• Korean Journal of Veterinary Research
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• v.31 no.4
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• pp.403-409
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• 1991

This experiment was carried out to develop a sensitive, rapid, solid-phase microtitre plate assay of progesterone using the monoclonal antibody to this hormone. Monoclonal antibody to progesterone was much higher titre and binding affinity about 10 times than conventional polyclonal antibody to progesterone. Dot-blot analysis of monoclonal antibody revealed a single precipitation band when reacted with anti-mouse IgM and anti-mouse K. A competitive reaction was used with a reaction time of 2 hours. The standard dose-response curve was linear through 1,000pg/well. This ELISA system approach is applicable to evaluation for the rapid assessment of luteal function and reproductive status in both clinical and research in a wide variety of species.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1997.04a
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• pp.95-95
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• 1997

Among the various receptor molecules discovered so far the ${\beta}$2-adrenergic receptors have been regarded as excellent model systems for the so called 7 transmembrane helix receptor and have been the focus of extensive studies. For the analysis of receptor structure and function a monoclonal antibody plays a crucial role, thus providing useful tools for the study of receptor. However, because of the minute quantity of receptor molecules which could be obtained from natural sources, the generation of specific monoclonal antibody against receptor molecules from the purified receptors has been regarded as virtually impractical in consideration of cost and experimental times. The purpose of the present study was to generate and characterize a monoclonal antibody against human ${\beta}$2-adrenergic receptor. For the production of antibody, C-terminal regions of the human ${\beta}$2-adrenergic receptor was produced as a fusion protein with Glutathion S-transferase (GST) in E. Coli. The expression of the fusion protein was identified by SDS-PAGE and Western blot using monoclonal anti-GST antibody. The fusion protein was purified to an apparent homogeniety by affinity chromatography with Glutathion Sepharose CL-4B and used as an antigen for the immunization of BALB/C mice. The Production of monoclonal antibody was achieved by fusion of the immunized spleen cells and SP/2-0 myeloma cells. Positive hybridomas were screened by ELISA and were cloned by two consecutive rounds of limiting dilution. The monoclonal antibody produced in this study (mAb${\beta}$C02) was IgM type and purified by immunoaffinity chromatography using anti-mouse IgM agarose as an affinity matrix. MAb${\beta}$C02 showed strong and specific immunoreactivity against both the fusion protein and human ${\beta}$2-adrenergic receptor in ELISA and Western blot. The molecular weight of immunoreactive band was 64 kDa and exactly coincided with the previously reported molecular weight of ${\beta}$2-adrenergic recepters. The results of the present study suggest that mAb${\beta}$C02 may be used for the study of receptor function and regulation in normal or nonphysiological status.

• YAKHAK HOEJI
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• v.47 no.3
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• pp.176-179
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• 2003

A library of unlimited number of novel lectins with diverse specificities has been previously generated by randomly mutating the carbohydrate-recognition domain of Maackia amurensis hemagglutinin (MAH). To establish the experimental environment capable of selecting high affinity mutant lectins in E. coli, phage display system was adapted. Carbohydrate binding capacity of two phagemid vectors, pComb3 and pComb8 displaying wild-type MAH lectin was assessed. Specific bindings of pComb3 and pComb8 phages expressing w.t. MAH to affinity-purified polyclonal anti-MAH antibody and to glycophorin was demonstrated. Both phages also showed strong hemagglutinating activity to intact but not sialidase-treated human erythrocytes, which is consistent to the specificity of native MAH. Taken together, two different phage display vectors successfully allowed the expression of active MAH as a fusion protein on the surface of bacteriophage, which will lead to preparation of unique plant lectins with high affinity toward a variety of carbohydrate chains.

• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2113-2120
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• 2018

Cross-reactive material 197 ($CRM_{197}$) is a non-toxic mutant of diphtheria toxin containing a single amino acid substitution of glycine 52 with glutamic acid. $CRM_{197}$ has been used as a carrier protein for poorly immunogenic polysaccharide antigens to improve immune responses. In this study, to develop a sandwich ELISA that can detect $CRM_{197}$ and $CRM_{197}$ conjugate vaccines, we generated a human anti-$CRM_{197}$ monoclonal antibody (mAb) 3F9 using a phage-displayed human synthetic Fab library and produced mouse anti-$CRM_{197}$ polyclonal antibody. The affinity ($K_D$) of 3F9 for $CRM_{197}$ was 3.55 nM, based on Bio-Layer interferometry, and it bound specifically to the B fragment of $CRM_{197}$. The sandwich ELISA was carried out using 3F9 as a capture antibody and the mouse polyclonal antibody as a detection antibody. The detection limit of the sandwich ELISA was <1 ng/ml $CRM_{197}$. In addition, the 3F9 antibody bound to the $CRM_{197}$-polysaccharide conjugates tested in a dose-dependent manner. This ELISA system will be useful for the quantification and characterization of $CRM_{197}$ and $CRM_{197}$ conjugate vaccines. To our knowledge, this study is the first to generate a human monoclonal antibody against $CRM_{197}$ and to develop a sandwich ELISA for $CRM_{197}$ conjugate vaccines.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.829-832
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• 2001

Phage display technique as a new antibody production method can express the protein on the minor coat of phage particle in a library constructed by utilizing a recombination of genes coding the variable regions of immunoglobulin. This new method is particularly advantageous in producing antibodies against toxic substances and compounds with low immunogenicities. We first confirmed the concept of antibody expression on the phage particle by selecting a positive control of the phage library (e.g., Griffin.l donated from MRC center in England). The library was then employed to produce antibodies specific to human serum albumin via repetitive bio-panning procedure. The mean affinity of the antibodies selected gradually increased along with the number of bio-panning, which demonstrated that the phage display method could produce monocloanl antibodies with high affinities.

