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
Circulating antisperm antibodies (AsAb) and immunosuppressive material in seminal plasma (SPIM)were determined by solid-phase enzyme staining assay and anticomplement test respectively in 686 patients with abortion (i...Circulating antisperm antibodies (AsAb) and immunosuppressive material in seminal plasma (SPIM)were determined by solid-phase enzyme staining assay and anticomplement test respectively in 686 patients with abortion (including 285 couples) . 241 fertile couple served as control. It’s found that the positive rate of AsAb in infertile patients was significantly higher than that in fertile control,being 36.6% vs 3.3% (P【0.001). AsAb was even more offen detected in recurrent aborting patients. Male patients whose spouses aborted 2-6 fetuses had significantly less SPIM than control, sperm count and sperm motility were also significantly decreased. But the incidence of pyospermia was significantly greater than that in control. It is concluded that AsAb and SPIM have played an important role in the development of recurrent abortions.展开更多
Goose parvovirus(GPV)can cause a highly contagious and fatal gosling plague(GP)disease in goslings and muscoy ducklings.Here,three goose-origin neutralizing single chain variable fragment(scFv)antibodies against GPV S...Goose parvovirus(GPV)can cause a highly contagious and fatal gosling plague(GP)disease in goslings and muscoy ducklings.Here,three goose-origin neutralizing single chain variable fragment(scFv)antibodies against GPV SYG-61 were isolated.The genes of scFv antibodies were derived from goslings immunized with GPV SYG-61,and scFvs were subcloned into a pBSD vector for the construction of pBSD-scFv libraries.The pBSD-scFv libraries were screened following three rounds using VP2(protective antigen of GPV)as the bait by flow cytometry(FCM).After screening,the 15 clones with high mean fluorescence intensity(MFI)were isolated and sequenced.These 15 scFvs were expressed by pET-28a(+)in E.coli.The specificity and affinity of the 15 purified scFvs were successfully confirmed by ELISA.In the preliminary neutralization experiment on primary goose embryo fibroblast(GEF)in vitro,three of the 15 purified scFvs(named scFv-10,scFv-11 and scFv-50)showed significant neutralizing capacities.The study generated the first goose-origin neutralizing scFv against GPV and laid the foundation for the appearance of full-length goose-origin neutralizing monoclonal antibody against GPV.展开更多
In order to evaluate effects of Chinese medicinal herb (CMH) on growth and immunization of laying male chicks,eleven CMHs were used in this study,which are Astragalus membranacens (AM),Schisandra chinensis (SC),Ligust...In order to evaluate effects of Chinese medicinal herb (CMH) on growth and immunization of laying male chicks,eleven CMHs were used in this study,which are Astragalus membranacens (AM),Schisandra chinensis (SC),Ligustrum Lucidum(LL),Codonopsis,Scutellaria baicalensis (SB),Atractylodes macrocephala(AMA),Haw,Ginger,Acanthopanax scenticosns (AS),Angelica and Lycium,added to basal diet as 1%,respectively,with the basal diet supplemented with 50 mg·kg^(-1) bacitracin zinc(BZ) as the control.The body weight (BW) of birds were recorded at 1,21 and 42 days after birth,individually.The birds were vaccinated 0.50 mL against Newcastle disease (ND) with La sota vaccinel (containing mineral oil as adjuvant) by i.m.inoculation at 21 d of age.All of birds was vaccinated with F48E9 NDV by i.m.inoculation at 49 day of age.Blood samples were taken via wing vein from each bird on the day receiving the vaccination (Day-1) and on 14,21,28,30 and 34 d after vaccination (Days 14,21,28,30 and 34). Results showed that bodyweight (BW) and bodyweight gain(BWG) of the trial groups are similar to the control group.No significant differences of relative weight (RW) of bursa and spleen were observed among trial groups except for AS (P<0.05).Serum antibody titers of SC,LL,Codonopsis,SB,AMA and Lycium groups were significantly increased (P<0.05) in contrast to these control group on day-21 or 28 after immunized ND La sota vaccinel,which suggested that SC,LL,Codonopsis,SB,AMA and Lycium could augment antibody formulation.Furthermore,Compared with the control,antibody titers in SC,Codonopsis,AS,Lycium,SB and AMA group were higher (P<0.05) after vaccinated with F48E9 NDV,which suggested that SC,Codonopsis,AS,Lycium,SB and AMA have activity of antivirus.In conclusion,CMH used in the present study have similar effect on BW and BWG of chicks compared with the control.Moreover,these eleven CMH have little influence on RW of immunized organ except AS.However,SC,Codonopsis,LL,Lycium,SB and AMA could augment antibody formulation.Furthermore,SC,Codonopsis,AS,Lycium,SB and AMA have activity of antivirus compared with the control.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
