• Title/Summary/Keyword: Monoclonal Antibody

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$NH_2-terminal$ Amino acid Sequence Analysis of Monoclonal Antibody by Electroblotting Method (Electroblotting을 이용한 단일클론항체의 $NH_2$-말단 아미노산 배열분석)

  • Nam, Kyung-Soo;Chang, Hyeun-Wook;Chung, Kyu-Charn
    • YAKHAK HOEJI
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    • v.34 no.1
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    • pp.11-14
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    • 1990
  • $NH_2-terminal$ amino acid sequence analysis of monoclonal antibody is very important to identify gene family and diversities of antigen-antibody recognition. When we used the PVDF (Polyvinylidene difluoride) membrane blotting method, we could easily analyze $NH_2-terminal$ sequence of monoclonal-antibody which specifically binds to phosphatidylinositol 4,5-biphosphate. PVDF membrane is an ideal solid-phase support for sequence analysis, especially when used with electroblotting method. This method is superior to continual method and will be applied to the sequence analysis of picomole quantities of proteins by gel electrophoresis.

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Development of Chromatographic Downstream Processing for the Purification of Monoclonal Antibody from Ascites Fluid: Part II Use of Single Hydroxylapatite Chromatographic Step (생쥐 복수로부터의 단세포군 항체분리를 위한 크로마토그라피 분리정제 방법의 개발 Part II. 히드록실아파타이트 크로마토그라피 단일 단계만의 사용)

  • Ahn, I.S.;Park, C.Y.
    • Microbiology and Biotechnology Letters
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    • v.17 no.3
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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.

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Production and Characterization of a Monoclonal Antibody against Human ${\beta}_2$-adrenergic receptor

  • Kang, Suk-Jo;Shin, Chan-Young;Song, Mi-Ryoung;Lee, Chung-Jae;Cheong, Jae-Hoon;Lee, Sang-Bong;Ko, Kwang-Ho
    • Biomolecules & Therapeutics
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    • v.5 no.4
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    • pp.344-350
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    • 1997
  • The purpose of the present study was to produce and characterize a monoclonal antibody against human ${\beta}_2$-adrenergic receptor. Male BALB/c mice were immunized with glutathione S-transferase (GST) fusion protein of the C-terminal portion of the human ${\beta}_2$-adrenergic receptor which was expressed in E.Coli. The immunized splenocytes were fused with myeloma SP2/0-Agl4 cells. The resulting hybridomas were screened for the production of a monoclonal antibody which can recognize human ${\beta}_2$-adrenergic receptor, and then subcloned by limiting dilution. The resulting monoclonal antibody was named as mAb$\beta$CO2. The mono-clonal antibody $\beta$CO2 was determined as IgM subtype and then purified by anti-mouse IgM-agarose affinity chromatography. The results of ELISA, Western blot, and immunocytochemistry showed that mAb$\beta$CO2 recognized human ${\beta}_2$-adrenergic receptor in the ${\beta}_2$-adrenergic receptor-GST fusion protein and human spider-moid carcinoma cell line A431 with highly specific immunoreactivity, The monoclonal antibody $\beta$CO2 may provide useful tools for the study of the $\beta$-adrenergic receptor of human and other species including rats.

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Development of Competitive Direct Enzyme-linked Immunosorbent Assay for the Detection of Gentamicin Residues in the Plasma of Live Animals

  • Jin, Yong;Jang, Jin-Wook;Lee, Mun-Han;Han, Chang-Hoon
    • Asian-Australasian Journal of Animal Sciences
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    • v.18 no.10
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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.

Production of Group Specific Monoclonal Antibody to Aflatoxins and its Application to Enzyme-linked Immunosorbent Assay

