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
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
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
Fetal liver tissues obtained from 28 human fetuses with gestation age from 3 to 6 months and fetal bone marrow from 35 human fetuses from 3 to 7 months were observed by immunochemical staining with anti-platelet GPⅡ ...Fetal liver tissues obtained from 28 human fetuses with gestation age from 3 to 6 months and fetal bone marrow from 35 human fetuses from 3 to 7 months were observed by immunochemical staining with anti-platelet GPⅡ b / Ⅲa monoclonal antibody and ABC technique. In the fetal liver, megakaryocytes were wholly located among growing fetal liver cells and near foci of hemopoiesis. Some megakaryocytes in the fetal liver were small7890- lymphoid-like megakaryocytes. The size of megakaryocytes both in the fetal liver (14.79 ± 4.52μm) and in the fetal bone marrow (16.08±7.39 μm) was small, which did not vary significantly over the gestation age ranging from 3 to 6 or 7 months. However, the maturation stage of megakaryocytes in the fetal liver shifted to more mature stage with the advancement of gestation, although the maturation stage of megakaryocytes in the fetal bone marrow did not change with the advancement of gestation from 4 to 7 months, the megakaryocyte in the fetal bone marrow was less mature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
The mechanisms for apoptotic induced by anti-DR-5 agnoistic mAb(mDRA-6)were studied with Jurkat and U937 cell.DNA fragmentation,activation of caspase-9,-3 and PARP were observed in Jurkat and U937 cell lines treated b...The mechanisms for apoptotic induced by anti-DR-5 agnoistic mAb(mDRA-6)were studied with Jurkat and U937 cell.DNA fragmentation,activation of caspase-9,-3 and PARP were observed in Jurkat and U937 cell lines treated by mDRA-6.However,in Jurkat cells,展开更多
Preliminary study has been made to test wether three strains McAb（1B1,5D<sub>6</sub>, 6D<sub>8</sub>）are against the same antigen determinant.Througn ELISA additive and competition expeiments...Preliminary study has been made to test wether three strains McAb（1B1,5D<sub>6</sub>, 6D<sub>8</sub>）are against the same antigen determinant.Througn ELISA additive and competition expeiments,itproved that these three strains are against different antigen determinant.The result of positiveserum antigen component analysis with 2 strains IBDV McAb showed that sample IBD positiveserum had obvious inhibition against combination of 5D<sub>6</sub> McAb with corresponding antigen.Theresults of substitution of corresponding component in ELISA inhibition experiment and compari-son of non-IBD serum （SPF chicken serum,ND,MD,IA positive serum）proved that IBD anti-serum was the only one showing inhibition against 5D<sub>6</sub> McAb.Comparison with AGP and electro-microscope observation showed that ELISA inhibition experiment was characterised by high-specificity,rapidity and sensitiveness.799 serum samples were tested with ELISA inhibition anddouble immunodiffusion（AGP） experiments.ELISA had gotten 486 positive,with positive rate of60.83%;and AGP 334。展开更多
In order to increase the expression of mdrl genetransfectcd using retroviral vcctors, we constructedtwo retroviral vectors (LmdrlSN and MSCV-mdrl)containing mdrl cDNA and compared the expressionof mdrl gene with anoth...In order to increase the expression of mdrl genetransfectcd using retroviral vcctors, we constructedtwo retroviral vectors (LmdrlSN and MSCV-mdrl)containing mdrl cDNA and compared the expressionof mdrl gene with another vector pHamdrl/A(Pastan et al. Proc Natl Acad Sci USA 85: 4486,1986) in NIH.3T3 cells. The LmdrlSN was made展开更多
In this study, various factors of ELISA for detection of sulfamethazine residues were explored, the coating antigen was diluted to 1：400, the best coating condition was at 4℃ overnight, the working concentration of ...In this study, various factors of ELISA for detection of sulfamethazine residues were explored, the coating antigen was diluted to 1：400, the best coating condition was at 4℃ overnight, the working concentration of HRP-IgG enzyme conjugate was 1 ： 7 000. The pre-incubation time and incubation time was 30 min and 120 min, respectively, the substrate solution working time was 20 min. Two moL · L^-1 H2SO4 was used to stop the reaction and checked. A standard curve of direct competitive ELISA had been established to detect the sulfamethazine residues in milk. The detection limit of this method was 1.97 ng · mL^-1. The mean concentration of sulfamethazine required to inhibit 30% antibody was 7.1 ng · mL^-1. The linear range of the detection was 5-200 ng · mL^-1. The recovery ratio was between 73.20% and 91.16%. The CV% of within array and between arrays was less than 10%.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
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.
