, sulfuric acid, 3,3',5,5'- tetramethylbenzidine (TMB), sodium hydrate, 1-bromo-7-heptanic acid, and ethyl acetate were purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). Unless otherwise stated, all other inorganic chemicals and organic solvents were of analytical-reagent grade or better.

Synthesis of 1-(heptyl-7-carboxylate)-3-(1-naphtoyl) indole

2.95 g (23.2 mmol) oxalyl chloride was added drop-wise to a suspension of 1.00 g (5.81 mmol) 1-naphtoic acid in 30 mL dichloromethane at 0°C. The reaction mixture was warmed to 40°C and stirred for 4 h. After cooling, the solvent and excess oxalyl chloride were removed in vacuo to give a brown residue (0.80 g; 4.20 mmol), which was used in the next step without further purification.

0.64 g (5.46 mmol) of indole in 5.0 mL of diethyl ether was added drop-wise to a stirred solution of 3.17 mL (8.01 mmol) of 3 M ethyl diethyl ether magnesium bromide in ether, diluted with 3 mL of diethyl ether, at 0°C. The solution was stirred for 30 min at room temperature and a solution of 0.80 g 1-naphtoyl chloride in 5 mL of ether was added drop-wise. The reaction mixture was stirred for 3 h at 40°C, quenched with saturated aqueous ammonium chloride, and stirred until the solid was broken into a fine suspension. The residue was washed with water and ether, then suspended in 20 mL of ethanol, to which was added 4 g sodium hydroxide and 10 mL of water. The mixture was stirred at room temperature overnight, and the solid was filtered off and washed with successive portions of water and ether. After drying in vacuo at 100°C, the solid was recrystallized with ethanol and 0.8 g (70%) of 3-(1-naphtoyl) indole was obtained. 1H NMR (400 MHz, CDCl3) d 7.40 (s, H), 7.34 (m, 1H), 7.35 (m, 1H), 7.41 (m, 1H), 7.46 (m, 1H), 7.50 (m, 1H), 7.55 (dt, 1H), 7.69 (dd, 1H), 7.93 (d, 1H), 8.00 (d, 1H), 8.23 (d, 1H), 8.45 (m, 1H); 13C NMR (100 MHz, CDCl3) d 110.0, 117.8, 122.9, 122.9, 123.7, 124.5, 125.9, 126.0, 126.3, 126.7, 126.8, 128.1, 130.1, 130.8, 133.8, 137.4, 138.6, 138.9, 192.0.

0.5 g sodium hydrate was added to a solution of 0.8 g (2.93 mmol) of 3-(1-naphtoyl) indole in 10.0 mL of tetrahydoxyfuran. The reaction mixture was stirred at room temperature and 0.79 mL (3.80 mmol) of 1-bromo-7-heptanic acid was added slowly. The solution was stirred at 80°C for 3 h. After cooling, the reaction mixture was diluted with water and extracted with three portions of ethyl acetate. The extracts were dried, and the solvent was removed in vacuo. Recrystallization with ethanol at 4°C produced 0.82 g (70%) 1-(heptyl-7-carboxylate)-3-(1-naphtoyl) indole as a yellow solid. The synthetic route for the compound is shown in Figure 1: 1H NMR (400 MHz, CDCl3) d 1.44 (m, 2H), 1.52 (m, 2H), 1.68 (m, 2H), 1.89 (quint, 2H), 2.18 (m, 2H), 4.12 (t, 2H), 4.45 (m, 2H), 7.40 (s, H), 7.34 (m, 1H), 7.35 (m, 1H), 7.41 (m, 1H), 7.46 (m, 1H), 7.50 (m, 1H), 7.55 (dt, 1H), 7.69 (dd, 1H), 7.93 (d, 1H), 8.00 (d, 1H), 8.23 (d, 1H), 8.45 (m, 1H); 13C NMR (100 MHz, CDCl3) d 25.4, 26.2, 29.1, 28.8, 29.6, 34.4, 47.1, 110.0, 117.8, 122.9, 122.9, 123.7, 124.5, 125.9, 126.0, 126.3, 126.7, 126.8, 128.1, 130.1, 130.8, 133.8, 137.4, 138.6, 138.9, 192.0.

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FIG. 1.

Synthesis procedure for 3-(1-naphthoyl) indole derivative and artificial antigen.

