Bovine rotavirus (BRV) is one of the common gastrointestinal diseases that can occur in calves, causing severe diarrhea. This study was conducted to investigate the effects of oral administration of single-domain antibodies, single variable domain of heavy chain of HCAb (VHH), on the prevention and treatment of rotavirus-induced diarrhea in calves. Thirty calves were divided into five experimental groups (negative control, positive control, group A, group B and group C). Except for the negative control group, the experimental groups were inoculated with BRV on the 4th day after birth. The VHH supplementation groups were fed with a substitute milk containing different concentrations of VHH (1%, 2.5% and 7.5%). Changes in body weight, clinical symptoms, serum antibodies, and virus detection in feces were observed for a total of 10 days in each experimental group. The results showed a higher survival rate in the VHH supplementation groups compared to the positive control group. Clinical symptoms caused by rotavirus were alleviated in the VHH supplementation groups. The detection rate of rotavirus in feces also decreased. Gross findings indicated that the severity of disease in the VHH supplementation groups was milder than that in the positive control group. Therefore, VHH could be proposed as a useful alternative for the prevention and treatment of rotavirus-induced diarrhea in calves.

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.

The immunological sperm separation method is economical compared to the existing sorting method, and it is promising for the development of new technologies by reducing sperm damage. Wholemom (WM) is a sex-regulating protein that comprises on immunoglobulin G coupled with magnetic nanoparticles (MNPs) that responds to surface proteins derived from the Y chromosome in cattle. Y sperms are restricted in motility as the WM aggregates them, and the magnet could separate the non-aggregated cells. This study was conducted to investigate the effect of WM treatment on the characteristics of bull sperm. After treating sperm with WM and incubation for 6 h, the motility parameters including total motility, progressive motility, velocity average path, velocity straight line, amplitude of lateral head displacement, and linearity were significantly higher in the WM treatment group than in the control group (p < 0.05). Sperm viability and acrosome reaction rates were similar in both groups during each incubation period (p > 0.05). In conclusion, the immunological sperm sexing procedure using a monoclonal antibody conjugated with MNPs did not affect the characteristics of bull sperm. This study suggests that compared to other techniques, the immunological method for sperm sexing could classify sperm quickly and efficiently without the use of expensive equipment.

A solid-phase competition enzyme-linked immunosorbent assay (ELISA), recombinant VP2 (rVP2) protein, and monoclonal antibody (mAb) were developed for the specific and sensitive detection of porcine parvovirus (PPV) antibodies in pig sera. A total of 1,544 sera samples were collected from breeding pig farms located in the Gyeongsangbuk-do Province in the Republic of Korea. The optimal operating conditions of SC-ELISA were as follows. The concentration of rVP2 proteins coated on the wells was 4 μg/mL, the swine sera were diluted 1:2, and the HRP-conjugated PPV VP2 mAb (9A8 clone) was used at 500 ng/mL. These results suggest that the SC-rVP-ELISA assay may be a valuable alternative to the current diagnostic tools used to detect PPV-specific monoclonal antibodies and broadly monitor PPV infections in domestic pigs at different breeding stages.

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.

In previous studies, we found the production of antibodies against cross-reactive bovine serum albumin (BSA) in D-galactose (D-gal) induced aging mouse models. We performed immunoblot analysis with mouse tissue lysates to investigate the changes in the overall autoantibody production in this animal model. And we were able to see the possibility of altering the activity of mouse natural antibodies in this process. In this study, we examined changes in existing natural antibodies in a D-gal-induced aging mouse model. Serum samples were collected from 3-week-old mice (3w), 13-week-old mice (13w), and 13-week-old mice that were treated with D-gal for 6 weeks (13wDG), beginning at the age of 8 weeks. Levels of immunoglobulins (IgM, IgG, and IgA) were quantitatively analyzed in serum samples. Tissue samples were obtained from skin, spleen, and ovary for Western blotting analyses. Natural antibody activity was examined by enzyme-linked immunosorbent assay analyses of anti- double-stranded DNA (dsDNA) antibody. Western blotting analyses using mouse tissue lysates showed that several protein bands detected by serum antibodies from 3w mice became increasingly thicker when detection was performed with serum samples from 13w and 13wDG mice, indicating quantitative increases in levels of natural antibodies. Relative amounts of total IgG, IgM, and IgA immunoglobulins sequentially increased in serum samples from 3w, 13w, and 13wDG mice. A similar tendency was observed regarding the levels of IgM and IgG antibodies against dsDNA. These results indicate increased levels of natural antibodies in the D-gal-induced aging mouse model. Therefore, this animal model could be useful for future natural antibody research.

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.

The present study aimed at evaluating serum immunoglobulin G (IgG) avidity to Porphyromonas gingivalis in elderly patients with mild and severe chronic periodontitis. The avidity of antibodies against P. gingivalis present in the sera of 18 patients with mild chronic periodontitis and 18 patients with severe chronic periodontitis was evaluated using an ammonium thiocyanate-dissociated enzyme-linked immunosorbent assay (ELISA). The results showed that the mean absorbance value in serum IgG antibody titers was significantly higher in the severe chronic periodontitis group than in the mild chronic periodontitis group (198 ± 35 ELISA unit [EU] vs. 142 ± 32 EU, p < 0.01). However, there was no significant difference between the two groups in antibody avidity (65 ± 57 EU vs. 54 ± 27 EU). These findings suggest that humoral immune responses to P. gingivalis between mild and severe chronic periodontitis in elderly patients are characterized by the differences in the quantity rather than the quality of the antibodies.

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.