The canine parvovirus (CPV) causes clinical signs, such as severe enteritis, dehydration, diarrhea, vomiting, leukopenia, and hair loss, which may lead to death. Vaccination is still the most important approach, as no specific treatment exists to prevent CPV. Monoclonal antibodies are valuable tools to study the pathogenic mechanisms of CPV and develop effective diagnostic reagents and pharmaceuticals. In this study, two monoclonal antibodies (MAbs) against CPV-2a were obtained through hybridoma technology by fusing myeloma cells and B cells from the spleens of mice immunized with CPV type 2a (CPV-2a). Two MAbs (CPV-330 and CPV-620) were studied on the reactivity of vaccine (CPV-2a) and field strains (CPV-new 2a, -2b, and -2c) by indirect immunofluorescence (IFA), hemagglutination inhibition test (HI), virus neutralization test (VN), and inhibition of virus growth test. Two MAbs showed similar antibody titers for HI and VN. On the other hand, CPV-330 inhibited the viral replication in Crandell-Rees Feline Kidney (CRFK) cells better than CPV-620. These CPV MAbs may provide valuable biological reagents to study the CPV pathogenic mechanisms and work as therapeutic antibodies.

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae is a highly contagious disease that leads to enormous economic losses in pig industry, worldwide. Of the many virulence factors produced by the causative bacterium, ApxA exotoxins have been considered as the most important contributor to the disease. The toxins are classified into four different types; ApxIA, ApxIIA, ApxIIIA and ApxIVA. Uniquely, ApxIVA is expressed across all serotypes of A. pleuropneumoniae only during in vivo infection in pigs. Active research focusing on resolving the precious roles and mechanisms of the toxins is still at its primitive stage. In this study, we report the development of monoclonal antibodies against the two major antigenic epitopes that were characterized in our previous study incorporating the in silico predictions and protein modeling analyses. Recombinant proteins of the selected epitopes were expressed and purified after molecular cloning of the corresponding partial genes in E. coli expression system. Subsequently, we generated hybridomas with lymphoid cells from the rats immunized with the recombinantly expressed proteins of Apx. Consequently, hybridomas exhibiting strong productivity of the monoclonal antibodies were selected for downstream verifications that tested for reactivity and specificity using Western blot and ELISA. Our results strongly suggest the potential application of the monoclonal antibodies developed in this study as useful reagents to further elaborate the mechanism of the A. pleuropneumoniae infection in pig.

American foulbrood (AFB) is caused by the bacterium Paenibacillus larvae, which is highly contagious and often lethal to honeybee broods. To control AFB, rapid diagnostic tools including those based on immunological methods are required. We produced several specific mouse monoclonal antibodies (MAbs) against P. larvae. Interestingly, a few of the MAbs were revealed to be an IgM-type antibody. To ascertain the effects of adjuvants on immunoglobulin isotype switching, BALB/c mice were immunized with various adjuvants, i.e., Freund's adjuvant (FA), Alum adjuvant, and AddaVax™ followed by the generation of hybridoma that secreted monoclonal antibodies to P. larvae. In the case of AddaVax™, all screened hybridoma clones secreted IgG-type MAbs, whereas hybridomas generated by Alum and FA secreted 91.25% (7/80) and 66.67% (11/33) respectively, IgG-type MAbs. Although the mechanism of incomplete immunoglobulin isotype switching associated with the P. larvae antigen needs further study, our results indicate that the applied adjuvants can have a significant effect on immunoglobulin isotype switching results.

In order to identify the specific antigens for pine wood nematode (PWN), we confirmed that one of the genes commonly found in the transcriptome, proteome and secretory proteins of PWN belonged to the Aldose Reductase (AR) family protein. 36.5 kDa PWN-AR1 was expressed and purified using Baculovirus Expression System. Total 1,546 hybridoma fusion library was generated and screened for specificity to PWN-AR1 by Enzyme Linked Immunosorbent assay (ELISA). Nine clones showed strong immunoreactivity to PWN-AR1 were limited-diluted. Total 864 limited-diluted clones were further screened using PWN-AR1 by ELISA and 34 monoclonal antibody (Mab) clones were selected. 34 Mab clones were further screened using PWN extracts and a standard PWN-infected pine tree extract by ELISA. Finally nine clones were selected and their immunoreactivities to 4 different nematodes were examined by ELISA. Seven clones pecifically recognized PWN while two clones recognized 4 nematodes. Our data suggested that PWN-AR1 is a PWN secretory enzyme while PWN is invading pine trees, Thus, PWN-AR1-Mabs could be used to develop diagnosis tools for PWN and its infected pine trees. (This work was supported by National Institute of Forest Science)

