Enzyme-Linked Immunosorbent Assay for Glycyrrhizin Using Anti

mouse immunized against a glycyrrhizin-bovine serum albumin conjugate with the hypoxanthine-aminopterin- thymidine-sensitive mouse myeloma cell line, ...
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Anal. Chem. 2001, 73, 5784-5790

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Enzyme-Linked Immunosorbent Assay for Glycyrrhizin Using Anti-Glycyrrhizin Monoclonal Antibody and an Eastern Blotting Technique for Glucuronides of Glycyrrhetic Acid Shaojie Shan, Hiroyuki Tanaka, and Yukihiro Shoyama*

Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higahshi-ku, Fukuoka 812-8582, Japan

Hybridomas secreting a monoclonal antibody against glycyrrhizin were produced by fusing splenocytes from a mouse immunized against a glycyrrhizin-bovine serum albumin conjugate with the hypoxanthine-aminopterinthymidine-sensitive mouse myeloma cell line, P3-X63Ag8-653. A very weak cross-reaction with glycyrrhetinic acid monoglucuronide and glycyrrhetic acid occurred. An enzyme-linked immunosorbent assay (ELISA) that had an effective measuring range of 20 -200 ng/mL of glycyrrhizin was established using this monoclonal antibody. In addition, a method named eastern blotting for the detection of glycyrrhizin was investigated. In this method, we developed a new way to separate the glycyrrhizin molecule into two functional parts using a simple and well-known chemical reaction. The sugar parts were oxidized by sodium periodate to give dialdehydes, which reacted with amino groups on the protein and covalently bound to the adsorbent membrane. The monoclonal antibody bound to the aglycone part of the glycyrrhizin molecule for immunostaining. This method was validated by immunocytolocalization of glycyrrhizin in fresh Glycyrrhiza root.

also been reported to be a promising drug for the treatment of AIDS or for protecting asymptomatic HIV carriers from AIDS.7-10 With the rapid development of the molecular biosciences and their biotechnological applications, immunoassays using monoclonal antibodies (MAbs) against drugs and low-molecular-weight bioactive compounds have become an important tool, because of their specificity, for receptor binding analyses, enzyme assays, and quantitative and qualitative analytical techniques in both animals and plants. Although immunological approaches for assaying glycyrrhetic acid (GA) using an anti-glycyrrhetic acid MAb have been investigated by Mizugaki et al.,11 no immunoassays for GC using MAbs have been reported. For a long time, we have been involved in studying the formation of MAbs against naturally occurring bioactive compounds and developing their applications. Recently, we published a rapid communication regarding the formation of MAbs against GC.12 In this paper, we report in detail on the preparation of the anti-GC MAbs, and their application in the development of an ELISA and an eastern blotting method for quantifying and identifying GC and its structurally related compounds. On the basis of the eastern blotting method, a new immunocytolocalization of GC in fresh Glycyrrhiza root was established.

Licorice (Glycyrrhiza spp.) is one of the most important drugs used in traditional Chinese medicine and is prescribed with other herbal medicines as an antitussive, an expectorant, a tastemodifying agent, and for relieving pain.1 These pharmaceutical properties are due to glycyrrhizin (GC), which has protein kinase inhibitory activity,2 anti-ulcer3 and anti-viral4 activities, and is now used in Japan as an antiallergenic5 and for liver protection.6 It has

EXPERIMENTAL SECTION Chemicals and Immunochemicals. GC, GA, and glycyrrhetic acid 3-O-β-glucuronide (GG) were purchased from Wako Pure Chemical Ind., Ltd. (Osaka, Japan). BSA and HSA were provided

* To whom correspondence should be addressed. Phone: 81-92-642-6580. Fax: 81-92-642-6580. E-mail: [email protected]. (1) The Japanese Pharmacopoeia, 14th ed.; Jiho: Totyo, 2000; pp 839-841. (2) Ohtsuki, K.; Ishida, N. Biochem. Biophys. Res. Comm. 1988, 157, 597604. (3) Doll, R.; Hill, I. D.; Hutton, C.; Underwood, D. L. Lancet 1962, 2, 793-796. (4) Pompei, R.; Flore, O.; Marccialis, M. A.; Pani, A.; Loddo, B. Nature 1979, 281, 689-690. (5) Kuroyanagi, T.; Saito, M. Jpn. J. Allergol. 1966, 15, 67-74. (6) Fujita, H.; Sakurai, T.; Toyoshima, S. Pharmacometrics 1978, 16, 637-645.

