• 대한의생명과학회지
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• 제11권4호
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• pp.429-434
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• 2005

Apolipoprotein E (apoE) restriction isotyping used oligonucleotides to amplify apoE gene sequences containing amino acid positions 112 and 158. The amplification products were digested with HhaI and subjected to electrophoresis on $4\%$ agarose gel. Each of the isoforms was distinguished by a unique combination of HhaI fragment sizes that enabled unambiguous typing of all homozygotic and heterozygotic combinations. HhaI cleaves at GCGC encoding 112arg (E4) and 158arg (E3, E4), but does not cut at GTGC encoding 112cys (E2, E3) and] 58cys (E2). DNA was isolated from 72 study participants and apoE genotypes were determined utilizing the polymerase chain reaction and restriction isotyping. In the entire group of subjects, $38 (52.8\%)$ had apo E4/4 or E3/4 (Group E4), $28(38.9\%)$ had the apo E3/3 genotype (Group E3) and $6(8.3\%)$ had apo E2/2 or E2/3 (Group E2). This genotypic information may help to identify individuals at increased risk for several diseases.

• 약학회지
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• 제49권6호
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• pp.484-489
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• 2005

Streptococcus pmeumoniae is the leading cause of pneumonia and bacterial meningitis. The current polysaccharide vaccine has been reported ineffective in elderly adults and children less than 2 years of age. Thus, in recent many researchers have been focused on a different approach, DNA vaccine. In our laboratory we developed a Streptococcus pneumoniae DNA (SPDNA) vaccine. This SPDNA vaccine was formulated by inserting the region encoding part of the capsule in the S. pneumoniae into the LAMP-1. In present work, with use of the SPDNA vaccine we attempted to establish a certain methodology useful for evaluation of effectiveness and immunoresponse of a DNA vaccine. Results showed that the subcutaneous route was the most effective for production of antisera specific for S. pneumoniae in mice. By isotyping analyses, IgM, IgGl, IgG2a, and IgG2b were determined. In addition, INF-$\gamma$ and IL-4 were predominantly detected. Combination of those data resulted in a pattern of IgGl < IgG2a=IgG2b and INF$\gamma\>$ >IL-4, which indicates the inmmunity towards the Thl response predominantly; furthermore, the SPDNA vaccination induced resistance of the CD4+T lymphocyte-depleted mice against disseminated pneumococcal infection. These data appear to be possibly due to activation of CDS8+T cell-activation. Taken together, this methodology can be applied for evaluating efficacy and mode of action of a DNA vaccine as minimum critera.

• 약학회지
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• 제56권6호
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• pp.366-371
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• 2012

In this present study, we determined whether or not there is an immunoadjuvant effect of ochnaflavone, a biflavone isolated from Lonicera japonica. As an antigenic source, the cell wall (CACW) of Candida albicans, a fungal pathogen, was used. CACW consists of 95% carbohydrate (mannan). In the experiments, BALB/c mice were immunized with emersion forms of CACW combined with or without ochnaflavone (Och) in the presence of IFA containing mineral oil or CACW alone. Then, the amounts of antisera collected from these mice groups were measured by the ELISA method. Data from these experiments showed that CACW combined with Och (CACW/Och/IFA) provoked the production of antisera app. 2.2 or 5 times more than the corresponding CACW/IFA or CACW alone (CACW/DPBS), respectively, in mice (P<0.05). We further examined the immune response type induced by Och. Analysis of the values of the IgG1/IgG2a ratios obtained from IgG isotyping revealed that Och induced Th2-immunity more dominantly than Th1. This finding was confirmed by cytokine profile. CACW/Och/IFA formulation induced IL-4 (Th2-type cytokine) more than IFN${\gamma}$ (Th1-type cytokine) as compared with CACW/IFA and CACW/DPBS formulations (P<0.05). All data combined, Och appears to have an immunoadjuvant activity that may convert Th1 immunity into Th2 immunity.

• 약학회지
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• 제50권1호
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• pp.33-39
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• 2006

We have previously reported the minimum criteria that can be applied to evaluate efficacy and safety of a DNA vaccine with use of Streptococcus pneumoniae DNA vaccine (SPDNA). The SPDNA was formulated by inserting the DNA sequences that are codons specific for the carbohydrate epitope in the capsule of S. penumoniae by phage display peptide library. Administration of the SPDNA into mice induced both humoral and cell-mediated immunities. The induction was protective even in the absence of CD4+ T lymphocyte in mice. Profiles of cytokine and isotyping of antibody displayed tendency of the Th1. In continuation of these studies, we examined if the efficacy of the SPNDA was provoked by the peptide recognized by codons specific for the capsule. Results showed that the peptide vaccine formulae (SPP) induced protective antibody in mice as did the SPDNA. Involvement of the cell-mediated immunity was also determined. Possible side effects of autoimmune diseases such as myositis and C3a production and tumor-formation were undetectable in mice given 7 times of SPDNA vaccination during entire of 92 days. Even after the frequent immunization, immunogenicity of the SPDNA was observed as determined for antibody production, suggesting that there was no immunotolerance provoked. All together, these examining factors would be applied to measurement of a DNA vaccine safety regarding the immunopathological aspect.

