• KSBB Journal
• /
• 제7권2호
• /
• pp.92-95
• /
• 1992

동물세포 대량 배양의 저혈청 배지를 개발하기 위하여 융합제작한 하이브리도마 KA112를 대상으로 혈청의 역할을 살펴본 다음, 혈청의 구성인자를 최고세포 농도를 결정하는 소모성 영양인자와 세포성장속도를 결정하는 성장 제어인자로 나눈 혈청 역할모델을 개발하여 실험 결과와 비교 검토하였다. 이 모델이 의하면 7%이상의 혈청 첨가시에는 혈청내의 소모성 인자가 그와 같은 역할을 맡게 되며, 3% 첨가시에는 성장 제어 인자의 고갈로 인해 세포성장속도가 제한을 받는다. 동시에 소모성 단일 인자가 혈청농도에 의하여 limiting하게 됨에 따라 최고 도달 세포 농도와 세포 성장 속도를 제어함을 설명할 수도 있다. 이러한 실험결과들은 혈청농도와 기저배지 성분들의 농도를 항상 균형있게 고려해 주어야 함을 보여주고 있다.

• 한국식품위생안전성학회지
• /
• 제13권4호
• /
• pp.419-424
• /
• 1998

Zearalenone 검출을 위하여 Z-M-26 hybridoma cell을 mouse의 복강에 투여한 후 생산, 정제한 항체와 합성한 Zearalenone-oxime-OVA conjugate를 이용하여 ELISA법을 확립하였다. Carbonyl buffer로 희석한 Zearalenone-oxime-OVA conjugate를 4$^{\circ}C$에서 하룻밤 coating 하고 1% BSA용액으로 하룻밤 blocking한 다음, 1,000배 희석한 항체를 Zearalenone 또는 시료와 혼합하여 하룻밤 반응시키는 것이 효과적이었다. 또한 2차 항체와 기질용액의 반응시간은 각각 1시간, 30분이 적당하였고, 발색된 반응액 450nm에서 측정하였다. 이 분석법의 결과 0.1~100 ppb의 Zearalenone이 측정 가능하였으며, 본 실험에서 확립한 indirect competitive ELISA법은 농산물 중 Zearalenone 분석에 효과적으로 활용할 수 있으리라 생각된다.

• 한국가금학회지
• /
• 제19권1호
• /
• pp.13-16
• /
• 1992

마사츄셋형 전염성 기관지염 바이러스(IBV)를 SPF 발육란의 뇨막강내에서 증식시켜 Sucrose 밀도구배 초원심분리에 의해 정제한 다음 BALB/c 마우스에 면역시켰다. 면역 마우스에서 채취한 비장세포와 마우스 골수암세포와 여러 차례 융합시험을 실시하였다. 많은 융합세포 중에서 IBV에 특이적으로 작용하는 단클론항체(monoclonal antibody ： MCA)를 산생하는 hybridoma클론은 2주밖에 얻지 못했다. 2주의 MCA는 모두 IgG형이었고 IBV중화능이나 혈구응집 억제능이 인정되지 않았다. 간접형광항체법으로 작성된 MCA를 이용하여 인공접종한 닭의 기관도말표본에서 10일간의 시험기간중 계속 IBV를 검출할 수 있었다.

