• Archives of Pharmacal Research
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• 제20권1호
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• pp.46-52
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• 1997

To obtain more sensitive immunoassay for methamphetamine (MA) determination, the optimum condition of enzyme-linked immunosorbent assay (ELISA) was investigated in regard to immunogens, antibody purification methods and coating tracers. Activated MA, N-(4-aminobutyl)methamphetamine (4-ABMA), was conjugated with bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) and used as immunogen. The antibodies were purified by protein G chromatography or various immunoaffinity chromatography-linked MA-protein ligands, such as MA-BSA, MA-KLH or MA-ovalbumin (OVA). Each purified antibody was characterized by means of sensitivity and cross-reactivity using the three MA-protein coating tracers, MA-BSA, MA-KLH and MA-OVA. The best sensitivity of each antibody was acquired with the MA-OVA tracer although the tracer concentration and the antibody titer level at optimum condition were varied. The antibody with high titer level did not always yield good sensitivity. At optimum condition, immunoaffinity chromatography-purified antibodies were better for sensitivity and for specificity than protein G-purified antibodies. The cross-reactivity of the purified antibodies seemed to be affected by immunogen structure and showed somewhat different patterns according to the immunoaffinity ligand utilized. These data show that the antibody purification method as well as choice of coating tracer and immunogen is essential for the sensitivity and specificity of EIA; the optimum condition for assay should be discovered using various methods and combinations.

• 산업식품공학
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• 제13권4호
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• pp.314-319
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• 2009

Metallothioneins (MTs) are low molecular weight, cysteine-rich, and heavy metal binding proteins, which could be induced with heavy metals such as Cd, Hg, Zn and Cu in liver, kidney, and in cultured cells. By using ion exchange chromatography on DE-52, MT was purified from the serum of carp induced with cadmium in order to produce antibody against MT. Polyclonal antibody produced against purified carp MT reacted well with MT in the serum of carp induced with cadmium, whereas control serum did not. This may indicate that the polyclonal antibody against the carp MT could be used for the preparation of biosensors to detect MT in fishes like carp.

• BMB Reports
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• 제33권2호
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• pp.188-192
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• 2000

Recombinant protein G has been utilized in the purification of antibodies from various mammalian species based on the interaction of antibodies with protein G. The interaction between immunoglobulin and protein G may not be restricted to the Fc protion of antibodies, as many different $F(ab)_2$ or Fab fragments can also bind to protein G. I found both FAb $F(ab)_2$ of 145-2C11, a hamster anti-mouse $CD3{\varepsilon}$ antibody, bound to the protein G-sepharose. Interestingly, Fab and $F(ab)_2$ of 145-2C11 did not bind to the protein A-sepharose. The binding of Fab and $F(ab)_2$ of 145-2C11 to protein G provided a useful method to remove proteases, chopped fragments of the Fc region, and other contaminating proteins. The remaining intact antibody in the protease reaction mixture can be removed by using a protein A-sepharose, because the Fab and $F(ab)_2$ portions of 145-2C11 did not bind to protein A-sepharose. The specific binding of Fab and $F(ab)_2$ portions of 145-sC11 to a protein G-sepharose (though not to a protein A-sepharose) and binding of intact 145-2C11 to both protein A- and G-sepharose will be useful in developing an effective purification protocol for Fab and $F(ab)_2$ portions of 145-2C11.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1997년도 춘계학술대회
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• pp.93-93
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• 1997

Analysis of protein is often frustrated by the inability to isolate large amounts of purified protein from a native source. To overcome this problem, fusion protein expression systems such as pGEX system have been widely used. Using pGEX system, the desired protein could be easily obtained in a large amount in E. coli, and then the fusion protein could be used for the study of the function of the given protein. To analyze and purify the GST fusion protein, anti-GST antibody could be used as one of the system of choice. However, the production and characterization of monoclonal anti-GST antibody has not been studied extensively yet. To produce monoclonal anti-GST antibody, GST was purified from E. coli transformed with pGEX-cs, one of the pGEX system and was used as an antigen. The monoclonal antibody was produced by fusion of the immunized spleen cells with SP2-0 myeloma cells. The antibody was characterized by ELISA, western blotting, etc. The monoclonal antibody produced in this study (mAb-GSTA) showed strong and specific immunoreactivity against not only GST but also GST-fusion proteins. Also, mAb-GSTA was successfully used for the immunoaffinity purification of the GST ${\beta}$-Rc.-third intracellular-loop fusion protein. The results of the present study suggest that mAb-GSTA may be used for the identification and purification of GST fusion proteins.