Canine parvovirus(CPV)is a high morbidity and lethality virus which causes severe enteric disease in dogs.In an attempt to gain canine-origin neutralizing antibodies against CPV,single chain variable fragment(scFv)bac...Canine parvovirus(CPV)is a high morbidity and lethality virus which causes severe enteric disease in dogs.In an attempt to gain canine-origin neutralizing antibodies against CPV,single chain variable fragment(scFv)bacteria display libraries against the protective antigen VP2 were constructed and screened.VP2 specific scFvs were selected following three rounds of screening procedures.Selected scFvs were characterized by FCM and ELISA.Seven scFvs showed high affinity and specific binding to CPV.Moreover,the neutralizing activity of the antibody was preliminarily identified by CPV(100 TCID_(50))in vitro and scFv-2 showed the neutralizing ability with a titer of 32768.This study generated the first canine-origin neutralizing scFv against CPV.The neutralizing scFv would be constructed into full-length antibody in the future.This study laid the foundation for the generation of an effective therapeutic reagent with long half-life and no immunological rejection for the prevention and treatment of CPV infection.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
Osteoporosis and osteopenia are major health issues that mainly affect elderly people,women after menopause and immobilized patients.Our previous studies have proved that sclerostin antibody(Scl-Ab)can dramatically en...Osteoporosis and osteopenia are major health issues that mainly affect elderly people,women after menopause and immobilized patients.Our previous studies have proved that sclerostin antibody(Scl-Ab)can dramatically enhance bone formation and reduce bone resorption in a severe osteoporosis rat model with the combination of ovariectomy(OVX)and hindlimb immobilization(HLS).However,the mechanism in the cellular level is unclear.The objective of this study is to assess the effect of Scl-Ab on osteocytic morphology change in a combined OVX and HLS rat model via quantification of long-and short-axis and the ratio and osteocyte volume in midshaft cortical bone.Four-month-old virgin female SD rats were divided into 7 groups(n=11 per group):Sham+Veh,Sham+HLS+Veh,Sham+HLS+Scl-Ab,OVX+Veh,OVX+Scl-Ab,OVX+HLS+Veh,OVX+HLS+Scl-Ab.HLS was performed 2 weeks after sham or OVX surgery;and treatment was initiated at the time of HLS.Scl-Ab(25 mg/kg)or vehicle was subcutaneously injected twice per week for 5 weeks.Femurs were harvested at the end of study and embedded in PMMA and polished for SEM imaging.Cortical bone mid shaft osteocyte number per bone area was quantified under 1K magnification;the ratios between long axis and short axis of osteocytes were quantified under 2K magnification;osteocyte dendrite number and surface area were quantified under 5K magnification.Osteocyte dendrites width was quantified using 10K magnification.All the quantification was done by ImageJ.We have reported that multiple morphological and structural changes in osteocytes,including a decreased osteocyte density and reduced osteocyte dendrite number in HLS,OVX or the combination group and Scl-Ab’s ability to abolish these unfavorable alterations.We continued our SEM analysis on osteocytes and discovered that the oval shape of osteocyte under HLS,OVX or HLS+OVX has been distorted toward a spindle-like shape,with relatively longer long axis and shorter short axis,assuming osteocyte has a perfect spheroid shape.The ratio between long-and short-axis showed an increased trend in OVX and HLS condition,but Scl-Ab inhibited these increases(P<0.001,P<0.01,respectively).The volume decreased in HLS,OVX group,but Scl-Ab maintained osteocytes’volume in HLS condition(P<0.001).It indicates that cortical bone responds to HLS and/or OVX and Scl-Ab treatment via multiple cellular mechanisms,including density of osteocyte,dendrite number and osteocyte shape.The change of osteocyte shape may imply an altered cytoskeleton system within osteocyte and a subsequent disruption of mechanosensing ability for osteocyte,which lead to bone loss macroscopically.These data suggest Scl-Ab’s therapeutic potential could be related with its ability to maintain osteocyte’s morphologic and structural changes induced by OVX,HLS or both.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