  • Kim, Sung-Hee;Cha, Sang-Ho;Karyn, Bischoff;Park, Sung-Won;Son, Seong-Wan;Kang, Hwan-Goo
    • Toxicological Research
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    • v.27 no.2
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    • pp.125-131
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    • 2011
  • Through the present study, we produced a monoclonal antibody against aflatoxin B1 (AFB1) using AFB1-carboxymethoxylamine BSA conjugates. One clone showing high binding ability was selected and it was applied to develop a direct competitive ELISA system. The epitope densities of AFB1-CMO against BSA and KLH were about 1 : 6 and 1 : 545, respectively. The monoclonal antibody (mAb) from cloned hybridoma cell was the IgG1 subclass with ${\lambda}$-type light chains. The $IC_{50}s$ of the monoclonal antibody developed for AFB1, AFB2, AFG1 and AFG2 were 4.36, 7.22, 6.61 and 29.41 ng/ml, respectively, based on the AFB1-KLH coated ELISA system and 15.28, 26.62, 32.75 and 56.67 ng/ml, respectively, based on the mAb coated ELISA. Cross-relativities of mAb to AFB1 for AFB2, AFG1 and AFG2 were 60.47, 65.97 and 14.83% in the AFB1-KLH coated ELISA, and 59.41, 46.66 and 26.97% in the mAb coated ELISA, respectively. Quantitative calculations for AFB1 from the AFB1-Ab ELISA and AFB1-Ag ELISA ranged from 0.25 to 25 ng/ml ($R^2$ > 0.99) and from 1 to 100 ng/ml ($R^2$ > 0.99), respectively. The intra- and inter-assay precision CVs were < 10% in both ELISA assay, representing good reproducibility of developed assay. Recoveries ranged from 79.18 to 91.27%, CVs ranged from 3.21 to 7.97% after spiking AFB1 at concentrations ranging from 5 to 50 ng/ml and following by extraction with 70% methanol solution in the Ab-coated ELISA. In conclusion, we produced a group specific mAb against aflatoxins and developed two direct competitive ELISAs for the detection of AFB1 in feeds based on a monoclonal antibody developed.

Characterization of the Monoclonal Antibody Specific to Human S100A2 Protein (인체 S100A2 단백질에 특이적인 단일클론 항체)

  • Kim, Jae Wha;Yoon, Sun Young;Kim, Joo Heon;Joo, Jong-Hyuck;Kim, Jin Sook;Lee, Younghee;Yeom, Young Il;Choe, Yong-Kyung;Choe, In Seong
    • IMMUNE NETWORK
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    • v.3 no.1
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    • pp.16-22
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    • 2003
  • Background: The S100A2 gene, also known as S100L or CaN19, encodes a protein comprised of 99-amino acids, is a member of the calcium-binding proteins of EF-hand family. According to a recent study, this gene was over-expressed in several early and malignant carcinomas compared to normal tissues. To elucidate the role of S100A2 protein in the process during carcinogenesis, production of monoclonal antibody specific to the protein is essential. Methods: First, cDNA sequence coding for ORF region of human S100A2 gene was amplified and cloned into an expression vector to produce GST fusion protein. Recombinant S100A2 protein and subsequently, monoclonal antibody to the protein were produced. The specificity of anti-S100A2 monoclonal antibody was confirmed by immunoblot analysis of cross reactivity to other recombinant proteins of S100A family (GST-S100A1, GST-S100A4 and GST-S100A6). To confirm the relation of S100A2 to cervical carcinogenesis, S100A2 protein in early cervical carcinoma tissue was immunostained using the monoclonal antibody. Results: GST-S100A2 recombinant protein was purified by affinity chromatography and then fusion protein was cleaved and S100A2 protein was isolated. The monoclonal antibody (KK0723; Korean patent pending #2001-30294) to the protein was produced and the antibody did not react with other members of EF-hand family proteins such as S100A1, S100A4 and S100A6. Conclusion: These data suggest that anti-S100A2 monoclonal antibody produced in this study can be very useful for the early detection of cervical carcinoma and elucidation of mechanism during the early cervical carcinogenesis.

