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Objective T9 provide candidate molecules for developing recombinant anti-idiotypic antibody (anti-Id) vaccine of gastric carcinoma by selection of recombinant anti-Id to monoclonal antibody ( McAb) MGb1 directed against the cancer with phage display technique.Methods Balb/c mice were immunized with MGb1 and the mRNA was isolated from the spleens of the immunized mice. The VL and VH cDNAs of the antibody were amplified separately by RT-PCR and assembled into ScFv DNAs with a linker DNA. The ScFv DNAs were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phage antibody ScFv library. After four rounds of biopanning to the library with MGb1, the MGb1-positive clones were selected from the enriched phages by ELISA. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA. Results The VL and VH cDNAs was about 320 bp and 340 bp, respectively. The ScFv DNA were about 750 bp. After four rounds panning to the phage antibody ScFv library with MGb1, 18 MGb1-positive phage clones displayed anti-Id ScFv were selected from 50 pre-selected phage clones, among which 4 clones displayed β or γ type anti-Id ScFv. Conclusion The phagedisplayed anti-Id ScFvs to McAb MGb1 are successfully selected by recombinant phage antibody technique, which might lay a foundation for screening the anti-Id ScFv possessing the characteristics of inducing anti-gastric carcinoma immunity.
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BACKGROUND: Disialoganglioside GD2 is a tumor-associated antigen that is overexpressed on tumor cells of neuroectodermal origin, such as melanoma, small cell lung carcinoma and neuroblastoma. Immunity against GD2 has anti-tumor activities, but GD2 is poorly immunogenic. Anti-idiotypic antibodies that mimic GD2 may induce more effective immune responses than GD2 antigen itself, because they are protein antigens and are known to be able to break immune tolerance. In our previous study, we produced anti-idiotypic antibodies mimicking GD2 (3A4 and 3H9), which induced humoral immunity. However, cellular immunity is essential to eradicate tumor cells in vivo as well as humoral immunity. In the present study, we investigated whether these anti-idiotypic antibodies 3A4 and 3H9 could induce cellular immunes responses. METHODS: BALB/C mice were immunized with anti-idiotypic antibody 3A4 or 3H9, or normal mouse IgG as a negative control. Lymphoproliferative responses, cytokine production responses, and delayed-type hypersensitivity reactions were measured in mice immunized with the anti-idiotypic antibodies. RESULTS: Both the anti-idiotypic antibody 3A4 and 3H9 induced GD2-specific lymphoproliferative responses and IFN-gamma production of lymph node lymphocytes in BALB/C mice. Only anti-idiotypic antibody 3H9 induced significant GD2-specific delayed-type hypersensitivity in the mice. CONCLUSION: These results show that anti-idiotypic antibodies 3A4 and 3H9 have the potentiality of inducing GD2-specific cellular immune responses that cannot be induced by the native antigen GD2 itself.
Subject(s)
Animals , Mice , Antibodies, Anti-Idiotypic , Hypersensitivity , Immune Tolerance , Immunity, Cellular , Immunity, Humoral , Immunoglobulin G , Lymph Nodes , Lymphocytes , Melanoma , Neural Plate , Neuroblastoma , Small Cell Lung CarcinomaABSTRACT
Objective To pave the way for developing recombinant antiidiotypic antibody(antiId)vaccine of gastric carcinoma by generating phagedisplayed antiId to monoclonal antibody MGd1 directed against the cancer. Methods Balb/c mice were immunized i.p. with MGd1 conjugated with KLH, and mRNA was isolated from the spleens of the immunized mice. VH and VL DNAs of the antibody were amplified separately by RTPCR and assembled into ScFv DNAs with a linker DNA by PCR. The ScFv DNAs were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E.coli TG1. The transformed cells were infected with M13KO7 helper phage to yield phage antibody ScFv library. After four rounds of panning to the library with MGd1, the MGd1positive clones were selected by ELISA from the enriched phages. The types of the antiId ScFv displayed on the selected phage clones were preliminary identified by competition ELISA. Results The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After four rounds of panning to the antibody library, 17 MGd1positive phage clones displayed antiId ScFv were selected from 40 enriched phage clones, among which 3 displayed ? or ? type antiId ScFv. Conclusion The successful generation of antiId ScFv to monoclonal antibody MGd1 by recombinant phage antibody library technology might lay a foundation for screening of novel candidate molecules for developing recombinant antiId vaccine of gastric carcinoma.