• 약학회지
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• 제48권6호
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• pp.345-351
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• 2004

ln the present work to determine effect of a Streptococcus pneumoniae conjugate vaccine, S.pneumoniae capsule attached to the surface protein (JY-Pol) was ex amined. This JY-Pol contained approximately 92% and 6% carbohydrate and protein, respectively. Gel electrophoresis revealed the presence of the surface protein in the JY-Pol. By the double immunodiffusion and isotyping ELISA analyses, administration of JY-Pol that was adsorbed to alum adjuvant (JY-Pol/Alum) into mice induced IgM, IgG, and IgA specific for the S.pneumoniae capsule. The ATCC capsular polysaccharide adsorbed to alum (ATCC-Pol/Alum) provoked only IgM in mice. In survival tests, mice that were immunized with the JY-Pol/Alum before intravenous challenge with live S.pneumoniae survived entire period of 46 day-observation, whereas all mice that received ATCC-Pol/Alum or only diluent instead of the vaccination died within 5 and 12 days, respectively. Results from footpad-edema test showed that JY-Pol/Alum formula provoked the cellular immunity as determined by swelling of the mouse footpad. These data indicate that the naturally conjugated JY- Pol enhances resistance of mice against disseminated pneumococcal disease due to S.pneumoniae by both humoral and cellular immune responses.

• 한국수정란이식학회지
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• 제11권1호
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• pp.71-80
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• 1996

The envelope of the rnannnalian oocyte plays crucial roles in sperm-oocyte interactions by providing sperm receptors, inducing acrosome reaction and preventing polyspermy. Understanding of properties of the zona pellucida (ZP) is essential for the artificial control of fertility in mammals. This study was carried out to produce and characterize monoclonal antibodies(MAbs) to porcine ZP proteins. Approximately 8,000 ZPs were obtained from follicular oocytes and dissolved in 40$\mu$l of double distilled water. Following immunization through foot-pad injections of Balb /c mice with a ZP solution, the popliteal lymph nodes were recovered at 2 weeks after the last injection. Hybridoma cell lines were established by fusing lymph node cells with P3X63 myeloma cells through selection using HAT medium and screening by immunofluorescence(IF) microscopy on the isolated ZP. Secreted MAbs were found to consist k chains and different heavy chains as evidenced by isotyping. Some of the MAbs demonstrated high specificity to the ZP in IF. The Mabs also showed positive cross reactivity with hamster and mouse eggs, while negative with bovine eggs. The results implicate that the MAbs can be used not only for identification of functional regions of the ZP, but also for elucidation of mechanisms involved in fertilization of mammals. The MAbs will provide basic information on biochemical anatomy of the ZP as well as can be candidates for the future contraceptive vaccines.

• 한국식품위생안전성학회지
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• 제8권4호
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• pp.205-214
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• 1993

The monoclonal antibody to chloramphenicol(CAP) was produced to develop an enzyme-linked immunosorbent assay(ELISA) for residual CAP. An immunogen(CAP-BSA) was prepared by immunogen, antibody titer was measured by indirect ELISA. Spleen cells form the immunized mouse were fused with SP2/OAg14 myeloma cells. Among hybridomas selected in HAT media, 6 clones shown high antibody titer to CAP were subjected to cloning by limit dilution, and all of the monoclonal antibodies(MCA1, 2, 3, 4, 5, 7 and 9) produced by each clone were identified as IgG1 by ELISA isotyping analysis. Competitive ELISA condition was established by using the purified monoclonal antibody MCA1 as primary antibody and CAP-HSA conjugate as coating antigen. Standard curve of CAP(n=28) showed that the lowest detection limit of CAP is 20ng/ml level. The cross-reactivities of the 6 monoclonal antibodies showed that CAP sodium succinate. CAP base, P-nitrophenol, and p-nitrobenzyl alcohol were 89∼178, 0.050∼2.237, 0.056∼0.794 and 0.013∼7.939%, respectively. No cross-reactivities were observed with phenylalanine, tyrosine, glutamine, thiamphenicol, neomycin, streptomycin, gentamicin, sulfamethazine, sulfathiazole, chlortetracycline and p-aminobenzoic acid.

