Two monoclonal antibodies (McAbs) against Aα chain's C terminus offibrinogen (Fg) have been prepared and designated SZ-78 and SZ-79. Both theantigens in binding assay and immunoblot analysis showed that the two M...Two monoclonal antibodies (McAbs) against Aα chain's C terminus offibrinogen (Fg) have been prepared and designated SZ-78 and SZ-79. Both theantigens in binding assay and immunoblot analysis showed that the two McAbs recognized the epitopes located in residues 549-560 of the Aαchain. The two McAbs couldaccelerate rate of fibrin polymer assembly both in the purified system and in the humanplasma. From the pictures of transmission electronmicroscope, the average diametersof the fibers increase significantly to an average diameters of 375 nm after incubationwith the McAbs, while it was only 75nm without addition of the McAbs. There were al-so more branchings of fibers with addition of McAbs. These observations demonstratethat the amino acid sequences ofα 549-560 in the COOH terminus of the Aα chain mayplay an important role in the assembly of a fibrin clot, presumably being involved in lat-eral aggregation of protofibrils. The preparation of the McAbs supplies a usuful probe for the investigation of the展开更多
Gp96, a member of HSP90 family, is a versatile molecular chaperone with various newly-discovered functions, for example to serve as a low affinity, high capacity calcium binding protein, a natural adjuvant for therape...Gp96, a member of HSP90 family, is a versatile molecular chaperone with various newly-discovered functions, for example to serve as a low affinity, high capacity calcium binding protein, a natural adjuvant for therapeutic cancer vaccines, a tumor rejection antigen, an immune regulator to pathological cell death. Its multi-functional and structural characteristics make it also an interesting target to develop antibody-based therapeutics. However, its low immunogenicity to mice, because of its high-sequence similarity among different species, is an obstacle to obtain valuable monoclonal antibodies (MAbs). This is a common problem for any low immunogenic proteins, whose sequences share close identity between mice and other species. Here, a new strategy of priming was employed by swine endogenous full-length gp96 and then boosting by E. coli-system heterologously expressed gp96 N-terminal fragment (N-355) to generate MAbs. Twelve different highly-specific MAbs against swine/human endogenous gp96 were successfully obtained. The binding activities of these MAbs were confirmed by enzyme-linked immunosorbent assay (ELISA), Western blot (WB), immunofluorescence and flow cytometry analysis. This provides some important reagents for further research and potential therapeutics. The methods employed can be used for MAb production of any sequence-highly-conserved proteins between mice and swine/human (or any other species).展开更多
Two artificial antigens were synthesized successfully by diazotizing method, sulfamethazine（SM2）-human serum albumin （HSA） was used for the immunogen, and SM2-ovalbumin（OVA） was used for the coating antigen. The...Two artificial antigens were synthesized successfully by diazotizing method, sulfamethazine（SM2）-human serum albumin （HSA） was used for the immunogen, and SM2-ovalbumin（OVA） was used for the coating antigen. The coupled reaction was successful by confirmation of the ultraviolet scanning spectrometer, and the conjugation ratio of SM2 with HSA and OVA was 9：1 and 15：1, respectively. Using cell-fusion and limiting dilution method to reclone 5 times to get 3 hybridoma strains, which could stably secret monoclonal antibody （Mab）, named CBT, BC4 and BB12. The subtype of BC4 Mab was IgG1 and chain, the molecular weight was 162 ku, the numbers of chromosomal were about 90, the affinity constant was 6.1 × 10^12 M^-1. No cross reactivity was seen between the Mab and the other 4 sulfonamides, as well as the 2 carries proteins. The Mab antibody had excellent stability.展开更多
Dendritic cells(DCs) are professinal antigen-presenting cells with the ability to initiate primary Tcell responses. While it iswell known that inflammatory stimuli induce the functional maturation of immature DCs, whe...Dendritic cells(DCs) are professinal antigen-presenting cells with the ability to initiate primary Tcell responses. While it iswell known that inflammatory stimuli induce the functional maturation of immature DCs, whether adhesion molecule selectins regulate DCmaturation is poorly understood. Using anti-P-selectin lectin-EGF domain monoclonal antibody (PsL-EGFmAb) that blocks the adhesionof P-, E-, and L-selectin, we demonstrate herein that selectins play important role in stimulating functional maturation of immature DCs.Immature DCs are generated from human cord blood CD34+hematopoietic stem/progenitor cells that were cultured in the presence of stemcell factor, Fms-like tyrosine-kinase-3 ligand, granulocyte-macrophage colony stimulating factor, and transform growth factor-β1. Whenstimulated with tumor necrosis factor-alpha(TNF-α), immature DCs differentiated into mature DCs, producing increased levels of costim-ulatory molecules and interleukin (IL)-12 and obtaining the ability to potently activate naive Tcells. Interestingly, in contrast to matureDCs derived from TNF-α-induced immature DC cultures without PsL-EGFmAb, immature DCs treated with PsL-EGFmAb for7 days werecompletely blocked their maturation, as evidenced by decreased expression of costimulatory molecules CD80, CD86, and CD83, inhibi-ted production of IL-12, and inability to activate naive Tcellsin vitro. Thus, blockade of selectins using PsL-EGFmAb will prove to bea valuable tool for the study of the molecuar mechanisms of DC maturation, as well as for the prevention and treatment of DC-mediated au-toimmunity.展开更多
