Characterization and Selection of 3-(1-Naphthoyl)-Indole Derivative-Specific Alpaca VHH Antibodies Using a Phage Display Library

Synthesis of 7-(3-(1-naphthoyl)-1H-indol-1-yl)-heptanoic acid and immunogen

Sodium hydrate, 0.5 g, was added to a solution of 0.8 g (2.93 mmol) of 3-(1-naphthoyl)-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-heptanoic 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 ethylacetate. The extracts were dried and the solvent was removed in vacuo. The solid was recrystallized with ethanol, and 0.82 g (70%) of 7-(3-(1-naphthoyl)-1H-indol-1-yl)-heptanoic acid was obtained as yellow solid: 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.

7-(3-(1-Naphthoyl)-1H-indol-1-yl)-heptanoic acid-KLH (Hep-KLH) protein conjugates as an immunogen and 7-(3-(1-naphthoyl)-1H-indol-1-yl)-heptanoic acid-BSA (Hep-BSA) as an immobilized antigen were synthesized following the method described by Chimalakonda et al.⁽¹⁵⁾

Animal immunization and acquisition of mononuclear cells

An alpaca was immunized with Hep-KLH. Specifically, the Hep-KLH having a concentration of 100 μg/mL was administered to the alpaca. After 1 week, the Hep-KLH having the same concentration was administered to the alpaca again. In this way, the alpaca was immunized with the Hep-KLH five times for 5 weeks. After another week, blood of the alpaca was extracted. Then, mononuclear cells were acquired from the blood as mentioned hereunder.

In brief, a blood cell separation solution was added to a lymphocyte separation tube. Then, the lymphocyte separation tube was centrifuged at 20,000 g for 10 min. The fraction containing the mononuclear cells was collected.

Phage library construction

Phage library construction was performed as described by Kim et al. with slight modifications.⁽⁹⁾ In brief, total RNA was extracted from the collected mononuclear cells using RNAiso plus. First, strand cDNA was synthesized with Prime Script II using oligo dT primers according to the manufacturer's protocols. The VHH-specific primers for polymerase chain reaction (PCR) amplification were used with the same primers as described previously.⁽⁹⁾ The VHH repertoire was amplified with two pairs of gene-specific primers in two consecutives reactions of PCR (recognition site; SfiI). The obtained PCR products were purified, digested with SfiI, and inserted into different SfiI sites of the phagemid vector in frame with the M13 gene III for expression of VHH-gIIIp fusion protein. Ligated samples were transformed into electrocompetent Escherichia coli HST02 to obtain the VHH primary library. After construction, the library size was determined by plating the transformation product on 2YT/ampicillin/glucose agar plates. Recombinant phages expressing VHH- g3p fusion protein were produced after the infection of VHH primary library with helper phage M13KO7. E. coli HST02 cells grown in 2YT/ampicillin/glucose medium at 30°C to log phage were infected with helper phage M13KO7 without shaking at 30°C for 1 h. Afterward, the bacterial culture was centrifuged at 1200 g for 15 min, the medium was replaced by 2YT containing 100 mg/mL ampicillin and 25 mg/mL kanamycin, and the culture was incubated overnight with shaking at 30°C. Subsequently, the solution was centrifuged and the supernatant was precipitated with PEG 6000 (20% polyethylene glycol 6000 in 2.5 M NaCl in water) at 4°C for 1 h. The pellet was resuspended in 1 mL PBS and used for biopanning.

Selection of specific phages that bound to 3-(1-naphthoyl)-indole derivatives

Immunotube was coated with 2 mL of Hep-BSA (20 μg/mL) dissolved in PBS at 4°C overnight. The tube was washed with PBS, blocked with 3% skim milk in PBS at room temperature for 1 hour, and then washed three times with PBS containing 0.1% Tween-20 (PBS-T). Phage solution (2 mL), containing 1 × 10¹¹ colony-forming units of phages suspended in PBS supplemented with 3% skim milk, was added to the tube and incubated at room temperature for 1 hour. The tube was washed five times with PBS-T, and bound phages were eluted in 1 mL of 100 mM triethylamine for 10 minutes, and then the eluate was neutralized with 1 M Tris-HCl buffer (pH 6.8). Eluted phages were then infected with fresh E. coli TG-1 cells and used for a second round of biopanning. The second biopanning was carried out with the same method as the first biopanning. The titer of the eluted phage was increased in the second round of panning (not shown).

Bacterial expression of VHH antibodies

VHH genes from phagemid vector were subcloned into pET22b(+) vector and transformed into E. coli BL21(DE3)pLysS. The transformed E. coli were cultured in 2TY medium containing 100 μg/mL ampicillin and 1 mM isopropyl-β-d-thiogalactopyranoside at 30°C overnight without shaking. After centrifugation (20,000 g, 10 min), the pellet was suspended in cold PBS and sonicated for 5 minutes using a SONIFIER 450 (Branson). After centrifugation (20,000 g, 10 min), the supernatant was collected.