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(7) Ito, M.; Nakashima, H.; Baba, M.; Pauwels, R.; Clercq, E. D.; Shigeta, S.; Yamamoto, N. Antiviral Res. 1987, 7, 127-137. (8) Mori, K.; Sakai, H.; Suzuki, S.; Akutsu, Y.; Ishikawa, M.; Aihara, M.; Yokoyama, M.; Sato, Y.; Okaniwa, S.; Endo, Y.; Sasaki, H.; Saito, S.; Hayashi, T.; Niikawa, K.; Suzuki, S.; Uchida, T.; Hiwatashi, K. Jpn. J. Clin. Pathol. 1989, 37, 1200-1208. (9) Endo, Y.; Mamiya, S.; Iwamoto, K.; Niitsu, H.; Ito, T.; Miura, A. Jpn. J. Clin. Pathol. 1990, 38, 188-192. (10) Mori, K.; Sakai, H.; Suzuki, S.; Akutsu, Y.; Ishikawa, M.; Tada, K.; Aihara, M.; Sawada, Y.; Yokoyama, M.; Sato, Y.; Endo, Y.; Suzuki, Z.; Sato, S.; Sasaki, H.; Yokoyama, S.; Hayashi, T.; Uchida, T.; Hiwatashi, K.; Ishida, N.; Fujimaki, M.; Yamada, K. J. Naturopathic Med. 1993, 4, 2. (11) Mizugaki, M.; Itoh, K.; Hayasaka, M.; Ishikawa, S.; Nozaki, S.; Nagata, N.; Hanadate, K.; Ishida, N. J. Immunoassay 1994, 15, 21-34. (12) Tanaka, H.; Shoyama, Y. Biol. Pharm. Bull. 1998, 21, 1391-1393. 10.1021/ac0106997 CCC: $20.00

