• Journal of Marine Bioscience and Biotechnology
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• v.1 no.4
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• pp.298-304
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• 2006

In this study, we establish a thin-layer chromatography (TLC) immunostaining method for detecting starfish gangliosides. A new monoclonal antibody (MAb) against AG-2, the major gangliosides molecular species of Acanthaster planci, was produced by fusing hybridoma with splenocytes immunized to liposomal AG-2. BALB/c male mice were injected with liposomal AG-2 antigen, and immunized. Their splenocytos were isolated and fused with hypoxanthine-aminopterine-thimidine (HAT)-sensitive mouse myeloma cells. Hybridomas producing MAb reactive to AG-2 were cloned using the limited dilution method. Established hybridomas were cultured in eRDF medium. Crude MAb produced from clone 8D4 was purified with a magnesium pyrophosphate column. Enzyme immunoassay and TLC immunostaining of AG-2 were performed using the purified MAb. Structurally related gangliosides did not cross-react with anti-AG-2 antibodies. The detection limit of TLC immunostaining was 50 ng of AG-2. The newly established immunostaining method was further developed for detecting AG-2 distribution and qualitative analysis in tissues and/or organs. Our results show that the majority of AG-2 is present in the stomach of male A. planci, while AG-2 is distributed not only in the stomach but also in the the pyloric caeca of female A. planci.

• Journal of Veterinary Clinics
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• v.25 no.5
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• pp.317-323
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• 2008

Canine trypsin-like immunoreactivity (cTLI), which is a mirror of the concentration of trypsin and trypsinogen, is a pancreas-specific enzyme and a suitable marker for canine pancreatitis and especially exocrine pancreatic insufficiency (EPI). To develop the immunochromatographic test kit, monoclonal antibodies that recognize cTLI were prepared. Anionic trypsin, cationic trypsin, and chymotrypsin from canine pancreas were successfully purified to homogeneity, using ammonium sulfate fractionation and benzamidine-affinity chromatography. The purification fold for anionic trypsin was 108 times when compared with that of the homogenation of pancreas. The molecular weights by SDS-PAGE analysis were approximately 23 kDa for chymotrypsin and approximately 20 kDa for cationic trypsin and anionic trypsin, respectively. Using the purified trypsin-like proteins, ten hybridomas which secret canine trypsin-specific monoclonal antibody were prepared. Klotz plot indicated that hybridomas, 5G2H10G4 and 2F4A11, have high affinity constant (Ka) of $4.1\;{\times}\;10^{9}$ and $1.8\;{\times}\;10^{9}$, respectively. Especially, 5F9H3 showed the cationic typsin-specific binding pattern and its Ka was determined to $4.5\;{\times}\;10^{9}$. The development of immunochromatographic test kit using these monoclonal antibodies against cTLI will be very useful in the diagnosis of canine EPI or canine pancreatitis.

• Korean Journal of Veterinary Research
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• v.37 no.2
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• pp.375-381
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• 1997

The purpose of the production of monoclonal antibodies aganist the Canine distemper virus(CDV) were perfect diagnosis and a new approach to treat canine distemper because the diagnosis and treatment of canine distemper were difficult. Canine distemper virus(CDV) was purified using saturated ammonium sulfate, and injected into hind footpads of BALB/c mouse. 12-15 days later, popliteal lymph node(PN) cells were harvested and fused with SP2/O myeloma cells. Characteristics of monoclonal antibodies were analysed. 1. 9 hybridomas produce the specific antibody against CDV. 2. 6 monoclonal antibodies are against intranuclear and cytoplasmic component of CDV, and 3 monoclonal antibodies are against cytoplasmic inclusions. 3. All monoclonal antibodies did not react with other 5 different viruses (CAV-I, CAV-II, CCV, CPV and CPIV) and react with another CDV-FXNO strain. 4. 3 monoclonal antibodies have neutralizing activity against CDV. 5. Antigenic difference was observed between CDV by IFA.

• Journal of Microbiology and Biotechnology
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• v.5 no.6
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• pp.353-358
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• 1995

Escherichia coli causes intestinal and extraintestinal infections and has been an indicator of fecal pollution in water and food. BALB/c mouse was immunized by injection of somatic E. coli (ATCC 8739) cells to produce monoclonal antibodies. Splenocytes of mouse were fused with myeloma cells (Sp2/0-Ag14). Two hybridomas secreting monoclonal antibodies were established after being cloned. In SDS-PAGE analysis of E. coli antigens 37 protein profiles appeared from 14 kDa to 182 kDa. Western blot analysis using polyclonal antibodies demonstrated that protein antigens of 41 kDa, 38.2 kDa and 31.7 kDa were immunodominant. Monoclonal antibodies DY-CM1 and DY-CM2 recognized 31.7 kDa and 2.0 kDa antigens in Western blot analysis, respectely.

• Animal cells and systems
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• v.2 no.3
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• pp.371-375
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• 1998

Twenty-one monoclonal anti-DNA autoantilndies were produced by fusing spleen cells from an autoimmune MRL/lpr mouse with SP2/0 myeloma cells. Hybridomas generated by the fusions were chosen for cloning on the basis of DNA binding by supernatant antibody. Each monoclonal antibody was purified to homogeneity and analyzed for the heavy and light chain isotypes and the binding specificity for single-stranded DNA, double-stranded DNA, and RNA. Sequence specificities and isoelectric points of the antibodies were also examined. All of the antibodies were lgG and tended to bind to both single-stranded and double-stranded DNA with a preference for the double-stranded form. Some of them also bound to RNA. Isoelectric points of the antibodies were shown to be high. The antibodies described in this report have characteristics of pathogenic anti-DNA antibodies.

