凡纳滨对虾主要过敏原-原肌球蛋白单克隆抗体的制备及其过敏原表位分析

目的制备凡纳滨对虾主要过敏原-原肌球蛋白的单克隆抗体(mAb),运用它们和虾过敏患者血清分析凡纳滨对虾原肌球蛋白的过敏原表位。方法纯化的凡纳滨对虾原肌球蛋白免疫Balb/c小鼠,间接ELISA和Western blot筛选并建立稳定分泌抗原肌球蛋白抗体的杂交瘤细胞株;腹水型mAbs经硫酸铵沉淀、Protein G亲和层析纯化;叠加ELISA分析mAbs的抗原结合表位;虾过敏患者血清lgE与mAbs的抑制Western blot和间接竞争ELISA分析原肌球蛋白的过敏原表位。结果共筛选出5株mAbs,它们之间叠加ELISA的叠加值均高于40%;其中B5和A5能显著抑制虾过敏患者血清IgE与原肌球蛋白的结合,抑制率分别为58.1%和48.6%,同时也能抑制66.7%和44.3%的血清IgE与虾蛋白粗提液反应。结论成功制备了5株分别结合原肌球蛋白不同抗原表位的单克隆抗体,其中B5和A5能结合其过敏原表位。     

凡纳滨对虾新过敏原烯醇化酶的鉴定

目的：鉴定凡纳滨对虾分子量为47 kD过敏原的性质。方法：采用丙酮沉淀法提取凡纳滨对虾总蛋白,通过SDS-PAGE、11例虾过敏患者血清IgE的Western blot。分析凡纳滨对虾中过敏原组份,运用基质辅助激光解析串联飞行时间质谱仪(Matrix-Assisted laser desorption ionization/time of flight mass spectrometry,MALDI-TOF/TOF-MS)鉴定凡纳滨对虾47 kD未知过敏原组分。结果：通过 SDS-PAGE电泳证明所提取的凡纳滨对虾总蛋白组分完全。Western blot 结果显示,凡纳滨对虾至少有14种与阳性血清反应的组分,其中,55%的虾过敏患者IgE与分子量为47 kD的蛋白分子发生特异性反应,质谱分析结果显示47 kD蛋白为烯醇化酶。结论：烯醇化酶是凡纳滨对虾的一种新的过敏原。     

凡纳滨对虾次要过敏原肌质钙结合蛋白的质谱鉴定及免疫交叉反应

目的:质谱鉴定凡纳滨对虾相对分子质量(Mr)为21 000的次要过敏原组分肌质钙结合蛋白(SCP),分析它与其他虾、蟹等甲壳类过敏原的免疫交叉反应,以阐明SCP可作为甲壳类食物过敏原检测、检验及脱敏的标准过敏原物质。方法:运用MALDI-TOF/TOF-MS鉴定凡纳滨对虾Mr为21000的过敏原组分,利用软件BLAST、ClustalW分析甲壳类食物中该蛋白的氨基酸序列同源性,同时用纯化的21 000过敏原免疫小鼠制备其特异性多克隆抗体,通过该抗体与其他8种甲壳类食物蛋白粗提液的Western blot法结果分析该过敏原的免疫交叉反应。结果:质谱鉴定结果显示,凡纳滨对虾21 000过敏原为肌质钙结合蛋白;氨基酸序列同源性分析显示其与斑节对虾、中国对虾、克氏原螯虾、细趾小龙虾、褐虾的SCP序列一致性为81%～100%;Western blot结果显示,针对SCP的特异性多克隆抗体与凡纳滨对虾、刀额新对虾、斑节对虾、口虾蛄、罗氏沼虾、克氏原螯虾、远海梭子蟹、锈斑鲟、中华绒螯蟹粗提液在SCP对应的21 000左右处均有反应条带。结论:凡纳滨对虾Mr为21 000的次要过敏原为肌质钙结合蛋白,其氨基酸序列与多种甲壳类具有很高的同源性以及较强的免疫交叉反应。     