Purification of VHH antibodies

The obtained supernatant was purified with His-trap column in accordance with the manufacturer's protocol. An elution solution was substituted with PBS, using PD-10 column. In this way, a solution containing the anti-3-(1-naphthoyl)-indole derivatives VHH antibody was obtained. The concentration of the obtained VHH antibody solution was quantified on the basis of the absorption measurement value at a wavelength of 280 nm. As a result, the concentration of the anti-3-(1-naphthoyl)-indole derivative VHH antibodies, NN01 and NN02, was 8 mg/mL and 10 mg/mL, respectively. The purified VHH antibodies in this study were much greater than conventional antibodies using the E. coli expression system.

Noncompetitive ELISAs

ELISA plates were coated with 100 μL/well of Hep-BSA (20 μg/mL) dissolved in PBS (pH 7.4) at 4°C overnight. 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 was added to each well and incubated for 1 hour at room temperature. After another three washing steps, 100 μL/well VHH antibodies solution with an appropriate dilution was added into each well of the plates. After 1 hour incubation at room temperature, the plates were rewashed three times, and the wells were incubated with HRP-conjugated anti-Histag antibody (0.2 μg/mL) for 45 min at room temperature. After washing three additional times, the color was developed by adding 100 μL/well freshly prepared substrate solution (composed of 9.5 mL pH 5.0 phosphate citrate buffer, 0.5 mL of 2 mg/mL TMB, and 32 mL of 3% (w/v) urea hydrogen peroxide), and the mixture was incubated for 15 minutes 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 (Tecan Group Ltd.).

Competitive ELISA

Competitive ELISA was carried out to determine the binding specificity of the obtained two VHH antibodies. The procedure was identical to that of noncompetitive ELISA except that 50 μL/well of VHH antibodies (NN01, NN02) diluted in PBS and 50 μL/well of an inhibitor, containing 3-(1-naphthoyl)-indole and its derivatives (Hep, 1-methyl-3-(1-naphthoyl)-indole, 1-ethyl-3-(1-naphthoyl)-indole, 1-propyl-3-(1-naphthoyl)-indole, and 1-heptyl-3-(1-naphthoyl)-indole) or raw materials (naphthoic acid and indole) dissolved in 10% DMSO–PBS were added after blocking. As a result, sigmoidal curves were fitted to a logistic equation from which IC₅₀ values (concentrations at which binding of the antibody to the coating antigen is inhibited by 50%) were determined.

Alpaca VHH phage display library

We constructed a VHH phage display library using mononuclear cells from alpaca immunized with Hep-KLH. The genes encoding the VHH antibodies were amplified using the same primers as described by Kim et al.⁽⁹⁾

Analysis and isolation of 3-(1-naphthoyl)-indole derivative-specific VHH phage clones

Phages from the library were cloned and screened for binding capacity using p- ELISA. Two clones from the VHH library showed specific binding to Hep-BSA. For the two clones, VHH antibodies were expressed in the periplasmic fractions of E. coli. The purity of purified VHH antibodies was verified by SDS-PAGE (not shown). Prominent bands were observed at ∼14 kDa, corresponding to the molecular weight calculated from amino acid sequences (Fig. 1). The amino acid sequences of the two VHH antibodies were different only in the framework region Nos. 3 and 4.

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FIG. 1.

Amino acid sequences of two anti-3-NI VHH antibodies (NN01 and NN02). Dots indicate unchanged amino acids. Bars indicate deletions.

In contrast, the IC₅₀ values of the two purified VHH antibodies were measured against 3-(1-naphthoyl)-indole, its derivatives, and raw material using c-ELISA. Results of the IC₅₀ of the two VHH antibodies (NN01 and NN02) for 3-(1-naphthoyl)-indole, its derivatives, and raw material are shown in Table 1. The results show that both of these antibodies recognize 3-naphthoylindole derivatives. However, neither of these antibodies recognize 3-(1-naphthoyl)-indole, naphthoic acid, and indole. The sensitivity of the NN01 VHH antibody was shown to be higher than that of NN02. About 300 nmol/L of Hep was detected using the obtained NN01 VHH antibody. In addition, about 2 μmol/L of 3-naphthoylindole derivatives such as 1-methyl-3-(1-naphthoyl)-indole, 1-ethyl-3-(1-naphthoyl)-indole, 1-propyl-3-(1-naphthoyl)-indole, and 1-heptyl-3-(1-naphthoyl)-indole was detected using the obtained NN01 VHH antibody.

The two VHH antibodies that were produced in this study will certainly be bound to JWH-018 and JWH-073, because the 3-(1-naphthoyl)-indole derivative used in this study and JWH-018 (or JWH-073) are different only from each other in length of alkyl chain.

In addition, these VHH antibodies (8–10 mg/500 mL culture volume) were obtained in much greater amounts than conventional antibodies (<1 mg/500 mL culture volume) using a culture of E. coli. This shows that it is easy to obtain a great amount of VHH antibodies in this manner.

In future studies, the two VHH antibodies obtained in this study will be easily used as an immunosensor for illegal drugs and evaluated on real samples.