• BMB Reports
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• 제55권3호
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• pp.142-147
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• 2022

Human pluripotent stem cells (PSCs) have been utilized as a promising source in regenerative medicine. However, the risk of teratoma formation that comes with residual undifferentiated PSCs in differentiated cell populations is most concerning in the clinical use of PSC derivatives. Here, we report that a monoclonal antibody (mAb) targeting PSCs could distinguish undifferentiated PSCs, with potential teratoma-forming activity, from differentiated PSC progeny. A panel of hybridomas generated from mouse immunization with H9 human embryonic stem cells (hESCs) was screened for ESC-specific binding using flow cytometry. A novel mAb, K312, was selected considering its high stem cell-binding activity, and this mAb could bind to several human induced pluripotent stem cells and PSC lines. Cell-binding activity of K312 was markedly decreased as hESCs were differentiated into embryoid bodies or by retinoic acid treatment. In addition, a cell population negatively isolated from undifferentiated or differentiated H9 hESCs via K312 targeting showed a significantly reduced expression of pluripotency markers, including Oct4 and Nanog. Furthermore, K312-based depletion of pluripotent cells from differentiated PSC progeny completely prevented teratoma formation. Therefore, our findings suggest that K312 is utilizable in improving stem cell transplantation safety by specifically distinguishing residual undifferentiated PSCs.

• 대한바이러스학회지
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• 제28권4호
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• pp.383-391
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• 1998

A murine monoclonal antibody (mAb) specific for the envelope glycoprotein gp120 of human immunodeficiency virus type-I (HIV -1) was chemically coupled to pokeweed antiviral protein (PAP) from Phytolacca americana. The immunotoxin was purified by FPLC using S200 colum. The purified immunotoxin efficiently bound to HIV-infected T cells as evidenced by fluorescenceactivated cell sorter analysis. The immunotoxin selectively killed human T lymphoid lines infected with $HIV-1_{IIIB}$ at less than 250 pM of the immunotoxin cells, while PAP or mAb alone did not have any significant effect on infected cells. The uninfected control T cell lines were not affected. Human cells infected with HIV-2 or other HIV-1 strains were not killed, suggesting that the killing depends completely on the antibody used for coupling. These in vitro results suggest that the PAP-mAb conjugate may be used to selectively remove cells expressing viral antigens from individuals infected with HIV.

• 대한수의학회지
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• 제37권3호
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• pp.547-554
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• 1997

Galectin-3 is known as an animal ${\beta}$-galactoside-binding lectin charicterized with S-type carbohydrate recognition domain. It plays a role in growth, adherence and movement of cells. It is, also, related to the cell transformation and metastasis of tumor cells. In this study, we have expressed and purified recombinant human galectin-3 (rHgalectin-3) using E coli system and asialofetuin affinity chromatography for the future development of monoclonal antibody to Hgalectin-3, which is suggested as the tumor marker for the gastric and thyroid gland cancers. Expressed protein was confirmed as the Hgalectin-3 by immunoblot with cross-reactive murine monoclonal antibody. Lectin activity and specificity of purified protein were, also, confirmed by the competitive inhibition with galectin-3 specific carbohydrate, lactose. Like physiological galectin-3, lectin activity of the molecule was not changed in nonreduced condition. Dimer formation, furthermore, was observed at high concentration of the protein even in the reduced condition, which is well known in physiological galectin-3. These results showed purified rHgalectin-3 has the same activity and molecular nature compared to the physiological galectin-3.

• Biomolecules & Therapeutics
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• 제3권4호
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• pp.266-272
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• 1995

