한수지 50(2), 169-174, 2017 Original Article Korean J Fish Aquat Sci 50(2),169-174,2017 넙치 (Paralichthys olivaceus) 의 immunoglobulin M 에대한단클론항체생산 김위식 김기홍 김춘섭 1 오명주 * 전남대학교수산생명의학과, 1 엔바이로젠 Production of Monoclonal Antibodies Against the Immunoglobulin M of Olive Flounder Paralichthys Olivaceus Wi-Sik Kim, Ki-Hong Kim, Choon-sup Kim 1 and Myung-Joo Oh * Department of Aqualife Medicine, Chonnam National University, Yeosu 59626, Korea 1 EnbioGene, Yeosu 59771, Korea Immunoglobulin M (IgM) was purified from olive flounder Paralichthys olivaceus sera using mannan-binding protein (MBP) and protein L affinity columns (designated as MBPIgM and ProLIgM, respectively). A monoclonal antibody (MAb) against olive flounder IgM was produced. The MBPIgM and ProLIgM had apparent molecular weights of 77, 73, and 28 kda in SDS-PAGE. Nine hybridomas secreting MAbs against olive flounder IgM were established: five MAbs for MBPIgM (1, 2, 3, 4, and 5) and four for ProLIgM (6, 7, 8, and 9). Western blotting indicated that seven MAbs recognized heavy (H; MAbs 1, 2, 3, 4, 5, 6, and 7) chains and one recognized light (L; MAb 9) chains of IgM, while MAb 8 did not recognize IgM. The results of enzyme-linked immunosorbent assay (ELISA) with bovine serum albumin (BSA, antigen) and the nine MAbs revealed that the optical density (OD) values of sera differed significantly between BSA- and non-immunized fish, despite some sera from non-immunized fish with slight high OD values. These results suggest that the MAbs produced in this study reacted specifically with the IgM from olive flounder. Key words: Immunoglobulin M, Olive flounder, Monoclonal antibody, Mannan-binding protein affinity column, Protein L affinity column 서론 (Paralichtys olivaceus),. 2015 45,759 ( 5,040 ) 53% (KOSIS, 2016).,,. enzyme-linked lmmunosorbent assay (ELISA),,, (Crowther, 1995; OIE, 2016). immunoglobulin A (IgA), IgD, IgE, IgG IgM, IgM IgG (Crowther, 1995; Peter, 2014). IgM, IgD IgT/IgZ, IgM,, (IgG) (Wilson and Warr, 1992; Kaattari and Piganelli, 1996; Pilström and Bengtén, 1996; Tort et al., 2003; Ye et al., 2013). (Yoshimizu et al., 1992; LaPatra, 1996; Kim et al., 2009; Kim et al., 2011), http://dx.doi.org/1657/kfas.2017.0169 Korean J Fish Aquat Sci 50(2) 169-174, April 2017 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens (http://creativecommons.org/licenses/by-nc//) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 10 February 2017; Revised 20 March 2017; Accepted 23 March 2017 *Corresponding author: Tel: +82. 61. 659. 7173 Fax: +82. 61. 659. 7170 E-mail address: ohmj@jnu.ac.kr Copyright 2017 The Korean Society of Fisheries and Aquatic Science 169 pissn:0374-8111, eissn:2287-8815
170 김위식ㆍ김기홍ㆍ김춘섭ㆍ오명주, (LaPatra, 1996; Watanabe et al., 1998; 2000; Kibenge et al., 2002). ELISA, 2 IgM. ELISA IgM (monoclonal antibody, MAb). IgM mannan binding protein (MBP) affinity column protein L affinity column. kda 116 66 53 38 M 1 2 77 73 재료및방법 30 넙치혈청분리 25 28 ( : - kg). 30 6,000 rpm 20. -80. 항 bovine serum albumin (BSA) 넙치혈청생산 BSA ( : - kg) 2 BSA 1 mg/200 µl 17-20 0, 7, 14, 21 (6,000 rpm, 4, 20 min), 2 phosphate-buffered saline (PBS, ph 7.5) 200 µl. 넙치 IgM 정제 1) MBP affinity column Shin et al. (2007) ImmunoPure IgM purification kit (Pierce, USA) IgM. ImmunoPure MBP column preparation buffer 5 ml binding buffer 20 ml column equilibration. binding buffer 1:1 column 4 30, binding buffer 42 ml. Elution buffer 3 ml column 1 1 ml. IgM (MB- PIgM) -80. 