• Korean Journal of Microbiology
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• v.47 no.4
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• pp.328-334
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• 2011

Leucine zipper motif consists of multiple periodic leucine residues, which forms amphipathic alpha helix. The hydrophobic nature of leucine zipper motif can dimerize proteins which contain this motif. Leucine zipper motif addition at C-terminus of single-chain Fv (ScFv) antibody induces its dimerization. Since the dimeric ScFv antibody contains two antigen binding sites (bivalency) like Y-shaped complete antibody, it could increase avidity. As a result, it could show higher antigen binding activity than monomeric ScFv antibodies. Based on this concept, monomeric chicken 8C3 ScFv antibody previously developed from chicken hybridoma was dimerized by the addition of leucine zipper motif at C-terminus of ScFv antibody. The dimeric 8C3 ScFv antibody specifically reacted with Eimerian sporozoite which causes Avian Coccidiosis. As expected, dimeric 8C3 ScFv antibody showed 3-folds higher antigen binding activity than monomer due to increased avidity. In addition, protien yields of dimer expression were 2-folds higher than monomer.

• Journal of Sericultural and Entomological Science
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• v.53 no.2
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• pp.92-96
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• 2015

It is known that silk protein supports effectively proliferation of cell such as insect cell and hybridoma cell. Although there are many varieties of Bombyx mori silkworm, the effect of silkworm varieties on cell proliferation has not been considered in detail. We studied that characteristics of silk cocoon obtained from Baegokjam, Kumokjam, Daeseongjam silkworm varieties and whether silk protein affected cell proliferation or not. Silk sericin was prepared under high temperature and high pressure condition. Silk fibroin was prepared using $CaCl_2:H_2O:EtOH$ with different dissolution time. As a result, there are differences in silk cocoon from different silkworm varieties about cell proliferation. The proliferation was accelerated in the presence of Baegokjam silk sericin and Kumokjam silk fibroin with 5hr dissolution time. We expect that silk proteins could be a preferable culture medium supplement for stimulating the proliferation of cell. Then, this results suggest silk as a new material for medium supplement replacing with fetal bovine serum.

• Journal of Veterinary Science
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• v.23 no.4
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• pp.55.1-55.12
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• 2022

Background: ASF was first reported in Kenya in 1910 in 1921. In China, ASF spread to 31 provinces including Henan and Jiangsu within six months after it was first reported on August 3, 2018. The epidemic almost affected the whole China, causing direct economic losses of tens of billions of yuan. Cause great loss to our pig industry. As ELISA is cheap and easy to operate, OIE regards it as the preferred serological method for ASF detection. P54 protein has good antigenicity and is an ideal antigen for detection. Objective: To identify a conservative site in the African swine fever virus (ASFV) p54 protein and perform a Cloth-enzyme-linked immunosorbent assay (ELISA) for detecting the ASFV antibody in order to reduce risks posed by using the live virus in diagnostic assays. Method: We used bioinformatics methods to predict the antigen epitope of the ASFV p54 protein in combination with the antigenic index and artificially synthesized the predicted antigen epitope peptides. Using ASFV-positive serum and specific monoclonal antibodies (mAbs), we performed indirect ELISA and blocking ELISA to verify the immunological properties of the predicted epitope polypeptide. Results: The results of our prediction revealed that the possible antigen epitope regions were A23-29, A36-45, A72-94, A114-120, A124-130, and A137-150. The indirect ELISA showed that the peptides A23-29, A36-45, A72-94, A114-120, and A137-150 have good antigenicity. Moreover, the A36-45 polypeptide can react specifically with the mAb secreted by hybridoma cells, and its binding site contains a minimum number of essential amino acids in the sequence 37DIQFINPY44. Conclusions: Our study confirmed a conservative antigenic site in the ASFV p54 protein and its amino acid sequence. A competitive ELISA method for detecting ASFV antibodies was established based on recombinant p54 and matching mAb. Moreover, testing the protein sequence alignment verified that the method can theoretically detect antibodies produced by pigs affected by nearly all ASFVs worldwide.

• Korean Journal of Food Science and Technology
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• v.42 no.3
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• pp.329-334
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• 2010

Escherichia coli O157:H7 causes hemolytic uremic syndrome and hemorrhagic colitis in humans. The objectives of this study were to produce monoclonal antibody(MAb) against E. coli O157:H7 and to develop an enzyme linked immunosorbent assay(ELISA) for the rapid detection of E. coli O157:H7 in agri-stockbreeding. The characterization of MAb produced from hybridoma cell (HKEC 4G8-5) was validated by ELISA and Western blot. The produced MAb was specific to E. coli O157:H7 and showed weak cross-reaction to Staphylococcus aureus. The detection limit of ELISA based on 4G8-5 MAb was $10^5\;cell/mL$. Although the ELISA could not detect E. coli O157:H7 in the meat and sprout samples inoculated with $1{\times}10^1\;cell$/10 g without enrichment, the same samples after enrichment for 6 hr were confirmed to be positive by ELISA. These results indicated that the ELISA combined with short enrichment (6 hr) is useful tool for rapid screening of E. coli O157:H7 in various samples.