In this study, PAT protein of genetically modified maize was prepared from the recombinant E. coli strain BL21 (DE3), and mice were immunized with the recombinant PAT protein. After cell fusion and cloning, two hybridoma cells (PATmAb-7 and PATmAb-12) were chosen since the monoclonal antibodies (Mabs) produced by them were confirmed to be specific to PAT protein in the indirect enzyme-linked immunsorbent assay (ELISA) and western blot tests. There were no cross-reactions of either Mabs to other GM proteins or to the extracts of non-GM maize. The ELISA based on the PATmAb-7 can sensitively detect 0.3 ng/g PAT protein in corn. These results indicate that the developed Mabs can be used as bio-receptors in the development of immunosensors and biosensors for the rapid and simple detection of GM corn adulterated in foods.

소나무재선충(pine wood nematode, PWN) 감염 소나무를 현장에서 신속하게 진단을 할 수 있는 방법은 현재 없다. 본 연구에서는 PWN특이적인 항원으로 알려진 PWN-GaLectin을 baculovirus발현체계로 발현시켜서 총 1,464개의 fusion hybridoma 세포 주 라이브러리 를 제작 하는 항원으로 이용하였다. 총 1,464개의 fusion hybridoma 세포 주 중, PWN-GaLectin에 대한 높은 반응을 보이는 62개의 fusion hybridoma 세포 주를 선별했다. 이들 중, 표준 PWN 감염소나무 PBS추출물과 PWN 단백질 추출물에 강한 반응을 보이는 세포 주 12개를 선 별하여 단클론 항체(monoclonal antibody, Mab) 분비세포 주 확립을 위한 limited dilution을 실시하였다. Mab분비세포 주 확립을 위해서 표 준 PWN 감염소나무 추출물에 대한 반응이 표준 정상 소나무 추출물 보다 높은 세포 주들을 선별했다. 그리고 추가로 PWN 단백질 추출물에 대 해서 3종의 비 병원성 선충 단백질 추출물 보다 높은 반응을 보이는 세포 주들도 선별, 확립했다. 본 연구에서 확립된 Mab들을 우리는 현장과 실 험실에서 사용할 수 있는 신속진단키트의 개발에 이용할 수 있을 것이다.

Pine wilt disease is currently the most deadly forest disease in the world and is known to be caused by infection of Bursaphelenchus xylophilus. Until now, no method has been developed to confirm the pine tres infected with pine wood nematodes. In order to develop a method to diagnose pine wood nematode infection, we produced a monoclonal antibody against Expansin B3, which was found to be secreted by pine wood nematodes. ELISA assay using various monoclonal antibody confirmed that the pine trees infected with the rewarming can be detected. Further studies using our antibodies may help to develop a diagnostic method that can quickly confirm infection of pine wood nematodes in the field.

Rotaviruses are enteric pathogens causing acute watery dehydrating diarrhea in humans and animals. The importance of group C rotavirus (GpC-RV) infections has not been established as the studies on the GpC-RV have been hampered by the lack of an in vitro culture system. However, diarrheal diseases associated with GpC-RV have been gradually increasing worldwide. In this study, VP6 gene of bovine GpC-RV Korean isolate was expressed, and monoclonal antibodies (mAbs) against VP6 were produced and characterized. The VP6 gene was cloned and expressed based on a baculovirus expression system. Indirect fluorescence antibody (IFA), polymer chain reaction (PCR), and Western blot assays were used to confirm expression of VP6 gene synthesized by the recombinant baculovirus. Eleven mAbs against VP6 were produced using expressed VP6. Cross-reactivity of the mAbs was assessed with recombinant VP6 proteins from porcine GpC-RV and human GpA-RV, or different serotypes of group A rotavirus strains by IFA test. Some mAbs reacted with intact porcine GpC-RV Cowden strain as well as bovine GpC-RV VP6 recombinant baculoviruses, but not with human and animal GpA-RV strains. The VP6-specific mAbs might be useful to develop immunodiagnostic tests such as rapid diagnostic kit, IFA and enzyme-linked immunosorbent assay (ELISA) for detection of GpC-RV.