Thirty-six cases of neuroblastoma and two hundredand twenty-nine cases with various kinds of leukemia inchildren were observed systematically. The applicationof monoclonal antibody in the differential diagnosis ofmeta...Thirty-six cases of neuroblastoma and two hundredand twenty-nine cases with various kinds of leukemia inchildren were observed systematically. The applicationof monoclonal antibody in the differential diagnosis ofmetastasis of neuroblastoma cells in bone marrow and leu-kemia cells was studied and some precautions should betaken for a rapid and accurate diagnosis. The accordantrate between clinical diagnoses and the results of thisstudy was 91%.展开更多
Porcine reproductive and respiratory syndrome virus(PRRSV)GP4 protein was prokaryotically expressed,and used as an antigen to immunize six-week-old BALB/c female mice.With conventional cell fusion method,an anti-PRRSV...Porcine reproductive and respiratory syndrome virus(PRRSV)GP4 protein was prokaryotically expressed,and used as an antigen to immunize six-week-old BALB/c female mice.With conventional cell fusion method,an anti-PRRSV GP4 protein monoclonal antibody(Mab)5F12 was successfully prepared.It was identified as IgG2b subclass and had better stability and specificity,which not only responded with recombinant PRRSV GP4 protein,but also with PRRSV.Phage display technique had varieties of applications,in particular,the identification of key antigen epitopes for the development of therapeutic and diagnostic reagents and vaccines.In this study,Mab-5F12 was used as the target for biopanning a 12-mer phage random peptide library.After four rounds of biopanning,two phage-displayed peptides,named P-A and P-G(AKFEVCSPVVLG and GVNQENMLHFSF)were identified that recognized Mab-5F12 specifically.Sequence analysis showed that one or more of the peptides exhibited partial sequence similarity to the native GP4 protein sequence,which corresponded to 69-80 and 84-95 aa segments of the HP-PRRSV GP4 protein.Furthermore,real-time quantitative RT-PCR and indirect immunofluorescence assay indicated consistently the abilities of P-A and P-G to block viral infection in Marc-145 cells and they could function as antiviral agents for PRRSV.展开更多
文摘Two monoclonal antibodies (McAbs) against Aα chain's C terminus offibrinogen (Fg) have been prepared and designated SZ-78 and SZ-79. Both theantigens in binding assay and immunoblot analysis showed that the two McAbs recognized the epitopes located in residues 549-560 of the Aαchain. The two McAbs couldaccelerate rate of fibrin polymer assembly both in the purified system and in the humanplasma. From the pictures of transmission electronmicroscope, the average diametersof the fibers increase significantly to an average diameters of 375 nm after incubationwith the McAbs, while it was only 75nm without addition of the McAbs. There were al-so more branchings of fibers with addition of McAbs. These observations demonstratethat the amino acid sequences ofα 549-560 in the COOH terminus of the Aα chain mayplay an important role in the assembly of a fibrin clot, presumably being involved in lat-eral aggregation of protofibrils. The preparation of the McAbs supplies a usuful probe for the investigation of the
基金Project(31030030) supported by the National Natural Science Foundation of China
文摘Gp96, a member of HSP90 family, is a versatile molecular chaperone with various newly-discovered functions, for example to serve as a low affinity, high capacity calcium binding protein, a natural adjuvant for therapeutic cancer vaccines, a tumor rejection antigen, an immune regulator to pathological cell death. Its multi-functional and structural characteristics make it also an interesting target to develop antibody-based therapeutics. However, its low immunogenicity to mice, because of its high-sequence similarity among different species, is an obstacle to obtain valuable monoclonal antibodies (MAbs). This is a common problem for any low immunogenic proteins, whose sequences share close identity between mice and other species. Here, a new strategy of priming was employed by swine endogenous full-length gp96 and then boosting by E. coli-system heterologously expressed gp96 N-terminal fragment (N-355) to generate MAbs. Twelve different highly-specific MAbs against swine/human endogenous gp96 were successfully obtained. The binding activities of these MAbs were confirmed by enzyme-linked immunosorbent assay (ELISA), Western blot (WB), immunofluorescence and flow cytometry analysis. This provides some important reagents for further research and potential therapeutics. The methods employed can be used for MAb production of any sequence-highly-conserved proteins between mice and swine/human (or any other species).
