• Korean Journal of Veterinary Service
• /
• v.34 no.1
• /
• pp.5-12
• /
• 2011

Infectious bursal disease (IBD) caused by infectious bursal disease virus (IBDV) is a highly contagious viral disease in chicken. It causes heavy economic loss by immune suppression and high mortality. The IBDV, designated Avibirnavirus in the Family Birnaviridae, has a double-stranded RNA genome formed by two segments, segment A and segment B. Segment A encodes a 108 KDa polypeptide that is self-cleaved to produce pVP2, VP3 and VP4, and later pVP2 is cleaved to VP2. The VP2 contains the antigenic regions responsible for elicitation of neutralizing antibodies and VP3 is a major immunogenic protein of IBDV. In this study, monoclonal antibodies (MAbs) specific for IBDV were produced and characterized. All 15 MAbs were specific for IBDV and did not react with other viruses used in this study. The protein specificity of MAbs was determined by comparing the reactivity patterns of each MAb with IBDV VP2 and VP234 recombinant baculoviruses and Western blot analysis. As a result, 7 MAbs (1F5, 2C8, 2F4, 3C7, 4C3, 6F11, 6G5) and 5 MAbs (2A4, 2G2, 3F5, 3G2, 4F10) were specific for VP2 and VP3, respectively. The protein specificity of 3 MAbs (2B8, 3F7, 3F8) were not determined. Five (2C8, 2F4, 4C3, 6F11, 6G5) of the VP2-specific MAbs had a neutralizing activity against IBDV. Some MAbs reacted with IBDV-infected bursa of Fabricius by indirect fluorescence antibody (IFA) and immunohistochemistry (IHC) assay. The MAbs produced in this study would be used for diagnostic reagents for the detection of IBDV infection.

• The Korean Journal of Physiology and Pharmacology
• /
• v.13 no.5
• /
• pp.373-378
• /
• 2009

Cerebellar Purkinje cells (PCs) play a crucial role in motor functions and their progressive degeneration is closely associated with spinocerebellar ataxias. Although immunohistochemical (IHC) analysis can provide a valuable tool for understanding the pathophysiology of PC disorders, the method validation of IHC analysis with cerebellar tissue specimens is unclear. Here we present an optimized and validated IHC method using antibodies to calbindin D28k, a specific PC marker in the cerebellum. To achieve the desired sensitivity, specificity, and reproducibility, we modified IHC analysis procedures for cerebellar tissues. We found that the sensitivity of staining varies depending on the commercial source of primary antibody. In addition, we showed that a biotin-free signal amplification method using a horseradish peroxidase polymer-conjugated secondary antibody increases both the sensitivity and specificity of ICH analysis. Furthermore, we demonstrated that dye filtration using a $0.22\;{\mu}m$ filter eliminates or minimizes nonspecific staining while preserving the analytical sensitivity. These results suggest that our protocol can be adapted for future investigations aiming to understand the pathophysiology of cerebellar PC disorders and to evaluate the efficacy of therapeutic strategies for treating' these diseases.

• Journal of Animal Science and Technology
• /
• v.47 no.1
• /
• pp.19-28
• /
• 2005

The objectives of the current study were to develop polyclonal antibodies in sheep against adipocyte plasma membrane(APM) proteins isolated from swine, to investigate tissue specificity, and to determine cytotoxic effects of antiserum on swine adipocytes. Plasma membrane proteins from adipocyte, brain, heart, kidney, liver, and spleen were isolated using a 32% sucrose gradient. Adult male sheep was immunized three times at three week interval with the purified swine APM proteins. Antiserum was taken from immunized sheep at 10, 12, and 14 days after the third immunization. Antiserum expressed strong reactivity with APM proteins determined by enzyme-linked immunosorbent assay(ELISA), and the reactivity could be detected at dilutions in excess of 1 : 81,000. Antiserum showed very low binding affinity with proteins isolated from brain, heart, kidney, liver, or spleen. Tissue specificity of the antiserum was reconfirmed by Western immunoblotting using anti-sheep immunoglobulin G•alkalinephosphatase conjugate as a secondary antibody. The reactivity of antiserum to the external surface of fixed swine adipocytes was confmned by an immunohistochemical technique using anti-sheep immunoglobulin G-FITC. Confluent swine adipocytes in culture were lysed by antiserum treatment and cytosolie lactate dehydrogenase(LDH) was released as a dose-dependent patterns while adipocytes treated with normal sheep serum maintained their integrity and expressed low level of LDH. These results implicate that fat contents in the pigs can be reduced by immunological methods.

