• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1997년도 춘계학술대회
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• pp.95-95
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• 1997

Among the various receptor molecules discovered so far the ${\beta}$2-adrenergic receptors have been regarded as excellent model systems for the so called 7 transmembrane helix receptor and have been the focus of extensive studies. For the analysis of receptor structure and function a monoclonal antibody plays a crucial role, thus providing useful tools for the study of receptor. However, because of the minute quantity of receptor molecules which could be obtained from natural sources, the generation of specific monoclonal antibody against receptor molecules from the purified receptors has been regarded as virtually impractical in consideration of cost and experimental times. The purpose of the present study was to generate and characterize a monoclonal antibody against human ${\beta}$2-adrenergic receptor. For the production of antibody, C-terminal regions of the human ${\beta}$2-adrenergic receptor was produced as a fusion protein with Glutathion S-transferase (GST) in E. Coli. The expression of the fusion protein was identified by SDS-PAGE and Western blot using monoclonal anti-GST antibody. The fusion protein was purified to an apparent homogeniety by affinity chromatography with Glutathion Sepharose CL-4B and used as an antigen for the immunization of BALB/C mice. The Production of monoclonal antibody was achieved by fusion of the immunized spleen cells and SP/2-0 myeloma cells. Positive hybridomas were screened by ELISA and were cloned by two consecutive rounds of limiting dilution. The monoclonal antibody produced in this study (mAb${\beta}$C02) was IgM type and purified by immunoaffinity chromatography using anti-mouse IgM agarose as an affinity matrix. MAb${\beta}$C02 showed strong and specific immunoreactivity against both the fusion protein and human ${\beta}$2-adrenergic receptor in ELISA and Western blot. The molecular weight of immunoreactive band was 64 kDa and exactly coincided with the previously reported molecular weight of ${\beta}$2-adrenergic recepters. The results of the present study suggest that mAb${\beta}$C02 may be used for the study of receptor function and regulation in normal or nonphysiological status.

• 대한의생명과학회지
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• 제5권1호
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• pp.1-10
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• 1999

본 연구에서는 혈장이나 양수에 있는 $\alpha$-fetoprotein (AFP)을 인식 할 수 있는 모노클로날 항체를 제조하고, 모노클로날 항체를 이용한 효소면역분석법을 개발하고자 하였다. 양수로부터 얻은 AFP를 쥐에 주사한 후 비장을 분리하여 종양세포 (Sp2/O-Ag-14)와 융합하였고, 하이브리도마 기술을 이용하여 모노클로날 항체를 제조하였다. 모노클로날 항체를 클로닝하였으며, 생성된 항체를 MabF22로 명명하였고, IgG1 중사슬과 k 경사슬의 isotype을 나타냈다. 또한 immunoblotting 방법과 ELISA로 특이도를 조사한 결과 모노클로날 항체는 AFP와만 반응하였고, 결합 친화상수는 0.8$\times$$10^{-10}$M이었다. 두 종류의 효소면역분석법 -경쟁적 또는 비경쟁적 분석 -을 이용하여 항체의 효용성을 조사하였으며, 두 방법 모두 AFP와 농도에 비례하여 반응하였다. 따라서 본 연구에서 생산된 모노클로날 항체는 연구목적으로 뿐만 아니라 AFP 농도를 측정하기 위한 면역진단시약의 개발에도 유용할 것으로 생각된다.

• The Korean Journal of Physiology and Pharmacology
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• 제24권2호
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• pp.185-191
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• 2020