본 연구에서는 V. parahaemolyticus를 신속정확하게 고감도로 검출하기 위해 단클론성 항체를 개발하고 이를 이용하여 일반적인 효소면역분석법(ELISA)과 효소면역분석법의 낮은 민감도를 보완할 수 있는 면역선택여과법(ISF)을 개발하여 민감도 및 효율성을 비교분석 하였다. 먼저heat killed V. parahaemolyticus (HKVP)를 준비하여 7주령 BALB/c mouse에 면역한 후 세포융합 및 클로닝을 통해 HKVP 4H9-9번 및 16번 2종의 hybridoma cell을 확보하였다. Western blot을 통해 보다 특이성이 높은 것으로 확인된 HKVP 4H9-9번 항체를 대량 생산정제하여 분석법을 확립한 결과 검출한계가 ID-ELISA법은 106 cell/mL, ISF법은 5 × 101 cell/mL로 나타나 ISF법의 민감도가 매우 높은 것으로 확인되었다. 분석효율성은 ID-ELISA법의 경우분석을 위해 9단계의 과정을 거쳐 총 16시간의 분석시간이 소요된 반면, ISF법의 경우 3단계의 과정을 거쳐 1시간 이내에 분석을 완료할 수 있는 것으로 확인되었다. 교차반응성의 경우 두 분석법 모두 일부 Vibrio spp. (IDELISA법:V. alginolyticus, ISF법: V. vulnificus)과 S. aureus에 반응성이 있는 것으로 확인되었지만 V. parahaemolyticus에 보다 강한 반응성을 나타내었기 때문에 V. parahaemolyticus에 특이적인 분석법인 것으로 확인되었다. 특히 ISF법은 ELISA법 보다 민감도가 높고 분석에 소요되는 시간도 짧아 V. parahaemolyticus를 신속하게 분석하는데 활용이 가능할 것으로 판단된다.

Norovirus (NoV) is an etiologic agent of human and animal acute gastroenteritis and is a member of the family Caliciviridae. NoV is classified based on nucleotide sequences of the VP1 gene into at least six genogroups (GI-GVI), among which GI, GII, and GIV are known to infect humans and GII is the most prevalent genogroup. In this study, VP1, the full gene of GII human NoV, was cloned from a human fecal sample and expressed using a baculovirus expression system. Human NoV VP1-specific monoclonal antibodies (MAbs) were produced using expressed recombinant VP1. Expressed VP1 in the recombinant virus was confirmed by polymerase chain reaction (PCR), indirect fluorescence antibody (IFA) test, and Western blot analysis. Eight hybridomas secreting VP1-specific MAbs against human GII NoV were generated and characterized. All of the MAbs produced in this study reacted with human GII NoV VP1-recombinant baculoviruses but not with other non-human calicivirus recombinant baculoviruses. These MAbs reacted specifically with human NoV GII.4-2009 virus-like particles (VLPs), and some MAbs showed cross-reactivity with other GII.4 variant VLPs. Expressed human GII NoV VP1-recombinant protein and MAbs specific to this protein can be used as useful reagents for detecting and characterizing human NoV.

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 40l 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.