• Biomedical Science Letters
• /
• v.5 no.1
• /
• pp.1-10
• /
• 1999

This study was attempted to generate a monoclonal antibody against human $\alpha$-fetoprotein (AFP) and to produce an immunoassay, recognizing AFP in plasma and amniotic fluid. AFP was purified from human amniotic fluid and used to immunize mice. Spleens were taken from the mice and the cells were fused with mouse myeloma cells (Sp2/0-Ag-14) for the production of monoclonal antibodies by employing the hybridoma technology. As a result, a hybridoma cell line producing anti-AFP monoclonal antibody was cloned out and designated as MabF22. From isotyping analysis, it was found that monoclonal antibody MabF22 was IgG type with IgG1 heavy chain and k light chain. The binding specificity of MabF22 was analyzed by immunoblotting as well as by ELISA. MabF22 was highly specific, reacting with only AFP-containing samples. The binding affinity was determined by ELISA (free-capture mode) and Scatchard analysis. As a result, the value of Kd was 0.8$\times$10$^{-10}$M. The validity of the MabF22 for AFP assay was examined by two kinds of ELISAs, i.e., non-competitive and competitive ELISA. Both assays revealed that MabF22 reacted well with AFP in sample in a concentration-dependent manner. Standard curve and antibody titration curve were obtained by using purified AFP and MabF22. These results indicate that the monoclonal antibody produced in this study would be useful not only for research purposes but also for further development of immune-diagnostic kit for the measurement of AEP concentration.

• Journal of Practical Agriculture & Fisheries Research
• /
• v.20 no.1
• /
• pp.89-96
• /
• 2018

The object of this study was to evaluate the change of antibody titers on virus strains after inoculation with commercial killed equine influenza (EI) vaccines in horses. Serum antibodies of 20 Thoroughbred yearlings were detected using hemagglutination inhibition test for 41 weeks. Second vaccination is inoculated 4 weeks after the initial vaccination. Most of antibody titers were not increased until 4 weeks after first vaccination. The highest titers were detected 6-10 weeks after vaccination. The titers were decreased slowly and maintained for 16 weeks after inoculation. We could barely detect the antibody 41 weeks after vaccination in most cases. Vaccine anergia were appeared in 3 horses (15%) but it depended on virus strains. A/Equine/La Plata/93(H3N8) strain that induce high and durable antibody responses was the most effective among three strains. This study presents the first comprehensive data on the endurance of antibody titers against EI. Our data also suggests that yearlings should be inoculated three times in order to maintaining optimal antibody titers against EI. We speculate the causes of anergia were vaccine break down or individual specificity. Further research is needed to investigate immunological unresponsiveness. This was the first study on strain of equine vaccine in Korea.

• Journal of Veterinary Clinics
• /
• v.8 no.1
• /
• pp.1-10
• /
• 1991

The B-lymphocyte differentiation from committed B-cell progenitors to antibody-secreting cells was discussed. B-cell progenitors derived from hematopoietic stem cells undergo the rearrangement of immunoglobulin(Ig) gene. The earliest cells as B-cell precursors have cytoplasmic Is(${\mu}$ chain). The entire Is molecule is expressed on the surface after synthesis of L chain. The resting B cells(Go stage) stimulated by binding antigen via Ig-receptors are activated(G$_1$ stage) and followed by proliferation(S stage), coupled with further selection(affinity maturation. class switch). The production of antibody against a particular antigen depends on the activation of B cells with surface Is capable of reacting with that antigen. This process does not occur in isolation but is controlled by helper and suppressor T cells and antigen presenting cells(APC). The mechanism of T cell-dependent B-cell response for production of antibody is largely explained by the cell to cell cooperation and soluble helper factors of T cells. 1) The antigen specific B cells and helper T cells are linked by Is-receptors, leading to the delivery of helper signals to the B cells. 2) Helper T cells recognize the processed antigen-derived peptides with the MHC class II molecules(la antigen) and is stimulated to secrete B-cell proliferation and differentiation factors which activate B cells of different antigenic specificity. The two models are shown currently 1) At low antigen concentration, only the antigen-specific B cell binds antigen and presents antigen-derived peptides with la molecules to helper T cells, which are stimulated to secrete cytokines(IL-4, IL-5, etc.) and 2) At high antigen concentration, antigen-derived peptides are presented by specific B cells, by B cells that endocytose the antigens, as well as by APC Cytokines secreted from helper T cells also lead to the activation of B cells and even bystander B cells in the on- vironmment and differentiate them into antibody-secreting plasma cells.

• Parasites, Hosts and Diseases
• /
• v.55 no.6
• /
• pp.623-630
• /
• 2017

Plasmodium lactate dehydrogenase (pLDH) is a strong target antigen for the determination of infection with Plasmodium species specifically. However, a more effective antibody is needed because of the low sensitivity of the current antibody in many immunological diagnostic assays. In this study, recombinant Plasmodium vivax LDH (PvLDH) was experimentally constructed and expressed as a native antigen to develop an effective P. vivax-specific monoclonal antibody (mAb). Two mAbs (2CF5 and 1G10) were tested using ELISA and immunofluorescence assays (IFA), as both demonstrated reactivity against pLDH antigen. Of the 2 antibodies, 2CF5 was not able to detect P. falciparum, suggesting that it might possess P. vivax-specificity. The detection limit for a pair of 2 mAbs-linked sandwich ELISA was 31.3 ng/ml of the recombinant antigen. The P. vivax-specific performance of mAbs-linked ELISA was confirmed by in vitro-cultured P. falciparum and P. vivax-infected patient blood samples. In conclusion, the 2 new antibodies possessed the potential to detect P. vivax and will be useful in immunoassay.