• 한국균학회지
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• 제27권4호통권91호
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• pp.299-303
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• 1999

Aspergillus ficuum이 발효조의 배양액에서 phytase를 가장 많이 분비 생산하는 시간은 5일째였다. 이 phytase를 ultrafiltration, cation exchange column, 그리고 anion exchange column으로 최종 정제하여 SDS-PAGE로 분석하여 본 결과 단일 band로 약90,000의 분자량을 가진 것으로 나타났다. 정제된 phytase를 사용하여 phytase에 대한 항체를 생산하였고 titer는 1/25,000로 결정하였다.

• 한국잠사곤충학회지
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• 제26권2호
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• pp.1-6
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• 1984

미포자충 포자의 정제법을 개발하고 포자표면의 혈청학적 반응을 구명하기 위하여 수행한 시험결과를 요약하면 다음과 같다. 1. 미포자충 포자의 분리정제는 예비원심-percoll 평형밀도원심-세척의 3단계법이 우수하였고, 분리정제된 포자는 극사추출에 나쁜 영향도 받지 않았다. 2. 포자의 형태는 K79 포자가 장타원형(장단경비 2.76)이고 N. bombycis는 단타원형(장단경비 2.07)으로 양포자가 상이하였다. 3. 형광항체 간접법에 의하여 항 N. bombycis 포자혈청에 대하여는 N. bomvycis 포자만이 강한 형광을 발현하였으나 항 K79 포자의 혈청에 대하여는 10배희석구에서는 양포자 모두 강하게, 20배 이상 희석구에서는 약하게 형광을 발현하였고 K79 포자의 특이항체가 포자외각에 인정되었다.

• 대한수의사회지
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• 제14권3호
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• pp.189-191
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• 1978

• 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.

• 한국가축번식학회지
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• 제12권1호
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• pp.51-62
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• 1988

hCG로 면역화된 생쥐의 비장세포와 골수종양세포(SP 2/0 Ag14)를 융합하여 hCG에 대한 단일클론항체를 생산하는 잡종세포(hybridoma)를 얻었다. 생산된 면역글로부린 type과 titer 그리고 면역분석법 이용시 감도 등을 조사함으로써 그 특성을 조사하였다. 배지나 복수내에 존재하는 항체를 순수분리 정제하기 위하여 gel-filtration, DEAE-ion exchange chromatography, 그리고 affinity chromatography 원리에 의한 방법 등을 사용하여 전기영동(SDS-PAGE)으로 순수도를 조사함으로 상호 비교하였다. 또한 hCG의 정량을 위하여 항체를 plastic tube에 피복시킨 것과 화학발광체로 표지된 항체를 이용한 소위 two-site immunochemiluminometric assay(ICMA)를 개발하여 생산된 항체의 이용가능성을 제시하였다.

• 한국초전도ㆍ저온공학회논문지
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• 제25권4호
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• pp.60-64
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• 2023

The manufacturing process of antibody drugs comprises two main stages: the upstream process for antibody cultivation and the downstream process for antibody extraction. The domestic bio industry has excellent technology for the upstream process. However, it relies on the technology of foreign countries to execute downstream process such as affinity chromatography. Furthermore, there are no domestic companies capable of producing the equipment for affinity chromatography. High gradient magnetic separation technology using a high temperature superconducting magnet as a novel antibody separation and purification technology is introduced to substitute for the traditional technology of affinity chromatography. A specially designed magnetic filter was equipped in the bore of the superconducting magnet enabling the continuous magnetic separation of nano-sized paramagnetic beads that can be used as affinity magnetic nano beads for antibodies. To optimize the magnetic filter that captures superparamagnetic nanoparticles effectively, various shapes and materials were examined for the magnetic filter. The result of magnetic separation experiments show that the maximum separation and recovery ratio of superparamagnetic nanoparticles are 99.2 %, and 99.07 %, respectively under magnetic field (3 T) and flow rate (600 litter/hr).