Objective:To establish the sensitive,specific,stable and convenient sgp130 ELISA kit.Methods:The mAb T2 against human gp130 was used as coating antibody;the other mAb T12 recognized different epitope with T2 was labeled by biotin,then a ELISA kit for detecting sgp130 was set up.Results:sgp130 ELISA kit is successfully established and its sensitivity is 10 ng/ml.After the kit is placed in 4℃ for 3 months,the kit's CV is less than ?7.6% and the retrievable rate is 95%～111%.These indicate that it has highly sensitivity,stability and accuracy.The normal serum concentration of sgp130 in healthy donors is 536.92～287.88(ng/ml),but there is higher in patients with hyperthyroidism(937.16?217.5) and chronic nephritis(806.45?138.47).Significant difference is found comparing with normal control(P

Objective To study the effect of acute hypervolemic hemodilution on expression of plasma bac-tericidaL/permeability-increasing protein (BPI) in patients undergoing total hip replacement. Methods Twenty ASA Ⅰ-Ⅱ patients undergoing elective total hip replacement were randomly divided into two groups (n=10 for thesia. The blood loss,blood transfusion and the time of operation were recorded. Venous blood samples were taken before anesthesia (T0) ,at the begining of operation (T1) ,30 min after operation (T2) ,and at the end of operation (T3) for determination of plasma bactericidal/permeability-increasing protein. Results The blood loss and the blood transfusion in HES group were significantly lower than that of LR group[blood loss: (560±90)ml vs (810±110) ml and blood transfusion: (200±100) ml vs (600±200) ml,t=5.562 and 5.657,P<0.001]. The plasma BPI concentrations in HES group were significantly increased at T2～T3 as compared to baseline value at T0 [(8.9±1.6)μg/L,(13.4±1.2)μg/L and (4.9±1.2)μg/L,P<0.05]. The plasma BPI concentrations in LR group were significantly increased at T2～T3 as compared to baseline value at T0 [(7.3±1.2)μg/L,(9.9±0.8) μg/L and (5.0±1.1)μg/L,P<0.05],but were lower than those in HES group (t=2.530 and 7.674,P=0.021 and 0.001 ). Conclusion Acute hypervolemic hemodilution with 200/0.5 hydroxyethyl starch can reduce blood transfusion during total hip replacement operation and also can increase the BPI level which would beneficial for the immunological function.

Objective To establish the sensitive,specific,stable and convenient immunoradio assay for detecting human soluble IL-6R?.Methods The hybridoma cell lines were obtained by fusing spleen cells of BALB/c mice that had been immunized with soluble IL-6R? protein to mouse myeloma cells sp2/0. Ascites were used to produce the monoclonal antibodies (mAbs). The mAbs were purified by protein G immunoaffinity method. The mAb SI10 was used as coating antibody, the other mAb H126 recognized different epitope from SI10 was labeled by 125I. Results The immunoradio assay for detecting soluble IL-6R? was set up. It has high stability and accuracy. The detecting limit is 10 ng/ml. The serum concentration of soluble IL-6R? is (81.96 ? 7.23) ng/ml in healthy donors and (237.58?70.96) ng/ml in patients with multiple myeloma. Significant difference was founded between two groups (P

Objective:To obtain a monoclonal antibody(mAb) against human CD80(B7 1) and to study of its biological effects.Methods:The hybridoma cell line was obtained by using the B lymphoma hybridoma technique after immunization of Balb/c mice with XG7 B7 the cells.Ascites were induced to produce the mAb.The specificity and affinity of mAb were verified B7 competition and FACS.Expression of B7 1 in PBLs,DCs,Raji and Daudi were studied by indirect immunofluorescence.Using counting and trypan blue staining,inhibitory effects of mAb on Raji and Daudi cells were analyzed.The neutralization activity of the 4E5 determined by MTT assay using PBLs as response cells.Results:The anti CD80(B7 1) was obtained.The expression of B7 1 in PBLs、DC、Raji ad Daudi was 10 2%,95 1%,96 7% and 89 2%,respectived 4E5 can inhibit the growth in Raji and Daudi cells and block the costimulatory signals of B7/CD28.Conclusion:4E5 is a specific and functional anti CD80(B7 1) and has high affinity for its ligand.It may be of significant value in basic studies and find clinical applications.

Objective:To prepare mouse anti human CD40L antigen monoclonal antibody and study its biological characteristics and functions.Methods:Using CD40L transfected cell as antigen,cell fusion,mAb screening,immunofluorescence,Western blot and competitive test,obtain two mouse anti human CD40 mAb.Their biological functions are evaluated by the analysis of the effect of the Daudi cell proliferation and the mixed lymphocyte reaction.Results:On the basis of phenotype analysis and competition test,it was evidenced that 1B1 and 4F1 recognized different epitopes of human CD40L antigen specially,and they could reverse the growth inhibition of Daudi cell mediated by CD40L transfected cells and reduce MLR in different extents.Conclusion:Two stable hybridomas murine anti human CD40L monoclonal antibodies have been obtained and antibodies(1B1?4F1)showed a obviously blocking function for CD40/CD40L signal.

