目的构建噬菌体抗体库并筛选抗BP180-NC16A抗体。方法以大疱性类天疱疮(BP)患者外周血淋巴细胞为基因来源,扩增多样性的轻链和重链Fd段基因,构建Fab段表面展示的噬菌体抗体库,用BP180分子的NC16A片段为抗原对抗体库进行筛选并对筛选得...目的构建噬菌体抗体库并筛选抗BP180-NC16A抗体。方法以大疱性类天疱疮(BP)患者外周血淋巴细胞为基因来源,扩增多样性的轻链和重链Fd段基因,构建Fab段表面展示的噬菌体抗体库,用BP180分子的NC16A片段为抗原对抗体库进行筛选并对筛选得到的抗体用ELISA,W estern b lot和免疫荧光等方法进行鉴定。结果经过轻、重链基因的重组和转化,获得了库容为5×107的噬菌体抗体库。以BP180-NC16A抗原进行"亲和吸附—洗脱—扩增"淘筛,获得2株特异性抗体。结论利用噬菌体抗体库技术成功制备了抗BP180抗体,为深入研究BP自身抗体、探讨新的治疗策略奠定了基础。展开更多
Background: Pemphigoid gestationis (PG) is a rare pregnancyassociated subepidermal immunobullous disease that targets hemidesmosomal proteins, particularly BP180. Clinically, PG can resemble the eruption known as poly...Background: Pemphigoid gestationis (PG) is a rare pregnancyassociated subepidermal immunobullous disease that targets hemidesmosomal proteins, particularly BP180. Clinically, PG can resemble the eruption known as polymorphic urticarial papules and plaques of pregnancy (PUPPP), and accurate differentiation between these 2 pruritic pregnancy dermatoses has important implications for fetal and maternal prognoses. Results of epitope mapping studies show that IgG autoantibodies in up to 90%of PG serum samples target the well-defined membrane-proximal NC16a domain of BP180. Objective: To examine the usefulness of a commercially available NC16a domain enzyme-linked immunosorbent assay in the serodiagnosis of PG and in the differentiation of PG from PUPPP. Participants: A total of 412 women consisting of pretreatment patients with PG (n=82), patients with PUPPP (n=164), and age-and sex-matched controls (n=166). Methods: All serum samples were assayed in duplicate. Receiver operating characteristic analyses were performed to determine a cutoff value for the diagnosis of PG and for differentiation from PUPPP and controls. Results: A cutoff value of 10 enzyme-linked immunosorbent assay units was associated with specificity and sensitivity of 96%. Conclusions: The NC16a enzyme-linked immunosorbent assay is highly sensitive and highly specific in differentiating PG from PUPPP, and it is potentially a valuable tool in the serodiagnosis of PG.展开更多
We report on a 24-year-old, male Ugandan patient with a 2-week history of itchy papules, vesicles, erosions, and crusts distributed on the entire body, accompanied by minor erosions on the palate, tongue, and lower li...We report on a 24-year-old, male Ugandan patient with a 2-week history of itchy papules, vesicles, erosions, and crusts distributed on the entire body, accompanied by minor erosions on the palate, tongue, and lower lip. Conjunctivae and genital mucosa were not involved. Circulating IgG and IgA autoantibodies were found against recombinant full-length BP180, BP180 4575, and the C-terminus of BP230. In addition, IgG reactivity was observed against the 16th noncollagenous region of the BP180 ectodomain, the cell-derived soluble ectodomain of BP180 (linear IgA disease antigen 1), and the α 3 and γ 2 chains of laminin 5. No reactivity was detected with type VII collagen, α 6β 4 integrin, and the p200 protein. Oral prednisolone and dapsone led to clearance of lesions that mostly healed with scarring and milia formation. Here, we describe a scarring mucocutaneous variant of an autoimmune blistering skin disorder that extends the current clinical and immunopathologic spectrum of this group of diseases.展开更多
We report a 73- year-old Japanese female who developed IgG autoantibodies against BP180 as well as desmoglein 3 (Dsg3). She showed tense blisters on the extremities without apparent mucosal involvement and a skin biop...We report a 73- year-old Japanese female who developed IgG autoantibodies against BP180 as well as desmoglein 3 (Dsg3). She showed tense blisters on the extremities without apparent mucosal involvement and a skin biopsy indicated subepidermal blisters with eosinophilic spongiosis. Her clinical and histologic features indicated the diagnosis of bullous pemphigoid while anti- Dsg3 IgG might not show an apparent pathogenic effect. Interestingly, titres of anti-Dsg3 IgG fluctuated in parallel with those of antiBP180 IgG throughout the course with two flares. Although the exact mechanism for autoantibody production is still unknown, the close link in the production of IgG autoantibodies against two independent skin antigens suggests a shared immunoregulatory mechanism against cutaneous autoantigens.展开更多
