• IMMUNE NETWORK
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• v.1 no.3
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• pp.187-195
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• 2001

The expression of recombinant proteins fused to 26 kDa glutathione S-transferase (GST) extracted from Schistosoma japonicum represents an attractive system for purifiying proteins of interest in a single step using GST-affinity chromatography. In addition, the GST-tag is used conveniently for detecting fused proteins since its high solubility as well as its relatively small size rarely interferes with the biological activity of the fused protein. In this regard, the GST system is frequently applied for tracing fusion proteins in both prokaryotic and eukaryotic cells to elucidate the physiological interactions and functional compartments of proteins. To provide a further tool in analyzing GST-fusion proteins, a new monoclonal antibody, with a high specificity to the S. japonicum GST was produced. Methods: BALB/c mice were immunized both with recombinant S. japonicum GST proteins, and by the fusion of splenocytes from these mice with myeloma cells. From this, a new anti -GST monoclonal antibody, termed SARAH, was generated. The specificity and reactivity of this antibody was confirmed by ELISA and by Western blot analysis. Results: SARAH showed a high reactivity to recombinant GST and GST fusion protein but not with native mammalian GST proteins as derived from other species including humans, cows, rabbits and rats. The applicability of SARAH was further demonstrated by confocal laser scanning microscopy, where GST proteins that were expressed transiently in mouse fibroblast cells, were specifically detected without interference of endogenous GST. Conclusion: SARAH is new monoclonal antibody with a high specificity to recombinant GST proteins but not to endogenous GST in mammalian cells.

• KSBB Journal
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• v.10 no.2
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• pp.204-211
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• 1995

A target-sensitive liposome was prepared by using a dioleoyl-phosphatidylethanolamine(DOPE) and a palmitic acid coupled antibody(p-IgG). For the preparation of stable PE-liposomes, the key factors such as antibody modification method with palmitic acrid, molar ratio of p-IgG to lipid and the amount of various additives, were examined. The optimum molar ratio of p-IgG to lipid was found to be $2.5{\times}10^{-4}$ and the final concentration of deoxycholate for the stable liposome formation was about 0.09%. Two kinds of target-sensitive liposomes, containing polyclonal anti-SRBC(Sheep Red Blood Cell)-antibody and monoclonal anti-${\beta}$-HCG(Human Chorionic Gonadotropin)-antibody, were successfully prepared. The destabilization of liposomes was examined by measuring the release of calcein entrapped in the liposome vesicles. Calcein was released only when the liposomes were contacted with the specific target cells. The calcein release with non-specific target cells was negligible. From this result, it is clear that p-IgG is indispensible for the maintenance of stable PE-liposome and the calcein release is mainly due to the specific interactions between the liposomes containing antibody and the target cells containing antigen.

• BMB Reports
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• v.33 no.1
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• pp.92-95
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• 2000

The HIV-1 p24(202-221) sequence ETINNEEEWDRVHPV HAGP contains a B-cell epitope with the earliest immune response and the highest antibody titer against anti-mouse sera obtained by immunization with p24 antigens. A novel mouse monoclonal antibody (mAb) was generated against the immunodominant B-cell epitope of the HIV-1 p24 capsid protein, p24(202-221). BALB/c mice were immunized with the four branched multiple antigenic peptide (MAP) containing the HIV-1p24(202-221) sequence, and antibody-secreting hybridoma were produced by fusion of mouse splenocytes with P3X63Ag8.653, mouse myeloma cells. One clone which produced the antigen-specific mAb named KI-24 (Isotype IgG1, light chain: ${\kappa}$) was identified. mAb KI-24 was highly specific for both the p24(202-221) and p24 proteins when analyzed by ELISA and Western blotting. Since p24(202-221) also contains a cytotoxic T-lymphocyte epitope, this specfic peptide epitope and the monoclonal antibody with specific reactivity against the p24 protein and p24(202-221) can be used in peptide vaccine development and p24 antigen detection from HIV patients.

• Journal of Microbiology and Biotechnology
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• v.17 no.4
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• pp.571-578
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• 2007

A monoclonal antibody (mab) against the antimicrobial sulfamethazine was prepared and characterized by an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA). Sulfamethazine in the range of 0.2 and 45ng/ml could be determined with the mab by IC-ELISA. cDNAs encoding a variable heavy chain and variable light chain of the mab were cloned to produce recombinant antibodies using phage display technology. Following phage rescue and three rounds of panning, a single-chain variable fragment (scFv) antibody with high sulfamethazine-binding affinity was obtained. ELISA analysis revealed that scFv antibody and parent mab showed similar, but not identical, characteristics. The $IC_{50}$ value by IC-ELISA with scFv antibody was 4.8ng/ml, compared with 1.6ng/ml with the parent mab. Performances of the assays in the presence of milk matrix were compared; the mab-based assay was less affected than the scFv-based assay. Sixty milk samples were analyzed by mab-based IC-ELISA, and four samples were sulfamethazine positive; these results were favorably correlated with those obtained by HPLC.

