• BMB Reports
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• v.38 no.3
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• pp.294-299
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• 2005

In this report, we describe the high-yield secretory expression of the recombinant human anti-HBsAg Fab fragment from Pichia pastoris that was achieved by co-integration of the genes encoding the heavy and light chains (both under the control of alcohol oxidase promoter) into the genome of the yeast cells. The fed-batch fermentations were carried out in a 5 L scale. Both chains of the Fab were successfully expressed upon methanol induction. The absorbance ($OD_{600}$) of the broth can reach 350~500 at the end of fed-batch phase. After the induction, the expression level of the recombinant Fab (soluble) reached 420~458 mg/L. The recombinant Fab fragment was purified from the crude culture supernatant by ion exchange chromatography and the purity of the recombinant Fab fragment was over 95%. The affinity activities of the crude fermentation supernatant and the purified Fab were analyzed by indirect ELISA, which showed that the purified recombinant Fab fragment had high affinity activity with hepatitis B surface antigen.

• Parasites, Hosts and Diseases
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• v.24 no.2
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• pp.187-193
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• 1986

Enzyme-linked immunosorbent assay(ELISA) using crude and affinity-purified antigens of adult worms of Paragonimus westermani was performed for infected cat sera with different worm burden, from preinfection to 18th week after infection. Crude antigen was used with supernatant of homogenated worms by freezing-thawing method, and the supernate was centrifuged for 1 hour at 10,000 rpm at $4^{\circ}C$. Affinity-purified antigen(antibody-bound antigen) was prepared from fractions(bound and unbound) of crude antigen by affinity chromatography on CNBr-activated sepharose 4B, and IgG as a ligand was prepared from paragonimiasis cat serum(6 months infected) obtained by ammonium sulfate ($40%{\sim}45%$ saturated) precipitation method. By SDS-PAGE, crude antigen showed 22 polypeptide fractions while purified antigen showed 4 fractions: 36, 400, 34, 700, 27, 600 and 11, 500 in molecular weights. All cats were divided into five groups($G_1-G_5$) by different worm burdens. The mean of recovered worms(${\pm}SD$) and the number of cats in each group are as follows: $G_1$, 2 worms(0) and 4 cats; $G_2$, 4.75 (${\pm}0.66$) and eight; $G_3$, 10.75(${\pm}1.92$) and four; $G_4$, 23.20(${\pm}3.43$) and five; $G_5$, 48(${\pm}12.63$) and five cats. The results were summarized as follows: 1. The antibody levels(OD value) increased by worm burden in $G_1$ to $G_4$ generally. However, individual antibody levels were not exactly related with worm burden in all groups, especially there was a wide difference in $G_4$ and $G_5$. These results suggested that the worm burden in $G_4$ (about $20{\sim}30$ worms) is enough to produce antibody maximum in cats of $2{\sim}3kg$ weight. 2. The antibody levels increased significantly(p<0.05) compared to control sera at the 3rd week in $G_1$ and $G_2$, at the 2nd week in $G_3$, and at the 1st week in $G_4$ and $G_5$. Especially in the 4th week, OD value increased more in $G_1$(p<0.01) and in $G_2$ to $G_5$(p<0.001). In the pattern of antibody levels by ELISA in each group, OD in $G_1$ increased to the 18th week continuously, in $G_2$ OD was maintained same after the 16th week, but in $G_3$ it decreased after the 16th week, and it was maintained same in $G_4$ and $G_5$ after the 14th week. 3. The antibody levels by ELISA with the affinity-purified antigen were higher than those with crude antigen in all groups generally. Especially, the difference of OD values between two antigens was larger from the 4th to the 10th week. In $G_1$ and $G_2$ OD with purified antigen was higher than that with crude one to the 18th week. It was also higher in $G_3$ than that with crude antigen to the 16th week and OD of $G_4$ and $G_5$ were higher before the 14th week than that with crude antigen, however became lower at the 16th week. Consequently, the antibody level in ELISA with affinity-purified antigen was more sensitive at the early weeks after infection and in light infection groups than that with crude antigen.

• Journal of environmental and Sanitary engineering
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• v.12 no.2
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• pp.59-64
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• 1997

For purification of a 70kDa toxic protein of mosquitocidal delta-endotoxin from B. thuringiensis strain H9B, immuno-affinity chromatography was performed. After separation of 70kDa toxic proteins from the delta-endotoxin of the strain H9B on SDS-PAGE, the 70kDa toxic protein was subcutaneously injected into rabbit for making a polyclonal antibody. A anti-70kDa toxic protein was purified by a column chromatography packed with protein A-sepharose 4B gels. The 70kDa toxic protein from delta-endotoxin of the strain H9B was also purified by an immuno-affinity chromatography packed with CNBr-activated sepharose 4B gels conjugated anti-70kDa toxic protein after elution with 1/10M citric acid-1/5M Na$_{2}$HPO$_{4}$ buffer(pH3.2) containing 0.5M NaCl. The 70kDa toxic protein was purified through only one step-separation system, was demonstrated by SDS-PAGE and immunoblot.

• IMMUNE NETWORK
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• v.16 no.4
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• pp.219-232
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• 2016

Production of high affinity antibodies for antigens is a critical component for the immune system to fight off infectious pathogens. However, it could be detrimental to our body when the antigens that B cells recognize are of self-origin. Follicular helper T, or Tfh, cells are required for the generation of germinal center reactions, where high affinity antibody-producing B cells and memory B cells predominantly develop. As such, Tfh cells are considered as targets to prevent B cells from producing high affinity antibodies against self-antigens, when high affinity autoantibodies are responsible for immunopathologies in autoimmune disorders. This review article provides an overview of current understanding of Tfh cells and discusses it in the context of animal models of autoimmune diseases and allograft rejections for generation of novel therapeutic interventions.