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BACKGROUND: Disialoganglioside GD2 is a tumor-associated antigen that is overexpressed on tumor cells of neuroectodermal origin, such as melanoma and neuroblastoma. Anti-idiotypic antibodies that mimic GD2 may induce more effective immune responses than GD2 antigen itself, because they are protein antigens and are known to be able to break immune tolerance. In this study, to explore the potential of anti-idiotypic antibodies as tumor vaccines, the ability of anti-idiotypic antibodies (Ab2) to induce anti-anti-idiotypic antibodies (Ab3) that bind to the original antigen GD2 was investigated. METHODS: Six monoclonal anti-idiotypic antibodies (1A8, 1G5, 2B6, 3A4, 3D6, 3H9) to monoclonal antibody M2058, which is a monoclonal antibody to GD2, were produced in mice. Three (1A8, 3A4, 3H9) of them were selected based on their ability to inhibit the binding of Ab1 to D142.34 (murine melanoma cell expressing GD2). These 3 different Ab2 were injected into rabbits, and rabbit Ab3 induced by each of them were characterized. RESULTS: Ab3-containing sera from two rabbits immunized with 1A8, 3A4, or 3H9 bound significantly (P<0.05) to D142.34 but not to B78.96 (GD2-negative cell), and bound significantly (P<0.05) to isolated GD2 but not to GD1a. Ab3-containing sera from two rabbits immunized with 3A4 or 3H9 inhibited significantly (P<0.05) the binding of Ab1 M2058 to D142.34, and inhibited significantly (P<0.05) the binding of Ab1 M2058 to the Ab2. CONCLUSION: These results suggest that anti-idiotypic antibodies 3A4 and 3H9 have a potential to be used as vaccines against tumors expressing GD2 by inducing GD2-specific antibodies (Ab3).
Subject(s)
Animals , Mice , Rabbits , Antibodies , Antibodies, Anti-Idiotypic , Cancer Vaccines , Immune Tolerance , Immunization , Melanoma , Neural Plate , Neuroblastoma , VaccinesABSTRACT
Objective:To generate phage displayed anti idiotypic antibody single chain variable fragments(anti Id ScFv)to monoclonal antibody MC3 directed against colorectal carcinoma.Methods:Balb/c mice were immunized i.p. with MC3(McAb against colorectal Carcinoma) conjugated with KLH,and mRNA was isolated from the spleens of the immunized mice.VH and VL DNAs of the antibody were amplified separately and assembled into ScFv DNAs with a linker DNA by PCR.The ScFv DNAs were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E.coli TG1.The transformed cells were infected with M13KO7 helper phage to yield phage antibody ScFv library.After four rounds of panning to the library with MC3,the MC3 positive clones were selected by ELISA from the preselected phages.Results:The VH,VL and ScFv DNAs were about 340,320 and 750 bp respectively.After four rounds of panning to the antibody library,15 MC3 positive phage clones displayed anti Id ScFv were selected from 50 enriched phage clones.Conclusion:The phage displayed anti Id ScFv to MC3 were successfully selected by phage antibody library technology,which might provide new putative candidate molecules for developing recombinant anti Id vaccine of colorectal carcinoma.
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Objective] To amplify and sequence the light chain of anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum. [Methods] By comparing the conserved regions at each end of the nucleotide sequences of murine germ line genes enco ding FR1 and FR4 regions of immunoglobulin light chain variable regions, we designed a set of primers for amplification of V L gene. The hybridoma cells secreting anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum were cultured and their genome DNAs were extracted and used as templates for PCR. The PCR product was then cloned into pUC19 vector. The recombinants were sequenced by Sanger′s method. The V L gene was compared with GenBank and published mouse V L genes. [Results] The full length of V L gene was 318 bp. The V L gene was a member of mouse Ig ? light chain subgroup IV and generated from rearrangement of germ line V and J? 4 genes. The V L gene sequence has been registered by GenBank(accession No. AF206720). [Conclusion] The obtained V L gene was a potentially functional gene of anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum .
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Objective To study the molecular mechanism of apoptosis of cells in egg granuloma induced by anti-idiotypic antibody NP30 of Schistosoma japonicum.Methods BALB/c mice were randomly divided into two groups. The mice of the experimental group were immunized by injecting NP30 intraperitoneally for three times, while the mice of control group were injected normal saline intraperitoneally. The mice were sacrificed respectively on the 39th, 49th, 64th, 108th, 112nd day after challenge with schistosome cercariae. The expressions of apoptosis-related gene Bax, Bcl-2, death receptor Fas, FasL (Fas ligand) and c-Fos were examined by the S-P method of immunohistochemistry,and Bax, mRNA and Fas mRNA investigated by the in-situ hybridization. Results The expressions of Bax, Fas, FasL and c-Fos were positive in granuloma cells of both groups. The expressions of Bax and FasL in experimental group were higher than those in control group (P
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Objective To study the effects of the monoclonal anti idiotypic antibody NP30 active immunization on egg granuloma formation and hepatic fibrosis in Schistosoma japonicum infection. Methods ICR mice were actively immunized with NP30 100 ?g ?3 ip. every 10 days while the mice in control group were injected with SP2/0 ascites ip. simultaneously. After cercariae challenging,the mice were killed at the 4th, 8th,12th, 16th, 20th and 24th week, respectively.Mouse livers were removed and stained histochemically with VG and subjected to immunohistochemical assay of collagen type Ⅰ,Ⅲ and fibronectin(FN).The volume of egg granulomas and the content of collagen type Ⅰ,Ⅲ and FN were determined quantitatively by NYD 1000 Image Analysis System. Results The volume of egg granulomas in NP30 immunized group was much smaller than that of control group from the 12th week after cercariae challenge. The cellular components of egg granulomas in NP30 immunized group were significantly different from those of the control group,exhibiting two types of atypical egg granulomas were found.VG stain revealed that the average optical density of collagen in hepatic granulomas of experimental group was lower than that of control group.Immunohistochemical assay revealed that the contents of collagen type Ⅰ,Ⅲ and fibronectin in egg granulomas of experimental group were lower than those of control group. Conclusion NP30 vaccination may induce both cellular and humoral protective immunity to modulate egg granulomas and suppress liver fibrosis of schistosomiasis japonica.