• Asian-Australasian Journal of Animal Sciences
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• 제16권4호
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• pp.541-548
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• 2003

The Immunoglobulin $IgG_1$ and $IgG_2$ isotype immune responses of domestic ruminants and mice to Cowdria. ruminantium live infection or by immunization with inactivated organisms were determined by the enzyme linked immunosorbent assay and Western blotting. Immunization of goats with inactivated elementary bodies (IEBs) led to a predominant $IgG_1$ isotype response. This indicated that a Th2 response was induced. After challenge, the IgG isotype responses were mixed whereby both $IgG_1$ and $IgG_2$ antibodies were detected. Two goats that survived virulent challenge had a predominant $IgG_2$ isotype response. In cattle live infection by natur l challenge or experiment led to a predominant $IgG_1$ isotype response. Immunization of cattle with IEBs however led to mixed IgG responses characterized by similar $IgG_1$ and $IgG_2$ ratios. In the mouse live infection led to a predominant $IgG_2$ isotype response. This indicated the mouse developed a true Th1 type cell mediated immune response when inoculated with live organisms. Immunization with inactivated organisms on the other hand led to a dominant $IgG_1$ response. It is evident from this work that the immune responses of ruminants and mice to C. ruminantium are different and that using mice as the experimental model for immune responses to Cowdria ruminantium. is not the appropriate.

• Parasites, Hosts and Diseases
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• 제39권1호
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• pp.67-76
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• 2001

This experiment was focused on the characterization of anti- Toxoplasma monoclonal antibodies (mAbs) and the effect of mAbs on the parasite invasion of mouse peritoneal macrophages. Twenty eight mAbs including M110, M556, R7A6 and M62l were characterized by Ab titer, immunoglobulin isotyping and western blot pattern. Antibody titer (optical density) of 4 mAbs. Ml 10. M556. R7A6 and M62l. were 0.53,0.67, 0.45 and 0.39 (normal mouse serum; 0.19) with the same IgGl isotypes shown by Enzyme-linked immunosorbent assay (ELISA). Western blot analysis showed that Ml 10. M556. R7A6 and M62l reacted with the 33 kDa (p30),31 kDa (p28),43 kDa and 36 kDa protein. Immuno-gold labelling of mAbs M110, M556, R7A6 and M621 reacted with the surface membrane, dense granules and parasitophorous vacuolar membrane (PVM) , rhoptries and cytoplasm of tachyzoite, respectively. For in vitro assay, preincubation of tachyzoties with four mAbs, Ml 10, M556, R7A6 and M62l resulted in the decrease of the number of infected macrophages (p < 0.05) and the suppression of parasite multiplication at 18 h post-infection. Four monoclonal antibodies including Ml 10 (SAGI) were found to have an important role in the inhibition of macrophage invasion and T. gondii multiplication in vitro, and these mAbs may be suitable for vaccine candidates, diagnostic kit and for chemotherapy.

• Parasites, Hosts and Diseases
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• 제30권4호
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• pp.289-298
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• 1992

Acanthnmoeba sp. YM-4의 영양형은 사상의 침상위족을 내어 전형적인 각canthamoeba 속의 특징이 관찰되었으며, 포낭(씨스트)은 외벽이 등근혐이었고 내벽이 단순한 구형이거나 다소 불규칙한 2개의 세포벽을 갖고 있었다. SDS-PAGE 결과, Acanthamoeha sp. Yhf-4는 약 16개의 주요 단백질 분획이 관찰되었는데, 2차원 전기영동 결과와 함께 Acanthamoeba sp. YM-4의 단백질 분획은 A. culbertsoni와 거의 같은 양상이 관찰되었다. Acanthamoeba sp. YM-4로 면역시킨 마우스의 비장세포와 myeloma 세포를 융합한 결과, 항체를 분비하는 17개 clone을 얻을 수 있었다. 생성된 단세포를 항체 중 isotyping을 실시하였는데 실험에 사용된 McAY 6, McAY 7, McAY 8, McAY 13, McAY 16 단세포군은 IgGl 항체를, McAY 10, McAY 11 단세포군은 19M 항체를 분비하였다. 생성된 단세포군 항체는 대부분이 아메바의 세포막에 있는 항원과 반응하였으며, EITB 실험결과, McAY 7 단세포군 항체는 43 kD에서 반응대가 관찰되었고, McAY 10 단세포군 항체는 55 kD과 105 kD에서 반응대가 관찰되었다. ELISA 방법을 이용한 단세포를 항체들과 여러 아메바 간의 교차반응실험 결과, McAY 7 단세포군 항체는 Acanthamoeba sp. YM-4에만 반응하였고, McAY 6과 McAY 10은 A. culbertsoni와도 반응하였다. 또한 McAY 11은 모든 아매바와 교차반응을 보였다.