• Journal of Microbiology
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• v.45 no.6
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• pp.572-577
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• 2007

To enhance therapeutic potential of murine monoclonal antibody, humanization by CDR grafting is usually used to reduce immunogenic mouse residues. Most humanized antibodies still have mouse residues critical for antigen binding, but the mouse residues may evoke immune responses in humans. Previously, we constructed a new humanized version (AKA) of mouse CC49 antibody specific for tumor-associated glycoprotein, TAG-72. In this study, to select a completely human antibody light chain against TAG-72, guided selection strategy using phage display was used. The heavy chain variable region (VH) of AKA was used to guide the selection of a human TAG-72-specific light chain variable region (VL) from a human VL repertoire constructed from human PBL. Most of the selected VLs were identified to be originated from the members of the human germline VK1 family, whereas the VL of AKA is more homologous to the VK4 family. Competition binding assay of the selected Fabs with mouse CC49 suggested that the epitopes of the Fabs overlap with that of CC49. In addition, they showed better antigen-binding affinity compared to parental AKA. The selected human VLs may be used to guide the selection of human VHs to get completely human anti-TAG72 antibody.

• Proceedings of the PSK Conference
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• 2003.04a
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• pp.316.2-317
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• 2003

Immunoglobulin Y (IgY) obtained from chicken as the immunization host brings several advantages to antibody production, such as improved yield, lower cost, longer stability, and higher specificity than mammalian immunoglobulin. In the present study, we attempted to purify Maackia fauriei lectin using antilectin IgY-affinity chromatography in order to produce a good yield and to reduce the purification time. (omitted)

• Molecules and Cells
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• v.39 no.3
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• pp.217-228
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• 2016

To generate a biobetter that has improved therapeutic activity, we constructed scFv libraries via random mutagenesis of several residues of CDR-H3 and -L3 of hu4D5. The scFv clones were isolated from the phage display libraries by stringent panning, and their antiproliferative activity against HER2-positive cancer cells was evaluated as a primary selection criterion. Consequently, we selected AH06 as a biobetter antibody that had a 7.2-fold increase in anti-proliferative activity ($IC_{50}$: 0.81 nM) against the gastric cancer cell line NCI-N87 and a 7.4-fold increase in binding affinity ($K_D$: 60 pM) to HER2 compared to hu4D5. The binding energy calculation and molecular modeling suggest that the substitution of residues of CDR-H3 to W98, F100c, A101 and L102 could stabilize binding of the antibody to HER2 and there could be direct hydrophobic interactions between the aromatic ring of W98 and the aliphatic group of I613 within HER2 domain IV as well as the heavy and light chain hydrophobic interactions by residues F100c, A101 and L102 of CDR-H3. Therefore, we speculate that two such interactions were exerted by the residues W98 and F100c. A101 and L102 may have a synergistic effect on the increase in the binding affinity to HER2. AH06 specifically binds to domain IV of HER2, and it decreased the phosphorylation level of HER2 and AKT. Above all, it highly increased the overall level of p27 compared to hu4D5 in the gastric cancer cell line NCIN82, suggesting that AH06 could potentially be a more efficient therapeutic agent than hu4D5.

• Journal of Microbiology and Biotechnology
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• v.16 no.8
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• pp.1316-1319
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• 2006

An antibody displayed phage collection (SBAE-2R), screened from a human synthetic phage antibody library (Fab 21ox), was used for the determination of dextran. The dextran-binding affinity was determined by serologically specific transmission electron microscopy (TEM) and a paper dipstick assay. The phage collection was distributed over the dextrancoated grids with 39$\pm$25 phages/$\mu$m$^2$ on the grids. Phages were not seen on dextran-coated grids exposed to the Fab 2lox phage library. The phage collection (SBAE-2R) produced 54$\pm$3 color normalized intensity (N.I.) from 125 ppm to 1,000 ppm of dextran and 5$\pm$1 (N.I.) for 63 ppm of dextran in a paper dipstick assay. This research extends the analytical options for dextran analysis by antibody displayed phage with a minimum of equipment usage.

• BMB Reports
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• v.28 no.1
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• pp.27-32
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• 1995

Polyclonal antibodies against human DNA-dependent RNA polymerase II (HPP II) were generated from chicken egg yolk after immunization with RNA polymerase II as an antigen. The antibodies from egg yolk (IgY) were purified and characterized. IgY showed a specificity against DNA-dependent RNA polymerase II, and was a polyclonal antibody against 12 subunits of polymerase II. An amount of 0.35 mg of IgY was obtained freman HPP II-Sepharose affinity column using 10 eggs from a chicken immunized against RNA polymerase II as an antigen. These antibodies can be used for isolating the genes for RNA polymerase II components, and for in vitro transcription assays using HP-RNA polymerase II.

• Journal of Microbiology and Biotechnology
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• v.20 no.8
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• pp.1179-1184
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• 2010

We report the generation of the first monoclonal antibody that specifically binds to the polysaccharide chitosan. Mice were immunized with a mixture of chitosans, and hybridoma clones were screened for specific binders, resulting in the isolation of a single clone secreting a chitosan-specific IgM, mAbG7. In ELISAs, the antibody could bind to chitosans of varying composition, but demonstrated the highest affinity for chitosans with lower degrees of acetylation (DA) and very poor binding to chitin. We tested the ability of the antibody to bind to chitosan in situ, using preparations of fungal cell walls. Immunofluorescence microscopy confirmed that the antibody bound strongly to the cell walls of fungi with high levels of chitosan, whereas poor staining was observed in those species with cell walls of predominantly chitin or cellulose. The potential use of this antibody for the detection of fungal contamination and the protection of plants against fungal pathogens is discussed.