© 2001 American Chemical Society Published on Web 11/13/2001

by Pierce (Rockford, IL). Peroxidase-labeled anti-mouse IgG was provided by Organon Teknika Cappel Products (West Chester, PA). Poly(vinylidene difluoride) (PVDF) membranes (ImmobilonN) were purchased from Millipore Corporation (Bedford, MA). Glass microfiber filter sheets (GF/A) were purchased from Whatman International Ltd. (Maidstone, England). All other chemicals were standard commercial products of analytical grade. Synthesis of Antigen Conjugates. GC-carrier protein conjugates were synthesized by a modification of the procedure used for solamargine13 that was based on the method of Erlanger and Beiser.14 A methanol solution (0.27 mL) of GC (5.2 mg) was added dropwise to a solution (0.4 mL) containing NaIO4 (5 mg) and stirred at room temperature for 1 h. Carbonate buffer (50 mM, pH 9.6, 1.4 mL) containing BSA (5 mg) was added to the above reaction mixture. The whole reaction mixture was adjusted to pH 9 with 1 M Na2CO3 solution and stirred at room temperature for 6 h. The reaction mixture was dialyzed against five changes of H2O for 3 days, and then lyophilized to produce GC conjugate (GC-BSA) (4.7 mg). GC-HSA conjugate was also synthesized by the same method. The hapten number in the GC-BSA conjugate was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) as previously described.15-17 Briefly, a small amount (1-10 pmol) of antigen conjugate was mixed with a 1000-fold molar excess of saturated sinapinic acid aqueous solution containing about 30% acetonitrile in 0.15% trifluoroacetic acid. Typically, 0.5-1.0 µL of the mixture was deposited on the gold-coated sample plate and air-dried. The mass spectra were acquired using a delayed extraction MALDI-TOF mass spectrometer (model Voyager Elite, PerSeptive Biosystems Inc., Framingham, MA) operated in the linear mode. The instrument was generally operated at an accelerating voltage of 20 kV and a grid voltage of 90.000% of the accelerating voltage. Even though the acquisition methods allow a delay to be specified for the extraction pulse (e.g., 300 ns), because of an inherent delay of 180 ns that exists between the nitrogen laser pulse (337-nm wavelength, 3-ns-wide pulses) and the extraction pulse, the actual time delay is the sum of the delay time entered into the experimental method and the inherent delay (i.e., 300 ns + 180 ns ) 480 ns, in this case). Flight times were measured using a 500 MHz transient digitizer board in the computer, and the data were analyzed using GRAMS/386 software (Galactic Industries Corp., Salem, NH). Immunization, Hybridization, and Purification of MAb. BALB/c female mice were immunized with the GC-BSA conjugate, and MAbs were generated using previously described protocols18 with some modifications. Immunizations were initiated by an intraperitoneal injection of 50 µg of conjugate dissolved in phosphate-buffered saline (0.15 M NaCl in 10mM potassium phosphate, pH 7.4; PBS) and emulsified in an equal volume of (13) Ishiyama, M.; Shoyama, Y.; Murakami, H.; Shinohara, H, Cytotechnology 1996, 18, 153-158. (14) Erlanger, B. F.; Beiser, S. M. Proc. Natl. Acad. Sci. U.S.A. 1964, 52, 6874. (15) Shoyama, Y.; Sakata, R.; Isobe, R.; Murakami, H. Org. Mass Spectrosc. 1993, 28, 987-988. (16) Shoyama, Y.; Fukada, T.; Tanaka, T.; Kusai, A.; Nojima, K. Biol. Pharm. Bull. 1993, 16, 1051-1053. (17) Goto, Y.; Shima, Y.; Morimoto, S.; Shoyama, Y.; Murakami, H.; Kusai, A.; Nojima, K. Org. Mass Spectrosc. 1994, 29, 668-671. (18) Sakata, R.; Shoyama, Y.; Murakami, H. Cytotechnology 1994, 16, 101-108.