Interstitial cells of Cajal (ICC) are known as the pacemaker cells of gastrointestinal tract, and it has been reported that acute gastroenteritis induces intestinal dysmotility through antibody to vinculin, a cytoskeletal protein in gut, resulting in small intestinal bacterial overgrowth, so that anti-vinculin antibody can be used as a biomarker for irritable bowel syndrome. This study aimed to determine correlation between serum anti-vinculin antibody and ICC density in human stomach. Gastric specimens from 45 patients with gastric cancer who received gastric surgery at Kangwon National University Hospital from 2013 to 2017 were used. ICC in inner circular muscle, and myenteric plexus were counted. Corresponding patient's blood samples were used to determine the amount of anti-vinculin antibody by enzyme-linked immunosorbent assay. Analysis was done to determine correlation between anti-vinculin antibody and ICC numbers. Patients with elevated anti-vinculin antibody titer (above median value) had significantly lower number of ICC in inner circular muscle (71.0 vs. 240.5, p = 0.047), and myenteric plexus (12.0 vs. 68.5, p < 0.01) compared to patients with lower anti-vinculin antibody titer. Level of serum anti-vinculin antibody correlated significantly with density of ICC in myenteric plexus (r = -0.379, p = 0.01; Spearman correlation). Increased level of circulating anti-vinculin antibody was significantly correlated with decreased density of ICC in myenteric plexus of human stomach.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.829-832
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• 2001

Phage antibody 생산방법은 유전자 조작에 의하여 phage coat에 항체를 발현시키는 방법이며 그 library를 이용하여 일반 hybridoma 방법에 의해 항체를 생산하기 어려운 성분 (예: 독성물질, 면역화가 잘 안되는 물질 등) 에 대해 적용할 수 있는 장점을 제공한다. 본 연구진은 Griffin.1 antibody library의 positive control을 통해 phage 표면에 항체가 발현되는 지의 여부를 ELISA test를 통하여 확인하였다. 또한 human serum albumin을 모델분석물질로 사용하여 $1{\sim}4$차까지의 bio-panning 을 실시하였고 각 단계에서 증폭된 phage를 가지고 ELISA test를 한 결과 신호가 점진적으로 증가하는 data를 얻을 수 있었다. 이것을 통해 phage library로부터 특정 항원에 대한 monoclonal antibody를 생산할 수 있다는 사실을 검증하였다.

• BMB Reports
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• 제30권5호
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• pp.315-319
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• 1997

It was discovered that monoclonal anti-erythropoietin (EPO) antibody CFC-6 can neutralize EPO-induced cell activation. To know the binding site of CFC-6, recombinant human erythropoietin (rhEPO) was digested with Glu-C, followed by a separation using high performance liquid chromato graphy (HPLC). Each HPLC fraction was blotted on the nitrocellulose membrane and the membrane was treated with anti-EPO antibody CFC-6 and anti-mouse antibody which is modified with peroxidase. Only one spot showed the color and the fraction was sequenced by Edman degradation. The results suggest that CFC-6 recognizes amino acid sequence V63-W-Q-G-L-A-L-L-S-E72 which is a part of helix II of the EPO molecule. Binding of CFC-6 to EPO may inhibit EPO binding to its receptor, which implies that the antibody binding site and the receptor binding site are close or overlapping.

• 대한미생물학회지
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• 제19권1호
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• pp.79-83
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• 1984

To determine the existence of anti-HLA antibodies finally in 220 human placental extracts to be proved negative antiserum by previous anti-HLA A,B,C antibody screening procedure, the present study was performed by fractionation of immunoglobulins using saturated ammonium sulfate and by simple batch method on DEAE cellulose. Thereafter using known 150 T-lymphocyte panels, complement-dependent microlymphocytotoxicity test was performed to observe the existence of anti-HLA antibodies and the degree of the antibody response of the concentrates. The following results were obtained: 1. Of total 141 placental sera concentrated 45 cases(31.9%) were showed significant anti-HLA A,B,C antibody response after concentration(Excellent, 19(13.5%), Good, 3(2.1%), Weak, 23(16.3%)). 2. Anti-HLA specificities of placental sera obtained after concentration were A2, A24, B13, B27, B44, B51, CN1, C7. 3. A new type C new-1 anti-HLA antibody that is only expressed in Korean people, was obtained. 4. 79 placental sera purrified by simple batch method using DEAE cellulose were showed negative anti-HLA antibody responses.

