几种常用的过敏原提取溶液对户尘螨放射过敏原吸附抑制实验荧光值的影响

目的 探讨几种常用的过敏原提取溶液对户尘螨（Dermatophagoides pterronyssinus）放射过敏原吸附抑制实验（RAST抑制实验）荧光值的影响。方法 将20例户尘螨过敏患者血清等量混合成阳性血清。其RAST实验荧光值为12505～24776。提取溶液分别是：SDS、PBS、NH4HCO3和Coca’S液。采用UniCAP 100过敏原检测系统进行RAST实验。结果 当SDS浓度为≥0．25％时,户尘螨过敏原RAST荧光值下降〉5％;当PBS浓度为1mol／L,0．1mol／L,0．01mol／L时,户尘螨过敏原RAST荧光值分别下降9．2％,2．9％和0．9％;用0．125mol／LNH4HCO3时,户尘螨过敏原RAST荧光值下降4．5％;使用Coca’s液时,户尘螨过敏原RAST荧光值下降3．4％。结论 在进行过敏原提取后,使用助溶剂复溶过敏原沉淀进行RAST抑制实验时,应当重视表面活性剂（如SDS）对RAST抑制实验的影响。另外,常用的0．1mol／L和0．01mol／L PBS,对RAST抑制实验无显著影响。Coca’s液和NH4HCO3对RAST抑制实验影响也不大。     

不同方法提取和处理中华绒螯蟹组织蛋白的过敏原组分分析

目的:分析不同方法提取和处理中华绒螯蟹组织的蛋白组分及过敏原组分,为检测中华绒螯蟹特异性IgE提供最佳抗原制备方法。方法:分别采用PBS提取法、丙酮抽提法和裂解液提取法提取中华绒螯蟹组织蛋白,并用裂解液提取法分别提取加热前、后的蟹蛋白;采用SDS-PAGE和双向电泳分析蛋白组分,采用Western blot分析过敏原组分。结果:不同方法提取的蛋白组分、sIgE与蛋白结合的强度和所识别的蛋白组分存在一定差异。PBS提取液蛋白主要分布在94、85、76、66、18kD,丙酮提取液蛋白主要分布在94、85、76、36 kD,裂解液提取液蛋白主要分布在200、125、51、43、38 kD。PBS提取液与sIgE结合的阳性反应条带主要分布在76、66、53、43、38、18 kD;丙酮提取液主要分布在94、76、66、50、43、38、18 kD;裂解液提取液主要分布在200、105、94、76、43、38、18 kD。加热前、后蟹蛋白过敏原组分差别不大。结论:裂解液提取法提取的中华绒螯蟹过敏原更适于制备测定特异性IgE的抗原。加热不影响蟹过敏原组分和与特异性IgE的结合活性。     

几种户尘螨过敏原点刺液的临床评价

评价户尘螨过敏原点刺皮试液用于诊断和筛查尘螨过敏的意义。方法：对219例患者进行户尘螨过敏原点刺试验和户尘螨sIgE检测。点刺试验所用点刺液为本课题组研制的点刺液（A 组）、ALK公司的户尘螨点刺液（B组）和ALK公司的混合螨（户尘螨和粉尘螨）点刺液（C组）,以sIgE为诊断标准。统计分析采用SPSS 11．0软件,对三组户尘螨过敏原点刺试验进行ROC曲线评价。结果：A、B和C组风团平均直径中位数为0.43cm、0.35cm和 0.28cm。所有入选者点刺试验后1h,A组有5例点刺试验后出现局部反应,2h左右自行缓解;有2例出现迟发反应,需1天左右才消退;B组和C组各有3例点刺试验后出现局部反应,B组和C组各有2例有迟发反应。所有患者均无局部皮疹、全身性不良反应。三组户尘螨过敏原点刺试验的ROC曲线下面积分别为0.765、0.801、0.782,说明户尘螨过敏原点刺皮试液对诊断户尘螨过敏具有较高准确性。以户尘螨sIgE为标准的点刺试验灵敏度和特异度：A、B 和C组点刺液的灵敏度分别为92.4％、87.0％和81.5％,特异度分别为60.6%、73.2%和74.8%。结论：户尘螨点刺液可作为用于尘螨过敏的诊断试剂,有良好的灵敏度和特异度,具有临床筛查和诊断意义。     

肿瘤细胞诱导T细胞下调表达腺苷脱氨酶

目的 蛋白质组学分析鉴定与肿瘤细胞共培养T细胞的表达差异蛋白.方法 双向电泳分离有/无肿瘤细胞共培养的T细胞蛋白,PDQuest软件分析筛选肿瘤细胞诱导表达异常的蛋白点,用基质辅助激光解析电离飞行时间质谱(MAL-DI-TOF MS)对酶解消化样品进行肽质量指纹图谱(PMF)分析,结合数据库搜索鉴定蛋白质.结果 PDQuest分析有/无肿瘤细胞共培养的T细胞蛋白双向电泳图谱,70余个蛋白点表达水平差异显著,其中与肿瘤细胞共培养的T细胞有一明显表达下调的蛋白点,质谱分析结合数据库检索表明该蛋白点为腺苷脱氨酶.结论 肿瘤细胞诱导T细胞下调表达腺苷脱氨酶.     

