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 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.

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

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 study the mRNA expression levels and clinical significance of omsomucoid 1-like protein 3 (ORMDL3) gene in the peripheral blood of recurrent wheeze children under 3 years old.Methods Peripheral blood specimens of 25 recurrent wheeze children including 14 non-atopy patients (group A) and 11 atopy patients (group B) that were registered in the First Affiliated Hospital of Nanjing Medical University,from Sep.2010 to Sep.2012 were enrolled based on the inclusion criteria and 24 non-allergic controls(the children with food allergy,drug allergy or ectema was excepted).The mRNA expression levels of ORMDL3 gene were detected by reverse transcription (RT)-PCR and clinical features were analyzed.Results The expression levels of ORMDL3 were up-regulated in the peripheral blood specimens of group B compared with group A (t =14.12,P ＜ 0.01).Compared with peripheral blood specimens from normal subjects,mRNA expression of ORMDL3 were significantly increased in recurrent wheeze children(t =10.29,5.73,P ＜0.01).The incidence of wheeze groups exist gender differences,male ＞ female.Wheeze usually with a high incidence in winter and spring.Conclusions The increase of ORMDL3 gene expression levels were correlated with recurrent wheeze under 3 years old especially in atopy group and may be involved in the pathogenesis of recurrent wheeze.

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.