中国人的虾、蟹致敏过敏原组分分析

目的：分析鉴定中国人群的虾、蟹致敏组分，确定主要过敏原及其致敏率，为深入研究食物过敏原检测及脱敏治疗提供依据。方法：通过46份虾蟹过敏症患者血清，对刀额新对虾、罗氏沼虾和锈斑鲟的蛋白粗提液进行Western blot 分析，统计数据得出其全部过敏原组分及其致敏率，并确定主要过敏原。结果：Western blot 结果显示，虾与过敏患者血清IgE的反应强于蟹，且虾、蟹间存在很多Mr相同的过敏原；32～38 kD原肌球蛋白（ TM）、40 kD精氨酸激酶（ AK）、60～80 kD血蓝蛋白（ Hc）和21 kD肌质钙结合蛋白（ SCP）是虾的主要致敏组分，TM、AK和Hc是虾、蟹共同的主要过敏原，其中TM的致敏率最高；与国外研究结果相比，AK、Hc和SCP的致敏率相对较高，且发现虾中48 kD蛋白组分（未知过敏原）也具有较高致敏率。结论：对中国人群而言，虾的致敏性强于蟹，且虾、蟹的主要致敏组分基本相同；中国人的虾蟹主要致敏组分及其致敏率与国外研究大致相同但略有差异；发现一种潜在的新型过敏原。     

河虾主要过敏组分的分离、纯化、鉴定及其致敏性分析

目的:鉴定河虾中的过敏原组分,对主要过敏原组分进行纯化并分析其致敏性强弱。方法:用磷酸盐缓冲液(PBS)制备河虾蛋白粗提液,将其与11例虾过敏症患者血清IgE进行Westernblot,鉴定各种分子量的河虾过敏原组分;河虾蛋白粗提液用硫酸铵沉淀、G-50凝胶层析和阴离子交换层析等方法纯化其主要过敏原组分,再用虾过敏患者血清IgE进行Westernblot鉴定;并将纯化的相对分子质量(Mr)为21000、36000、800003种主要过敏原组分与虾过敏症患者血清IgE做间接ELISA,以分析其致敏性强弱。结果:West-ernblot结果显示,河虾蛋白粗提液出现9个阳性条带;其中3种主要过敏原组分21000、36000、80000与患者血清的反应率为36.4%,63.6%,45.5%;将这3种Mr的蛋白组分分别做间接ELISA,结果显示21000、36000、80000过敏原组分与11例虾过敏症患者的混合血清IgE结合的吸光值都显著高于河虾蛋白粗提液。结论:河虾中至少存在9个过敏原组分;21000、36000、8000等3种蛋白组分为河虾的主要过敏原组分,其中以36000过敏原组分致敏率和致敏性最强。进一步研究将探明21000、36000、80000等3种河虾过敏原组分的共同抗原表位,以期为明确食物过敏原检测、临床诊断和虾过敏原疫苗设计提供基础。     

虾类和蟹类过敏原的交叉反应性研究

目的：分析虾类和蟹类过敏原交叉反应性，探讨其在食物过敏症的预防、诊断和治疗中的意义。方法：采用SDS-PAGE分析虾蟹的蛋白组分，Westernblot分析其过敏原组分，Westernblot和ELISA抑制试验研究虾类和蟹类的过敏原交叉反应性。结果：虾类、蟹类过敏原蛋白组分相似性与其种属关系相关，相对分子量（Mr）在20000、36000、38000、44000、68000、75000、85000处具有相同条带；Mr为36000的蛋白是虾的主要过敏原，胁为36000、66000、85000的蛋白是蟹的主要过敏原；青蟹蛋白和日本沼虾蛋白对其余虾蟹过敏原有抑制作用，并且随着抑制物浓度增加抑制效应增强。结论：虾类和蟹类过敏原有相似性，其中Mr在36000处相互之间存在较强的交叉反应。     