The analysis of membrane receptors for hormones and neurotransmitters has progressed considerably by pharmacological and biochemical means and more recently through the use of specific antibodies. To generate and characterize a moloclonal antibody against $\beta$-adrenergic receptor, a synthetic $\beta$2-adrenergic receptor peptide (Phe-Gly-Asn-Phe-Trp-Cys-Phe-Trp-Thr-Ser-lle-Asp-Val-Leu) which may comprise part of $\beta$-adrenergic receptor ligand binding pocket was coupled to Keyhole Limpet Hemocyanin (KLH) and used as an immunogen. Male BALB/C mice were immunized with this antigen and the immunized spleen was fused with myeloma SP2/0-Ag14 cells to produce monoclonal antibodies. Two clones were obtained but one of monoclonal antibodies, mAb5G09, was used throughout in this study because the other clone, mAb5All showed weak immunoreactivity against KLH as well. The mouse monoclonal antibody mAb5G09 produced in this study showed immunoreactivity to peptide-KLH conjugates and also to human A43l cells and guinea pig lung $\beta$2-adrenergic receptor as revealed by ELISA and western blot. In the course of determination of the effects of mAb5G09 on $\beta$-receptor ligand binding, it was observed that mAb5G09 specifically bound $\beta$-adrenergic radioligand [$^3$H]dihydroalprenolol (DHA) with a dissociation constant (Kd) of 60 nM. The [$^3$H]DHA binding activity of mAb5G09 had characteristics of immunoglobulins and the binding activity was not observed in the control anti-KLH monoclonal antibody. The monoclonal antibody, mAb5G09 produced in this study may provide useful models for the study of the structure of receptor binding sites.

• BMB Reports
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• 제31권1호
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• pp.58-63
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• 1998

In this study, a mouse monoclonal antibody (mAb) against gp41(584-618), the immunodominant epitope protein, was generated. For this purpose, BALB/c mice were immunized with double branched multiple antigenic peptides derived from the HIV-1 gp41(584-618) sequence, and antibody-secreting hybridoma were produced by fusion of mice splenocytes with SP2/0 myeloma cells. One clone producing an antigen specific mAb, termed KI-41(isotype IgG1) was identified, whose specific reactivity against gp41(584-618) could be confirmed by ELISA and Western blot analysis. Epitope mapping revealed the recognition site of the mAb KI-41 to be located around the sequence RILAVERYLKDQQLLG, which comprises the N-terminal region within the immunized gp41(584-618) peptied. Since this mAb recognizes this specific epitope within the HIV-1 gp41 without any cross-reactivity to other immunodominant regions in the HIV-2 gp35, KI-41 will provide some alternative possibilities in further applications such as the development of indirect or competitive ELISA for specific antibody detection in HIV-1 infection or for other basic researches regarding the role and function of HIV-1 gp41.

• IMMUNE NETWORK
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• 제8권1호
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• pp.7-12
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• 2008

Background: Members belonging to the interferon-lambda (IFN-${\lambda}$) family exert protective action against viral infection; however, the mechanisms of their action have remained elusive. To study IFN-${\lambda}$ biology, such as endocytosis of IFN-${\lambda}$, we produced monoclonal antibodies (Abs) against human IFN-${\lambda}$ and examined their usefulness. Methods: We purified recombinant human IFN-${\lambda}$1 expressed in Escherichia coli by using affinity columns. Then, we generated hybridoma cells by fusing myeloma cells with splenocytes from IFN-${\lambda}$1-immunized mice. For evaluating the neutralizing activity of the monoclonal Abs against IFN-${\lambda}$1, we performed RT-PCR for the MxA transcript. In order to study the binding activity of IFN-${\lambda}$ and the monoclonal Ab complex on HepG2 cells, we labeled the monoclonal Ab with rhodamine and determined the fluorescence intensity. Results: Four hybridoma clones secreting Abs specific to IFN-${\lambda}$1 were generated and designated as HL1, HL2, HL3, and HL4. All the Abs reacted with IFN-${\lambda}$1 in the denatured form as well as in the native form. Abs produced by HL1, HL3, and HL4 did not neutralize the induction of the MxA gene by IFN-${\lambda}$1. We also demonstrated the binding of the HL1 monoclonal anbitody and IFN-${\lambda}$ complex on HepG2 cells. Conclusion: Monoclonal Abs against IFN-${\lambda}$1 were produced. These Abs can be used to study the cellular binding and internalization of IFN-${\lambda}$.

• Parasites, Hosts and Diseases
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• 제31권3호
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• pp.193-200
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• 1993

국내의 CUPtOSPOTidiUm 인체감염 실태를 조사하기 위하여 연세대학 세브란스 병원을 찾은 230명의 외래 환자 분변을 수거하였다. Acid-fast 염색, auramine-rhodamine 염색과 CWptosporidiwpowumoocyst에 특이적인 단클론 항체를 이용한 동정법을 이용하였다. 230명의 환자 중 48명(21%)이 AF 염색법에 의하여 50명(22%)이 AR 염색법에 의하여, 그리고 23명(10%)이 단클론 항체를 이용하는 형광현미경법으로 각각 Cryptosporidium에 감염된 것으로 조사되어 국내에서도 Cryptosporidium 인체 감염이 존재하고 있는 것으로 나타났다.