2) Protein L affinity column Protein L resin (GenScript, USA) column binding buffer (20 mm Na 2 HPO 4, 0.15 M NaCl, ph 8), binding buffer 1:1 Fig. 1. SDS-PAGE analysis of olive flounder Paralichthys olivaceus IgM purified with mannan-binding protein (MBP) and protein L affinity columns. Lanes are M, molecular marker; 1, IgM purified with MBP affinity column; 2, IgM purified with protein L affinity column. column. 4 15 binding buffer, elution buffer (0.1 M glycine, ph ) 1M Tris-HCl (ph 8.5). IgM (ProLIgM) -80. MAb 생산 IgM (MBPIgM ProLIgM) complete Freund s adjuvant (Gibco, USA) 100 L 2 BALB/c 1, 2 IgM 2. 1 IgM 3, polyethylene glycol (Roche, Germany) SP2/0 myeloma fetal bovine serum 10% HAT (0.1 mm hypoxanthine, 4 10-4 mm aminoprotein, 1.6 10-2 mm thymidine in Dulbecco s modified eagle medium) suspension 96 well plate 37 CO 2. hybridoma IgM ELISA 3. MAb isotyping Ig isotyping ELISA kit (BD Biosciences, USA).
넙치 IgM 에대한단클론항체생산 171 M 1 2 3 4 5 1 2 3 4 5 M 6 7 8 9 6 7 8 9 kda kda 100 75 H 100 75 H 50 50 37 37 25 20 25 20 L MBPIgM ProLIgM Fig. 2. Western blotting of purified olive flounder Paralichthys olivaceus IgMs (MBPIgM and ProLIgM) immunostained with 5 anti- MBPIgM monoclonal antibodies (MAbs). M, molecular marker; 1-5, MAbs against MBPIgM; MBPIgM, olive flounder IgM purified with mannan-binding protein affinity column; ProLIgM, olive flounder IgM purified with protein L affinity column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 와 western blotting Laemmli (1970) SDS-PAGE. IgM, IgM (MBPIgM ProLIgM) 12% polyacrylamide gel loading 100 V. gel coomassie brilliant blue. Western blotting Towbin et al. (1979). IgM nitrocellulose membrane (Bio-Rad, USA), MAb (1 ) alkaline phosphatase (AP) goat anti-mouse IgG (Sigma, USA, 2 ) AP substrate kit (Bio-Rad, USA). ELISA IgM MAb hybridoma MAb IgM ELISA. Hybridoma Kim et al. (2008) ELISA. IgM (MBPIgM ProLIgM) coating buffer (M carbonate-bicarbonate, ph 9.6) ELISA plate (Nunc, Denmark) 50 µl (250 ng/well), 4 overnight coating MBPIgM ProLIgM Fig. 3. Western blotting of purified olive flounder Paralichthys olivaceus IgMs (MBPIgM and ProLIgM) immunostained with 4 anti- ProLIgM monoclonal antibodies (MAbs). M, molecular marker; 6-9, MAbs against ProLIgM; MBPIgM, olive flounder IgM purified with mannan-binding protein affinity column; ProLIgM, olive flounder IgM purified with protein L affinity column.. Tween-20 5% PBS (T-PBS) 3 5% skim milk 380 µl 25 1 blocking. 1 hybridoma 50 µl, 2 5% skim milk 1,000 peroxidase conjugated IgG (Younginfrontier, Korea) 50 µl. 25 1. T-PBS 5 ELISA (100 mm Na 2 HPO 4, 50 mm citric acid, 1 mg/ml o-phenylenediamine, 3% H 2 O 2 ) well 50 µl 25 15. well 2 N H 2 SO 4 50 µl microplate photometer (Multiskan, USA) 492 nm (optical density, OD). MAb IgM BSA ELISA Kim et al. (2007, 2011). ELISA plate 5 µg BSA/50 µl well, 37 overnight coating. 1 (BSA ) 5% skim milk (40 ) 1 50 µl, 2 MAb 50 µl, 3 peroxidase conjugated
172 김위식ㆍ김기홍ㆍ김춘섭ㆍ오명주 CON1 CON2 1 3 4 5 CA 6 8 9 CON1 CON2 2 7 Fig. 4. Antibody detection enzyme-linked lmmunosorbent assay with bovine serum albumin (antigen) and 10 monoclonal antibodies [1-9, commercial antibody (CA)]. and, sera from BSA immunization of olive flounder Paralichthys olivaceus; and cont2, sera from non-immunization of olive flounder.