特异性过敏原TBt多克隆抗体的制备与鉴定

目的:制备特异性苦荞主要过敏原TBt的多克隆抗体,为深入研究TBt分子中各结构域的功能奠定基础.方法:通过盐析,离子交换层析,分子筛层析等方法从苦荞种子中纯化出过敏蛋白TBt,免疫新西兰兔子,制备多克隆抗体.用间接ELISA、Western blot检测该抗体的效价和特异性；竞争ELISA研究IgG抗体对TBt过敏患者血清IgE的抑制作用.结果:间接ELISA法检测所制备抗体的效价达1∶256000左右；Western blot显示该抗体能与TBt蛋白特异结合；竞争ELISA表明制备的IgG抗体能够特异性抑制养麦过敏患者血清1gE与过敏蛋白的结合.结论:制备的TBt多抗血清具有较高的效价和良好的特异性,该多克隆抗体可用于TBt的免疫活性鉴定,为下一步研究该蛋白的分子特征及免疫治疗奠定了基础.     

青霉素类药物之间交叉过敏反应

目的 :探讨青霉素类抗生素之间交叉过敏反应性 ,并分析交叉过敏反应与药物化学结构之间的关系。方法 :收集青霉素过敏者血清 397例。采用放射过敏原吸附试验法 (radioallergsorbenttest,RAST) ,检测青霉素过敏病人血清中 8种主、次要抗原决定簇 ,分别为青霉素G(BPO、BPA)、青霉素V(PVO、PVA)、氨苄西林 (APO、APA)和阿莫西林 (AXO、AXA)特异性IgE抗体 ,分析血清中特异性IgE抗体的种类与过敏药物的关系。结果 :397例青霉素过敏病人血清特异性IgE抗体总阳性 2 34例 ( 5 8.9% )。 1～ 8种抗原决定簇特异性IgE抗体的阳性率分别…     

荨麻疹患者血清特异性IgE过敏原检测结果排序分析

目的 了解荨麻疹患者血清特异性IgE检测过敏原的情况.方法 回顾国内核心期刊发表的关于荨麻疹患者通过血清特异性IgE检测过敏原的文献报道,归纳总结引起人们过敏的过敏原主要为哪些.结果 吸入性过敏原以尘螨、粉螨最高,约占19.14 %.食入性以虾最高,约占11.03 %.结论 荨麻疹病因比较复杂,过敏原是大多数荨麻疹患者发病的主要因素,通过了解人们对过敏原过敏情况,为临床治疗和预防提供依据.     

日本柳杉花粉变应原CJP-6的重组表达和免疫反应性鉴定

目的:获得日本柳杉花粉主要变应原CJP-6的编码基因,构建其原核表达载体进行表达,并对重组CJP-6(rCJP-6)进行免疫活性鉴定。方法:从日本柳杉花粉中提取总RNA,采用RT-PCR的方法扩增CJP-6编码基因,将其克隆入pET-19b表达载体。转入大肠杆菌BL21 Star(DE3)pLysS,经IPTG诱导表达,以Ni2+亲和层析柱对重组变应原进行纯化,采用Dot blot和ELISA方法检测重组变应原rCJP-6与对日本柳杉花粉、尘螨和蒿草花粉过敏患者的血清中的IgE反应活性。结果:重组变应原与日本柳杉花粉过敏患者血清中的IgE具有较高的结合活性,rCJP-6与尘螨、蒿草花粉过敏患者血清中的IgE也具有很高的反应性,而且反应性与日本柳杉花粉过敏的患者血清相似。结论:制备并获得了具有IgE结合活性的重组日本柳杉花粉变应原CJP-6,日本柳杉花粉变应原是中国过敏反应性疾病患者潜在的变应原,为国内过敏反应性疾病的临床诊断和免疫治疗及进一步的实验研究奠定了基础。     

Immunological analysis of allergenic cross-reactivity between peanut and tree nuts