DETECTION SYSTEM OF STREPTOCOCCUS MUTANS IN SALIVA USING MONOCLONAL ANTIBODY (Monoclonal Antibody를 이용한 Streptococcus mutans 검출 방법의 임상적 적용에 관한 연구)

  • Hong, Hi-Jung;Kim, Jong-Soo;Kim, Yong-Kee
    • JOURNAL OF THE KOREAN ACADEMY OF PEDTATRIC DENTISTRY
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    • v.36 no.4
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    • pp.522-530
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    • 2009
  • The purpose of this study was to evaluate the clinical effectiveness of new streptococcus detection system which used monoclonal antibody against Streptococcus mutans. 92 children aged between 2 and 8 were involved in this experiment and their saliva samples were collected for testing. Streptococcus mutans were measured by both monoclonal antibody-based detecting system (Saliva-$check^{TM}$ Mutans) and dip slide detecting system($Dentocult^{TM}$-SM). The results showed that Saliva-$check^{TM}$ Mutans levels had a significant correlation with dfs rate of subjects and the two test kits, Saliva-$check^{TM}$ Mutans and $Dentocult^{TM}$-SM were shown to have a good correlation although they were based on different mechanism.

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Demonstration of TCM-9 Monoclonal Antibody in Follicular Neoplasm of Thyroid (갑상선의 여포상 종양의 감별에 있어서 TCM-9의 발현양상)

  • Kim, Yun-Jung;Shim, Jung-Weon;Ahn, Hye-Kyung;Park, Young-Euy
    • The Korean Journal of Cytopathology
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    • v.7 no.2
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    • pp.134-137
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    • 1996
  • Monoclonal antibody(TCM-9) against human thyroid cancers have been studied by screening with human thyroid cancers, normal and benign thyroid tissue, and normal human serum protein. A monoclonal antibody(TCM-9) that is known to have strong specificity for human thyroid cancer but not for Graves' disease, adenoma or normal thyroid does not bind to native or mature human thyroglobulin(Tg). We used to TCM-9 antibody by immunohistochemical staining on 5 follicular cancer, 2 follicular adenoma, 1 follicular neoplasm with suspicious invasion, 2 papillary cancer to ascertain being of help in differentiation between follicular carcinoma and adenoma. Reactivity of TCM-9 was observed in follicular carcinoma and papillary carcinoma but not observed in follicular adenoma. Thus TCM-9 is a novel monoclonal antibody against the thyroid cancer.

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Production of monoclonal antibody to 45 kDa somatic protein of Trichuris suis (돼지편층의 45kDa 항원단백질에 대한 단클론항체 생산)

  • Lee, Jong-Kyung;Kim, Jung-Tae;Seo, Hun-Su;Park, Jong-Yeol;Yun, Hee-Jeong
    • Korean Journal of Veterinary Research
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    • v.44 no.4
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    • pp.625-635
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    • 2004
  • Trichnuris suis does not excrete eggs during larval stage as well as in particular adult stage, It is impossible to diagnose by use of fecal examination method in those periods. Therefore, serological diagnostic method can be very useful for those stages. In order to produce monoclonal antibody, specific somatic and secretory-excretory (SE) antigens of T. suis were identified and analyzed by SDS-PAGE and Western blot. Monoclonal antibody-producing hybridoma cells were cloned, which were made of popliteal lymph node of BALB/c mice immunized with a 45 kDa somatic antigen of T. suis. Five clones (1B9, 2C4, n2C5, 2D7 and 2D8) showing strong responses to T. suis antigens were selected and the isotype identified. All monoclonal antibodies were IgG1 isotype and the light chains were k chain. Established monoclonal antibodies reacted specifically to somatic and SE antigens of T. suis and did not cross-reacted to antigens of ascaris suum, trichuris vulpis, or Trichinella spiralis. The sensitivity of somatic and SE antigens against these monoclonal antibodies were significant (p<0.01) associated with those of positive and negative sera.

Characteristics and application of monoclonal antibody to progesterone I. Production of monoclonal antibody to progesterone (Progesterone의 단크론성 항체에 관한 특성 및 활용에 관한 연구 I. 단크론성 항체의 생산)

  • Kang, Chung-boo;Kim, Yong-hwan
    • Korean Journal of Veterinary Research
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    • v.30 no.4
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    • pp.511-513
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    • 1990
  • Monoclonal antibody to progesterone was produced using the antigen $11{\alpha}$-hydroxyprogesterone hemisuccinate conjugated to bovine serum albumin. Hybridomas secreting antibody to progesterone were detected by radioimmunoassay and enzyme-linked immunosorbent assay and cloned in soft agar. Two stable monoclonal antibodies which were highly specific to progesterone were obtained, so it may be advantageously used to study on several physiological functions of progesterone including immunological research.

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