• Bulletin of the Korean Chemical Society
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• v.30 no.5
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• pp.1101-1106
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• 2009

Multivalent and multi-specific antibodies can provide valuable tools for bio-medical research, diagnosis and therapy. In antigen-antibody interactions, the avidity of antibodies depends on the affinity and the number of binding sites.$^1$ As artificial multivalent antibody agents, single chain Fv-streptavidin fusion tetramer proteins $(scFv-SA)_4$ have been previously tested.$^{1,\;2}$ Although, the Fab domain is known to be more stable than scFv in animal models,$^{3,\;4}$ it has never been used to make a multivalent agent with a streptavidin fusion. In this study, we prepared tetra-valent $(Fab-cSA)_4$ by fusing Fab with core streptavidin (cSA). This molecule was made using inclusion body production, refolding and chromatography purification. Affinities of the Fab-cSA tetramer and a scFv-cSA tetramer to a cell surface antigen were compared by ELISA using biotin-HRP. The Fab-cSA tetramer showed higher binding avidity than the scFv-cSA tetramer. The higher binding avidity of the Fab-cSA tetramer demonstrates its potential as a therapeutic agent for target-specific antibody therapy.

• IMMUNE NETWORK
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• v.4 no.1
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• pp.16-22
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• 2004

Background: To develop a novel treatment strategy for hepatitis B virus infection, a major cause of liver chirosis and cancer, we aimed to make human monoclonal antibodies inhibiting RNase H activity of P protein playing in important role in HBV replication. In this regard, phage display technology was employed and demonstrated as an efficient cloning method for human monoclonal antibody. So this study analysed the usability of human monoclonal antibody as protein based gene therapy. Methods: RNase H of HBV was expressed as fusion protein with maltose binding protein and purified with amylose resin column. Single chain Fv (scFv) phage antibody library was constructed by PCR cloning using total RNAs of PBMC from 50 healthy volunteers. Binders to RNase H were selected with BIAcore 2000 from the constructed library, and purified as soluble antibody fragment. The affinity and sequences of selected antibody fragments were analyzed with BIAcore and ABI automatic sequencer, respectively. And finally RNase H activity inhibiting assay was carried out. Results: Recombinant RNase H expressed in E. coli exhibited an proper enzyme activity. Naive library of $4.46{\times}10^9cfu$ was screened by BIAcore 2000. Two clones, RN41 and RN56, showed affinity of $4.5{\times}10^{-7}M$ and $1.9{\times}10^{-7}M$, respectively. But RNase H inhibiting activity of RN41 was higher than that of RN56. Conclusion: We cloned human monoclonal antibodies inhibiting RNase H activity of P protein of HBV. These antibodies can be expected to be a good candidate for protein-based antiviral therapy by preventing a replication of HBV if they can be expressed intracellularly in HBV-infected hepatocytes.

• Molecules and Cells
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• v.38 no.3
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• pp.195-201
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• 2015

Antibodies are powerful defense tools against pathogens but may cause autoimmune diseases when erroneously directed toward self-antigens. Thus, antibody producing cells are carefully selected, refined, and expanded in a highly regulated microenvironment (germinal center) in the peripheral lymphoid organs. A subset of T cells termed T follicular helper cells (Tfh) play a central role in instructing B cells to form a repertoire of antibody producing cells that provide life-long supply of high affinity, pathogenspecific antibodies. Therefore, understanding how Tfh cells arise and how they facilitate B cell selection and differentiation during germinal center reaction is critical to improve vaccines and better treat autoimmune diseases. In this review, I will summarise recent findings on molecular and cellular mechanisms underlying Tfh generation and function with an emphasis on T cell costimulation.

• International Journal of Oral Biology
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• v.44 no.1
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• pp.14-19
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• 2019

The present study aimed at evaluating serum immunoglobulin G (IgG) avidity to Porphyromonas gingivalis in elderly patients with mild and severe chronic periodontitis. The avidity of antibodies against P. gingivalis present in the sera of 18 patients with mild chronic periodontitis and 18 patients with severe chronic periodontitis was evaluated using an ammonium thiocyanate-dissociated enzyme-linked immunosorbent assay (ELISA). The results showed that the mean absorbance value in serum IgG antibody titers was significantly higher in the severe chronic periodontitis group than in the mild chronic periodontitis group ($198{\pm}35ELISA$ unit [EU] vs. $142{\pm}32EU$, p < 0.01). However, there was no significant difference between the two groups in antibody avidity ($65{\pm}57EU$ vs. $54{\pm}27EU$). These findings suggest that humoral immune responses to P. gingivalis between mild and severe chronic periodontitis in elderly patients are characterized by the differences in the quantity rather than the quality of the antibodies.

• YAKHAK HOEJI
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• v.44 no.6
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• pp.588-594
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• 2000

To investigate the effects of iota-carrageenan (CAR) and/or alum on the adjuvancity as well as the structural difference of oligosaccharide on the IgG2b in the adjuvant effect, C57BL/6 mice were immunized twice with fetuin as a model antigen. CAR alone showed no significant effect on induction of antibody except IgG1. In contrast, Alum-CAR (after mixing of antigen-Alum, CAR adjuvant was prepared) and CAR-Alum (after formulation of antigen-CAR, Alum adjuvant was prepared) enhanced production of antibody, especially, IgG2b. After separation of IgG2b, changes of glycosylation were investigated using enzymelinked lectin assay. High affinity of IgG2b to N-acetylneuraminic acid, galactose and mannose-specific lectin were induced by CAR-Alum adjuvant, however, the affinity of IgG2b induced by CAR-Alum to GlcNAc and GalNAc-specific lectin were much less than that induced by Alum-CAR.