• 제목/요약/키워드: IgA purification

검색결과 47건 처리시간 0.028초

Biochemical Application of IgG Fc-binding peptide: From Biochip to Targeted Nano Carrier

  • Chung, Sang Jeon
    • 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.

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Development of Chicken Immunoglobulin Y for Rapid Detection of Cronobacter muytjensii in Infant Formula Powder

  • Kim, Yesol;Shukla, Shruti;Ahmed, Maruf;Son, Seokmin;Kim, Myunghee;Oh, Sejong
    • 한국축산식품학회지
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    • 제32권6호
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    • pp.706-712
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    • 2012
  • The present study was aimed to produce a chicken polyclonal antibody against Cronobacter muytjensii and to develop an immunoassay for its detection. Purification of anti-C. muytjensii IgY from egg yolk was accomplished using various methods such as water dilution and salt precipitation. As a result, sodium dodecyl sulfate-polyacrylamide gel electrophoresis produced two bands around 30 and 66 kDa, corresponding to a light and a heavy chain, respectively. Indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was performed to determine the effectiveness of the chicken IgY against C. muytjensii. The optimum conditions for detecting C. muytjensii by indirect ELISA and checkerboard titration of the antigen revealed an optimum average absorbance at the concentration of 18 ${\mu}g/mL$, having ca. $10^8$ coated cells per well. The anti-C. muytjensii IgY antibody had high specificity for C. muytjensii and low cross-reactivity with other tested pathogens. In this assay, no cross-reactivity was observed with the other genera of pathogenic bacteria including Escherichia coli O157:H7, Salmonella Typhimurium, Staphylococcus aureus, Bacillus cereus, Enterobacter aerogenes, Salmonella Enteritidis and Listeria monocytogenes. In addition, detection of C. muytjensii in infant formula powder showed a low matrix effect on the detection curve of IC-ELISA for C. muytjensii, with similar detection limit of $10^5$ CFU/mL as shown in standard curve. These findings demonstrate that the developed method is able to detect C. muytjensii in infant formula powder. Due to the stable antibody supply without sacrificing animals, this IgY can have wide applications for the rapid and accurate detection of C. muytjensii in dairy foods samples.

An effective immunoaffinity clean-up method for multi-DDT residue analysis

  • Hong, Ji-Youn;Hong, Jee-Eun;Lee, Eun-Ah;Park, Song-Ja;Lho, Dong-Seok;Kim, Jong-Hyun;Choi, Myung-Ja
    • 대한약학회:학술대회논문집
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    • 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.1
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    • pp.290.3-291
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    • 2003
  • To increase detection sensitivity for multi-DDT residues (o,p-/p,p-DDT, o,p-/p,p-DDE, o,o-/o,p-DDD) analysis, a highly selective sample clean-up method was introduced prior to GC/MS analysis using immunoaffinity column. The immunoaffinity matrix was prepared by coupling IgG fraction of DDT antiserum to cyanogens bromide activated Sepharose 4B. Three DDT antisera (DDA-1, DDHP-2, DDCP-3) were test for affinity column ligand that obtained by imunizing respective DDT immunogen to rabbits, and IgG was purified using protein A affinity purification. (omitted)

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J-Chain의 순수분리에 관한 연구 (Purification of J-Chain)

  • Kang, Yoon-Se;Kang, Shin-Sung
    • 한국동물학회지
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    • 제19권2호
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    • pp.63-70
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    • 1976
  • 免疫抗體分子의 基本 構造成分인 H-chain, L-chain외에 重合抗體分子 속에서만 발견되는 이른바, J-chain의 構造와 機能을 밝히기 위한 前段階로 J-chain의 純粹分離를 試圖하였다. 우선 多發性骨髓腫 患者의 血淸으로부터 重合型 IgA를 純粹分離한후, 이를 환원시켜 L-J-chain 混合物을 얻은 다음 3가지 方法, 1) 제조용 디스크 전기영동법 2) 脫鹽 透析法, 3) 이온-교환 크로마토그라피법으로 J-chain을 순수분리할 수 있었다. 위 3가지 방법으로 분리한 J-chain의 物理化學的 및 生化學的 성상은 동일하였으며, 사용한 세가지 방법 중 脫鹽 透析法이 가장 간편하고 효과적인 方法임을 알았다.

