• Archives of Pharmacal Research
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• v.20 no.1
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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.

• Food Engineering Progress
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• v.13 no.4
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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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• v.33 no.2
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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.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1997.04a
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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.

• The Korean Journal of Mycology
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• v.27 no.4 s.91
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• pp.299-303
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• 1999

Phytase(myo-inositol-hexakis phosphate 3-phosphohydrolase, E C 3.1.3.8) sequentially hydrolyzes phytate to myo-inositol and inorganic phosphate. Phytase of Aspergillus ficuum was purified to homogeneity using ultrafiltration, cation exchange column and anion exchange column. It's molecular weight is estimated as around 90,000 by SDS-PAGE. Antibody against the phytase was produced by immunizing mice with the purified phytase. The titer of the antibody was determined to be 1/25,000.

• Journal of Sericultural and Entomological Science
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• v.26 no.2
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• pp.1-6
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• 1984

The present study was made to establish the purification method of mature microsporidian spores by iso-density equilibrium technique using percoll and the serological discrimination by an indirect fluorescent antibody technique. The purification of new microsporidian spores took effect in the three steps purification method(precentrifugation-percoll iso-density equilibrium centrifugation-rising). There were clear differences in the size of spores between the new microsporidia and N. bombycis. The spores of N. bombycis is short elliptical of 2.07 in a ratio of length to width in diameter while that of new microsporidia is characterized with long elliptical shape which shows a ratio of 2.76 in length to width in diameter. The specific antigens of new microsporidia K79 spores was showed in the spores wall by the indirect fluorescent antibody reaction, and it was affected by the antibody against N. bombycis which antiserum was diluted in 1:20. It means that the new microsporidia K79 is serologically not identical to N. bombycis.

• Journal of the korean veterinary medical association
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• v.14 no.3
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• pp.189-191
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• 1978

• Proceedings of the Korean Vacuum Society Conference
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• 2013.02a
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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.

• Korean Journal of Animal Reproduction
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• v.12 no.1
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• pp.51-62
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• 1988

Spleen cells of mouse immunized with hCG were fused with myeloma cell (SP 2/0 Ag 14) to produce monoclonal antibody against hCG. Several clones of hybridoma secreting monoclonal antibody were established and antibodies were characterized in terms of titer, subisotyping and sensitivity in immunoassay. Several methods, for the purification of anti¬bodies, based on gel-filtration, DEAE-ion exchange chromatography and affinity chromatography. were applied and compared each other by the result of SDS-PAGE. Two-site immunochemiluminometric assay (ICMA) involving the use of an excess concentration of a specific monoclonal antibody passively adsorbed onto the walls of plastic tubes and a chemiluminescence labelled antibody conjugate were de¬veloped for the determination of hCG as a preliminary study.

• Progress in Superconductivity and Cryogenics
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• v.25 no.4
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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).