| BP is an autoimmune subepidermal bullous disease typically presents with highly pruritic normal-looking skin or urticaria-like lesions of limbs and trunk on which tense blisters may develop after a variable period of time.BP is diagnosed on the basis of clinical,histologic,and immunologic findings.Serologic diagnosis of pemphigoid has been carried out by Blister disease laboratory of Institute of Dermatology,Chinese Academy of Medical Sciences for five years.Therefore,this study was undertaken to evaluate diagnostic value of serological methods(IIF-SSS,BP180NC16a ELISA,immunoblotting)in BP and the correlation of autoantibodies in BP and CXCL10 were also analyzed.Part 1 Comparative study of IIF-SSS and BP180 NC16a-ELISA for diagnosis of bullous pemphigoidObjective:To evaluate the efficacy and correlation of IIF-SSS and BP 180 NC16a-ELISA in the diagnosis of BP.Methods:Sera from BP patients(n=174)and control subjects(n=129)were assayed by IIF-SSS and BP180-NC16a ELISA.A11 BP sera were obtained at presentation before initiation of systemic immunosuppressive therapy.The results of IIF-SSS and NC16a-ELISA were compared.Results:The sensitivities and specificities for IIF-SSS,BP180 NC16a-ELISA were 93.67%,100%;96.55%,96.12%,respectively.The BP180 NC16a-ELISAAUC value is 0,978.The correlation coefficient between IIF-SSS and BP180 NC16a-ELISA was 0.147.Conclusion:The results of IIF-SSS can be used as an important criterion for the diagnosis of BP.BP180 NC16a-ELIS A provides clinic with abundant diagnostic information and objective,quantitative and reproducible datas,however.Part 2 Comparative study of IIF-SSS and DIF for diagnosis of bullous pemphigoidObjective:To evaluate the efficacy and correlation of IIF-SSS and DIF in the diagnosis of BP.Methods:Sera from BP patients(n=44)and control subjects(n=58)were assayed by IIF-SSS and DIF.A11 BP sera were obtained at presentation before initiation of systemic immunosuppressive therapy.The results of IIF-SSS and DIF were compared.Results:The sensitivities and specificities for IIF-SSS,DIF were 97.72%,100%;84.09%,91.3%,respectively.Conclusion:IIF-SSS can be used as first choice for the diagnosis of BP,and staining patterns in IIF-SSS can classify autoimmune subepidermal blistering diseases.The staining patterns should be analyzed when DIF is used as a diagnostic tool for BP.Part 3 Preparation of human epidermal side antigen by thermal separation and western blot analysis in BPObjective:To prepare human epidermal side antigen through thermal separation,and to evaluate the value of western blot in the diagnosis of BP.Methods:Fresh human skin was obtained from surgical specimens.Excess fat and connective tissue were removed and the specimens were cut into pieces of approximately 2×2 cm.These were incubated in thermal separation buffer for 4min.The epidermis was gently separated by forceps and grinded to powder within liquid nitrogen,and then was lysed by epidermis extract solution(sample:extract solution =1:4w/v)and homogenate 10 times to shear DNA in ice condition.Supernatant was collected by centrifugation at 100000×g for 30 min and boiled for 10 min in a water bath.Human epidermal side antigen was identified by Western blot.Sera from BP patients(n=22)and control subjects(n=25)were assayed by epidermal side antigen-IB.A11 BP sera were obtained at presentation before initiation of systemic immunosuppressive therapy.The result of epidermal side antigen-IB was analyzed.Results:After the above experiment process,the human epidermal side antigen was prepared successfully.The sensitivity and specificity for epidermal side antigen-IB were 86.36%,100%.Epidermal side antigen-IB results showed that a protein band of 230KDa were seen in 4 patients(18.18%),180KD band in 18 patients(81.81%),120KD band in 1 patient(4.54%),and 97KD band in 1 patients(4.54%).Conclusion:The main protein band in BP was 180KD.The sensitivity of epidermal side antigen-IB was lower than that of BP 180 NC16a-ELISA,which means that the result of the epidermal side antigen-IB was often negative when the titer of the autoantibody was low.Part 4 Preparation of recombinant protein of BP180 NC16a and COOH-terminal Objective:To prepare recombinant protein of BP 180 NC16a and BP 180 COOH-terminal through prokaryotic expression system.Methods:The successfully constructed recombinant p-GEX-6P-1 BP180NC16a and Pet15bBP180 COOH-terminal plasmid were transformed into E.coli BL21(DE3).Transformant was screened and induced by IPTG overnight.Thallus was collected by centrifugation and crashed by sonication.Supernatant was collected by centrifugation and purified by GST affinity chromatography(BP180NC16a)and Precipitation was lysed by inclusion body solublization buffer,and then supernatant was collected by centrifugation and purified by Ni affinity chromatography(BP 180 COOH-terminal).Recombinant protein of BP 180 NC16a and COOH-terminal were identified by Western blot.Results:After the above experiment process,the recombinant protein of BP180NC16a and COOH-terminal were purified successfully.Conclusion:In this experiment,recombinant p-GEX-6P-1 BP180NC16a expression plasmid and recombinant Pet15bBP180 COOH-terminal expression plasmid were transformed into E.coli BL21(DE3),and BP 180 NC16a and COOH-terminal recombinant protein were induced by IPTG and purified by affinity chromatography.The recombinant protein of BP 180 NC16a and COOH-terminal were identified by Western-blot.The successful preparation of the recombinant protein of BP 180 NC16a and COOH-terminal laid a foundation for the further experiments.Part 5 Detection of NC16a and COOH-terminal epitope and correlation analysis with CXCLI0Objective:To detection NC16a and COOH-terminal autoantibodies in BP patients and control subjects,and the correlation of autoantibodies in BP and CXCL10 were analyzed.Methods:Sera from BP patients(n=22)and control subjects(n=25)were assayed by BP180 NC16a-IB,BP180 COOH-IB,and BP patients were also assayed by CXCL10 ELISA.The results of BP180 NC16a-IB,BP180 COOH-IB and CXCL10 were analyzed.Results:The sensitivities and specificities for BP 180 NC16a-IB and BP 180 COOH-IB were 90.90%,4.54%;100%,100%respectively.The correlation coefficient between CXCL10 and BP180NC16a Pearson was 0.152 in BP patients.There was no statistical significance(P=0.5>0.05),while CXCL10 and BP 180 NC16a ELISA values decreased in 5 cases of BP when the condition is improved,and CXCL10 concentration varies with individual.Conclusion:Autoantibody against BP180 NC16a play a major role in the pathogenesis of BP,and there was correlation between BP 180 NC16a autoantibody and CXCL10,while IP-10 expression varied greatly which is affected by many factors. |
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