• BMB Reports
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• 제32권5호
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• pp.474-479
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• 1999

Using a hybridoma technique, spleen cells of Balb/c mice immunized with human chorionic gonadotropin (hCG) were fused with NS-1 mouse myeloma cells. Two hybrid cell lines, clones KS-8 and KS-19, secreting monoclonal antibodies to hCG, were isolated. KS-8 and KS-19 belong to the immunoglobulin $G_1$ subclass. With the aid of a double-antibody radioimmunoassay, it was established that the KS-8 monoclonal antibody recognizes an immunodeterminant of the $\beta$-subunit of hCG, whereas the KS-19 monoclonal antibody recognizes an epitope present on the $\alpha$-subunit of hCG. The KS-8 monoclonal antibody specifically reacts with human chorionic gonadotropin and shows cross-reactivity of less than 0.3% to other related human glycoprotein hormones. On the other hand, using a hemagglutination test based on antibody-induced agglutination of sheep red blood cells coated with hCG, It was shown that only the KS-19 monoclonal antibody was capable of inducing a positive reaction, although both monoclonal antibodies had similar binding capacity to the coated cells. The results from the dual screening procedures demonstrate that KS-8 and KS-19 monoclonal antibodies show high sensitivity in two different assays, and are hence useful for the qualitative and quantitative determination of hCG by both radioimmunoassay and hemagglutination inhibition tests.

• 한국미생물·생명공학회지
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• 제17권3호
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• pp.269-272
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• 1989

In order to obtain monoclonal antibody from ascites fluid at sufficiently high purity using a single hydroxylapatite chromatography (HA) a further optimization on its operating variables was carried out. By adjusting the pH of the eluent, the sodium phosphate buffer, to 6.0 from 6.8 and adding CaCl$_2$to 1 mM at the column inlet, the elution molarities (M$_{elu}$) for the desired monoclonal antibody and contaminating proteins can be distinguished from each other with enough resolution. Previously these two groups of proteins co-eluted at the same time at pH 6.8 and without CaCl$_2$. This sin81e step hydroxylapatite chromatography yields the desired antibody pure enough for diagnostic use.

• 한국동물학회지
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• 제35권1호
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• pp.29-36
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• 1992

The present study describes the production of monoclonal antibodies against cultured chick myoblast to pursue critical proteins in muscle cell fusion. Among a panel of monoclonal antibodies, three, Mll-3H 13, Mll-3Hl8 and Mll-3H35 were inhibited movblast fusion. A single 101-kDa antigen reactive with monoclonal antibody Mll-3H35 was detected by radioimmu-noprecipitation or by immunoblotting. During the course of myogenesis, the level of the protein remarkably decreased as the cells there differentiated. These results suggest that the protein platys a direct role in the process of myoblast fusion mechanism.

• BMB Reports
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• 제28권4호
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• pp.353-358
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• 1995

Epitope tagging is the process of fusing a set of amino acid residues that are recognized as an antigenic determinant to a protein of interest. Tagging a protein with an epitope facilitates various immunochemical analyses of the tagged protein with a specific monoclonal antibody. The monoclonal antibody H8 has subtype specificity for an epitope derived from the preS2 region of hepatitis B virus surface antigen. Previous studies on serial deletions of the preS2 region indicated that the preS2 epitope was located in amino acid residues 130~142. To test whether the amino acid sequence in this interval is sufficient to confer on proteins the antigenicity recognizable by the antibody H8, the set of amino acid residues in the interval was tagged to the amino terminal of ${\beta}$-galactosidase and to the carboxyl terminal of the truncated $p56^{lck}$ fragment. The tagged ${\beta}$-galactosidase, expressed in Escherichia coli, maintained the enzymatic activity and was immunoprecipitated efficiently with H8. The tagged $p56^{lck}$ fragment, synthesized in an in vitro translation system, was also immunoprecipitated specifically with H8. These results demonstrate that the amino acid sequence of the preS2 region can be used efficiently for the epitope tagging approach.

