RNase A 와 B 에 대한 Mouse Immunoglobulin의 생성 및 분리조건

전숙영,이광표,김하형 중앙대학교 약학연구소 1997 약학 논총 Vol.11 No.-

Ribonuclease A, B (RNase A, B) are the enzymes catalyse he hydrolysis of 3',5'-phosphodiester linkages of ribonucleic acids. RNase B(M.W. 15.5KDa) differs from RNase A(M.W. 13.6KDa) in that RNase B has five glycoforms consisting of Man_9GlcNAc_2, to Man_9GlcNAc_2 at the single glycosylation site (Asn-34). It has not been fully studied that the difference of glycosylation between RNase A and RNase B would make difference in production of polyclonal antibody when it was used as antigen. In the present study, male Balb/c mice aged 6-7weeks were used, and three mice per group were immunized intraperitoneally with 0.2ml of emulsion mixture containing 100ug RNase A or B in 0.01ml of PBS and 0.18ml of Freund's adjuvant, and immunized with PBS as a control. To identify the production of polyclonal antibody, total protein amount was detected by use of UV- spectrophotometer and tested 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In order to know the affinity of polyclonal antibody to RNase A, B and to measure the concentration of polyclonal antibody. ELISA method had been applied using the mouse monoclonal antibody 6 isotypes (IgG1, IgG2a, IgG2b, IgG3, IgA, IgM) and monoclonal anti-goat/sheep IgG peroxidase. Protein A column was prepared to test the possibility of separation of the polyclonal antibody to each immunoglobulin class. As a result, the glycosylation between RNase A and B did not have an effect on production pattern of polyclonal antibody from ascites of mice immunized with RNase A or B. Second, in the present study, the polyclonal antibody made from mice immunized with RNase A or RNase B should be isolated to immunoglobulin class or subclass by use of various binding or elution buffer.

Polyclonal gammopathy related to renal bleeding in a peritoneal dialysis patient

조은미,문혜현,황영주,이성진,고철우,조민현 대한소아청소년과학회 2013 Clinical and Experimental Pediatrics (CEP) Vol.56 No.7

Polyclonal gammopathy represents the diffuse activation of B cells and is usually related to inflammation or immune-related diseases. However, the mechanisms leading to polyclonal gammopathy are essentially speculative. Generally, infectious, inflammatory, or various other reactive processes may be indicated by the presence of a broad-based peak or band in the gamma region on serum protein electrophoresis results. A 15-year-old girl, who had been receiving peritoneal dialysis, presented with polyclonal gammopathy and massive gross hematuria. Renal artery embolization was performed, after which the continuous bleeding subsided and albumin-globulin dissociation resolved. This is a rare case of polyclonal gammopathy related to renal bleeding.

Polyclonal gammopathy related to renal bleeding in a peritoneal dialysis patient

Cho, Eun-Mi,Moon, Hye-Hyun,Hwang, Young-Ju,Lee, Seung-Jin,Ko, Cheol Woo,Cho, Min Hyun The Korean Pediatric Society 2013 Clinical and Experimental Pediatrics (CEP) Vol.56 No.7

Polyclonal gammopathy represents the diffuse activation of B cells and is usually related to inflammation or immune-related diseases. However, the mechanisms leading to polyclonal gammopathy are essentially speculative. Generally, infectious, inflammatory, or various other reactive processes may be indicated by the presence of a broad-based peak or band in the gamma region on serum protein electrophoresis results. A 15-year-old girl, who had been receiving peritoneal dialysis, presented with polyclonal gammopathy and massive gross hematuria. Renal artery embolization was performed, after which the continuous bleeding subsided and albumin-globulin dissociation resolved. This is a rare case of polyclonal gammopathy related to renal bleeding.

Polyclonal Antibody to a 37-kDa Recombinant Protein Derived from Bovine 20α-Hydroxysteroid Dehydrogenase

Purevjargal Naidansuren,Kwan-Sik Min 한국동물번식학회 2012 Reproductive & developmental biology Vol.36 No.2

We prepared the polyclonal antibody anti-20α-hydroxysteroid dehydrogenase (anti-20α-HSD) against the recombinant full-length protein bovine 20α-HSD in Escherichia coli. The specificity of anti-20α-HSD was demonstrated using Chinese hamster ovary (CHO) cells transfected with recombinant bovine 20α-HSD and bovine placental tissues. According to western blot analysis, anti-20α-HSD specifically recognizes the 37-kDa protein bovine 20α-HSD. The protein is not present in untransfected CHO cells. Anti-20α-HSD also recognizes a specific protein in the ovaries and placenta of other animals. Immunostaining was used to detect expression of bovine 20α-HSD protein in the cultured luteal cells during the estrous cycle later.