胆囊癌组织的蛋白质组研究

目的: 通过分离并鉴定胆囊癌和胆囊良性组织的差异表达蛋白质,以发现可能用于早期诊断的胆囊癌肿瘤标志物。方法: 提取人胆囊癌和胆囊良性组织的总蛋白质,用双向电泳分离蛋白并进行比较。选择在胆囊癌组织中明显差异表达的蛋白点,行质谱分析。结果: 获得了分辨率和重复性均很好的凝胶蛋白图谱。对筛选出的在胆囊癌组织中明显差异表达的46个蛋白点,共有17个蛋白点被成功鉴定,其中在胆囊癌组织中高表达的为9个,低表达的为8个。结论: 胆囊癌组织相对于胆囊良性组织蛋白存在明显的差异,通过蛋白质组学方法筛选并鉴定出的这些蛋白质可能成为用于胆囊癌早期诊断和治疗的分子靶点。     

恶性胶质瘤细胞SHG-44诱导分化后的差异蛋白质组双向电泳分析

目的用双向电泳分析诺帝诱导胶质瘤细胞SHG-44分化后的差异蛋白质组,为进一步了解这些差异蛋白质的作用打下基础。方法将诺帝诱导胶质瘤细胞SHG-44分化后的总蛋白及其相应的空白对照组细胞总蛋白进行双向电泳分离,重复3次后用PDQuest7.1软件比较分析蛋白质表达差异并获得差异蛋白质的相对分子质量、等电点等信息。结果诺帝诱导胶质瘤细胞SHG-44分化后有23个差异蛋白点,其中21个蛋白点表达下调,2个蛋白点表达上调。结论诺帝诱导胶质瘤细胞SHG-44分化后大部分蛋白质表达下调,推测诺帝诱导胶质瘤细胞SHG-44分化时蛋白表达以抑制作用为主。     

霍乱弧菌O1群有毒株和无毒株的蛋白质谱特征分析

目的 利用双向电泳和质谱鉴定技术探讨环境和人体中霍乱弧菌蛋白表达的差异.方法 利用适当的裂解液处理霍乱弧菌,提取全菌蛋白;采用pH梯度等电聚焦对全菌蛋白进行双向电泳;考马斯亮蓝染色后获得双向电泳图谱,并利用ImageMaster 2D Elite 5.0图像分析软件进行分析,所得的数据采用SPSS15.0进行统计分析,找出差异蛋白;在此基础上,胰蛋白酶消化这些特殊差异蛋白,并进行质谱(MALDI-TOF-MS)分析.结果 获得1032±22个蛋白斑点,蛋白主要集中在等电点(PI)4.00～7.20之间,重复胶的匹配点数为1025±24,匹配率为96.30%;发现了有明显差异的21个蛋白点,并用质谱鉴定了其中4种蛋白.结论 获得了环境和人体中霍乱弧菌蛋白的差异表达蛋白.     

霍乱弧菌O1群稻叶型流行株和非流行株的蛋白质谱特征分析

目的探讨霍乱弧菌01群稻叶型流行株和非流行株的蛋白表达的差异。方法利用裂解液处理霍乱弧菌,提取全菌蛋白;采用pH梯度等电聚焦对菌蛋白进行双向电泳;考马斯亮蓝染色后获得的双向电泳图谱,并利用ImageMaster2DElite5．0图象分析软件进行分析,找出差异蛋白;在此基础上,胰蛋白酶消化这些特殊差异蛋白,并进行质谱（MALDI-TOF—MS）分析。结果获得了（1081±16）个蛋白斑点,蛋白主要集中在pI4．00—7．20之间,重复胶的匹配点数为（1057±28）,匹配率为97．85％;发现了有明显差异的21个蛋白点,并进行了质谱鉴定。结论获得了霍乱弧菌01群稻叶型流行株和非流行株的差异表达蛋白。     

SELDI蛋白质芯片筛选差异蛋白分子的二级鉴定

 目的 探讨以SELDI蛋白质芯片筛选后的细胞差异表达蛋白的分离和鉴定方法及其意义。 方法 以经体外培养及10μmol/L褪黑素药物干预的内皮祖细胞差异表达蛋白质为研究对象,分别采用Tricine-SDS-PAGE和双向凝胶电泳方法进行差异蛋白分离及结果比较,以FT-MS二级质谱鉴定。 结果 经Tricine-SDS-PAGE分离后,选取分子量为53ku左右的趋势性差异表达蛋白进行FT-MS分析,质谱鉴定为微管蛋白-α3（吻合度评分为87）。经双向凝胶电泳分离后,于凝胶近酸性端、分子量在72～95ku之间区域,选取蛋白表达量较高的一点,质谱鉴定其为人热休克蛋白90-α（吻合度评分为91）。结论Tricine-SDS-PAGE对于大致35～62ku范围的蛋白分离较清晰、重复性好且样品用量少,2-DE对最低上样量有较严格要求,但它能提供分子量、等电点双重参数来更准确定位所感兴趣蛋白点,且对大分子量蛋白的分离效果更佳,经质谱鉴定结果理想。     