Freund’s complete adjuvant. Two weeks later, the injection was repeated with the same amount of immunogen, but emulsified in Freund’s incomplete adjuvant. Two weeks later, a booster dose of 100 µg of conjugate dissolved in PBS without adjuvant was administered intraperitoneally. On the third day after the final immunization, the splenocytes were isolated and fused with a logarithmically growing hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line, P3-X63-Ag8-653, by the poly(ethylene glycol) (PEG) method.19 Hybridomas producing MAbs reactive to GC were cloned by the limited dilution method.20 Established hybridomas were cultured in enriched RPMI1640Dulbecco’s-Ham’s F12 (eRDF) medium supplemented with 10 µg/ mL of insulin, 35 µg/mL of transferrin, 20 µM of methanolamine and 25 nM of selenium (ITES).21 Each MAb was purified using a protein G FF column (0.46 × 11 cm, Pharmacia Biotech, Uppsala, Sweden). The culture medium (200 mL) containing the IgG was adjusted to pH 7 with 1 M Tris solution and loaded onto the column. After washing the column with 20 mM phosphate buffer (pH 7), the absorbed IgG was eluted with 100 mM of citrate buffer (pH 3). The eluted IgG was neutralized with 1 M Tris solution, then dialyzed against three changes of PBS (pH 7.4), and finally lyophilized. Analytical Procedure. Direct ELISA Using GC-HSA. The reactivity of MAbs to GC-HSA (average 5 molecules of GC/ molecule of HSA) was determined by an ELISA. A 100-µL portion of GC-HSA (1 µg/mL in 50 mM carbonate buffer) was allowed to adsorb onto the wells of a 96-well immunoplate (NUNC Roskilde, Denmark). The plate was treated with 300 µL of PBS containing 5% skim milk (SPBS) for 1 h to reduce nonspecific adsorption, washed three times with PBS containing 0.05% Tween20 (TPBS), and reacted with 50 µL of MAb for 1 h. The plate was washed three times with TPBS, and then the MAb was combined with 100 µL of a 1:1000 dilution of peroxidase-labeled anti-mouse IgG for 1 h. After washing the plate three times with TPBS, 100 µL of substrate solution, 100 mM citrate buffer (pH 4.0) containing 0.003% H2O2 and 0.3 mg/mL of 2,2-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS) (Wako, Tokyo, Japan) was added to each well and incubated for 15 min. The absorbance at 405 nm was measured using a microplate reader (model 450 microplate reader BIO-RAD Laboratories, CA). All reactions were carried out at 37 °C. Competitive ELISA. GC-HSA (100 µL, 1 µg/mL) was adsorbed onto the wells of a 96-well immunoplate and then treated with 300 µL SPBS for 1 h to reduce nonspecific adsorption. A 50-µL portion of various concentrations of GC dissolved in 10% of methanol was incubated with 50 µL of IgG solution for 1 h. The plate was washed three times with TPBS, and then the MAb was combined with 100 µL of a 1:1000 dilution of peroxidase-labeled anti-mouse IgG for 1 h. After washing the plate three times with TPBS, 100 µL of substrate solution was added to each well and was incubated for 15 min. The absorbance at 405 nm was measured using a microplate reader. The cross-reactivities of GC and related compounds were determined according to Weiler and Zenk’s equation.22 (19) Galfre, G.; Milstein, C. Methods Enzymol. 1981, 73, 3-46. (20) Goding, J. W. J. Immunol. Methods 1980, 39, 285-308. (21) Murakami, H.; Masui, H.; Sato, G. H.; Sueoka, N.; Chow, T. P.; Kano-Sueoka, T. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 1158-1162. (22) Weiler, E. W.; Zenk, M. H. Phytochemistry 1976, 15, 1537-1545.

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Figure 1. Direct determination of the hapten number in the glycyrrhizin-BSA (GC-BSA) conjugate by matrix-assisted laser desorption/ ionization TOF mass spectrometry. [M + H]+ and [M + 2H]2+ are single- and double-protonated molecules of GC-BSA, respectively.

Eastern Blotting for GC and its Related Compound. Glycyrrhetic acid, GG, and GC (1 µg each) were applied to a silica gel TLC plate and developed with n-butanol-water-acetic acid (7:2:1 by volume). The developed TLC plate was dried and then sprayed with a blotting solution mixture of 2-propanol-methanolwater (1:4:20 by volume). It was placed on a stainless steel plate and covered with a piece of PVDF membrane. After covering with a glass microfiber filter sheet, the whole assembly was pressed evenly for 50 s with a 120 °C hot plate as previously described23 with some modifications. The PVDF membrane was separated from the TLC plate and dried. The blotted PVDF membrane was dipped into water containing NaIO4 (10 mg/mL) and stirred at room temperature for 1 h. After washing with water, 50 mM carbonate buffer solution (pH 9.6) containing BSA (1%) was added, and stirred at room temperature for 3 h. After washing the PVDF membrane with PBS, the membrane was treated with SPBS for 3 h to reduce nonspecific adsorption. The PVDF membrane was immersed in anti-GC MAb and stirred at room temperature for 1 h. After washing the PVDF membrane twice with TPBS and water, a 1:1000 dilution of peroxidase-labeled goat anti-mouse IgG in PBS containing 0.2% gelatin (GPBS) was added, and the mixture was stirred at room temperature for 1 h. The PVDF membrane was washed twice with TPBS and water and then exposed to freshly prepared 1 mg/mL 4-chloro-1-naphthol-0.03% H2O2 in PBS for 10 min at room temperature. The reaction was stopped by washing with water, and the immunostained PVDF membrane was allowed to dry. RESULTS AND DISCUSSION Direct Determination of Hapten-Carrier Protein Conjugate by MALDI-TOF Mass Spectrometry. It is known that the hapten number in an antigen conjugate is important for immunization against small molecular compounds. We have previously described an analysis of hapten-carrier protein conjugates by MALDI-TOF (23) Taki, K.; Kasama, T.; Handa, S.; Ishikawa, D. Anal. Biochem. 1994, 223, 232-238.