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In this study the ability of the monoclonal anti-idiotypic antibody NP30 was tested as a substitute of diagnostic antigen in detecting antibody of Schistosoma japonicum from human sera by use of ELISA. The results showed that the seropositive rate was 98% with NP30 in the group of acute infection, which was comparable to 94% with gut associated antigens (GAA)and 98% with the soluble egg antigens (SEA); 87% with NP30 in the group of chronic infection which was comparable to 86% with GAA but lower than that of 98% with SEA. The false positive rate was about 3% for all three diagnostic antigens. The results also showed that the geometric mean titer (GMT) of antibody to NP30 was higher than that to GAA but lower than that to SEA in the acute infection group and the GMT of antibody to NP30 was lower than both those to GAA and to SEA in the chronic infection group,suggesting that the antibody to NP30 appeared earlier and decayed more quickly during the process of infection. The authors suggested that NP30 could be used for the diagnosis of schistosomiasis japonica.
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Objective To study the immune regulation of antisense peptide in rats by observing immune function of activity fragments of thyrotropin receptor (TSHR) and their corresponding antisense peptides. Methods TSHR peptides TR1, TR2, TR3 and their antisense peptides RT1, RT2, RT3, and three pairs of complementary peptides were injected into rats of different groups respectively, and the serum levels of TT_3, TT_4, TSHR antibody (TRAb), thyroid stimmulating antibody, thyroid blocking antibody and TSH antibody (TSHAb) and pathological changes in thyroid tissue were investigated. Results Serum TRAb could be induced when each of three fragments of TSHR was injected into rats; TRAb and TSHAb were induced by RT1 or RT2; epithelial hyperplasia and lymphocytic infiltration observed in thyroid tissue of rats injected with TR2 could be abated by injecting RT2 subsequently. Conclusion The results suggest that all 3 TSHR fragments are shown to be immunogenic and are capable to induce TRAb; both RT1 and RT2 show their effect on immune regulation and are idiotypic of TSHR peptides; On the other hand, the humoural and cell immunities are ameliarated by injection of antisense peptides. Therefore, it is possible that antisense peptides may be involved in immune regulation via immune network.
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Hightiter anti-idiotypic antibodies(Ab_2)sera and two monoclonal anti-idiotypic antibodies (McAb_2)were repared by immunizating syngeneic BALB/c mice wth a complex of 7A_4 monoclonalantibody(McAb_1)against the a eterminant of the Hepatitis B Surface antiger(HBsAg)。Both Ab_2sera and McAb_2 recognize the idiotype of aratope n 7A_4McAb_1.Ab_2 sera contain the antibodiesbinding specially to anti-HBs from other species(guinea pig、onkey、rabbit and goat)and the twoMcAb_2 can not bind to these anti-HBs.Therefore it is thought that the two McAb_ re noninternalimage(Ab_2?)anti-idiotypic antibodies,this study proves that the McAb_1 against he eterminantof the HBsAg are different in their paratope,The McAb_2 can be used to stinguish the parotope onthe McAb_1 as a biological probe.
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By immunizing Balb/c mice with immunoglobulin IgG which was isolated from the D_3 monoclonal antibody specific to the retinal ganglion neuronotrophic factor (RGNTF-McAb) with a Phast System gel electrophoretic method, we have developed anti-idiotypic antibodies against the RGNTF-McAb. A MTT colorimetric microassay designed to measure the effect of conditioned media from hybridomas containing antibodies on the growth activities of retinoblastoma (Rb) cells, was used to screen for idiotypic antibodies from these hybridomas. The results showed that G_4 hybridoma produced an anti-idiotypic antibody which could bind to the D_3 RGNTF-McAb and neutralize the inhibitory function of D3 on Rb cells, and possessed the trophic activity similar to RGNTF in promoting the growth activity of Rb cells. Immunohistochemical studies with anti-idiotypic antibody revealed that Rb cells and embryonic and neonatal rat retinal ganglion cells possessed coarse immunoreactive positive granules distributed on their cell surface membrane. These granules are identified as the receptors for RGNTF. Therefore, we concluded that the G_4 monoclonal antibody is not only an anti-idiotypic antibody but also an anti-paratopic antibody, It may be very useful in studying the mechanism of action of RGNTF and has a widespread applications in future.