BACKGROUND: Peanut and tree nut allergy is characterized by a high frequency of life-threatening anaphylactic reactions and typically lifelong persistence. Peanut allergy is more common than tree nut allergy, but many subjects develop hypersensitivity to both peanuts and tree nuts. Whether this is due to the presence of cross-reactive allergens remains unknown. OBJECTIVE: The aim of this study was to investigate the presence of allergenic cross-reactivity between peanut and tree nuts. METHODS: Western blotting and ELISA were performed using sera from subjects with or without peanut and tree nut allergy to assess immunoglobulin E (IgE) reactivity to peanut and tree nut extracts. Inhibition ELISA studies were conducted to assess the presence of allergenic cross-reactivity between peanut and tree nuts. RESULTS: Western blot and ELISA results showed IgE reactivity to peanut, almond, Brazil nut, hazelnut and cashew nut for peanut- and tree nut-allergic subject sera. Raw and roasted peanut and tree nut extracts showed similar IgE reactivities. Inhibition ELISA showed that pre-incubation of sera with almond, Brazil nut or hazelnut extracts resulted in a decrease in IgE binding to peanut extract, indicating allergenic cross-reactivity. Pre-incubation of sera with cashew nut extract did not cause any inhibition. CONCLUSION: These results show that multiple peanut and tree nut sensitivities observed in allergic subjects may be due to cross-reactive B cell epitopes present in different peanut and tree nut allergens. The plant taxonomic classification of peanut and tree nuts does not appear to predict allergenic cross-reactivity.     

Functional analysis of cross-reactive immunoglobulin E antibodies: peanut-specific immunoglobulin E sensitizes basophils to tree nut allergens

BACKGROUND: Peanut and tree nuts are a major cause of food-induced anaphylaxis with an appreciable mortality. Co-sensitization to peanuts and tree nuts is a common clinical observation and may be because of peanut-specific serum IgE antibodies that cross-react with tree nut allergens. It is, however, unclear whether these cross-reactive IgE antibodies are involved in effector-cell activation. OBJECTIVE: To determine if cross-reactivity of peanut-specific IgE antibodies with tree nuts can cause effector cell activation using an in vitro basophil activation assay. METHODS: Two peanut allergic subjects with positive specific IgE for peanut and tree nuts (as measured by CAP-FEIA) were studied. Basophil activation to peanut and tree nuts, as indicated by CD63 expression, was assessed by flow cytometry to confirm co-sensitization to peanut and tree nuts. Inhibition ELISA using sera from the subjects was performed to detect peanut-specific IgE antibodies that cross-reacted with tree nut proteins. To determine whether cross-reactive tree nut allergens can induce effector-cell activation, peanut-specific antibodies were affinity purified from the subject sera and used to resensitize non-peanut/tree nut allergic donor basophils stripped of surface IgE. Basophil activation was then measured following stimulation with peanut and tree nut extracts. RESULTS: The two peanut allergic subjects in this study showed positive basophil activation to the peanut and tree nut extracts. Inhibition ELISA demonstrated that pre-incubation of the peanut allergic subject sera with almond, Brazil nut and hazelnut extracts inhibited IgE binding to peanut extract. IgE-stripped basophils from non-peanut/tree nut allergic subjects resensitized with affinity-purified peanut-specific antibodies from the peanut allergic subject sera became activated following stimulation with peanut, almond and Brazil nut extracts, demonstrating biological activity of cross-reactive IgE antibodies. CONCLUSION: Peanut-specific IgE antibodies that cross-react with tree nut allergens can cause effector-cell activation and may contribute to the manifestation of tree nut allergy in peanut allergic subjects.     

IgE antibody response to vertebrate meat proteins including tropomyosin.

BACKGROUND: Although meat is a main source of proteins in western diets, little information is available regarding allergy to vertebrate meats or the allergens implicated in these reactions. OBJECTIVE: To evaluate the in vitro IgE antibody response to different vertebrate meats in suspected meat-allergic subjects, as well as the possible role of tropomyosin in meat allergy and to analyze the cross-reactivity between vertebrate meats and the effect of heating on the IgE-binding to meat proteins. METHODS: Fifty-seven sera from suspected meat-allergic subjects were tested by grid blot to extracts of beef, lamb, pork, venison, chicken, and turkey and to four mammalian tropomyosins of different origins. RESULTS: Meat-allergic subjects have IgE antibodies to proteins in different mammalian meats (43/57 subjects); cross-reactivity with avian meat was limited: less than 50% (19/43) of meat positive sera reacted to chicken. In contrast, most of the poultry-positive sera also reacted to different mammalian meats. In general, there was stronger IgE reactivity to raw meats in comparison to cooked meats; an exception was six cases in which IgE reactivity to cooked poultry was stronger. Weak IgE reactivity to tropomyosin was detected in only 2/57 sera tested. CONCLUSIONS: Suspected meat-allergic subjects have serum IgE directed to meat proteins. In vitro cross-reactivity among mammalian meats appears to be important, while cross-reactivity to poultry is limited indicating mammalian-specific proteins. Although cooking in general denatures meat proteins rendering them less allergenic, in some cases the process of cooking may result in the formation of new allergenic moieties. The muscle protein tropomyosin is not an important vertebrate meat allergen.     

