The production of polyclonal and monoclonal antibodies in mice using novel immunization methods

Hong, Tsai-Hsia; Chen, Shui-Tsung; Tang, Tswen-Kei; Wang, Sheng-Chang; Chang, Tong

doi:10.1016/0022-1759(89)90236-6

Cited by 28 publications

(17 citation statements)

References 5 publications

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“…Splenic lymphocytes from immunized mice were fused to BALB/c myeloma FO obtained from the American Type Cell Culture (ATCC) laboratories. Hybridomas were selected in HAT-10% fetal calf serum (FCS)-RPMI medium as previously described by Hong et al 39 Quanti®cation of cell viability About 10 5 CHSE-214 cells/ml were seeded in a 60-mm petri dish and cultured for more than 20 h. To test if antibodies could prevent IPNV E1-S from inducing cell death, confluent cells in 60-mm-diameter plastic tissue culture plates (Nunc) were pretreated with either 1 mg/ml anti-virion polyAb of E1-S or 1 mg/ml anti-Mab of VP3 (8-42-E7 and 8-42-B9) for 2 h, and then these infected-CHSE-214 cells (MOI 1) were incubated for one of the following time periods: 0, 16, or 32 h. For assayed groups using genistein (100 mg/ml) or cycloheximide (1 mg/ ml) (CHX) to prevent cell death, confluent cells were cultured in 60-mm-diameter plastic tissue culture plates (Nunc) that had been treated with 100 mg/ml genistein and 1 mg/ml CHX, then infected with MOI 1 of IPNV E1-S for one of the following time periods: 0, 6, 12, or 24 h in 1% FBS MEM at 188C. At the completion of the various culture periods, cell layers contained in the culture vessels were washed with PBS, and the monolayers were treated with 0.5 ml of 0.1% trypsin-EDTA (GIBCO) for from 1 to 2 min.…”

Section: Cells and Virusmentioning

confidence: 99%

Induction of apoptotic death in cells via Bad gene expression by infectious pancreatic necrosis virus infection

Hong

2002

Cell Death Differ

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A Bcl-2 related family member, Bad, promotes cell death, and its function is regulated by phosphorylation. In this study, we show how the IPNV elicits the induction of Bad gene expression and promotes host apoptotic death. Anti-IPNV-E1S polyclonal and anti-VP3 monoclonal antibodies are used to neutralize the virus that blocks the prime death signal via the virus receptor. In the viability assay, each antibody could also enhance cell viability during IPNV infection. We tested tyrosine kinase inhibitors on IPNVinfected cells in order to assess their effect on blocking the death signal. With 100 mg/ml genistein treatment, Bad-like gene expression was blocked, either by rescuing the IPNVinfected CHSE-214 cells or by blocking internucleosomal DNA cleavage; but the tyrphostin group did not block Bad expression. For CHSE-214 cells, treatment with the protein synthesis-inhibitor, cycloheximide (1 mg/ml), blocked new protein synthesis via activated tyrosine kinase during IPNV infection. We found that Bad protein expression could be blocked, and apoptotic death prevented. Together, these results demonstrate that the IPNV exerts up-regulation of a pro-apoptotic death gene (Bad), the expression of which serves to trigger apoptotic cell death. Our data also suggests that the IPNV induces apoptotic death via a viral receptor which triggers death effector Bad gene expression, possibly through a tyrosine kinase-dependent pathway.

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Section: Cells and Virusmentioning

confidence: 99%

Induction of apoptotic death in cells via Bad gene expression by infectious pancreatic necrosis virus infection

Hong

2002

Cell Death Differ

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“…Because pGH is very homologous to the GH of mouse, rat, and rabbit, but less homologous to that of human (69%), we could not generate efficient monoclonal antibodies from nuce or rats against pGH; on the other hand, the commercially available kit for human GH was not appropriate to our stody. We could only obtain polyclonal antiserum from rabbit by a novel inttaspleiucal immunization protocol (Hong et al, 1989). Thus, a sensitive ELISA is a Umitation in our hands.…”

Section: Discussionmentioning

confidence: 99%

“…Rabbit anti-pGH seram was obtained by immunizing rabbits inttasplenicaUy (Hong et al, 1989) and was kindly provided by W.C. Chang. In the enzyme-linked immunosorbent assay (ELISA), standard pGH (a gift from Intemational Mineral Corp.) at several known concenttations and culture medium samples were furst coated onto 96-well plates.…”

Section: Elisa For Pghmentioning

confidence: 99%

Long-Term Expression of the Biologically Active Growth Hormone in Genetically Modified Fibroblasts after Implantation into a Hypophysectomized Rat