Bullous pemphigoid (BP) is associated with autoantibodies to the 180-kDa BP antigen (BP180), and the antigenic site exists on noncollagenous 16a (NC16a) domain of BP180. We now report a male BP patient whose IgG autoa...Bullous pemphigoid (BP) is associated with autoantibodies to the 180-kDa BP antigen (BP180), and the antigenic site exists on noncollagenous 16a (NC16a) domain of BP180. We now report a male BP patient whose IgG autoantibodies did not react against the NC16a domain of BP180 by either immunoblotting or ELISA, whereas they did react with BP180 extracted from normal human keratinocytes. Anti-BP180 cicatricial pemphigoid was ruled out due to the lack of conjunctival mucosal involvement and the absence of scarring in the oral cavity. Our findings indicate that there is an antigenic reactive region other than NC16a on the extracellular domain of BP180. There have been few reports describing detailed clinical features of BP caused by autoantibodies targeting antigenic sites other than the NC16a domain. We conclude that it is difficult to differentiate their clinical features from those associated with autoantibodies targeting the NC16a domain of BP180.展开更多
Background: The NC16A immunodominant region of the bullous pemphigoid (BP) antigen BP180 has been used to develop several enzyme-linked immunosorbent assays (ELISAs) as diagnostic tools for BP autoantibody detection. ...Background: The NC16A immunodominant region of the bullous pemphigoid (BP) antigen BP180 has been used to develop several enzyme-linked immunosorbent assays (ELISAs) as diagnostic tools for BP autoantibody detection. Objectives: Because BP180 autoantibody reactivity is not restricted toNC16A, we have investigated the possibility of developing an ELISA based on selected epitopes additional to this immunodominant region. Methods: Initially 78 BP sera were tested using an NC16A ELISA and IgG reactivity was detected in 64 BP sera (82%). The 14 NC16A-negative BP sera were then analysed by immunological screening against seven BP180-specific epitopes. Recombinant phages displaying BP180 epitopes were grown as plaques, blotted onto a nitrocellulose filter and incubated with BP sera. Results: Three and five NC16A-negative BP sera reacted with epitopes AA 1080-1107 and AA 1331-1404 of the BP180 ectodomain, respectively. Thus, a novel ELISA with GST-1080 and GST-1331 (GST-1080/1331) was developed: 32 of 78 BP sera (41%) proved positive by this assay. The combined use of ELISAs with GST-NC16A and GST-1080/1331 detected IgG reactivity in 72 of 78 BP sera, increasing the sensitivity from 82%to 92%. In addition, autoreactivity against the three extracellular epitopes appeared to be related to the presence of both skin and mucosal involvement as assessed by Fisher‘s exact probability test. Conclusions: Our findings further characterize the autoimmune response in BP by identifying a subgroup of NC16A-negative patients who react with different BP180 extracellular epitopes. The developed ELISA system appearsmore sensitive than the ELISA based on NC16A alone and also informative about the epitope profile of BP patients.展开更多
文摘目的构建噬菌体抗体库并筛选抗BP180-NC16A抗体。方法以大疱性类天疱疮(BP)患者外周血淋巴细胞为基因来源,扩增多样性的轻链和重链Fd段基因,构建Fab段表面展示的噬菌体抗体库,用BP180分子的NC16A片段为抗原对抗体库进行筛选并对筛选得到的抗体用ELISA,W estern b lot和免疫荧光等方法进行鉴定。结果经过轻、重链基因的重组和转化,获得了库容为5×107的噬菌体抗体库。以BP180-NC16A抗原进行"亲和吸附—洗脱—扩增"淘筛,获得2株特异性抗体。结论利用噬菌体抗体库技术成功制备了抗BP180抗体,为深入研究BP自身抗体、探讨新的治疗策略奠定了基础。
文摘Background: Pemphigoid gestationis (PG) is a rare pregnancyassociated subepidermal immunobullous disease that targets hemidesmosomal proteins, particularly BP180. Clinically, PG can resemble the eruption known as polymorphic urticarial papules and plaques of pregnancy (PUPPP), and accurate differentiation between these 2 pruritic pregnancy dermatoses has important implications for fetal and maternal prognoses. Results of epitope mapping studies show that IgG autoantibodies in up to 90%of PG serum samples target the well-defined membrane-proximal NC16a domain of BP180. Objective: To examine the usefulness of a commercially available NC16a domain enzyme-linked immunosorbent assay in the serodiagnosis of PG and in the differentiation of PG from PUPPP. Participants: A total of 412 women consisting of pretreatment patients with PG (n=82), patients with PUPPP (n=164), and age-and sex-matched controls (n=166). Methods: All serum samples were assayed in duplicate. Receiver operating characteristic analyses were performed to determine a cutoff value for the diagnosis of PG and for differentiation from PUPPP and controls. Results: A cutoff value of 10 enzyme-linked immunosorbent assay units was associated with specificity and sensitivity of 96%. Conclusions: The NC16a enzyme-linked immunosorbent assay is highly sensitive and highly specific in differentiating PG from PUPPP, and it is potentially a valuable tool in the serodiagnosis of PG.