Identification of shared and unique immunoglobulin E epitopes of the highly conserved tropomyosins in Blomia tropicalis and Dermatophagoides pteronyssinus

BACKGROUND: Tropomyosin belongs to a class of highly conserved proteins in invertebrates and vertebrates. The invertebrate tropomyosins are allergenic in man with high IgE cross-reactivity and have been therefore referred to as pan-allergens. OBJECTIVES: This study aimed to clone and identify the IgE epitopes of tropomyosin from Blomia tropicalis (Blo t 10) mite. Cross-reactivity between the IgE epitopes of Blo t 10 and Der p 10 was also evaluated. METHODS: Blo t 10 was isolated using mouse anti-Der p 10 antibodies. Allergenicity of the cloned Blo t 10 was confirmed by skin prick test (SPT) and enzyme-linked immunosorbent assay (ELISA). Dose-dependent inhibition assay was performed to determine the degree of IgE cross-reactivity between Blo t 10 and Der p 10. Overlapping polymerase chain reaction-derived cDNA were generated and expressed as glutathione-S-transferase (GST) recombinant proteins in Escherichia coli and used to identify shared and unique IgE epitopes of Blo t 10 and Der p 10. RESULTS: The cloned Blo t 10 shared up to 96% amino acid identity to tropomyosin of other mites. SPT and ELISA IgE-immunoassay showed recombinant Blo t 10 sensitization rates of between 20% and 29% in atopic subjects. Results of SPT and dose-dependent inhibition assays showed that some allergic individuals had unique IgE epitopes for Blo t 10. IgE epitope mapping of Blo t 10 revealed that the epitopes were mainly located at N- and C-termini of the molecule. The results of ELISA inhibition assays of overlapping recombinant fragments indicated that the unique IgE epitopes of Blo t 10 were located at the C-terminal. CONCLUSION: Although Blo t 10 and Der p 10 are highly conserved (shared 95% amino acids identity) and significantly cross-reactive, unique IgE epitopes do exist. The results suggest the potential deficiency of using only one of these highly conserved allergens as diagnostic or therapeutic reagents.     

Cross-reactive and species-specific immunoglobulin E epitopes of plant profilins: an experimental and structure-based analysis

BACKGROUND: Profilins are cross-reactive plant allergens responsible for multiple pollen sensitization and pollen-associated food allergy. While it is assumed that profilins from different species are immunologically equivalent, some studies suggest partial or even lacking IgE cross-reactivity between certain profilins. OBJECTIVE: We aimed to obtain a semi-quantitative assessment of the contributions of conserved and species-specific epitopes to IgE binding of plant profilins. METHODS: We compared model structures of profilins from timothy, mugwort, celery and bell pepper with crystal structures of birch and latex profilins. We predicted potential conformational epitopes that consisted of contiguous patches of at least 20% surface-exposed residues. Celery and timothy profilins were purified from their natural sources, and profilins from birch, mugwort, bell pepper and latex were expressed in Escherichia coli. The structural integrity of all purified proteins was confirmed by circular dichroism spectroscopy. IgE ELISAs and ELISA inhibitions using sera from 22 profilin-sensitized allergic patients were carried out. RESULTS: Peptide backbone conformations of all six profilins were highly similar. Nine variable epitopes and two containing high proportions of conserved residues were predicted. IgE from all sera bound to all tested profilins and the amounts were highly correlated. However, IgE inhibition experiments revealed that up to 60% of total IgE binding was mediated by species-specific epitopes. The extent of cross-reactivity among profilins from timothy, birch, latex and celery was greater than cross-reactivity to mugwort and bell pepper profilins. This pattern was mirrored by sequence similarities among one of the predicted variable epitopes. Patients with IgE to cross-reactive epitopes displayed allergic reactions to a greater number of plant foods than patients having IgE directed to species-specific epitopes. CONCLUSION: The large extent of cross-reactivity among plant profilins justifies using a single profilin for diagnosis. However, the fine specificity of IgE directed to variable epitopes may influence the clinical manifestation of profilin sensitization.     