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
Hemorrhagic stroke,the second leading cause of stroke,is a severe medical emergency that often leads to severe disability or death;however,the causal relationship between antibody-mediated immune responses and hemorrh...Hemorrhagic stroke,the second leading cause of stroke,is a severe medical emergency that often leads to severe disability or death;however,the causal relationship between antibody-mediated immune responses and hemorrhagic stroke remains unknown.This study aimed to investigate the potential causal relationship between antibody-mediated immune responses to infectious agents and hemorrhagic stroke using the two-sample Mendelian randomization(MR)method.Comprehensive analyses were conducted using publicly available data from genome-wide association study(GWAS),which involved the whole genomes of 9724 European participants and 46 antibody measurement phenotypes,and summary statistics from the FinnGen dataset R12(including intracerebral hemorrhage and subarachnoid hemorrhage)were used.The causal relationship between the aforementioned immune responses and hemorrhagic stroke was analyzed using inverse-variance weighting,MR-Egger regression,weighted median,weighted mode,simple mode,and MR-pleiotropy residual sum and outlier(MR-PRESSO),while various sensitivity analyses were performed to assess heterogeneity and pleiotropy in the study findings.Results showed that human herpes virus 7(HHV-7)U14 antibody levels(OR:0.877,95%CI:0.797-0.964,P=0.007)exerted a protective effect against hemorrhagic stroke,and Chlamydia trachomatis(CT)tarp-D F2 antibody levels(OR:0.937,95%CI:0.885-0.992,P=0.025)had a potential protective effect;additionally,Epstein-Barr virus(EBV)ZEBRA antibody levels(OR:1.062,95%CI:1.012-1.114,P=0.014),human herpesvirus 6(HHV-6)p101k antibody levels(OR:1.054,95%CI:1.002-1.108,P=0.042),and cytomegalovirus(CMV)pp150 antibody levels(OR:1.086,95%CI:1.002-1.176,P=0.045)were potential risk factors for the disease.No significant pleiotropy or heterogeneity was observed in any of the MR analyses.Collectively,these findings confirmed a significant causal relationship between antibody-mediated immune responses and hemorrhagic stroke,and this study contributed to a deeper understanding of the potential mechanisms underlying hemorrhagic stroke onset.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Circulating antisperm antibodies (AsAb) and immunosuppressive material in seminal plasma (SPIM)were determined by solid-phase enzyme staining assay and anticomplement test respectively in 686 patients with abortion (including 285 couples) . 241 fertile couple served as control. It’s found that the positive rate of AsAb in infertile patients was significantly higher than that in fertile control,being 36.6% vs 3.3% (P【0.001). AsAb was even more offen detected in recurrent aborting patients. Male patients whose spouses aborted 2-6 fetuses had significantly less SPIM than control, sperm count and sperm motility were also significantly decreased. But the incidence of pyospermia was significantly greater than that in control. It is concluded that AsAb and SPIM have played an important role in the development of recurrent abortions.
基金Supported by the National Key R&D Program of China(2017YFD0501102,20I7YFD050I103-03 and 2017YFD0501004)Science and Technology Department of Heilongjiang Province(GX18B018)Education Department ofHeilongjiang Province(TSTAU-R2018017)。
文摘Goose parvovirus(GPV)can cause a highly contagious and fatal gosling plague(GP)disease in goslings and muscoy ducklings.Here,three goose-origin neutralizing single chain variable fragment(scFv)antibodies against GPV SYG-61 were isolated.The genes of scFv antibodies were derived from goslings immunized with GPV SYG-61,and scFvs were subcloned into a pBSD vector for the construction of pBSD-scFv libraries.The pBSD-scFv libraries were screened following three rounds using VP2(protective antigen of GPV)as the bait by flow cytometry(FCM).After screening,the 15 clones with high mean fluorescence intensity(MFI)were isolated and sequenced.These 15 scFvs were expressed by pET-28a(+)in E.coli.The specificity and affinity of the 15 purified scFvs were successfully confirmed by ELISA.In the preliminary neutralization experiment on primary goose embryo fibroblast(GEF)in vitro,three of the 15 purified scFvs(named scFv-10,scFv-11 and scFv-50)showed significant neutralizing capacities.The study generated the first goose-origin neutralizing scFv against GPV and laid the foundation for the appearance of full-length goose-origin neutralizing monoclonal antibody against GPV.