• IMMUNE NETWORK
• /
• 제2권4호
• /
• pp.233-241
• /
• 2002

Background: Monoclonal antibodies (mAb) of rodent origin are produced with ease by hybridoma fusion technique, and have been successfully used as therapeutic reagents for humans after humanization by genetic engineering. However, utilization of these antibodies for therapeutic purpose has been limited by the fact that they act as immunogens in human body causing undesired side effects. So far, there have been several attempts to produce human mAbs for effective in vivo diagnostic or therapeutic reagents including the use of humanized xenomouse that is generated by mating knockout mice which lost Ig heavy and light chain genes by homologous recombination and transgenic mice having both human Ig heavy and light gene loci in their genome. Methods: Genomic DNA fragments of mouse Ig heavy and light chain were obtained from a mouse brain ${\lambda}$ genomic library by PCR screening and cloned into a targeting vector with ultimate goal of generating Ig knockout mouse. Results: Through PCR screening of the genomic library, three heavy chain and three light chain Ig gene fragments were identified, and restriction map of one of the heavy chain gene fragments was determined. Then heavy chain Ig gene fragments were subcloned into a targeting vector. The resulting construct was introduced into embryonic stem cells. Antibiotic selection of transfected cells is under the progress. Conclusion: Generation of xenomouse is particularly important in medical biotechnology. However, this goal is not easily achieved due to the technical difficulties as well as huge financial expenses. Although we are in the early stage of a long-term project, our results, at least, partially contribute the successful generation of humanized xenomouse in Korea.

• BMB Reports
• /
• 제42권11호
• /
• pp.731-736
• /
• 2009

The generation of functional recombinant antibodies from hybridomas is necessary for antibody engineering. However, this is not easily accomplished due to high levels of aberrant heavy and light chain mRNAs, which require a highly selective technology that has proven complicated and difficult to operate. Herein, we attempt to use an alkaline phosphate (AP)-fused form of single-chain variable fragment (scFv) for the simple identification of a hybridoma-derived, functional recombinant antibody. As a representative example, we cloned the scFv gene from a hybridoma-producing mouse IgG against branched-chain keto acid dehydrogenase complex-E2 (BCKD-E2) into an expression vector containing an in-frame phoA gene. Functional recombinant antibodies were easily identified by conventional enzyme-linked immunosorbent assay (ELISA) by employing scFv-AP fusion protein, which also readily serves as a valuable immuno-detective reagent.

• KSBB Journal
• /
• 제5권4호
• /
• pp.365-371
• /
• 1990

To improve the growth of mouse hybridoma 2c3.1 secreting anti-Hepatitis B surface antigen monoclonal antibody (anti-HBsAg MAb), we had constructed an enriched medium and observed the effects of fetal bovine serum and serum-free supplements including human serum albumin, 'insulin and transferrin', and monoethanolamine. For further enhancement of growth, the concentrations of two major energy sources, glucose and glutamine, were strengthened with various ratios in the enriched medium. Maximum cell growth and monoclonal antibody production obtained in various ratios of glucose/glutamine with an inoculation concentration of 2$\times$105 cells/ml were 0.73$\times$106-4.62$\times$106 cells/ml and 65.1-422.6 $\mu\textrm{g}$/ml, respectively. Glutamine was round to be a major energy source and a limiting nutrient in comparison to glucose for 2c3.1 cell cultivation in enriched media with low serum.

• 한국가금학회지
• /
• 제15권3호
• /
• pp.199-206
• /
• 1988

국내에서 분리한 강독전염성후두기관염 바이러스 (ILTV)에 대한 세포융합방법에 의해 단크론성항채(MCA) 생산을 시도한 결과 총 8회의 세포융합을 통하여 총 1017개의 융합세포가 생산되었으며 그중 ILTV와 특이적으로 작용하늘 항체를 생산하는 3주의 Hybridoma를 작성하였다. 이 3주의 MCA는 모두 IgG형에 속하였으며 마우스 복강내접종하여 생산된 복수항체외 형광항체가는 $10^5$－$10^6$에 달하였고 약독 및 강독 ILTV에 차이가 없이 작용하였으며 중화능력은 인정되지 않았다. 이 MCA를 이용하여 간접형광항체법으로 인공감염계에서 ILTV 검출을 시도한 결과. 기관 및 안점막의 도말표본에서 감염후 10일 까지 진단이 가능하였으며 표준 양성혈청을 이용한 형광항체법이나 핵내봉입체 검출방법에 비해서 진단효율이 높았다.