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DEAE-Trisacryl 크로마토그래피법에 의한 IgG1 Type 쥐 단일클론 항체의 분리정제 (Purification of IgG1 Type Mouse Monoclonal Antibodies with DEAE-Trisacryl Chromatography)

  • 최태부;정용근
    • 한국미생물·생명공학회지
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    • 제16권5호
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    • pp.335-342
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    • 1988
  • 하이브리도마를 쥐의 복강이나 in-vitro에서 배양한 뒤 생산된 IgG1 type의 쥐 단일클론 항체를 정제하기 위하여 음이온 교환 크로마토그래피를 이용하였다. 배양이 끝난 배지를 원심분리하여 세포를 제거하고 50-60% ammonium sulfate로 침전물을 만든 다음 0.025M Tris-HCI(pH8.2)용액으로 투석하여 salt가 제거된 sample을 DEAE-Trisacryl M에 부하하였다. Column에 결합된 항체는 30-40mM NaCl 을 포함하는 0.025M Tris-HCI(pH8.2)용액으로 용출하였다. 혈청농도가 높은 배지 (10% FBS)에서는 50% ammonium sulfate 처리로 90% 이상의 항체가 회수되었으나 저혈청 배지 (2% FBS)에서는 60% ammonium sulfate 처리에도 회수율이 84%에 그쳤다. 후자의 경우 한외여과법 (ultrafiltration)을 이용하여 항체 회수율을 91%까지 증가시킬 수 있으나 농축된 항체를 크로마토그래피로 정제하였을 때 그순도가 ammonium sulfate 침전법에 비해 낮아졌다. 하이브리도마 Alps 25-3, HCGK, A4W, KW를 여러가지 배양조건에서 배양한 뒤 생산된 항체를 DEAE-Trisacryl M chromatography를 이용하여 정제해 본 결과 대체로 순도 70-80%의 항체를 얻을 수 있었고 이때 항체 회수율은 65% 선이었다. 항체의 순도를 높이기 위해서는 affinity chromatography 혹은 gel filtration과 같은 2차적인 방법이 필요 할 것으로 보이며 한 예로 affinity chromatography를 이용하여 순도 95%의 항체를 얻었다.

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Human T-cell Leukemia Virus Type I (HTLV-I) 의 Gag-Pro Transframe 단백질 정제를 위한 재조합 DNA 의 제작 (Construction of Recombinant DNA for Purification of the Gag-Pro Transframe Protein of Human T-cell Leukemia Virus Type I (HTLV-I) )

  • 남석현
    • 미생물학회지
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    • 제30권6호
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    • pp.466-471
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    • 1992
  • HTLV-I 의 gag-pro 유전자 중첩영역내에서 -1 ribosomal frameshifting 이 일어나는 자리를 결정하기 위하여 gag-pro 중첩영역의 일부를 SP6 promoter 를 가진 백터내에 클로닝하였다. 그 결과 닭의 prelysozyme 에서 유래한 5개의 아미노산을 코드하는 합성유전자와 141 bp 로된 gag-pro 중첩영역의 뒤에 Straphylococcus aureus 의 protein A 유전자단편이 연결된 hybrid 유전자를 보유한 플라스미드를 제작하였다. 이 DNA 클론을 주형으로 SP6 RNA polymerase 의 작용에 의해 한종류의 mRNA 를 다량으로 합성하였다. Invitro 에서 합성된 mRNA 로 무세포계에서 단백질을 합성한 결과 21 kDal 의 단백질이 생성되었고 IgG-Sepharose 를 사용한 affinity chromatography 로 합성된 단백질을 순수하게 정제할 수 있었다. 본연구에서 설명한 in vitro 실험계는 Gag-Pro transframe 단백질의 신속한 정제 및 일차구조의 결정에 유익하게 사용될 것으로 보이며 이와 같은 실험의 결과 mRNA 에서 ribosomal frameshifting 이 일어나는 정확한 site 를 결정할 수 있을 뿐 같은 실험의 결과 mRNA 에서 ribosomal frameshifting 이 일어나는 정확한 site 를 결정할 수 있을 뿐 아니가 pro 유전자의 발현에 필요한 frameshift 를 유도하는 tRNA 의 동정도 가능하게 될 것이다.

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Purification and Anti-pathogenic Properties of Immunoglobulin Concentrates from Porcine Blood

  • Jung, Tae-Hwan;Choi, Jae-Hwan;Koh, Kyung-Chul;Jeon, Woo-Min;Han, Kyoung-Sik
    • 한국축산식품학회지
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    • 제37권5호
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    • pp.743-751
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    • 2017
  • During slaughtering, animal blood is typically discarded, resulting in water pollution. However, this discarded blood has valuable components, such as immunoglobulin (Ig). Although several studies have been conducted to develop methods for effective recycling of slaughterhouse blood, they have not been commercially utilized in Korea. Here, we extracted an Ig-rich fraction from porcine blood that was then subjected to various in vitro tests, including pathogen growth inhibition, antigenic cross-reactivity, and anti-toxin activity. The porcine immunoglobulin concentrate (PIC) was effectively purified by eliminating other components, such as albumin, and consisted of approximately $63.2{\pm}2.9%$ IgG and $7.2{\pm}0.4%$ IgM on a protein basis. The results showed that it significantly suppressed the growth of pathogenic bacteria, and bound to all tested pathogens, including both gram-positive and gram-negative species, although the degree of activity differed according to strain. The PIC bound to two types of lipopolysaccharide (LPS) obtained from Escherichia coli O111:B4 and Salmonella enterica serotype typhimurium in a concentration-dependent manner. In addition, the PIC restored the proliferation activity of the lymphoblast K-562 cells when co-incubated with pathogenic LPS. These results confirm that the PIC prepared in this study is a potentially valuable functional food material or diet supplement as an alternative to antibiotics that can protect animals from pathogenic bacteria.