• Microbiology and Biotechnology Letters
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• v.17 no.5
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• pp.481-488
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• 1989

A serum-free medium that could be used for the large-scale culture of mouse hybridoma to produce monoclonal antibodies was developed. The medium was based on a 1:1 mixture of Iscove's Modified Dulbecco's Medium and Ham's F-12, supplemented with insulin 10$\mu\textrm{g}$/$m\ell$, transferrin 10$\mu\textrm{g}$/$m\ell$, ethanolamine 10$\mu$M and selenium 30nM (designated EBM (enriched basal medium) with the supplements). The effect of various supplements of steroid hormones, vitamins, lipid and mineral salts was investigated and their optimal concentration was determined to replace fetal calf serum (PCS). These components were added respectively and then added by way of two or three combination to discern of which component combination was effective to the culture of hybridoma. As a result, serum-free medium KM3 (EBM with BSA 100$\mu\textrm{g}$/$m\ell$, mineral cocktail and 0.05% PEG) was deter-mined. The hybridoma Alps 25-3 cultured in this medium showed almost the same growth rate as in medium added with 2% fetal bovine serum. However, the antibody concentration from KM3 cultures was 80% of that obtained from culture with FCS. KM3 was also examined for the culture of other mouse hybridomas, KW, A4W & HCGK, and it was confirmed that it could support the growth of these hybridomas and the production of monoclonal antibodies.

• Journal of fish pathology
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• v.26 no.3
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• pp.241-254
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• 2013

In order to establish bio-marker systems for the screening of endocrine-disrupting chemicals contaminated in various environment, Vitellogenin(Vtg) bio-marker have been developed to detect Scorpion fish's(Sebastiscus marmoratus) Vtg. Vtg has been induced by administration of estradiol into S. marmoratus, and purified by gel filtration and ion-exchange chromatography from serum of the fish. After immunization of the purified Vtg into BALB/c mouse, hybridomas secreting anti-Vtg antibodies have been produced. The size of induced Vtg in the serum was about 440 kDa by gel filtration using Sepharose CL-6B. By SDS-PAGE analysis, the main band of Vtg, however, was at 175 kDa, and several minor bands have been detected with the main band. Eight different monoclonal antibodies have been produced from established hybridomas and the antibodies did not cross-react with sera from different species of fishes tested in this study except with that of Sebastes hubbsi. These results suggested that the monoclonal antibody of S28 and S15 can used as capture and tracer antibodies for ELISA and ICG assays. The detection systems developed in this study can be used as Bio-marker assays to check endocrine disrupting activity of various chemicals as well as to detect known endocrine disrupting chemicals contaminated in environment.

• BMB Reports
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• v.42 no.11
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• pp.731-736
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• 2009

The generation of functional recombinant antibodies from hybridomas is necessary for antibody engineering. However, this is not easily accomplished due to high levels of aberrant heavy and light chain mRNAs, which require a highly selective technology that has proven complicated and difficult to operate. Herein, we attempt to use an alkaline phosphate (AP)-fused form of single-chain variable fragment (scFv) for the simple identification of a hybridoma-derived, functional recombinant antibody. As a representative example, we cloned the scFv gene from a hybridoma-producing mouse IgG against branched-chain keto acid dehydrogenase complex-E2 (BCKD-E2) into an expression vector containing an in-frame phoA gene. Functional recombinant antibodies were easily identified by conventional enzyme-linked immunosorbent assay (ELISA) by employing scFv-AP fusion protein, which also readily serves as a valuable immuno-detective reagent.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.34 no.s01
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• pp.48-54
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• 1989

An immunoassay techniques using monoclonal antibodies have been developed of the quantitative analysis of endogenous hormones in plants. In order to be useful for immunoassay, both a high degree of specificity and a high affinity are required. A system is described for production of hybridomas which secrete antibodies against the plant hormone. Using the system we were able to produce hybridmas with the desired antibody specificity by cell fusion and culture method. For a number of obvious reasons, monoclonal antibodies(mAb) were superior to polyclonal antibodies.

• Journal of Periodontal and Implant Science
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• v.37 no.1
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• pp.11-21
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• 2007

Heat shock protein (HSP) is one of cellular protein commonly present in major periodontopathogenic bacteria as well as mammalian cells. The protein may play a role in the immunopathogenesis by modulating autoimmune reaction due to its high level of sequence homology between bacteria and human counterpart. Hence, identifying immunodomiant epitope of bacteria HSP that is cross-reactive to periodontopathogenic bacteria with a specificity to human HSP may comprise a critical strategy for development of a periodontal vaccine. The present study was performed to establish clones producing monoclonal antibody reactive to Porphyromonas gingivalis (p. gingivalis) HSP with a specificity to human HSP. 4 different hybridomas were cloned producing monoclonal IgG antibodies to P, gingivalis HSP and evaluated for their reactivity and specificity to other periodontopathogenic bacteria as well as to human HSP. These four monoclonal antibodies reacted with p. gingivalis HSP only with specificities to other bacteria tested and human HSP as well. The antigenic epitopes producing the 4 monoclonal antibody may be potentially developed as vaccine candidates. Further investigations are under way to identify more clones producing monoclonal antibodies reactive to P, gingivalis HSP and to other periodontopathogenic bacteria as well, while maintaining specificities to human counterpart.