Synthesis of immunogen (1-(heptyl-7-carboxylate)-3-(1-naphtoyl) indole-protein conjugate)

Antigen was prepared following the method described by Cervino and associates with some modifications.⁽¹¹⁾ Briefly, 1-(heptyl-7-carboxylate)-3-(1-naphtoyl) indole was suspended in dimethyl sulfoxide, and then N-hydroxysuccinimide was added and reacted for 1 h at room temperature. Dicyclohexylcarbodiimide was added to the solution and reacted for 1 h at room temperature. Then the mixture was stirred for 8 h at room temperature. After being centrifuged at 8000 r/min for 5 min, the supernatant was added drop-wise to KLH solution dissolved in PBS (50 mM, pH 7.4) and kept for 8 h at room temperature. After being centrifuged (3000 r/min, 60 min), the obtained supernatant was dialyzed with 0.1 M sodium bicarbonate buffer (pH 8.5) for 10 h. The dialyzed protein solution was diluted with supplied 5% TNBSA solution 500-fold in 0.1 M sodium bicarbonate buffer (pH 8.5). To the protein solution, TNBSA solution was added and then incubated at 37°C for 2 h. The mixture solutions of 0.5 mL of 10% SDS and 0.25 mL of 1 N hydrochloric acid were added to each sample to stop and stabilize the reaction. Then absorbance of the solution at 335 nm was measured, and the concentration of primary amines was determined by calculation from the extinction coefficient or by comparison to amino acid standards. The synthetic route for the immunogen is shown in Figure 1.

Cell line

The mouse myeloma cells (P3X63-Ag8.653) were cultured in RPMI1640 medium supplemented with 15% fetal calf serum in a 37°C humidified incubator.

Hybridoma preparation

Every 100 μL of the prepared adjuvant emulsion (a 1:1 emulsion of the immunogen [100 μg per mouse]) and complete Freund's adjuvant was injected intraperitoneally into female A/J mice (8 weeks of age). The mice received the same injections 2 weeks later. After another 2-week interval, the mice were injected intraperitoneally with 100 μL booster solution containing 100 μg immunogen for the third immunization. Immunity was assessed by non-competitive enzyme-linked immunosorbent assay (ncELISA).

The fusion of myeloma cells was carried out using standard methodology. The hybridoma cells of positive wells were cloned by limiting dilution method.

Monoclonal antibody preparation

The cloned hybridoma cells were injected into the A/J mice to produce ascites. The mice were sacrificed 2 weeks after the injection. Ascites were extracted and centrifuged at 5000 rpm for 30 min at room temperature. The supernatants were purified by affinity chromatography using a protein A sepharose CL-4B.

Non-competitive ELISA assays

Ninety-six-well polystyrene plates were incubated with 100 μg/well of 1-(heptyl-7-carboxylate)-3-(1-naphtoyl) indole-BSA conjugate in PBS (pH 7.4) overnight at room temperature. The plates were washed and the unbound sites were incubated with 1% BSA in PBS (pH 7.4). After washing three times with 200 μL PBS buffer (pH 7.4), 200 μL of 1.0% BSA in the PBS solution were added to each well and incubated for 1 h at room temperature. After another three washing steps, 100 μL/well MAbs with an appropriate dilution were added into each well of the plates. After 1 h incubation at room temperature, the plates were rewashed three times and the wells were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (0.2 μg/mL) for 45 min at room temperature. After washing three times, the color was developed by adding 100 μL/well of freshly prepared substrate solution (composed of 9.5 mL phosphate-citrate buffer (pH 5.0), 0.5 mL 2 mg/mL TMB (dissolved by ethanol), and 32 mL 3% (w/v) urea-hydrogen peroxide, and the mixture was incubated for 15 min at room temperature. Then, 25 μL/well of the stop solution (1 M sulfuric acid) was added to each well. Finally, the absorbance of each well was determined at 492 nm with a microplate reader.

Competitive ELISA

Competitive ELISA was carried out to determine antibody sensitivity of mouse sera, cell culture supernatants, and monoclonal antibodies purified from ascites. The procedure was identical to that of non-competitive ELISA, except 50 μL/well of MAb diluted in PBS and 50 μL/well of analytes dissolved in 10% DMSO-PBS were added after blocking. Sigmoidal curves were fitted to a logistic equation from which IC50 values (concentrations at which binding of the antibody to the coating antigen are inhibited by 50%) were determined.