文摘In order to evaluate effects of Chinese medicinal herb (CMH) on growth and immunization of laying male chicks,eleven CMHs were used in this study,which are Astragalus membranacens (AM),Schisandra chinensis (SC),Ligustrum Lucidum(LL),Codonopsis,Scutellaria baicalensis (SB),Atractylodes macrocephala(AMA),Haw,Ginger,Acanthopanax scenticosns (AS),Angelica and Lycium,added to basal diet as 1%,respectively,with the basal diet supplemented with 50 mg·kg^(-1) bacitracin zinc(BZ) as the control.The body weight (BW) of birds were recorded at 1,21 and 42 days after birth,individually.The birds were vaccinated 0.50 mL against Newcastle disease (ND) with La sota vaccinel (containing mineral oil as adjuvant) by i.m.inoculation at 21 d of age.All of birds was vaccinated with F48E9 NDV by i.m.inoculation at 49 day of age.Blood samples were taken via wing vein from each bird on the day receiving the vaccination (Day-1) and on 14,21,28,30 and 34 d after vaccination (Days 14,21,28,30 and 34). Results showed that bodyweight (BW) and bodyweight gain(BWG) of the trial groups are similar to the control group.No significant differences of relative weight (RW) of bursa and spleen were observed among trial groups except for AS (P<0.05).Serum antibody titers of SC,LL,Codonopsis,SB,AMA and Lycium groups were significantly increased (P<0.05) in contrast to these control group on day-21 or 28 after immunized ND La sota vaccinel,which suggested that SC,LL,Codonopsis,SB,AMA and Lycium could augment antibody formulation.Furthermore,Compared with the control,antibody titers in SC,Codonopsis,AS,Lycium,SB and AMA group were higher (P<0.05) after vaccinated with F48E9 NDV,which suggested that SC,Codonopsis,AS,Lycium,SB and AMA have activity of antivirus.In conclusion,CMH used in the present study have similar effect on BW and BWG of chicks compared with the control.Moreover,these eleven CMH have little influence on RW of immunized organ except AS.However,SC,Codonopsis,LL,Lycium,SB and AMA could augment antibody formulation.Furthermore,SC,Codonopsis,AS,Lycium,SB and AMA have activity of antivirus compared with the control.
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Supported by the National Key R&D Program of China(2017YFD0501102,2016YFD0501003)Science and Technology Department of Heilongjiang Province(GX18B018)Education Department of Heilongjiang Province(TSTAU-R2018017)。
文摘Canine parvovirus(CPV)is a high morbidity and lethality virus which causes severe enteric disease in dogs.In an attempt to gain canine-origin neutralizing antibodies against CPV,single chain variable fragment(scFv)bacteria display libraries against the protective antigen VP2 were constructed and screened.VP2 specific scFvs were selected following three rounds of screening procedures.Selected scFvs were characterized by FCM and ELISA.Seven scFvs showed high affinity and specific binding to CPV.Moreover,the neutralizing activity of the antibody was preliminarily identified by CPV(100 TCID_(50))in vitro and scFv-2 showed the neutralizing ability with a titer of 32768.This study generated the first canine-origin neutralizing scFv against CPV.The neutralizing scFv would be constructed into full-length antibody in the future.This study laid the foundation for the generation of an effective therapeutic reagent with long half-life and no immunological rejection for the prevention and treatment of CPV infection.
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%.
