170 김위식ㆍ김기홍ㆍ김춘섭ㆍ오명주, (LaPatra, 1996; Watanabe et al., 1998; 2000; Kibenge et al., 2002). ELISA, 2 IgM. ELISA IgM (monoclonal antibody, MAb). IgM manna

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1 한수지 50(2), , 2017 Original Article Korean J Fish Aquat Sci 50(2), ,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), ,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), Korean J Fish Aquat Sci 50(2) , April 2017 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens ( 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: Fax: address: ohmj@jnu.ac.kr Copyright 2017 The Korean Society of Fisheries and Aquatic Science 169 pissn: , eissn:

2 170 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 M 재료및방법 30 넙치혈청분리 ( : - kg). 30 6,000 rpm 항 bovine serum albumin (BSA) 넙치혈청생산 BSA ( : - kg) 2 BSA 1 mg/200 µl , 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) ) 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 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, mm aminoprotein, 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).

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.

3 넙치 IgM 에대한단클론항체생산 171 M M kda kda H H 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 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 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

4 172 김위식ㆍ김기홍ㆍ김춘섭ㆍ오명주 CON1 CON CA 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.

5 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 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 (

6 174 김위식ㆍ김기홍ㆍ김춘섭ㆍ오명주 ). References Bang JD, Kim JW, Lee SD, Park SI, Chun SG, Jeong CS and Park JW Humoral immune response of flounder to Edwardsiella tarda: the presence of various sizes of immunoglobulins in flounder. Dis Aquat Org 26, Björck L Protein L. A novel bacterial cell wall protein with affinity for Ig L chains. J Immunol 140, Crowther JR ELISA. Theory and practice. Humana Press Inc., New Jersey, U.S.A. Jang HN, JK Woo, YH Cho, SB Kyong and SH Choi Characterization of monoclonal antibodies against heavy and light chains of flounder (Paralichthys olivaceus) immunoglobulin. J Biochem Mol Biol 37, Kaattari SL and Piganelli JD The specific immune system: humoral defense. In the fish immune system: organism, pathogen and environment. Iwama G and Nakanishi T, eds. Academic Press, San Diego, U.S.A., Kibenge MT, Opazo B, Rojas AH and Kibenge FSB Serological evidence of infectious salmon anaemia virus (ISAV) infection in farmed fishes, using an indirect enzymelinked immunosorbent assay (ELISA). Dis Aquat Org 51, Kim CS, Jang MS, Kim WS, Kim JO, Kim DW, Kim DH, Han HJ, Jung SJ and Oh MJ Evaluation of the stability of IgM and specific antibody response of sevenband grouper Epinephelus septemfasciatus for application of antibodydetection ELISA. J Fish Pathol 22, Kim WS, Mochizuki M, Nishizawa T and Yoshimizu M Detection of specific antibodies against infectious hematopoietic necrosis virus from rainbow trout sera by ELISA using two novirhabdoviruses. Fish Pathol 43, Kim WS, Jang MS, Jung SJ, Kim SR, Park MA, Lee JH, Myeong JI and Oh MJ Specific antibody response of olive flounder Paralichthys olivaceus by water temperature. J Fish Pathol 24, Kim WS, Nishizawa T and Yoshimizu M Non-specific adsorption of fish immunoglobulin M (IgM) to blocking reagents on ELISA plate wells. Dis Aquat Org 78, KOSIS (Korean statistical information service ) Result of fish farm trends survey in 2015[Internet]. Retrieved from jsp?vwcd=mt_etitle&parmtabid=m_01_01 on March Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, LaPatra SE The use of serological techniques for virus surveillance and certification of finfish. Annu Rev Fish Dis 6, Li Q, Zhan W, Xing J and Sheng X Production, characterization and applicability of monoclonal antibodies to immunoglobulin of Japanese flounder (Paralichthys olivaceus). Fish Shellfish Immun 23, Nilson BH, Lögdberg L, Kastern W, Björck L and Akerström B Purification of antibodies using protein L-binding framework structures in the light chain variable domain. J Immunol Methods 164, Nishida H, Enokida T, Hiramatsu N, Hara A and Yoshimizu M Purification of Immunoglobulin M (IgM) in serum of Japanese flounder (Paralichthys olivaceus). Bull Faculty Fish Hokkaido Univ 49, Peter P The immune system. Garland Science, Connecticut, U.S.A. Pilström L and Bengtén E Immunoglobulin in fish-genes, expression and structure. Fish Shellfish Immun 6, Shin GW, Kim YR, Shin YS, Lee EG, Oh MJ, Yoshida T and Jung TS Purification of two different immunoglobulins (Igs) from olive flounder Paralichthys olivaceus and analysis of Lactococcus garvieae antigens by the Igs. Fish Pathol 42, Tort L, Balasch JC and Mackenzie S Fish immune system. A crossroads between innate and adaptive responses. Num 22, Towbin H, Staehelin T and Gordon J Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets; procedure and some applications. Proc Natl Acad Sci USA 76, Watanabe K, Nishizawa T and Yoshimizu M Selection of brood stock candidates of barfin flounder using an ELISA system with recombinant protein of barfin flounder nervous necrosis virus. Dis Aquat Org 41, Watanabe K, Suzuki S, Nishizawa T, Suzuki K, Yoshimizu M and Ezura Y Control strategy for viral nervous necrosis of barfin flounder. Fish Pathol 33, Wilson MR and Warr GW Fish immunoglobulins and the genes that encode them. Annu Rev Fish Dis 2, World organization for animal health (OIE) Manual of diagnostic tests and vaccines for terrestrial animals[internet]. Retrieved from on May Ye J, Kaattari IM, Ma C and Kaattari S The teleost humoral immune response. Fish Shellfish Immunol 35, Yoshimizu M, Direkbusarakom S, Nomura T, Ezura Y and Kimura T Detection of antibody against Aeromonas salmonicida in the serum of salmonid fish by the enzyme linked immunosorbent assay. Fish Pathol 27,

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untitled 3. 농업환경연구과 과제구분 기본연구 수행시기 전반기 연구과제 및 세부과제 수행 기간 소 속 책임자 농가에 적합한 부식성곤충 대량 사육기술 개발 12~ 13 농업환경연구과 곤충팀 이영혜 1) 부식성 곤충 먹이 제조 기술 개발 12~ 13 농업환경연구과 곤충팀 이영혜 색인용어 부식성곤충, 장수풍뎅이, 계통, 먹이제조 ABSTRACT In first check,