Production of Polyclonal Antibody against α-Fetoprotein and Polyclonal Antibody-Based Competitive Enzyme-Linked Immunosorbent Assay for α-Fetoprotein

Yoon,Michung THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 1997 Journal of biomedical laboratory sciences Vol.3 No.2

인간 α-fetoprotein (AFP)은 간암, 위암, 생식기종양 및 신경관이상인 환자를 검사하고 진단하는데 유용한 지표로 알려져 있다. 본 연구에서는 사람의 AFP를 분리정제하여 폴리클로날 항체를 생산하고 인간 혈장과 양수내의 AFP를 측정하기 위한 경쟁적 효소면역분석법을 개발하고자 하였다. 친화 크로마토그래피법과 SDS-polyacrylamide 전기영동법을 이용하여 양수로부터 AFP를 분리하였다. 정제된 AFP를 토끼에 주사하여 폴리클로날 항체를 생산하였으며, 이중면역확산법과 Western blot 분석법을 사용하여 본 연구실에서 제조된 항체의 항원 특이성이 대단히 높음을 확인하였다. AFP와 항혈청을 이용하여 표준곡선을 얻었으며, 민감도는 5ng/ml이었고, 작용범위는 5∼1,000ng/ml이었다. 분석내 CV는 4.5%이었고, 분석간 CV는 8.5%이었다. 따라서 이러한 결과로 보아 본 연구에서 개발된 경쟁적 효소면역분석법이 AFP를 측정하기에 적절하며, 간암 등의 기초연구에도 많은 기여를 할 것으로 생각된다. α-Fetoprotein (AFP) has been a useful marker in screening and/or monitoring patients with hepatocellular carcinoma, gonadal germ cell tumor, gastric carcinoma and neural tube defects. In the present study, it was attempted to produce anti-human AFP polyclonal antibodies and to develop a competitive enzyme-linked immunosorbent assay (ELISA) for the measurement of AFP in human plasma and amniotic fluid. AFP was isolated from amniotic fluid using an isolation procedure consisting of affinity chromatography and preparative polyacrylamide gel electrophoresis. The antibody directed against AFP was raised in rabbits. Double immunodiffusion and Western blotting methods showed that the antiserum was highly specific, reacting with only AFP-containing samples. Standard curve was obtained by using purified AFP and specific antiserum. The assay sensitivity was 5ng/ml and the working range was 5∼1,000ng/ml. The within-assay and between-assay coefficient of variance (CV) was 4.5% and 8.5%, respectively. These results indicate that the assay is valuable for the measurement of AFP and found to be simple, reproducible, and accurate.

지방세포 원형질막 단백질에 대한 다클론 항체의 수동면역이 수컷 흰쥐의 체조성에 미치는 영향

백경훈,최창본 한국동물자원과학회 2002 한국축산학회지 Vol.44 No.1

본 연구는 흰쥐 지방세포 원형질막 단백질에 대한 항혈청의 면역주사가 흰쥐 체지방 함량에 미치는 영향을 규명하기 위하여 실시되었다. 20마리의 Sprague-Dawley 흰쥐를 대조구와 항체처리구(10두/구)로 완전 임의배치하였고, 대조구에는 생리식염수를, 항체처리구에는 면양으로부터 생산해 낸 흰쥐 지방세포 원형질막단백질에 대한 다클론항체를 면역주사하였다. 항체의 수동면역(복강주사)은 실험동물의 피하지방(21.9%)과 신지방+장간막지방+정소외막지방조직(36.0%)을 유의적으로(각각 P=0.0054, P=0.0019) 감소시켰다. 항체처리구의 체중은 항체처리기간동안 감소하였지만, 처리 후 1주를 경과하면서 다시 정상체중으로 회복되었다. 혈중 glucose 농도와 근육내 조지방 및 조단백질의 수준은 항체의 처리로 인한 유의적인 차이를 나타내지 않았다. 이러한 결과들은 면양에서 생산한 지방세포 원형질막 단백질에 대한 다클론 항체들이 육생산동물의 체지방 함량을 조절할 수 있다는 가능성을 시사해 주고 있다. 앞으로 본 연구결과의 실용화를 위하여 실용적인 측면에서의 연구가 필요할 것으로 생각된다. The current study was conducted to investigate the effects of administration of antiserum against adipocyte plasma membrane(AMP) proteins into rats on body fat mass. Twenty(20) male adult Sprague-Dawley rats were randomly allocated into either control or antiserum treatment group(10 rats/treatment) and immunized with physiological saline(control group) and polyclonal antiserum(treatment group), respectively, raised in sheep against rat APM proteins(5times, 2day interval). All animals were killed 4weeks after last injection. Intraperitoneal(i.p.) administration of antiserum significantly(P=0.0054 and P=0.0019, respectively) reduced subcutaneous(21.9%) and perirenal + mesentric + epididymic(36.0%) adipose tissue mass in rats of treatment group. Although body weights of antiserum treated rats were decreased during immunization, the rats recovered their body weight after 1 week of treatment. There were no significant changes in the level of blood glucose and in the contents of muscle protein and fat in antiserum treated animals. Current results indicate that polyclonal antibodies against APM proteins could be used to manipulate body fat mass in meat animals as well as laboratory animals. Further studies, however, are necessary for the practical applications of the current results.