文摘Osteoporosis and osteopenia are major health issues that mainly affect elderly people,women after menopause and immobilized patients.Our previous studies have proved that sclerostin antibody(Scl-Ab)can dramatically enhance bone formation and reduce bone resorption in a severe osteoporosis rat model with the combination of ovariectomy(OVX)and hindlimb immobilization(HLS).However,the mechanism in the cellular level is unclear.The objective of this study is to assess the effect of Scl-Ab on osteocytic morphology change in a combined OVX and HLS rat model via quantification of long-and short-axis and the ratio and osteocyte volume in midshaft cortical bone.Four-month-old virgin female SD rats were divided into 7 groups(n=11 per group):Sham+Veh,Sham+HLS+Veh,Sham+HLS+Scl-Ab,OVX+Veh,OVX+Scl-Ab,OVX+HLS+Veh,OVX+HLS+Scl-Ab.HLS was performed 2 weeks after sham or OVX surgery;and treatment was initiated at the time of HLS.Scl-Ab(25 mg/kg)or vehicle was subcutaneously injected twice per week for 5 weeks.Femurs were harvested at the end of study and embedded in PMMA and polished for SEM imaging.Cortical bone mid shaft osteocyte number per bone area was quantified under 1K magnification;the ratios between long axis and short axis of osteocytes were quantified under 2K magnification;osteocyte dendrite number and surface area were quantified under 5K magnification.Osteocyte dendrites width was quantified using 10K magnification.All the quantification was done by ImageJ.We have reported that multiple morphological and structural changes in osteocytes,including a decreased osteocyte density and reduced osteocyte dendrite number in HLS,OVX or the combination group and Scl-Ab’s ability to abolish these unfavorable alterations.We continued our SEM analysis on osteocytes and discovered that the oval shape of osteocyte under HLS,OVX or HLS+OVX has been distorted toward a spindle-like shape,with relatively longer long axis and shorter short axis,assuming osteocyte has a perfect spheroid shape.The ratio between long-and short-axis showed an increased trend in OVX and HLS condition,but Scl-Ab inhibited these increases(P<0.001,P<0.01,respectively).The volume decreased in HLS,OVX group,but Scl-Ab maintained osteocytes’volume in HLS condition(P<0.001).It indicates that cortical bone responds to HLS and/or OVX and Scl-Ab treatment via multiple cellular mechanisms,including density of osteocyte,dendrite number and osteocyte shape.The change of osteocyte shape may imply an altered cytoskeleton system within osteocyte and a subsequent disruption of mechanosensing ability for osteocyte,which lead to bone loss macroscopically.These data suggest Scl-Ab’s therapeutic potential could be related with its ability to maintain osteocyte’s morphologic and structural changes induced by OVX,HLS or both.
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%.
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.
基金Supported by the National Natural Science Foundations of China(82271340,82071368)。
文摘Hemorrhagic stroke,the second leading cause of stroke,is a severe medical emergency that often leads to severe disability or death;however,the causal relationship between antibody-mediated immune responses and hemorrhagic stroke remains unknown.This study aimed to investigate the potential causal relationship between antibody-mediated immune responses to infectious agents and hemorrhagic stroke using the two-sample Mendelian randomization(MR)method.Comprehensive analyses were conducted using publicly available data from genome-wide association study(GWAS),which involved the whole genomes of 9724 European participants and 46 antibody measurement phenotypes,and summary statistics from the FinnGen dataset R12(including intracerebral hemorrhage and subarachnoid hemorrhage)were used.The causal relationship between the aforementioned immune responses and hemorrhagic stroke was analyzed using inverse-variance weighting,MR-Egger regression,weighted median,weighted mode,simple mode,and MR-pleiotropy residual sum and outlier(MR-PRESSO),while various sensitivity analyses were performed to assess heterogeneity and pleiotropy in the study findings.Results showed that human herpes virus 7(HHV-7)U14 antibody levels(OR:0.877,95%CI:0.797-0.964,P=0.007)exerted a protective effect against hemorrhagic stroke,and Chlamydia trachomatis(CT)tarp-D F2 antibody levels(OR:0.937,95%CI:0.885-0.992,P=0.025)had a potential protective effect;additionally,Epstein-Barr virus(EBV)ZEBRA antibody levels(OR:1.062,95%CI:1.012-1.114,P=0.014),human herpesvirus 6(HHV-6)p101k antibody levels(OR:1.054,95%CI:1.002-1.108,P=0.042),and cytomegalovirus(CMV)pp150 antibody levels(OR:1.086,95%CI:1.002-1.176,P=0.045)were potential risk factors for the disease.No significant pleiotropy or heterogeneity was observed in any of the MR analyses.Collectively,these findings confirmed a significant causal relationship between antibody-mediated immune responses and hemorrhagic stroke,and this study contributed to a deeper understanding of the potential mechanisms underlying hemorrhagic stroke onset.