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Fetal liver tissues obtained from 28 human fetuses with gestation age from 3 to 6 months and fetal bone marrow from 35 human fetuses from 3 to 7 months were observed by immunochemical staining with anti-platelet GPⅡ b / Ⅲa monoclonal antibody and ABC technique. In the fetal liver, megakaryocytes were wholly located among growing fetal liver cells and near foci of hemopoiesis. Some megakaryocytes in the fetal liver were small7890- lymphoid-like megakaryocytes. The size of megakaryocytes both in the fetal liver (14.79 ± 4.52μm) and in the fetal bone marrow (16.08±7.39 μm) was small, which did not vary significantly over the gestation age ranging from 3 to 6 or 7 months. However, the maturation stage of megakaryocytes in the fetal liver shifted to more mature stage with the advancement of gestation, although the maturation stage of megakaryocytes in the fetal bone marrow did not change with the advancement of gestation from 4 to 7 months, the megakaryocyte in the fetal bone marrow was less mature
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.
文摘The mechanisms for apoptotic induced by anti-DR-5 agnoistic mAb(mDRA-6)were studied with Jurkat and U937 cell.DNA fragmentation,activation of caspase-9,-3 and PARP were observed in Jurkat and U937 cell lines treated by mDRA-6.However,in Jurkat cells,
文摘Preliminary study has been made to test wether three strains McAb（1B1,5D<sub>6</sub>, 6D<sub>8</sub>）are against the same antigen determinant.Througn ELISA additive and competition expeiments,itproved that these three strains are against different antigen determinant.The result of positiveserum antigen component analysis with 2 strains IBDV McAb showed that sample IBD positiveserum had obvious inhibition against combination of 5D<sub>6</sub> McAb with corresponding antigen.Theresults of substitution of corresponding component in ELISA inhibition experiment and compari-son of non-IBD serum （SPF chicken serum,ND,MD,IA positive serum）proved that IBD anti-serum was the only one showing inhibition against 5D<sub>6</sub> McAb.Comparison with AGP and electro-microscope observation showed that ELISA inhibition experiment was characterised by high-specificity,rapidity and sensitiveness.799 serum samples were tested with ELISA inhibition anddouble immunodiffusion（AGP） experiments.ELISA had gotten 486 positive,with positive rate of60.83%;and AGP 334。
文摘In order to increase the expression of mdrl genetransfectcd using retroviral vcctors, we constructedtwo retroviral vectors (LmdrlSN and MSCV-mdrl)containing mdrl cDNA and compared the expressionof mdrl gene with another vector pHamdrl/A(Pastan et al. Proc Natl Acad Sci USA 85: 4486,1986) in NIH.3T3 cells. The LmdrlSN was made
基金Supported by Key Items of National Technology Research Project(2002BA518A06)Heilongjiang Provincial Science and Technology Tackle Key Problem Project(10541021)Harbin Technical Fund(2003AA6CN179)
文摘In this study, various factors of ELISA for detection of sulfamethazine residues were explored, the coating antigen was diluted to 1：400, the best coating condition was at 4℃ overnight, the working concentration of HRP-IgG enzyme conjugate was 1 ： 7 000. The pre-incubation time and incubation time was 30 min and 120 min, respectively, the substrate solution working time was 20 min. Two moL · L^-1 H2SO4 was used to stop the reaction and checked. A standard curve of direct competitive ELISA had been established to detect the sulfamethazine residues in milk. The detection limit of this method was 1.97 ng · mL^-1. The mean concentration of sulfamethazine required to inhibit 30% antibody was 7.1 ng · mL^-1. The linear range of the detection was 5-200 ng · mL^-1. The recovery ratio was between 73.20% and 91.16%. The CV% of within array and between arrays was less than 10%.