• Parasites, Hosts and Diseases
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• 제51권1호
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• pp.93-98
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• 2013

A field applicable diagnostic technique, the dipstick assay, was evaluated for its sensitivity and specificity in diagnosing human Schistosoma mansoni infection. A monoclonal antibody (mAb) against S. mansoni adult worm tegumental antigen (AWTA) was employed in dipstick and sandwich ELISA for detection of circulating schistosome antigen (CSA) in both serum and urine samples. Based on clinical and parasitological examinations, 60 S. mansoni-infected patients, 30 patients infected with parasites other than schistosomiasis, and 30 uninfected healthy individuals were selected. The sensitivity and specificity of dipstick assay in urine samples were 86.7% and 90.0%, respectively, compared to 90.0% sensitivity and 91.7% specificity of sandwich ELISA. In serum samples, the sensitivity and specificity were 88.3% and 91.7% for dipstick assay vs. 91.7% and 95.0% for sandwich ELISA, respectively. The diagnostic efficacy of dipstick assay in urine and serum samples was 88.3% and 90.0%, while it was 90.8% and 93.3% for sandwich ELISA, respectively. The diagnostic indices of dipstick assay and ELISA either in serum or in urine were statistically comparable (P>0.05). In conclusion, the dipstick assay offers an alternative simple, rapid, non-invasive technique in detecting CSA or complement to stool examinations especially in field studies.

• Journal of Microbiology and Biotechnology
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• 제29권1호
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• pp.114-126
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• 2019

This paper introduces three ways to determine host-guest complexation of cucurbit[7]uril (CB[7]) with homocysteine (Hcy). After preincubating Hcy and cysteine (Cys) with CB[7], Ellman's reagent (DTNB) was used to detect Hcy and Cys. Only Cys reacted with DTNB and Hcy gave a retarded color change. This suggests that the -SH group of Hcy is buried inside CB[7]. Human cystathionine ${\gamma}-lyase$ (hCGL) decreased the level of Hcy degradation after preincubating Hcy and CB[7]. These results suggest that the amount of free Hcy available was decreased by the formation of a Hcy-CB[7] complex. The immunological signal of anti-Hcy monoclonal antibody was decreased significantly by preincubating CB[7] with Hcy. The ELISA results also show that ethanethiol group ($-CH_2CH_2SH$) of Hcy, which is an epitope of anti-Hcy monoclonal antibody, was blocked by the cavity in CB[7]. Overall, CB[7] can act as a host by binding selectively with Hcy, but not Cys. The calculated half-complexation formation concentration of CB[7] was 58.2 nmol using Ellman's protocol, 97.9 nmol using hCGL assay and 87.7 nmol using monoclonal antibody. The differing binding abilities of Hcy and Cys towards the CB[7] host may offer a simple and useful method for determining the Hcy concentration in plasma or serum.

• BMB Reports
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• 제57권4호
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• pp.188-193
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• 2024

Gastric cancer (GC), a leading cause of cancer-related mortality, remains a significant challenge despite recent therapeutic advancements. In this study, we explore the potential of targeting cell surface glucose-regulated protein 94 (GRP94) with antibodies as a novel therapeutic approach for GC. Our comprehensive analysis of GRP94 expression across various cancer types, with a specific focus on GC, revealed a substantial overexpression of GRP94, highlighting its potential as a promising target. Through in vitro and in vivo efficacy assessments, as well as toxicological analyses, we found that K101.1, a fully human monoclonal antibody designed to specifically target cell surface GRP94, effectively inhibits GC growth and angiogenesis without causing in vivo toxicity. Furthermore, our findings indicate that K101.1 promotes the internalization and concurrent downregulation of cell surface GRP94 on GC cells. In conclusion, our study suggests that cell surface GRP94 may be a potential therapeutic target in GC, and that antibody-based targeting of cell surface GRP94 may be an effective strategy for inhibiting GRP94-mediated GC growth and angiogenesis.