• YAKHAK HOEJI
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• v.45 no.2
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• pp.180-189
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• 2001

We have reported that water-extracted Korean mistletoe (KM-110) had various biological activities such as antitumor and immunomodulatory activity, and the pectin fraction (KML-C) of the extract was one of major factors related to its biological functions. In this paper, we produced murine monoclonal antibody (mAb) against KML-C. The cAbs obtained were largely classified into two groups according to specificity to KML-C and ML-I, a pectin from European mistletoe. One group mAbs (9H7-D10 and 3C2-lH4) strongly reacted with KML-C, but not ML-I. In contrast, another group cAbs (8Bll-2C5, BE12-3E9 and 5E10-Fl) reacted with both KML-C and ML-1. The subisotypes of these mobs were shown to be IgGl (9H7-lD10, 3C2-lH4 and 8Bll-2C5) or IgM (8E12-3E9 and 5E10-Fl). To develop an assay system for determination of the amount of KML-C, we established the sandwich ELISA (enzyme-linked immunosorbent assay) method using these mAbs and horse radish peroxidase (HRP)-labelled cAbs. In various combinations of the cAbs for coated antibody and detection antibody, the sandwich ELISA quantitatively detected KML-C, showing the detection limit ranging from 7-5,000 ng/ml. Especially reproducibility (C.V) of the sandwich ELISA, in which 8E12-3E9 was used for coating antibody and 8Bll-2C5-HRP for detection antibody, was 4.59-5.83 in intra assay, and 3.9-9.4 in inter assay.

• Biomolecules & Therapeutics
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• v.2 no.4
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• pp.303-309
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• 1994

The analysis of membrane receptors for hormones and neurotransmitters has progressed considerably by pharmacological and biochemical means and more recently through the use of specific antibodies. Two kinds of antibodies could be produced, one is from synthetic peptides and the other from proteins such as purified receptor. Anti-peptide antibodies gave some advantages; epitope is evident and also receptor purification in quantity is not prerequisite. It can be also applied to the study of receptor structure-activity relationship. The purpose of the present study was 1) to produce and characterize a polyclonal antibody against a synthetic $\beta$2-adrenergic receptor peptide(Phe-Gly-Asn-Phe-Trp-Cys-Phe-Trp-Thr-Ser-Ile-Asp-Val-Leu) and 2) to determine the effects of this antibody on the $\beta$-adrenergic receptor ligand interaction. The peptide sequence contains an amino acid residue such as Asp-113 which was identified as one of important component for receptor-ligand interaction in site-directed mutagenesis studies. Production of antibody was performed by immunization of rabbits through popliteal lymph node with the peptide coupled with Keyhole Limpet Hemocyanin (KLH). The titer of antibody against this peptide was 1 : 1000. The anti-peptide antibody was able to detect a 67 kDa protein band in western blot corresponding to the molecular weight of the $\beta$-adrenergic receptor in partially purified receptor fraction derived from guinea pig lung. The antisera inhibited the specific binding of [$^3$H]dihydroalprenolol to $\beta$-adrenergic receptor in a concentration-dependent manner. The results from this study suggest that the peptide sequence selected in the present study is important for the receptor ligand interaction.

• IMMUNE NETWORK
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• v.3 no.2
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• pp.118-125
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• 2003

Background: Disialoganglioside GD2 is a tumor-associated antigen that is overexpressed on tumor cells of neuroectodermal origin, such as melanoma and neuroblastoma. Anti-idiotypic antibodies that mimic GD2 may induce more effective immune responses than GD2 antigen itself, because they are protein antigens and are known to be able to break immune tolerance. In this study, to explore the potential of anti-idiotypic antibodies as tumor vaccines, the ability of anti-idiotypic antibodies (Ab2) to induce anti-anti-idiotypic antibodies (Ab3) that bind to the original antigen GD2 was investigated. Methods: Six monoclonal anti-idiotypic antibodies (1A8, 1G5, 2B6, 3A4, 3D6, 3H9) to monoclonal antibody M2058, which is a monoclonal antibody to GD2, were produced in mice. Three (1A8, 3A4, 3H9) of them were selected based on their ability to inhibit the binding of Ab1 to D142.34 (murine melanoma cell expressing GD2). These 3 different Ab2 were injected into rabbits, and rabbit Ab3 induced by each of them were characterized. Results: Ab3-containing sera from two rabbits immunized with 1A8, 3A4, or 3H9 bound significantly (P<0.05) to D142.34 but not to B78.96 (GD2-negative cell), and bound significantly (P<0.05) to isolated GD2 but not to GD1a. Ab3-containing sera from two rabbits immunized with 3A4 or 3H9 inhibited significantly (P<0.05) the binding of Ab1 M2058 to D142.34, and inhibited significantly (P<0.05) the binding of Ab1 M2058 to the Ab2. Conclusion: These results suggest that anti-idiotypic antibodies 3A4 and 3H9 have a potential to be used as vaccines against tumors expressing GD2 by inducing GD2-specific antibodies (Ab3).