• Parasites, Hosts and Diseases
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• v.24 no.2
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• pp.145-158
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• 1986

This study was undertaken to purify cystic fluid (CF) antigen of Taenia solium metacestodes by affinity chromatogaphy using specific monoclonal antibody(McAb) and to characterize the antigenicity of the purified antigen. The hybridoma cell lines, prepared by fusion between mouse plasmacytoma and spleen cells from BALB/c mice immunized with CF, secreted antibodies reacting to various helminthic antigens. Majority of cell lines reacted to CF only but some also reacted to parenchymal antigen of T. solium metacestodes, adult T. saginata, sparganum, hydatid cystic fluid, Paragonimus westermani and Clonorchis sinensis, either in combination with CF, other antigens or independently. Cloned cells derived from monoclonal lines also produced antibodies reacting either to CF only or to other helminthes in combination or independently. These results indicated that CF of T. solium metacestodes contained proteins which possessed antigenic determinants not only specific to CF but also cross reactive with the afore-mentioned helminthes. CF of T. solium metacestodes was purified by affinity chromatography using the McAb which reacted to CF and parenchymal antigens. The affinity-purified antigen (A-Ag) and unbound pool CF (U-Ag) were separated. A-Ag showed 2 protein bands by disc-PAGE whereas CF exhibited 6 bands and U-Ag consisted of all bands CF had. The diagnostic significance of A-Ag was evaluated by ELISA in human neurocysticercosis and other helminthic and neurologic diseases. By A-Ag, the levels of the specific IgG antibody, as shown by absorbance in sera and CSF, were lower than those of CF and U-Ag. Accordingly, the sensitivity was about 70% of CF and U-Ag. However, the nonspecific positive reactions to CF and U-Ag, observed in sparganosis, T. saginata infection and paragonimiasis did not occur when A-Ag was used. These results indicated that the affinity-purified A-Ag had the higher specificity but the lower sensitivity as a diagnostic antigen in cysticercosis, probably because it only detected a single or limited numbers of monospecific antibodies among the diverse polyclonal antibodies produced in the patients with neurocysticercosis.

• Parasites, Hosts and Diseases
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• v.29 no.3
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• pp.293-310
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• 1991

Clonorchis sinensis is a common parasite of man in Korea. Researches on the specific antigens of C. sinensis would be valuable not only because those elucidate the molecular characteristics of this fluke but also because it is applicable to immunodiagnosis. Although many monoclonal antibodies have been used in the field of parasite immunology, few articles on monoclonal antibodies against C. sinensis have been published so far. The aim of this study was to analyse C. sinensis antigens recognized by monoclonal antibodies, and to set up ELISA-inhibition test using C. sinensis specific monoclonal antibodies for improved specificity of immunodiagnostic tests. By fusion between spleen cells of the mice immunized with C. sinensis water-soluble crude adult worm antigens and plasmacytoma cells of mouse origin, 29 hybridoma clones secreting anti-C. sinensis monoclonal antibodies were made, and 8 clones among those were found specific. After cell cloning, isotypes of 6 selected specific monoclonal anti- bodies were determined to be IgGl, IgG2b and IgA. Four exposed antigenic determinants of natural infection were recognized by different specific monoclonal antibodies. By enzyme-immunoelectrotransfer blot, 10 KD, 34 KD antigenic determinants were found to be reacted with CsHyb 0714-20, CsHyb 0605-10 monoclonal antibodies, respectively, The antigenic determinant recognized by CsHyb 0714-20 monoclonal antibody was revealed to be located at the surface and parenchyme of a parasite by indirect immunoauorescent antibody technique, and those reacted with CsHyb 0605-10, CsHyb 0714-25 monoclonal antibodies were found at the parenchyme and intestine. The antigenic determinant reacted with CsHyb 0605-23 monoclonal antibody was found mainly around the uterine eggs. Four antigenic determinants recognized by specific monoclonal antibodies were all found to be present in the early eluted fractions of C. sinensis antigens separated by Sephadex G-200 gel filtration. By conventional ELISA, 75% of clonorchiasis cases were found positive, but 7.1% of normal controls and 37.5% of paragonimiasis cases showed false positives. However, by ELISA-inhibition test using C. sinensis specific monoclonal antibody (CsHyb 0605-23), 77.1% of clonorchiasis cases were found positive, and there were no false positives in normal controls or paragonimiasis cases, indicating 100% specificity. The ELISA- inhibition test using monoclonal antibodies was found to have same sensitivity and definitely high specificity in comparison with conventional ELISA for serodiagnosis of human clonorchiasis.