• Asian-Australasian Journal of Animal Sciences
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• 제18권10호
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• pp.1498-1504
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• 2005

Competitive direct ELISA was developed to detect gentamicin residues. Mice immunized with gentamicin-keyhole limpet hemocyanin (KLH) conjugate developed good antiserum titers, which gradually increased with booster injections, indicating immunization was successfully processed. Monoclonal antibody against gentamicin was prepared using hybridoma cells cloned by limit dilution of fused cells. IgG was purified from ascites fluid of hybridoma cell-injected mice through ammonium sulfate precipitation and Sephadex G-25 gel filtration. After the gel filtration, fractions of high antibody titer were further purified through affinity chromatography on protein A/G column. Monoclonal antibody against gentamicin was confirmed as IgG1, which has kappa light chain. Cross-reactivities ($CR_{50}$) of gentamicin monoclonal antibody to other aminoglycosides (kanamycin, neomycin, and streptomycin) were less than 0.005%, indicating the monoclonal antibody was highly specific for gentamicin. Standard curve constructed through competitive direct ELISA showed measurement range (from 80 to 20% of B/$B_0$ ratio) of gentamicin was between 1 and 40 ng/ml, and 50% of B/$B_0$ ratio was about 4 ng/ml. The gentamicin concentration rapidly increased to 1,300 ng/ml after the intramuscular administration up to 2 h, then sharply decreased to less than 300 ng/ml after 4 h of withdrawal, during which the elimination half-life ($t_{1/2}$) of gentamicin in the rabbit plasma was estimated to be 1.8 h. Competitive direct ELISA method developed in this study using the prepared monoclonal antibody is highly sensitive for gentamicin, and could be useful for detecting gentamicin residues in plasma of live animals.

• Molecules and Cells
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• 제39권6호
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• pp.460-467
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• 2016

Bacteriophytochromes are phytochrome-like light-sensing photoreceptors that use biliverdin as a chromophore. To study the biochemical properties of the Deinococcus radiodurans bacteriophytochrome (DrBphP) protein, two anti-DrBphP mouse monoclonal antibodies (2B8 and 3H7) were generated. Their specific epitopes were identified in our previous report. We present here fine epitope mapping of these two antibodies by using truncation and substitution of original epitope sequences in order to identify minimized epitope peptides. The previously reported original epitope sequences for 2B8 and 3H7 were truncated from both sides. Our analysis showed that the minimal peptide sequence lengths for 2B8 and 3H7 antibodies were nine amino acids (RDPLPFFPP) and six amino acids (PGEIEE), respectively. We further characterized these peptides in order to investigate their reactivity after single deletion and single substitution of the original peptides. We found that single-substituted 2B8 epitope (RDPLPAFPP) and dual-substituted 3H7 epitope (PGEIAD) showed significantly increased reactivity. These two antibodies with high reactivity for the short modified peptide sequences are valueble for developing new peptide tags for protein research.

• IMMUNE NETWORK
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• 제1권1호
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• pp.14-25
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• 2001

Background: Bone marrow stromal cells (BMSCs) express many cell surface molecules, which regulate the proliferation and differentiation of immune cells within the bone marrow. Methods: To identify cell surface molecules, which can regulate cell proliferation through cell interaction, monoclonal antibodies (MoAbs) against BMSCs were produced. Among them, 1H8 MoAb, which recognized distinctly an 80 kDa protein, abolished myeloma cell proliferation that was induced by co-culturing with BMSCs. Results: IL-6 gene expression was increased when myeloma or stromal cells were treated with 1H8 MoAb. In addition, the expression of IL-6 receptor and CD40 was up-regulated by 1H8 treatment, suggesting that the molecule recognized by 1H8 MoAb is involved in cell proliferation by modulating the expression of cell growth-related genes. Myeloma cells contain high levels of reactive oxygen species (ROS), which are related to gene expression and tumorigenesis. Treatment with 1H8 decreased the intracellular ROS level and increased PAG antioxidant gene concomitantly. Finally, 1H8 induced the tyrosine phosphorylation of several proteins in U266. Conclusion: Taken together, 1H8 MoAb recognized the cell surface molecule and triggered the intracellular signals, which led to modulate gene expression and cell proliferation.

• 약학회지
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• 제45권2호
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• pp.180-189
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• 2001

We have reported that water-extracted Korean mistletoe (KM-110) had various biological activities such as antitumor and immunomodulatory activity, and the pectin fraction (KML-C) of the extract was one of major factors related to its biological functions. In this paper, we produced murine monoclonal antibody (mAb) against KML-C. The cAbs obtained were largely classified into two groups according to specificity to KML-C and ML-I, a pectin from European mistletoe. One group mAbs (9H7-D10 and 3C2-lH4) strongly reacted with KML-C, but not ML-I. In contrast, another group cAbs (8Bll-2C5, BE12-3E9 and 5E10-Fl) reacted with both KML-C and ML-1. The subisotypes of these mobs were shown to be IgGl (9H7-lD10, 3C2-lH4 and 8Bll-2C5) or IgM (8E12-3E9 and 5E10-Fl). To develop an assay system for determination of the amount of KML-C, we established the sandwich ELISA (enzyme-linked immunosorbent assay) method using these mAbs and horse radish peroxidase (HRP)-labelled cAbs. In various combinations of the cAbs for coated antibody and detection antibody, the sandwich ELISA quantitatively detected KML-C, showing the detection limit ranging from 7-5,000 ng/ml. Especially reproducibility (C.V) of the sandwich ELISA, in which 8E12-3E9 was used for coating antibody and 8Bll-2C5-HRP for detection antibody, was 4.59-5.83 in intra assay, and 3.9-9.4 in inter assay.