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mass spectrometry that can directly describe the suitability of hapten number for immunization.15-17 Figure 1 shows the MALDITOF mass spectrum of the GC-BSA conjugate. A broad peak coinciding with the conjugate of GC and BSA appeared around m/z 70,021. Using a molecular weight of 66 433 for BSA, the calculated value of the GC component (MW 823) is 3588, indicating at least four molecules of GC conjugated with the BSA. This hapten number was estimated to be enough for immunization when compared to the previous result using solamargine.13 The number of GC moieties contained in the GC-HSA conjugate was determined by its spectrum to be approximately five molecules. Production and Characteristics of MAbs against GC. The hyper-immunized BALB/c mice used to derive the cell clones described in this paper yielded splenocytes that were fused with P3-X63-Ag8-653 myeloma cells by the established procedure in our laboratory.18 Four hybridomas producing MAbs reactive to GC were obtained. All of the MAbs were classified as IgG1 that had λ light chains. The reactivities of the MAbs to GC were tested by direct ELISA using various concentrations. The results showed that MAbs 5A5 and 5A8 had higher reactivities than MAbs 4G6 and 5B4, indicating that 5A5 and 5A8 had stronger affinity for GC-HSA. Assay Sensitivity and Assay Specificity. A competitive binding assay was established in which the MAb binds either free GC or GC-HSA conjugate adsorbed onto a polystyrene microtiter plate. Under these conditions, the full measuring range of the assay extended from 20 to 200 ng/mL as indicated in Figure 2. Since cross-reactivity is the most important factor in determining the value of an antibody and dominates the specificity of an immunoassay, the assay specificity was tested by determining the cross-reactivities of the MAbs with various related compounds. The cross-reactivities of the MAbs were examined by competitive ELISA and calculated using the method of Weiler and Zenk22 (Table 1). MAb 5A8 had 4.6% cross-reactivity with GG and 2.1% with GA. MAb 5A5 cross-reacted with GG and GA at 46.3% and 18.7%, respectively, although it had the higher reactivity. MAbs

Table 2. Variations among ELISA Runs for the Analysis of GCa CV (%) GC concn (ng/mL)

intra-assay

interassay

0 25 50 100 200

1.03 2.27 2.48 3.53 1.77

7.94 2.65 4.73 7.12 6.46

a The measured values were mean ( SD for four plates and three replicate wells for each concentration within one plate from four consecutive days. The variations in replicates from well to well and plate to plate are defined as intraassay variation and interassay variation, respectively.

Figure 2. Standard curve of inhibition by GC using MAb 5A8 in competitive ELISA. Various concentrations of GC were incubated with MAb 5A8 (5 µg/mL) in a 96-well immunoplate precoated with GCHSA (1 µg/mL). Error bars, ( 1 std dev; A/A0, A0 is the absorbance with no GC present; and A is the absorbance with GC present. The data represent the means of four replicates. Table 1. Cross-Reactivities (%) of Anti-GC MAbsa compound

5A8

5A5

4G6

5B4

glycyrrhizin glycyrrhetic acid 3-O-glucuronide glycyrrhetic acid 11-deoxo-18β-glycyrrhetic acid 18R-liquiritic acid 18β-liquiritic acid 11-deoxo-18β-liquiritic acid deoxycholic acid ursolic acid oleanolic acid hederagenin betulin lupeol cholic acid cholesterol ginsenoside Rb1 saikosaponin a solamargine solasonine digitonin ergosterol β-sitosterol

100 4.36 2.13 2.32