Biochemical Application of IgG Fc-Binding Peptide: From Biochip to Targeted Nano Carrier

  • Chung, Sang J.
    • 한국진공학회:학술대회논문집
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    • 한국진공학회 2013년도 제44회 동계 정기학술대회 초록집
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    • pp.110-111
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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 penetrating peptide (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 (Fig. 1). Fig. 1. Fluorescent microscopic images of SKBR3 breast cancer cells (A~C) and MCF10A breast cells (D~F) treated with Cy3-trastuzumab/fFcBP-Pf_Fn complexes. Trastuzumab and FcBP-Pf_Fn, which were labeled with Cy3 (Cy3-trastuzumab) and fluorescein (fFcBP-Pf_Fn), respectively, selectively targeted SKBR3 over MCF10A.

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자성 미세입자에의 항체 고정화 방법이 면역결합반응에 미치는 영향 (Effect of Antibody Immobilization Method to Magnetic Micro Beads on its Immunobinding Characteristics)

  • 최효진;황상연;장대호;조형민;강정혜;성기훈;주재범;이은규
    • Korean Chemical Engineering Research
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    • 제44권1호
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    • pp.65-72
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    • 2006
  • 자력을 이용한 분리기술의 장점을 이용하여 여러 가지 불순물들이 섞여 있는 현탁 용액으로부터 자성입자를 이용하여 목적 단백질만을 얻어낼 수 있는 기술의 가능성에 대하여 알아보고자 하였다. 자성입자를 이용하는 경우에는 (1) 자성입자 표면에 리간드 고정화, (2) ligand(리간드)와 목적 물질과의 특이적 결합, (3) 자력을 이용한 자성입자의 분리, (4) 목적 물질의 탈착 그리고 (5) 자성입자의 재사용 순서로 진행되어 여러 단계의 공정을 단순화시킬 수 있다. 이러한 자성입자를 이용한 방법은 효율성, 단순성, 까다롭지 않은 조건, 자동화의 용이성, 비용의 저렴함으로 인해 관심이 증대되고 있다. 본 연구에서는 표면이 카르복실기로 처리된 자성입자에 IgG 항체를 고정화시킨 후 IgG를 목적 단백질로 하여 이를 분리해내고자 하였다. 이를 위하여 자성입자 표면에 다른 물질과의 비특이적 결합이 일어나지 않을 조건에 대하여 확인해 보았으며, IgG 항체를 배향성 고정화시켜 자성입자가 목적 단백질과 보다 효과적으로 결합하여 목적 단백질인 IgG만을 선택적으로 분리해 낼 수 있는지에 대하여 알아보았다. 높은 pH를 사용할 경우 비특이적 흡착을 줄일 수 있었으며, IgG 항체 고정화된 자성입자를 사용할 경우 목적 단백질인 IgG와 선택적으로 반응함을 알 수 있었다. IgG 항체의 고정화에서 Fc지역 C-terminus에 인접해 있는 탄수화물 부분을 이용하여 배향성을 준 경우, IgG 항체 내의 아민기와 고정화시키는 비배향성 고정화 방법보다 항원과의 결합능력이 약 2배 높았다.

Large scale purification and characterization of recombinant human autotaxin/lysophospholipase D from mammalian cells

  • Song, Yuanda;Dilger, Emily;Bell, Jessica;Barton, William A.;Fang, Xianjun
    • BMB Reports
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    • 제43권8호
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    • pp.541-546
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    • 2010
  • We utilized a mammalian expression system to purify and characterize autotaxin (ATX)/lysophospholipase D, an enzyme present in the blood responsible for biosynthesis of lysophosphatidic acid. The human ATX cDNA encoding amino acids 29-915 was cloned downstream of a secretion signal of CD5. At the carboxyl terminus was a thrombin cleavage site followed by the constant domain (Fc) of IgG to facilitate protein purification. The ATX-Fc fusion protein was expressed in HEK293 cells and isolated from conditioned medium of a stable clone by affinity chromatography with Protein A sepharose followed by cleavage with thrombin. The untagged ATX protein was further purified to essential homogeneity by gel filtration chromatography with a yield of approximately 5 mg/liter medium. The purified ATX protein was enzymatically active and biologically functional, offering a useful tool for further biological and structural studies of this important enzyme.