• Journal of Microbiology and Biotechnology
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• 제28권8호
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• pp.1376-1383
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• 2018

The hepatitis B virus (HBV) envelope contains small (S), middle (M), and large (L) proteins. PreS1 of the L protein contains a receptor-binding motif crucial for HBV infection. This motif is highly conserved among 10 HBV genotypes (A-J), making it a potential target for the prevention of HBV infection. In this study, we successfully generated a neutralizing human monoclonal antibody (mAb), 1A8 (IgG1), that recognizes the receptor-binding motif of preS1 using a phage-displayed human synthetic Fab library. Analysis of the antigen-binding activity of 1A8 for different genotypes indicated that it can specifically bind to the preS1 of major HBV genotypes (A-D). Based on Bio-Layer interferometry, the affinity ($K_D$) of 1A8 for the preS1 of genotype C was 3.55 nM. 1A8 immunoprecipitated the hepatitis B virions of genotypes C and D. In an in vitro neutralization assay using HepG2 cells overexpressing the cellular receptor sodium taurocholate cotransporting polypeptide, 1A8 effectively neutralized HBV infection with genotype D. Taken together, the results suggest that 1A8 may neutralize the four HBV genotypes. Considering that genotypes A-D are most prevalent, 1A8 may be a neutralizing human mAb with promising potential in the prevention and treatment of HBV infection.

• 한국진공학회:학술대회논문집
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• 한국진공학회 2013년도 제44회 동계 정기학술대회 초록집
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• pp.84-84
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• 2013

FcBP consisting of 13 amino acids specifically binds to Immunoglobulin G Fc domain. Initially, we utilized this peptide for preparation of antibody chip as a PEG composite for enhanced solubility. After then, the peptide conjugate was immobilized on agarose resin, resulting in highly efficient affinity column for antibody purification. The efficiency was comparable to commercial Protein A column. Recently, this peptide was conjugated with cell penetratingpeptide (CPP) on a backbone of GFP, affording antibody transducer, which carries antibody into live cells by simple mixing of antibody and the transducer in cell culture media. Antibody transduction into cells was monitored by live cell imaging. More recently, the FcBP was fused to ferritin cage, which consists of 24 ferritin protein molecules. The FcBP-ferritin cage showed greatly increased binding affinity to human IgG. Its binding was analyzed by QCM and SPR analysis. Finally, it was selectively delivered by Herceptin to SKBR3, a breast cancer cell, over MCF10A, non-tumorigenic cells.

• Agricultural and Biosystems Engineering
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• 제5권1호
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• pp.21-24
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• 2004

Antibody based biosensors are very selective and ultra-sensitive. Antigen-antibody reactions have been used in immunoassays. In this research, a biosensor which uses antigen-antibody reaction was developed to measure and detect rat IgG. Because the antigen-antibody reaction is a physical bounding between antigen and antibody, there are several ways to measure an antigen-antibody reaction. Among the methods, impedance analysis has short measuring time and possibilities of analyzing various properties of the reaction using frequency analysis. Rat IgG could be detected with developed biosensor and impedance analyzer. The biosensor showed good repeatability and availability of detecting concentration changes of rat IgG.

• IMMUNE NETWORK
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• 제2권3호
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• pp.175-181
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• 2002

Background: S100A6 is a calcium-binding protein overexpressed in several tumor cell lines including melanoma with high metastatic activity and involved in various cellular processes such as cell division and differentiation. To detect S100A6 protein in patient' samples (ex, blood or tissue), it is essential to produce a monoclonal antibody specific to the protein. Methods: First, cDNA coding for ORF region of human S100A6 gene was amplified and cloned into the expression vector for GST fusion protein. We have produced recombinant S100A6 protein and subsequently, monoclonal antibodies to the protein. The specificity of anti-S100A6 monoclonal antibody was confirmed using recombinant S100A recombinant proteins of other S100A family (GST-S100A1, GST-S100A2 and GST-S100A4) and the cell lysates of several human cell lines. Also, to identify the specific recognition site of the monoclonal antibody, we have performed the immunoblot analysis with serially deleted S100A6 recombinant proteins. Results: GST-S100A6 recombinant protein was induced and purified. And then S100A6 protein excluding GST protein was obtained and monoclonal antibody to the protein was produced. Monoclonal antibody (K02C12-1; patent number, 330311) has no cross-reaction to several other S100 family proteins. It appears that anti-S100A6 monoclonal antibody reacts with the region containing the amino acid sequence from 46 to 61 of S100A6 protein. Conclusion: These data suggest that anti-S100A6 monoclonal antibody produced can be very useful in development of diagnostic system for S100A6 protein.