넙치 IgM 에대한단클론항체생산 173 IgG 50 µl.. IgM MAb (Diagnostics Ltd., Scotland) ELISA. 결과및고찰 ELISA, IgM MBP protein L affinity column IgM, IgM (MBPIgM ProLIgM) MAb. MBP protein L affinity column Ig SDS-PAGE, MB- PIgM ProLIgM 77, 73, 28 kda (Fig. 1). ProLIgM 77, 73, 28 kda 57 kda (data not shown). IgM SDS- PAGE heavy (H) chain light (L) chain 75 kda 23 kda (Nishida et al., 1998), 77, 72 kda 28, 26 kda (Jang et al., 2004), 74 kda 24 kda (Li et al., 2007), 68, 66 kda 27, 25 kda (Shin et al., 2007). Ig IgM Ig IgM. Shin et al. (2007) MBP affinity column Ig 68 kda (H chain) 25, 27 kda (L chain) MBP affinity column.. Protein L IgM, IgG, IgA, IgD IgE L chain (Björck, 1988; Nilson et al., 1993), ( IgM ) IgM protein L. MBPIgM ProLIgM SP2/0 myeloma hybridoma. Hybridoma ELISA, 9 MAb. MBPIgM 5 MAb (MBPIgM- MAb:1, 2, 3, 4, 5) ProLIgM 4 MAb (ProLIgM- MAb:6, 7, 8, 9). 9 MAb isotyping, H chain IgG1 (1, 2, 3, 4, 5, 9), IgG2a (7), IgG2b (6, 8), L chain kappa (data not shown). 9 MAb IgM western blotting, MBPIgM-MAb 5 MBPIgM ProLIgM H chain (Fig. 2). ProLIgM-MAb, 6 7 MBPIgM ProLIgM H chain, 9 L chain (Fig. 3). 8 H chain L chain. MAb ELISA, BSA 0, 7, 14, 21 BSA (Fig. 4). ProLIgM-MAb 4 (6, 7, 8, 9) BSA, (BSA ). Kim et al. (2011) BSA ( :15 ) 14 BSA, 21 BSA. Kim et al. (2011) OD BSA., ( IgM MAb ) OD, MAb IgM. MAb 6 7, 8, 9 IgM MAb BSA BSA 0.1-1.1 OD (Fig. 4). MBPIgM-MAb 5 (1, 2, 3, 4, 5) 3 ProLIgM-MAb (7, 8, 9) BSA. BSA BSA OD (8-0.8) (Fig. 4). BSA OD ELISA 5% skim milk (Kim et al., 2007) IgM.. Ig sephacryl S-300 gel filtration (Bang et al., 1996), salting-out, ionexchange chromatography gel filtration (Nishida et al., 1998), immune affinity column (Jang et al., 2004; Shin et al., 2007), MBP affinity column (Shin et al., 2007). protein L affinity column IgM. Protein L affinity column (salting-out, ion-exchange chromatography gel filtration, immune affinity column ) IgM. ProLIgM MAb MBPIgM-MAb IgM. 사사 2015 (
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