넙치(Paralichthys olivaceus)의 immunoglobulin M에 대한 단클론 항체 생산
Production of monoclonal antibodies against the immunoglobulin M of olive flounder Paralichthys olivaceus
2
조회
0
다운로드
1
인용
0
참고문헌
키워드
초록
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.
Abstract
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.
참고문헌 (27)
- Bang JD, "Humoral immune response of flounder to Edwardsiella tarda : the presence of various sizes of immunoglobulins in flounder", Dis Aquat Org, 26, pp.197~203 (1996)
- Björck L, "Protein L. A novel bacterial cell wall protein with affinity for Ig L chains", J Immunol, 140, pp.1194~1197 (1988)
- Crowther JR., "ELISA. Theory and practice" (1995), Humana Press Inc
- Han-Na Jang, "Characterization of Monoclonal Antibodies against Heavy and Light Chains of Flounder (Paralichthys olivaceus) Immunoglobulin", BMB reports, 37(3), pp.314~319 (2004), 한국생화학분자생물학회
- Kaattari SL, "the fish immune system: organism, pathogen and environment", pp.207~254 (1996), Academic Press
- Kibenge MT, "Serological evidence of infectious salmon anaemia virus(ISAV)infection in farmed fishes, using an indirect enzymelinked immunosorbent assay(ELISA)", Dis Aquat Org, 51, pp.1~11 (2002)
- 김춘섭, "항체검출 ELISA 적용을 위한 능성어 IgM의 안정성 및 특이 항체 반응 평가", 한국어병학회지, 22(3), pp.335~342 (2009), 한국어병학회
- Kim WS, "Detection of specific antibodies against infectious hematopoietic necrosis virus from rainbow trout sera by ELISA using two novirhabdoviruses", Fish Pathol, 43, pp.112~116 (2008)
- 김위식, "수온에 따른 넙치의 특이 항체 반응", 한국어병학회지, 24(1), pp.39~45 (2011), 한국어병학회
- Kim WS, "Non-specific adsorption of fish immunoglobulin M(IgM)to blocking reagents on ELISA plate wells", Dis Aquat Org, 78, pp.55~59 (2007)
- "Result of fish farm trends survey in 2015" (2016)[URL]
- Laemmli UK, "Cleavage of structural proteins during the assembly of the head of bacteriophage T4", Nature, 227, pp.680~685 (1970)
- LaPatra SE, "The use of serological techniques for virus surveillance and certification of finfish", Annu Rev Fish Dis, 6, pp.15~28 (1996)
- Li Q, "Production, characterization and applicability of monoclonal antibodies to immunoglobulin of Japanese flounder(Paralichthys olivaceus)", Fish Shellfish Immun, 23, pp.982~990 (2007)
- Nilson BH, "Purification of antibodies using protein L-binding framework structures in the light chain variable domain", J Immunol Methods, 164, pp.33~40 (1993)
- Nishida H, "Purification of Immunoglobulin M(IgM)in serum of Japanese flounder(Paralichthys olivaceus)", Bull Faculty Fish Hokkaido Univ, 49, pp.157~164 (1998)
- Peter P., "The immune system" (2014), Garland Science
- Pilström L, "Immunoglobulin in fish-genes, expression and structure", Fish Shellfish Immun, 6, pp.243~262 (1996)
- Shin GW, "Purification of two different immunoglobulins(Igs)from olive flounder Paralichthys olivaceus and analysis of Lactococcus garvieae antigens by the Igs", Fish Pathol, 42, pp.19~28 (2007)
- Tort L, "Fish immune system. A crossroads between innate and adaptive responses", Num, 22, pp.277~286 (2003)
- Towbin H, "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets; procedure and some applications", Proc Natl Acad Sci USA, 76, pp.4350~4354 (1979)
- Watanabe K, "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, pp.219~223 (2000)
- Watanabe K, "Control strategy for viral nervous necrosis of barfin flounder", Fish Pathol, 33, pp.445~446 (1998)
- Wilson MR, "Fish immunoglobulins and the genes that encode them", Annu Rev Fish Dis, 2, pp.201~221 (1992)
- "Manual of diagnostic tests and vaccines for terrestrial animals" (2016)[URL]
- Jianmin Ye, "The teleost humoral immune response", Fish & Shellfish Immunology, 35(6), pp.1719~1728 (2013), Elsevier BV[DOI]
- Yoshimizu M, "Detection of antibody against Aeromonas salmonicida in the serum of salmonid fish by the enzyme linked immunosorbent assay", Fish Pathol, 27, pp.73~82 (1992)