Cross-reactivity between terrestrial snails (Helix species) and house-dust mite (Dermatophagoides pteronyssinus). II. In vitro study

Epidemiologic and in vitro data have shown that the association of housedust mite (HDM) allergy and snail allergy in the same patients was due to cross-reactivity between HDM and snail allergenic components. However, the cross-reacting allergen(s) have not yet been identified. In vitro reactivity of seven patients' sera to the various extracts and hemolymph of four different Helix snail species was analyzed by IgE detection on immunodots and Western blots. Cross-reactivity between snails and Dermatophagoides pteronyssinus was assessed by immunodot and ELISA inhibition in two patients. Heterologous inhibition of the snail immunodot and ELISA was observed in one serum. Western blotting showed a specific binding on all four snail species extracts: molecular weights of snail allergens ranged from <21 to 200 kDa. Marked individual differences were observed in the seven sera under study: most sera demonstrated IgE recognition of multiple bands, illustrating that no single allergen is responsible for cross-reactivity between snail and mite. These results confirm that cross-reactivity exists between snails of the Helix genus and HDM. This cross-reactivity, involving more than a single allergen, may be of clinical significance in atopic patients allergic to D. pteronyssinus. The identity of the cross-reacting allergens remains to be determined. Potential candidates include the thermostable minor allergens of D. pteronyssinus, tropomyosin and hemocyanin. Diseases, Environment.     

Immunological characterization of a recombinant tropomyosin from a new indoor source, Lepisma saccharina

BACKGROUND: The presence of specific IgE antibodies to invertebrates is common among patients with rhinitis and asthma. Tropomyosin has been described as an invertebrate cross-reactive allergen. We have recently characterized an allergenic extract from silverfish (Lepisma saccharina). Since this insect could be a new source of tropomyosin in the indoor environment, we have thought important to clone and characterize the tropomyosin from it. METHODS: Recombinant tropomyosin was cloned and characterized by means of immunoblotting with tropomyosin-specific monoclonal antibodies, rabbit polyclonal antibodies and IgE from allergic patients. Its allergenic activity was investigated in histamine release assays. Immunoblotting and ELISA inhibition were carried out to identify the natural tropomyosin in the silverfish extract and to study the cross-reactivity among other arthropod tropomyosins. RESULTS: Tropomyosin-specific antibodies recognized in immunoblotting the natural tropomyosin in the insoluble fraction of silverfish extract. The silverfish tropomyosin (Lep s 1) was cloned and fully expressed. It shared high homology with other arthropod tropomyosins. rLep s 1 was recognized by tropomyosin-specific monoclonal and polyclonal antibodies and by IgE of allergic patients. It was able to inhibit the IgE binding to the insoluble fraction of silverfish extract, and to induce histamine release by an arthropod-allergic serum. Inhibition experiments revealed IgE cross-reactivity between rLep s 1 and other arthropod tropomyosins. CONCLUSION: rLep s 1 is the first allergen cloned and characterized from silverfish extract. It enabled us to identify the natural counterpart in the insoluble fraction of silverfish extract, suggesting that the tropomyosin is not readily extractable with a classic aqueous extraction procedure. rLep s 1 displayed biological activity, suggesting that it could be regarded as a useful tool to study the role of silverfish tropomyosin in the sensitization to invertebrate allergic sources.