• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.51 no.3
• /
• pp.328-331
• /
• 2018

We developed and subsequently characterized mouse monoclonal antibodies (MAbs) against nervous necrosis virus (NNV, RGNNV genotype). We established six hybridoma clones secreting MAbs against NNV antigen: 2B1, 2B11, 2C12, 13C1-1, 13C1-2 and 14D11. All six MAbs belonged to the IgG2a isotype with a kappa light chain and their reactivity recognized against the 41 kDa coat protein of NNV by Western blot analysis. The affinity constants of the six MAbs were measured by enzyme-linked immunosorbent assay (ELISA). All six MAbs reacted with two NNV isolates (SgNag05 and Gemunodo06), while no reactivity was observed with five know fish viruses, namely marine birnavirus, infectious pancreatic necrosis virus, viral hemorrhagic septicemia virus, hirame rhabdovirus, and infectious hematopoietic necrosis virus. Moreover, high ELISA optical density (OD) values (0.87-1.42) were observed in the brain tissues of NNV-infected sevenband grouper, while low OD values (less than 0.12) were recorded in the brain tissues of uninfected fish. These results suggest that these six MAbs are highly competent and useful for the detection of NNV with the RGNNV genotype.

• Korean Journal of Veterinary Research
• /
• v.30 no.4
• /
• pp.511-513
• /
• 1990

Monoclonal antibody to progesterone was produced using the antigen $11{\alpha}$-hydroxyprogesterone hemisuccinate conjugated to bovine serum albumin. Hybridomas secreting antibody to progesterone were detected by radioimmunoassay and enzyme-linked immunosorbent assay and cloned in soft agar. Two stable monoclonal antibodies which were highly specific to progesterone were obtained, so it may be advantageously used to study on several physiological functions of progesterone including immunological research.

• Journal of Embryo Transfer
• /
• v.11 no.1
• /
• pp.71-80
• /
• 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.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.2
• /
• pp.226-229
• /
• 2012

Bacterial hemoglobin from Vitreoscilla (VHb) is recognized as a good fusion protein for the soluble expression of foreign protein. In this study, we generated a monoclonal antibody (MAb) against VHb for its detection. For the rapid screening of MAb, a protein chip technology based on the Alexa-488 (A488) dye labeling method was introduced. In order to fabricate the chip, the VHb protein was chemically coupled to the chip surface and then the culture supernatants of 84 hybridoma cell lines were spotted onto the VHb chip. The bound MAbs were measured by A488-modified anti-mouse IgG. A single spot (MAb A10) exhibited significantly high signal intensity. The immunoblot analysis evidenced that the MAb A10 can detect VHb-fused proteins with high specificity.

• Journal of fish pathology
• /
• v.32 no.1
• /
• pp.45-48
• /
• 2019

We developed and subsequently characterized mouse monoclonal antibodies (MAbs) against recombinant VP28 structural protein (rVP28) of white spot syndrome virus (WSSV). We established six hybridoma clones secreting MAbs against rVP28: 15A11, 20G6, 31H2, 34H6, 38D1 and 43A1. All six MAbs recognized the 25 kDa of protein in gill homogenates of WSSV-infected shrimp by western blot analysis, while no reactivity was observed in gill homogenates of normal shrimp. Moreover, high enzyme-linked immunosorbent assay (ELISA) optical density (OD) values (0.8-2.68) were observed in the hemolymphs from WSSV-infected shrimp, while low OD values (less than 0.24) were recorded in the hemolymphs from normal shrimp, by using these six MAbs produced in this study. These results suggest that these six MAbs are useful for the detection of WSSV.