• BMB Reports
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• v.31 no.1
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• pp.58-63
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• 1998

In this study, a mouse monoclonal antibody (mAb) against gp41(584-618), the immunodominant epitope protein, was generated. For this purpose, BALB/c mice were immunized with double branched multiple antigenic peptides derived from the HIV-1 gp41(584-618) sequence, and antibody-secreting hybridoma were produced by fusion of mice splenocytes with SP2/0 myeloma cells. One clone producing an antigen specific mAb, termed KI-41(isotype IgG1) was identified, whose specific reactivity against gp41(584-618) could be confirmed by ELISA and Western blot analysis. Epitope mapping revealed the recognition site of the mAb KI-41 to be located around the sequence RILAVERYLKDQQLLG, which comprises the N-terminal region within the immunized gp41(584-618) peptied. Since this mAb recognizes this specific epitope within the HIV-1 gp41 without any cross-reactivity to other immunodominant regions in the HIV-2 gp35, KI-41 will provide some alternative possibilities in further applications such as the development of indirect or competitive ELISA for specific antibody detection in HIV-1 infection or for other basic researches regarding the role and function of HIV-1 gp41.

• Microbiology and Biotechnology Letters
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• v.49 no.2
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• pp.148-156
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• 2021

Single-chain antibodies against epidermal growth factor receptor variant III (EGFRvIII) are potentially promising agents for developing antibody-based cancer treatment strategies. We described in our previous study the successful expression of an anti-EGFRvIII scFv antibody in Escherichia coli. However, we could also observe the formation of insoluble aggregates in the periplasmic space, limiting the production yield of the active product. In the present study, we investigated the mechanisms by which growth conditions could affect the expression of the soluble anti-EGFRvIII scFv antibody in small-scale E. coli NiCo21(DE3) cultures, attempting to maximize production. The secreted scFv molecules were purified using Ni-NTA magnetic beads and protein characterization was performed using SDS-PAGE and western blot analyses. We used the ImageJ software for protein quantification and determined the antigen-binding activity of the scFv antibody against the EGFRvIII protein. Our results showed that the highest percentage of soluble scFv expression could be achieved under culture conditions that combined low IPTG concentration (0.1 mM), low growth temperature (18℃), and large culture dish surface area. We found moderate-yield soluble scFv production in the culture medium after lactose-mediated induction, which was also beneficial for downstream protein processing. These findings were confirmed by conducting western blot analysis, indicating that the soluble, approximately 30-kDa scFv molecule was localized in the periplasm and the extracellular space. Moreover, the antigen-binding assay confirmed the scFv affinity against the EGFRvIII antigen. In conclusion, our study reveals that low-speed protein expression is preferable to obtain more soluble anti-EGFRvIII scFv protein in an E. coli expression system.

• Journal of Microbiology and Biotechnology
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• v.34 no.6
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• pp.1222-1228
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• 2024

Protein-specific antibodies are essential for various aspects of protein research, including detection, purification, and characterization. When specific antibodies are unavailable, protein tagging is a useful alternative. Small epitope tags, typically less than 10 amino acids, are widely used in protein research due to the simple modification through PCR and reduced impact on the target protein's function compared to larger tags. The 2B8 epitope tag (RDPLPFFPP), reported by us in a previous study, has high specificity and sensitivity to the corresponding antibody. However, when attached to the C-terminus of the target protein in immunoprecipitation experiments, we observed a decrease in detection signal with reduced immunity and low protein recovery. This phenomenon was not unique to 2B8 and was also observed with the commercially available Myc tag. Our study revealed that C-terminal tagging of small epitope tags requires the addition of more than one extra amino acid to enhance (restore) antibody immunities. Moreover, among the amino acids we tested, serine was the best for the 2B8 tag. Our findings demonstrated that the interaction between a small epitope and a corresponding paratope of an antibody requires an extra amino acid at the C-terminus of the epitope. This result is important for researchers planning studies on target proteins using small epitope tags.