• 한국미생물·생명공학회지
• /
• 제14권3호
• /
• pp.219-223
• /
• 1986

사람 선유아세포 인터페론의 정제에 사용되는 단 clone성 항체생산 세포주를 조작하기 위하여 BA-LB/C mouse의 복강과 꼬리정맥을 통하여 HuIFN-$\beta$를 면역화시키고 그 비장세포(spleen cells) 와 NS-O 세포주를 세포융합 시켰다. 융합된 1300 hybrids를 ELISA방법으로 선별하고 soft agarose 방법과 limiting dilution방법으로 subcloning하여 높은 항체를 생성하는 것으로 판명된 11 hybrids를 재선별 하였다. 재선별된 11 hybrids 각각의 항체형 (Ig type)을 조사하고 최종 Protein A-sepharose와 친화성이 높은 IgG 2a/형의 clone # 4-1-19와 clone # 551-4-1을 선별하여 배양된 세포를 각각 nude(nu/nu) mouse 및 BALB/c mouse 복강에 접종배양 하였다. 이들 mouse복강액으로 부터 얻은 ascites fluid를 protein A-sepharose를 이용한 affinity column분획으로 항체를 정제하였으며 ascites fluid $m{\ell}$당 약 4mg의 정제된 항체를 얻을 수 있었고 SDS-polyacrylamide gel상에서 전기영동 시킨 결과 분자량 14-16만 dalton으로 추정되는 항체를 확인할 수 있었다.

• BMB Reports
• /
• 제29권2호
• /
• pp.127-132
• /
• 1996

Germinal centers (GCs) are formed in peripheral lymphoid tissues in response to protein antigens. In order to see if immunoglobulin isotype switching takes place in GC B-cells, we isolated GC B-cells (PNA positive cells) from mouse popliteal lymph nodes by a flow cytometer after the staining of lymph node cells with PNA-FITC and anti-B220-PE, and determined the expression of ${\gamma}1$ germline transcript and ${\gamma}1$ mRNA by RT-PCR. ${\gamma}1$ germline transcript and ${\gamma}1$ mRNA were amplified specifically in cDNAs from hybridoma expressing IgG1 or splenocytes stimulated LPS plus IL-4. Germinal center B-cells formed in popliteal lymph nodes of mice immunized with chicken ovalbumin were isolated 7 days after immunization. We sorted GC B-cells five times. Immunoglobulin ${\gamma}1$ germline transcripts were expressed in germinal center B-cells in three out of five sorts whereas two out of five sorts did not express ${\gamma}1$ germline transcripts in GC B-cells. The contents of GC B-cells ranged from 5 to 7% of total lymph node cells in most flow cytometric analyses but those of two sorted cells which did not express ${\gamma}1$ germline transcripts were out of normal range. These results imply that isotype switching of immunoglobulins may take place in GCs.

• 한국어업기술학회:학술대회논문집
• /
• 한국어업기술학회 2000년도 춘계수산관련학회 공동학술대회발표요지집
• /
• pp.446-446
• /
• 2000

Specific polyclonal and/or monoclonal antibodies (MAbs) to immunoglobulins (Igs) and their subunits have proved to be valuable tools in immunological research and in immunological assays. In this study, we developed and characterized MAbs against flounder serum Igs. To obtain the pure flounder serum Igs, mouse IgG (mIgG) was immunized to flounder. Flounder Igs were purified by using mIgG-agarose affinity column chromatography. The structure of purified flounder Ig was observed, on denatured SDS-PAGE, to be composed of two heavy chains (77 and 72 kd) and two light chains (28 and 26 kd). MAbs were produced by fusion of myeloma cells (SP2/0) with Balb/c mouse spleen cells previously primed with the flounder Igs. Finally, three hybridoma clones, FIM 511, FIM 519 and FIM 562 were established to recognize both 2 heavy chains, 26 kd of light chain and 28 kd of light chain, respectively. On the other hand, the flounder immune sera collected on the weekly basis were tested on ELISA and immunoblot analysis whether boosting effect is present in flounder humoral immune system. As a result, the secondary immune response in flounder was ascertained on ELISA, but not on immunoblot analysis. Further, we observed an alteration of serum protein levels following immunization. Our MAbs and basic information on flounder humoral immune system obtained in this study will be helpful to control and monitor the efficiency of fish vaccines and therapeutic process of flounder diseases.