Objective:To prepare the monoclonal antibody(mAb) against human B7 1 and analyse its biological characteristics.Methods:The B lymphocytes hybridization technique was applied by using XG7 B7 cell,a multiple myeloma(MM) cell line transfected with human B7 1 gene,as immunogen;the specificity and the antigen binding activity of mAbs were identified by flow cytometry and Western blot analysis;its biological effects on human PBTC and human B lymphoma cell line were examined by 3H TdR incroporation and annexin V satining.Results:Four mouse anti human B7 1(B7 1) hybridoma(1F11,3H8,6H2,7B10) were obtained.They secrete continuosly and steadily specific anti human B7 1(B7 1) mAb and their subclasses belong to IgG1 and IgM respectively;three of four mAbs could inhibit the proliferation of response cells(the human peripheral blood T lymphocytes),stimulated by costimulatory molecule B7 1.Furthermore,it was found that these mAbs induced the apoptosis of human B lymphoma cell line,Raji,which express naturally human B7 1 molecule,by using annexin V staining analysis after 24 hours of mAb treatment.Conclusion:Sucessefully obtained four mouse anti human B7 1 functional monoclonal antibodies,which have a potential value in anti allogenetic graft rejection and in the therapeutic approach of B lymphoma.

Aim To prepare the monoclonal antibodies (mAbs) against human CD28 and to study its biological feature. Methods The hybridoma cell lines were obtained by fusing spleen cells of Blab/c mice that had been immunized with murine lymphoma cells transfected with full-length huaman CD28 cDNA to myeloma cells Sp2/0. Ascites were induced to produce the mAbs. The specificity and affinity of the mAb 18G8 was verified by CD28 competitive inhibitory test and FACS. Reactivities of mAb 18G8 to PBTC, U266, 8226, Jurkat and Daudi cell were studied by indirect immunofluorescence staining. mAb 18G8-inducing proliferation of peripheral blood T cells (PBTCs) was determined by [3H]thymidine incorporation test. Results Five hybridoma cell lines were obtained. mAb 18G8 secreted by one of the them, belong to mouse IgG2a. It recognized a epitope different from which recognized by the standard mAb(clone CD28.2). The Reactivitrates of the mAb 18G8 to PBTC, U266, 8266, Jurkat and Daudi cells were 70.2％ , 99.3％ , 98.6％ , 76.4％ and 1.9％ , respectively, similar with CD28.2. It was indicated that different antigen epitopes expressed on all above cells. mAb 18G8 could promote the PBTC proliferation in vitro(SI=7). It was indicated that The substitution of mAb 18G8 for B7-1 molecule could also mediate the costimulatory signals. Conclusion 18G8 is a specific and functional anti-CD28 mAb it may be of significant value in basic studies and clinical application.

Objective:To construct lentiviral vector specific for mouse B7-1 RNA interference and study lentivirus-mediated B7-1 gene silencing effects in L929 fibroblast cells.Methods:Three candidate sequences for B7-1 RNAi selected from coding sequence of mouse B7-1 transcription were used to design short hairpin RNA ( shRNA ) templates and then cloned into lentiviral expression plasmid followed with correctness identification of inserted sequence by DNA sequencing.Recombinant lentivirus were prepared by co-transfecting lentiviral expression vector and packaging plasmids into 293T cells.Then the resulting culture supernatant containing infectious lentiviral particles was pooled and centrifuged via ultra-centrifugation.Infectious titer of the preparations was determined by detecting the expression of GFP in 293T cells after transfected by lentivirus.Cultured L929 cells were transfected with lentivirus to deter-mine transduction efficiency and silencing efficacy of B7-1 expression by flow cytometry.Transducted L929 cells were then screened using puromycin to generate stable cell clones followed by flow cytometry analysis of GFP and B7-1 expression.A mixed reaction system consisting of stable B7-1 silencing L929 cells and mouse splenic T cells was used to analyze ability of the established cell line to trigger T cells proliferation.Results: Lentiviral expression vector for mouse B7-1 RNAi was correctly constructed with inserted sequences as designed.Recombinant RNAi lentivirus were prepared with titers ranging (3-5) ×108 TU/ml and efficacy to mediate GFP transgene expression and B7-1 silencing.B7-1 expression and the ability to trigger T cells proliferation of stable L929 cells were suppressed significantly ( P<0.05 ).Conclusion: We generated lentiviral vector specific for mouse B7-1 RNAi with high performance of transduction efficiency as well as B7-1 silencing efficacy and the recombinant RNAi lentivirus can mediated stable B7-1 gene silencing in L929 cells and inhibition of T cells proliferation induced by B7-1/CD28 co-stimulatory signal.

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