文摘Two artificial antigens were synthesized successfully by diazotizing method, sulfamethazine（SM2）-human serum albumin （HSA） was used for the immunogen, and SM2-ovalbumin（OVA） was used for the coating antigen. The coupled reaction was successful by confirmation of the ultraviolet scanning spectrometer, and the conjugation ratio of SM2 with HSA and OVA was 9：1 and 15：1, respectively. Using cell-fusion and limiting dilution method to reclone 5 times to get 3 hybridoma strains, which could stably secret monoclonal antibody （Mab）, named CBT, BC4 and BB12. The subtype of BC4 Mab was IgG1 and chain, the molecular weight was 162 ku, the numbers of chromosomal were about 90, the affinity constant was 6.1 × 10^12 M^-1. No cross reactivity was seen between the Mab and the other 4 sulfonamides, as well as the 2 carries proteins. The Mab antibody had excellent stability.
文摘Dendritic cells(DCs) are professinal antigen-presenting cells with the ability to initiate primary Tcell responses. While it iswell known that inflammatory stimuli induce the functional maturation of immature DCs, whether adhesion molecule selectins regulate DCmaturation is poorly understood. Using anti-P-selectin lectin-EGF domain monoclonal antibody (PsL-EGFmAb) that blocks the adhesionof P-, E-, and L-selectin, we demonstrate herein that selectins play important role in stimulating functional maturation of immature DCs.Immature DCs are generated from human cord blood CD34+hematopoietic stem/progenitor cells that were cultured in the presence of stemcell factor, Fms-like tyrosine-kinase-3 ligand, granulocyte-macrophage colony stimulating factor, and transform growth factor-β1. Whenstimulated with tumor necrosis factor-alpha(TNF-α), immature DCs differentiated into mature DCs, producing increased levels of costim-ulatory molecules and interleukin (IL)-12 and obtaining the ability to potently activate naive Tcells. Interestingly, in contrast to matureDCs derived from TNF-α-induced immature DC cultures without PsL-EGFmAb, immature DCs treated with PsL-EGFmAb for7 days werecompletely blocked their maturation, as evidenced by decreased expression of costimulatory molecules CD80, CD86, and CD83, inhibi-ted production of IL-12, and inability to activate naive Tcellsin vitro. Thus, blockade of selectins using PsL-EGFmAb will prove to bea valuable tool for the study of the molecuar mechanisms of DC maturation, as well as for the prevention and treatment of DC-mediated au-toimmunity.
文摘Thirty-six cases of neuroblastoma and two hundredand twenty-nine cases with various kinds of leukemia inchildren were observed systematically. The applicationof monoclonal antibody in the differential diagnosis ofmetastasis of neuroblastoma cells in bone marrow and leu-kemia cells was studied and some precautions should betaken for a rapid and accurate diagnosis. The accordantrate between clinical diagnoses and the results of thisstudy was 91%.
基金Supported by the National Natural Science Foundation of China(31372438,31200122)
文摘Porcine reproductive and respiratory syndrome virus(PRRSV)GP4 protein was prokaryotically expressed,and used as an antigen to immunize six-week-old BALB/c female mice.With conventional cell fusion method,an anti-PRRSV GP4 protein monoclonal antibody(Mab)5F12 was successfully prepared.It was identified as IgG2b subclass and had better stability and specificity,which not only responded with recombinant PRRSV GP4 protein,but also with PRRSV.Phage display technique had varieties of applications,in particular,the identification of key antigen epitopes for the development of therapeutic and diagnostic reagents and vaccines.In this study,Mab-5F12 was used as the target for biopanning a 12-mer phage random peptide library.After four rounds of biopanning,two phage-displayed peptides,named P-A and P-G(AKFEVCSPVVLG and GVNQENMLHFSF)were identified that recognized Mab-5F12 specifically.Sequence analysis showed that one or more of the peptides exhibited partial sequence similarity to the native GP4 protein sequence,which corresponded to 69-80 and 84-95 aa segments of the HP-PRRSV GP4 protein.Furthermore,real-time quantitative RT-PCR and indirect immunofluorescence assay indicated consistently the abilities of P-A and P-G to block viral infection in Marc-145 cells and they could function as antiviral agents for PRRSV.