Polyclonal Antibody to a 37-kDa Recombinant Protein Derived from Bovine 20α-Hydroxysteroid Dehydrogenase

Purevjargal Naidansuren,Kwan-Sik Min 한국동물생명공학회(구 한국동물번식학회) 2012 Reproductive & Developmental Biology(Supplement) Vol.36 No.2s

We prepared the polyclonal antibody anti-20α-hydroxysteroid dehydrogenase (anti-20α-HSD) against the recombinant full-length protein bovine 20α-HSD in Escherichia coli. The specificity of anti-20α-HSD was demonstrated using Chinese hamster ovary (CHO) cells transfected with recombinant bovine 20α-HSD and bovine placental tissues. According to western blot analysis, anti-20α-HSD specifically recognizes the 37-kDa protein bovine 20α-HSD. The protein is not present in untransfected CHO cells. Anti-20α-HSD also recognizes a specific protein in the ovaries and placenta of other animals. Immunostaining was used to detect expression of bovine 20α-HSD protein in the cultured luteal cells during the estrous cycle later.

Carrier-Adjuvant로써 Cholera Toxin을 사용한 항 Fumonisin 항체생산과 이를 이용한 ELISA 개발

정순관(Soon Kwan Chung),이문한(Mun Han Lee),이항(Hang Lee),류판동(Pan Dong Ryu),조명행(Myung Haing Chio),이영재(Young Jae Lee),이혜숙(Hye Sook Lee),박종명(Jong Myung Park),심영화(Young Hwa Sim),김재명(Jae Myung Kim),임종섭(Jong Seop 한국예방수의학회 1995 예방수의학회지 Vol.19 No.4

Murine polyclonal antibody reactive with fumonisin B₁(FB₁) was produced by immunization with FB₁-cholera toxin(CT) conjugate as a carrier-adjuvant and an emzyme-linked immunosorbent assay (ELISA) was developed to detect fumonisin in feedstuffs. The immunogen, FB₁-CT, and coating antigen, FB₁-ovalbumin(OA), were prepared by coupling FB₁ to CT or OA with 2% glutaraldehyde. Mice were immunized by injecting FB₁-CT conjugate intraperitonially 3 times on 14 day intervals and a large volume of ascites containing antibody to fumonisins was produced by injecting pristane and myelorna cells into peritonium. Antibody titers were measured by indirect ELISA after each immunization and antibody in ascites was partially purified by ammonium sulfate precipitation and gel filtration. A standard curve of FB₁(n=8) was aquired by competitive indirect ELISA using the partially purified ascitic antibody and FB₁-OA as a coating antigen, and the standard curve showed that the lowest detection limit of FB₁ is 100ng/mL level(p<0.01). The ascitic antibody cross-reacted with FB₂(97%), but no cross-reactivities were observed with tricarballylic acid, aflatoxins(B₁, B₂, G₁, and G₂), zearalenone, T-2 toxin, and ochratoxin A Competitive ELISA curves in the presence of organic solvents used to extract FB₁ from feedstuffs showed that the concentrations of organic solvents up to 18.5% methanol and 12.5% acetonitrile had no prominent effects on the ELISA system developed, suggesting that the diluted organic solvent extract of the sample may be used for the ELISA without solvent evaporation. When FB₁ from spiked corns was extracted with 50% acetontrile and subjected to the ELISA after the concentration of acetonitrile was reduced to 12.5% by dilution with PBS, the recovery rates of FB₁ in corns containing 1, 2.5 and 5 μg/g of FB₁ were 194%, 107% and 82.1%, respectively. Therefore, the ELISA system developed in this study using the polyclonal antibody to FB1 could be applied to determine fumonisins in feedstuffs.