• Journal of fish pathology
• /
• v.33 no.2
• /
• pp.171-175
• /
• 2020

We developed and subsequently characterized mouse monoclonal antibodies (mAbs) against marine birnavirus (MABV). Eight hybridoma clones secreting mAbs against MABV were established. All eight mAbs (8G6, 11C3, 15E3, 17H6, 32A6, 35A7, 38B5, and 47E3) were reacted with viral protein 3 of MABV in MABV-infected CHSE-214, whereas, no reactivity was observed in normal CHSE-214 by western blot analysis. Moreover, these eight mAbs were strongly reacted with MABV, and no cross-reactivity has been observed against other five fish viruses (hirame rhabdovirus, infectious hematopoietic necrosis virus, nervous necrosis virus, spring viraemia of carp virus, and viral hemorrhagic septicemia virus), although five mAb (11C3, 15E3, 17H6, 32A6, and 38B5) reacted with both MABV and infectious pancreatic necrosis virus by enzyme linked immunosorbent assay (ELISA). These results indicate that the mAbs can be of value in MABV detection.

• Journal of fish pathology
• /
• v.32 no.2
• /
• pp.59-67
• /
• 2019

We developed and subsequently characterized mouse antibodies (MAbs) against viral hemorrhagic septicemia virus (VHSV, genotype IVa), the causative agent of VHS. Five hybridoma clones secreting MAbs against VHSV were established. The MAbs recognized the glycoprotein (MAbs 2C10, 18H4, 23H6, and 30B7) and nucleocapsid protein (15E10) of VHSV by western blot analysis. All five MAbs reacted with VHSV-infected cells and tissue homogenates of VHSV-infected olive flounder (Paralichthys olivaceus) by western blot analysis. Whereas, no reactivity was observed in normal cells and tissue homogenates of normal olive flounder. Moreover, these MAbs reacted with VHSV, but did not react with other fish viruses (infectious hematopoietic necrosis virus, hirame rhabdovirus, spring viraemia of carp virus, infectious pancreatic necrosis virus, marine birnavirus, and nervous necrosis virus) by enzyme linked immunosorbent assay (ELISA). These results indicate that the MAbs are specific to VHSV and can be of value in VHSV detection.

• Journal of Food Hygiene and Safety
• /
• v.32 no.1
• /
• pp.75-81
• /
• 2017

For people who have a food allergy the only way to manage the allergy is to avoid the food allergen. The mackerel is one of the major food allergens, but no immunoassay for the rapid and simple detection of mackerel has been reported. The objectives of this study are to develop and characterize monoclonal antibodies (MAbs) specific to mackerel using thermal stable-soluble proteins (TSSP) as an immunogen and to characterize the MAbs by indirect enzyme-linked immunosorbent assay (iELISA). The mice immunized with mackerel TSSP and showing high titer were used for cell fusion and cloning. The characterization of MAbs produced from hybridoma cells obtained was confirmed by indirect ELISA and western blot. Four MAbs were confirmed to be specific to mackerel without cross-reaction to other marine products and livestock products in the both methods. The iELISA and western blot based on the MAbs can sensitively detect 1% mackerel protein in other marine products. These results support that immunochemical methods based on the MAb produced could be used as rapid means to detect low levels of mackerel and to identify mackerel adulterated in food.

• 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 Food Hygiene and Safety
• /
• v.31 no.1
• /
• pp.21-27
• /
• 2016

The objectives of this study are to produce monoclonal antibodies (MAbs) against Vibrio parahaemolyticus and to develop an immuno-selective filtration (ISF) method for the rapid and sensitive detection of V. parahaemolyticus. The characterization of the MAb produced from HKVP 4H9-9 hybridoma cell was validated by enzyme-linked immunosorbent assay (ELISA) and western blot. The produced MAb was specific to V. parahaemolyticus and showed weak cross-reaction to V. alginolyticus, V. vulnificus and Staphylococcus aureus. After optimization of the method, $5{\times}10^1cell/mL$ of V. parahaemolyticus in a pure culture could be detectable. Although weak cross-reactivity to V. vulnificus, V. alginolyticus and Staphylococcus aureus was observed, the ISF was confirmed to be highly specific to V. parahaemolyticus. Especially, the ISF showed the most sensitivity compared to the immunoassays currently reported is easier to perform and quicker than ID-ELISA.