• 대한소아치과학회지
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• 제36권4호
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• pp.522-530
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• 2009

Monoclonal antibody를 이용한 Saliva-$check^{TM}$ Mutans키트의 타액 Streptococcus mutans검출방법으로서의 활용도와 임상적 우식지수 및 기존의 세균배양방법과의 상관관계를 알아보기 위해 2008년 2월에서 5월 중 평촌키즈웰치과에 내원한 만 2세에서 만 8세 사이의 92명의 아동을 대상으로 Streptococcus mutans 검출검사를 실시하였으며 또한 우식에 영향을 미치는 다른 요인인 치면세균막 pH와 타액완충능력검사를 실시하여 다음과 같은 결론을 얻었다. 1. Saliva-$check^{TM}$ Mutans 검사결과 양성을 나타낸 아동은 27명으로 29.3%이었고, 음성을 나타낸 아동은 65명으로 70.65%이었다. 우식경험유치면률은 음성 아동 13.89%, 양성 아동 25.23%으로 나타났다. 2. Monoclonal antibody를 이용한 검사방법 인 Saliva-$check^{TM}$ Mutans와 기존의 세균 배양방법인 $Dentocult^{(R)}$-SM은 각각 상이한 검사방법을 이용함에도 불구하고 검사결과 높은 상관관계를 보였다(p<0.01). 3. Saliva-$check^{TM}$ Mutans검사와 치면세균막 pH검사와는 역상관관계를 나타내었으나(p<0.01),타액완충능 검사와는 상관관계가 나타나지 않았다(p>0.05). 이상의 결과로 보았을 때 Monoclonal antibody를 이용한 검사방법인 Saliva-$check^{TM}$ Mutans는 구강 내 Streptococcus mutans를 측정하는 방법으로 적당하며 또한 검사에 필요한 시간을 대폭 줄일 수 있고 사용방법도 매우 간편하게 개발되어 환자들에게 적용함에 있어 효과적이라고 사료되었다.

• IMMUNE NETWORK
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• 제3권2호
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• pp.118-125
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• 2003

Background: Disialoganglioside GD2 is a tumor-associated antigen that is overexpressed on tumor cells of neuroectodermal origin, such as melanoma and neuroblastoma. Anti-idiotypic antibodies that mimic GD2 may induce more effective immune responses than GD2 antigen itself, because they are protein antigens and are known to be able to break immune tolerance. In this study, to explore the potential of anti-idiotypic antibodies as tumor vaccines, the ability of anti-idiotypic antibodies (Ab2) to induce anti-anti-idiotypic antibodies (Ab3) that bind to the original antigen GD2 was investigated. Methods: Six monoclonal anti-idiotypic antibodies (1A8, 1G5, 2B6, 3A4, 3D6, 3H9) to monoclonal antibody M2058, which is a monoclonal antibody to GD2, were produced in mice. Three (1A8, 3A4, 3H9) of them were selected based on their ability to inhibit the binding of Ab1 to D142.34 (murine melanoma cell expressing GD2). These 3 different Ab2 were injected into rabbits, and rabbit Ab3 induced by each of them were characterized. Results: Ab3-containing sera from two rabbits immunized with 1A8, 3A4, or 3H9 bound significantly (P<0.05) to D142.34 but not to B78.96 (GD2-negative cell), and bound significantly (P<0.05) to isolated GD2 but not to GD1a. Ab3-containing sera from two rabbits immunized with 3A4 or 3H9 inhibited significantly (P<0.05) the binding of Ab1 M2058 to D142.34, and inhibited significantly (P<0.05) the binding of Ab1 M2058 to the Ab2. Conclusion: These results suggest that anti-idiotypic antibodies 3A4 and 3H9 have a potential to be used as vaccines against tumors expressing GD2 by inducing GD2-specific antibodies (Ab3).