• Biomedical Science Letters
• /
• v.21 no.1
• /
• pp.23-31
• /
• 2015

Recently, specific antibodies have been used extensively to diagnose and treat various diseases. It is essential to assess the efficacy and specificity of antibodies, especially the in vivo environment. Anti-HER-2/neu mAb was evaluated as a possible transporting agent for radioimmunotherapy. The monoclonal antibody was successfully radio-labeled with $^{131}I$. In vitro binding assays were performed to confirm its targeting ability using another radio-iodine, $^{125}I$. Binding percentage of $^{125}I$ labeled anti-HER-2/neu mAb in HER-2/neu expressing CT-26 cells was found to be 4.5%, whereas the binding percentage of $^{125}I$ labeled anti-HER-2/neu mAb in wild-type CT-26 was only 0.45%. In vivo images were obtained and analyzed through $\gamma$-camera and an optical fluorescent modality, IVIS-200. $\gamma$-camera images showed that $^{131}I$ labeled anti-HER-2/neu mAb accumulated in HER-2/neu CT-26 tumors. Optical imaging based on near infrared fluorescence labeled anti-HER-2/neu mAb showed higher fluorescence intensities in HER-2/neu CT-26 tumors than in wild-type CT-26 tumors. Anti-HER-2/neu mAb was found to specifically bind to its receptor expressing tumor. Our study demonstrates that in vivo imaging technique is a useful method for the evaluation of an antibody's therapeutic and diagnostic potentials.

• IMMUNE NETWORK
• /
• v.4 no.1
• /
• pp.23-30
• /
• 2004

Background: A human orthologue of mouse S100A6-binding protein (CacyBP), Siah-1-interacting protein (SIP) had been shown to be a component of novel ubiquitinylation pathway regulating $\beta$-catenin degradation. The role of the protein seems to be important in cell proliferation and cancer evolution but the expression pattern of SIP in actively dividing cancer tissues has not been known. For the elucidation of the role of SIP protein in carcinogenesis, it is essential to produce monoclonal antibodies specific to the protein. Methods: cDNA sequence coding for ORF region of human SIP gene was amplified and cloned into an expression vector to produce His-tag fusion protein. Recombinant SIP protein and monoclonal antibody to the protein were produced. The N-terminal specificity of anti-SIP monoclonal antibody was conformed by immunoblot analysis and enzyme linked immunosorbent assay (ELISA). To study the relation between SIP and colon carcinogenesis, the presence of SIP protein in colon carcinoma tissues was visualized by immunostaining using the monoclonal antibody produced in this study. Results: His-tag-SIP (NSIP) recombinant protein was produced and purified. A monoclonal antibody (Korea patent pending; #2003-45296) to the protein was produced and employed to analyze the expression pattern of SIP in colon carcinoma tissues. Conclusion: The data suggested that anti-SIP monoclonal antibody produced here was valuable for the diagnosis of colon carcinoma and elucidation of the mechanism of colon carcinogenesis.

• The Korean Journal of Mycology
• /
• v.17 no.2
• /
• pp.82-90
• /
• 1989

Detection of antibody against pathogenic fungi in serum specimens of the patients with pulmonary tuberculosis or other lung diseases has been carried out(male) using the indirect fluorescence antibody technique and immunodiffusion tests. Immunodiffusion tests revealed that 104(36.5%) out of 285 patients examined showed a positive precipitin reaction against one or more of fungal antigens. The majority of ID positive patients 64(61.5%) reacted with Aspergillus fumigatus antigen and 49(47.1%) patients reacted with Candida albicans antigen ID positive reaction to A. fumigatus was found little more frequently among male patients, while Candida albicans reactors were found more frequently among female patients. Age distribution of ID positive reactors was high(49.1-43.3%) in age group of 40-59 years, but least or none in age group of less than 30 years. Age of fungal mycelium used as antigen did not effect sensitivity of the indirect flubrescence (IF) technique in detecting antibody to A. fumigatus. Antibody class against A. fumigatus that showed highest titer was IgG and thus FITC labeled anti-IgG immunoglobulin shoul be preferable. As relatively large amount of cell wall components of Aspergilli shared antigenically, a considerable cross-reaction was observed among A. fumigatus, A. flavus and A. niger, but not much with C. albicans. While (IF) has much better sensitivity when compared with ID, relative specificity of the latter procedure cannot to be overried, so that they could be batter used together in order to obtain quantitative measurement of antibody with relative specificity.