Chen¹,

Chang²,

Chen³

et al. 1995

Human Gene Therapy

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We employed the hypophysectomized rats as an animal model to explore the feasibility of using genetically engineered fibroblast cells for growth hormone gene therapy. An internal ribosome entry site (IRES)-directed bicistronic retroviral vector, PSN, which contained a porcine growth hormone (pGH) cDNA at the first cistron and a Neo(r) gene at the second cistron was used to infect primary rat embryo fibroblast (REF) cells. The infected cells (5 x 10(6) cells/rat) were injected directly into the peritoneum of syngeneic hypophysectomized rats. We demonstrate that the implanted PSN-infected REF cells could secrete biologically active pGH in vivo, leading to significant growth of the tibia at day 15 and day 57 post-implantation. We also treated the PSN-infected REF cells with collagen to form a tissue-like structure. The skin-like discs were grafted underneath the skin on the back of rats and cells were retrieved at different times. Using two criteria, semiquantitative reverse transcription-polymerase chain reaction on the pGH RNA extracted from the explants and G418 resistance conferred from the explanted cells, we demonstrate that pGH was expressed in the implanted fibroblasts up to 70 days. Despite the fact that the total pGH RNA level was reduced in the explants of long-time post-implantation, which was probably due to the reduction of transduced cells retained in the explants, the specific efficiencies of pGH RNA expression from these explants were maintained as high as the primary PSN-infected REF prior implantation. These results suggest that fibroblast cells are capable of expressing the foreign genes persistently in vivo.

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“…Intrasplenic immunization was proven to give a strong response and resulted in a high frequency of specific MAbs [10]. To pick up the positive hybridoma secreting immunoglobulin against the immunogen, the IFA on susceptible BHK monolayer infected by optimum dose of FMDV was chosen to screen the parental and monoclonal hybridoma cells.…”

Section: Vp1 and P29 Elisamentioning

confidence: 99%

“…The first and second immunizations were intraperitoneal injections. The third immunization and last injections three days before fusion were given to the anesthetic mice via intrasplenic route [10]. Animals were bled and sera neutralization titers were examined to monitor the individual immune response.…”

mentioning

confidence: 99%

Study on the Porcinophilic Foot-and-Mouth Disease Virus I. Production and Characterization of Monoclonal Antibodies against VP1

Cheng¹,

Liang

et al. 2006

J. Vet. Med. Sci.

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ABSTRACT. Monoclonal antibodies (MAbs) reported here were produced against the porcinophilic foot-and-mouth disease virus (FMDV) that caused the devastating swine disease on 1997 in Taiwan. A panel (25) of MAbs were found to react with VP1 of O/Taiwan/97 (O/ 97) by ELISA with various potencies. The biological identities of these VP1 reacting MAbs, such as neutralization activity, isotype and capability to distinguish between two serotype O FMDVs, O/97 and O/Taiwan/KM1/99 (O/99), were further analyzed. Eleven out of the total eighteen O/97 neutralizing MAbs were able to neutralize heterologous O/99. Eight O/97 neutralizing and five non-neutralizing MAbs could differentiate two serotype O FMDVs by immunofluorescence assay (IFA) implied that these thirteen MAbs recognized O/ 97 specific epitope(s). Furthermore, reactivities of the VP1 reacting MAbs with a 29 amino acids synthetic peptide (P29) representing the βG-βH loop of VP1 were analyzed by ELISA and fourteen were found positive. MAb clone Q10E-3 reacting strongest with VP1 and P29, neutralizing both but not differentiating two serotype O viruses suggested that the antibody binding site might involve the RGD motif and its C terminal conserved region on βG-βH loop. MAbs with diverse characters presented in this study were the first raised against porcinophilic FMDV. The complete set of MAbs may be used for further studies of vaccine, diagnostic methods, prophylaxis, etiological and immunological researches on FMDV. KEY WORDS: ELISA, foot-and-mouth disease virus, monoclonal antibody, neutralizing activity, VP1.J. Vet. Med. Sci. 68(8): 859-864, 2006 All cloven-hoofed species are susceptible to foot-andmouth disease (FMD) that is considered the most contagious animal disease. The outbreaks of FMD in Argentina, Europe, Asia, South Africa and Uruguay [8] have brought to world attention the devastating effects of the disease in a naïve population and economic costs of control and eradication. The swine-adapted strain of FMD virus which occurred in Taiwan in 1997 killed more than 4 million pigs, hit related industries heavily and made Taiwan losing FMD free status [27]. To regain the free status, an eradication campaign has been implemented since then.The FMD virus, O/Taiwan/97 (O/97), produced high morbidity and mortality in swine but did not affect cattle [5]. The distinct host tropism was demonstrated that the mutated 3A coding region of O/97 played a major role in the attenuation of this virus in bovine [3]. Besides, limited biologic character of this porcinophilic virus was known. With the high specificity of antigen (Ag) recognition, monoclonal antibody (MAb) has been applied on the field of determination of antigenic epitope, diagnostic development, vaccine research and studies of viral pathogenesis for years. Therefore, production of MAbs against the porcinophilic FMDV would be beneficial to both basic and applied researches.This paper described the production of a panel of MAbs against porcinophilic O/97 strain FMDV. Their reactivities with VP1 were determi...

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The production of polyclonal and monoclonal antibodies in mice using novel immunization methods

Cited by 28 publications

References 5 publications

Induction of apoptotic death in cells via Bad gene expression by infectious pancreatic necrosis virus infection

Induction of apoptotic death in cells via Bad gene expression by infectious pancreatic necrosis virus infection

Long-Term Expression of the Biologically Active Growth Hormone in Genetically Modified Fibroblasts after Implantation into a Hypophysectomized Rat

Study on the Porcinophilic Foot-and-Mouth Disease Virus I. Production and Characterization of Monoclonal Antibodies against VP1

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