Ascidian embryos have become an important model for embryological studies, offering a simple example for mechanisms of cytoplasmic components segregation. It is a well-known example that the asymmetric segregation of mitochondria into muscle lineage cells occurs during ascidian embryogenesis. However, it is still unclear which signaling pathway is involved in this process. To obtain molecular markers for studying mechanisms involved in the asymmetric distribution of mitochondria, we have produced monoclonal antibodies, Mito-1, Mito-2 and Mito-3, that specifically recognize mitochondriarich cytoplasm in cells of the ascidian Halocynthia roretzi embryos. These antibodies stained cytoplasm like reticular structure in epidermis cells, except for nuclei, at the early tailbud stage. Similar pattern was observed in vital staining of mitochondria with DiOC2, a fluorescent probe of mitochondria. Immunostaining with these antibodies showed that mitochondria are evenly distributed in the animal hemisphere blastomeres at cleavage stages, whereas not in the vegetal hemisphere blastomeres. Mitochondria were transferred to the presumptive muscle and nerve cord lineage cells of the marginal zone in the vegetal hemisphere more than to the presumptive mesenchyme, notochord and endoderm lineage of the central zone. Therefore, it is suggested that these antibodies will be useful markers for studying mechanisms involved in the polarized distribution of mitochondria during ascidian embryogenesis.

Ascidian embryos have become an important model for embryological studies, offering a simple example for mechanisms of cytoplasmic components segregation. It is a well-known example that segregation of mitochondria into muscle lineage cells occurs during ascidian embryogenesis. It is, however, still unclear what signaling and molecular event control polarized distribution of mitochondria in the early ascidian embryonic development. To obtain molecular markers for studying mechanisms involved in polarized distribution of mitochondria, we have produced monoclonal antibodies, Mito-1, Mito-2 and Mito-3, that specifically recognize mitochondria-rich cytoplasm in all cells of the ascidian Halocynthia roretzi embryos. These antibodies stained cytoplasm like a mesh structure in epidermis cells, except for nuclei, at the early tailbud stage. Similar pattern was observed in vital staining of mitochondria with DiOC2, a fluorescent probe of mitochondria. These antibodies showed that mitochondria were distributed evenly in the animal hemisphere blastomeres at cleavage stages, whereas did not in the vegetal hemisphere blastomeres. Mitochondria were transferred more into cells of the marginal zone, such as muscle and nerve cord lineage cells, than into cells of the central zone, such as mesenchyme, notochord and endoderm lineage, in the vegetal hemisphere. Therefore, it is suggested that these antibodies may be useful as markers for analysing mechanisms involved in polarized distribution of mitochondria during ascidian embryogenesis.

무척추동물에서 척추동물에 이르기까지 대부분의 난생동물들의 난황 단백질의 전구체를 vitellogenin(VTG)이라 한다. 난생 척추동물에서 VTG는 간에서 합성되어 혈액을 통해 난세포로 전이된다. 암컷 어류는 정상적인 생식주기에서 난황단백전구체 형성이 시작되면 혈중 농도는 급격히 증가하게 된다. 수컷도 VTG의 유전자를 가지고 있기 때문에 낮은 수준의 내인성 에스트로겐으로 아주 적은 양의 단백질이 있을 수 있다. 그렇지만 수컷에 외인성에스트로겐 유사물

IAA에 대한 단크론 항체를 생산하고 이를 이용하여 생체중의 내생 IAA를 정량분석하기 위해 ELISA를 개발하고 본법을 사용하여 담배 종자 발아중 내생 IAA함량을 정량분석하였다. 그 결과는 1. IAA에 대한 단크론 항체 생산 세포주 3가지를 선발 작성하였으며 이 세포주로부터 생산되는 항체는 모두 IgG1 타입의 면역 글로블린이었다. 2. 상기 항체를 사용하여 ELISA를 수행하여 표준곡선을 작성하였던 바 검출 한계는 1pmol이었으며 검출 범위는 500pmol이었다 3. 표준곡선으로부터 작성한 Scatchard plot에 의한 친화 상수와 결합상수는 6.7 1010 L/M과 6 1010 L/M이었다. 4. 여러가지 IAA유사물질과 교차반응에 의해서 본 mAb는 특이성이 매우 높고 RIA에 의해서 고역가임을 확인하였다 5. 발아중인 담배종자로부터 면역측정에 의해서 내생 IAA를 정량분석하였다 6. 상기의 결과에 따라서 본 mAb를 이용하여 생체중의 내생 IAA를 간편하게 정밀 분석할 수 있음을 확인하였다.