Production of Polyclonal Antibody against α-Fetoprotein and Polyclonal Antibody-Based Competitive Enzyme-Linked Immunosorbent Assay for a-Fetoprotein

Michung Yoon(윤미정) 대한의생명과학회 1997 Biomedical Science Letters Vol.3 No.2

인간 α-fetoprotein (AFP)은 간암, 위암, 생식기종양 및 신경관이상인 환자를 검사하고 진단하는데 유용한 지표로 알려져 있다. 본 연구에서는 사람의 AFP를 분리정제하여 폴리클로날 항체를 생산하고 인간 혈장과 양수내의 AFP를 측정하기 위한 경쟁적 효소면역분석법을 개발하고자 하였다. 친화크로마토그래피법과 SDS-polyacrylamide 전기영동법을 이용하여 양수로부터 AFP를 분리하였다. 정제된 AFP를 토끼에 주사하여 폴리클로날 항체를 생산하였으며, 이중면역확산법과 Western blot 분석법을 사용하여 본 연구실에서 제조된 항체의 항원 특이성이 대단히 높음을 확인하였다. AFP와 항혈청을 이용하여 표준곡선을 얻었으며, 민감도는 5ng/ml이었고, 작용범위는 5~1,000ng/ml이었다. 분석내 CV는 4.5%이었고, 분석간 CV는 8.5%이었다. 따라서 이러한 결과로 보아 본 연구에서 개발된 경쟁적 효소면역분석법이 AFP를 측정하기에 적절하며, 간암 등의 기초연구에도 많은 기여를 할 것으로 생각된다. α-Fetoprotein (AFP) has been a useful marker in screening and/or monitoring patients with hepatocellular carcinoma, gonadal germ cell tumor, gastric carcinoma and neural tube defects. In the present study, it was attempted to produce anti-human AFP polyclonal antibodies and to develop a competitive enzyme-linked immunosorbent assay (EUSA) for the measurement of AFP in human plasma and amniotic fluid. AFP was isolated from amniotic fluid using an isolation procedure consisting of affinity chromatography and preparative polyacrylamide gel electrophoresis. The antibody directed against AFP was raised in rabbits. Double immunodiffusion and Western blotting methods showed that the antiserum was highly specific, reacting with only AFP-containing samples. Standard curve was obtained by using purified AFP and specific antiserum. The assay sensitivity was 5ng/ml and the working range was 5~1,000ng/ml. The within-assay and between-assay coefficient of variance (CV) was 4.5% and 8.5%, respectively. These results indicate that the assay is valuable for the measurement of AFP and found to be simple, reproducible, and accurate.

Production of and Applications for a Polyclonal IgY Diagnostic Reagent Specific for Mycobacterium avium subsp. paratuberculosis

신성재,Seung-Sub Lee,Elizabeth J. B. Manning,Michael T. Collins 한국미생물학회 2009 The journal of microbiology Vol.47 No.5

Antibodies specific to the cell surface antigens of Mycobacterium avium subsp. paratuberculosis (MAP) have multiple useful applications, e.g. organism detection, immunoconcentration, and cell visualization. The aim of this study was to produce and compare polyclonal antibodies for such research and diagnostic purposes. Three polyclonal antibodies to MAP were produced using sera from immunized rabbits and chickens plus naturally infected cows. Cross-reactive antibodies in each MAP antibody preparation were removed by absorption with heterologous mycobacterial and non-mycobacterial cells. The specificity of each resulting polyclonal antibody preparation was evaluated by ELISA to multiple bacterial cell wall extract antigens. After absorption, chicken anti-MAP IgY had the highest specificity of the three antibody preparations. FITC-labeled anti-MAP IgY was used to effectively locate MAP in macrophages 12 h post-infection. Also, immuno- magnetic beads coated with anti-MAP IgY enhanced recovery of MAP from bacterial suspensions in comparison with non-antibody coated beads. Anti-MAP IgY provides a novel new reagent with broad diagnostic and research applications requiring specific concentration, detection, and quantification of MAP.