文摘We report on a 24-year-old, male Ugandan patient with a 2-week history of itchy papules, vesicles, erosions, and crusts distributed on the entire body, accompanied by minor erosions on the palate, tongue, and lower lip. Conjunctivae and genital mucosa were not involved. Circulating IgG and IgA autoantibodies were found against recombinant full-length BP180, BP180 4575, and the C-terminus of BP230. In addition, IgG reactivity was observed against the 16th noncollagenous region of the BP180 ectodomain, the cell-derived soluble ectodomain of BP180 (linear IgA disease antigen 1), and the α 3 and γ 2 chains of laminin 5. No reactivity was detected with type VII collagen, α 6β 4 integrin, and the p200 protein. Oral prednisolone and dapsone led to clearance of lesions that mostly healed with scarring and milia formation. Here, we describe a scarring mucocutaneous variant of an autoimmune blistering skin disorder that extends the current clinical and immunopathologic spectrum of this group of diseases.
文摘We report a 73- year-old Japanese female who developed IgG autoantibodies against BP180 as well as desmoglein 3 (Dsg3). She showed tense blisters on the extremities without apparent mucosal involvement and a skin biopsy indicated subepidermal blisters with eosinophilic spongiosis. Her clinical and histologic features indicated the diagnosis of bullous pemphigoid while anti- Dsg3 IgG might not show an apparent pathogenic effect. Interestingly, titres of anti-Dsg3 IgG fluctuated in parallel with those of antiBP180 IgG throughout the course with two flares. Although the exact mechanism for autoantibody production is still unknown, the close link in the production of IgG autoantibodies against two independent skin antigens suggests a shared immunoregulatory mechanism against cutaneous autoantigens.
文摘Bullous pemphigoid (BP) is associated with autoantibodies to the 180-kDa BP antigen (BP180), and the antigenic site exists on noncollagenous 16a (NC16a) domain of BP180. We now report a male BP patient whose IgG autoantibodies did not react against the NC16a domain of BP180 by either immunoblotting or ELISA, whereas they did react with BP180 extracted from normal human keratinocytes. Anti-BP180 cicatricial pemphigoid was ruled out due to the lack of conjunctival mucosal involvement and the absence of scarring in the oral cavity. Our findings indicate that there is an antigenic reactive region other than NC16a on the extracellular domain of BP180. There have been few reports describing detailed clinical features of BP caused by autoantibodies targeting antigenic sites other than the NC16a domain. We conclude that it is difficult to differentiate their clinical features from those associated with autoantibodies targeting the NC16a domain of BP180.
文摘Background: The NC16A immunodominant region of the bullous pemphigoid (BP) antigen BP180 has been used to develop several enzyme-linked immunosorbent assays (ELISAs) as diagnostic tools for BP autoantibody detection. Objectives: Because BP180 autoantibody reactivity is not restricted toNC16A, we have investigated the possibility of developing an ELISA based on selected epitopes additional to this immunodominant region. Methods: Initially 78 BP sera were tested using an NC16A ELISA and IgG reactivity was detected in 64 BP sera (82%). The 14 NC16A-negative BP sera were then analysed by immunological screening against seven BP180-specific epitopes. Recombinant phages displaying BP180 epitopes were grown as plaques, blotted onto a nitrocellulose filter and incubated with BP sera. Results: Three and five NC16A-negative BP sera reacted with epitopes AA 1080-1107 and AA 1331-1404 of the BP180 ectodomain, respectively. Thus, a novel ELISA with GST-1080 and GST-1331 (GST-1080/1331) was developed: 32 of 78 BP sera (41%) proved positive by this assay. The combined use of ELISAs with GST-NC16A and GST-1080/1331 detected IgG reactivity in 72 of 78 BP sera, increasing the sensitivity from 82%to 92%. In addition, autoreactivity against the three extracellular epitopes appeared to be related to the presence of both skin and mucosal involvement as assessed by Fisher‘s exact probability test. Conclusions: Our findings further characterize the autoimmune response in BP by identifying a subgroup of NC16A-negative patients who react with different BP180 extracellular epitopes. The developed ELISA system appearsmore sensitive than the ELISA based on NC16A alone and also informative about the epitope profile of BP patients.