建立人肾小管上皮细胞蛋白质组双向电泳的分离体系

背景:双向电泳分离技术是蛋白质组学研究的核心技术之一,但蛋白质样品的分离效果受各种实验条件的影响较大。因此,针对不同来源的蛋白样品进行实验条件的优化可获得具有较高分辨率的双向电泳图谱。目的:拟建立优化的人肾小管上皮细胞株蛋白质组双向电泳分离体系。方法:常规培养人肾小管上皮细胞株HK-2细胞并裂解提取全蛋白,按标准条件对蛋白质进行双向电泳分离,并对各个关键因素进行优化。等电聚焦采用缓慢升压模式,电泳参数根据Bio-Rad公司的预设方案进行调整。改良硝酸银法进行蛋白质斑点染色。采集电泳图谱并分析双向电泳图谱中蛋白斑点的数量、图像分辨率及背景条纹的变化。结果与结论:通过对实验条件的筛选和优化,成功建立了具有较高的分辨率和重复性的人肾小管上皮细胞蛋白质组双向电泳分离体系。其中,优化后的裂解液配方成分为1%TBP,4%CHAPS,0.2%Bio-Lyte,40mmol/LTris,8mol/L尿素,2mol/L硫脲;采用pH4～7的IPG胶条;上样方式选择被动的水化上样。等电聚焦过程中使用预设的缓慢升压模式,充分聚焦后选用合适的电压模式进行SDS-PAGE电泳,然后采用改良硝酸银法进行染色,最终获得了满意的蛋白质组双向电泳图谱。     

Comparison by electroblotting of IgE-binding components in extracts of house dust mite bodies and spent mite culture

Eight Dermatophagoides pteronyssinus extracts (three culture extracts, four mite body extracts, and the World Health Organization International Standard [IS]) were investigated by side-by-side sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by electrotransfer to nitrocellulose, and by probing with a pooled serum from mite-allergic subjects. Representative body and mite culture extracts were compared by probing with individual sera, and both types of extract were also compared by RAST-inhibition studies. Extracts from the same source differed in the molecular weight (MW) of some of their IgE-binding components. In general, most IgE-binding components in culture extracts and the IS were in the 14 to 35 kd MW region, whereas extracts from mite bodies and one culture extract contained more IgE-binding components of higher MW (35 to 110 kd). Comparison of representative mite body and culture extracts by use of 22 separate sera resolved 26 and 19 IgE-binding components in the two extracts, respectively. Patterns of RAST inhibition produced by both types of extracts when they were used either as the inhibitor or as the allergosorbent demonstrated qualitative differences between the two types of extracts. These results demonstrate that mite extracts may differ considerably in their allergenic composition and emphasize the need for standardization of mite allergenic extracts and the reexamination of the suitability of the D. pteronyssinus IS.     

Application of the first international standard of Dermatophagoides pteronyssinus (house dust mite) in the evaluation of allergen extracts produced from two different source materials

The first international standard (IS) of the house dust mite Dermatophagoides pteronyssinus was used to compare side-by-side two allergen extracts prepared from two different house dust mite sources, namely whole mite cultures (WMC) and purified mite bodies (PMB), by employing several biochemical and immunochemical methods. In most methods employed, IS and WMC resembled more to each other than to PMB. These similarities comprehend protein contents, protein patterns, antigen and allergen patterns as well as total in vitro activity obtained in RAST inhibition (RI). The differences between WMC and PMB could be reproduced in different batches. Standardization in relation to IS of D. pteronyssinus yields activity ratios ranging from 2.4 to 70.1 between WMC and PMB depending on the variation of RI employed, indicating quantitative differences between the two preparations. When coupled to BrCN activated paper disks and employed in direct RAST, both preparations yielded almost identical binding values of specific IgE. Also in skin prick test, WMC and PMB produced very similar results in 12 mite-allergic patients. It was not possible to differentiate between the two preparations when used in concentrations which are usually employed for diagnostic purposes. These findings demonstrate the diversity of allergens in mite extracts derived from different sources and reveal the problems which are involved with the use of the WHO D. pteronyssinus IS.     

Investigations of patients allergic to the house dust mite Dermatophagoides pteronyssinus by crossed radioimmunoelectrophoresis using purified mite bodies and whole mite culture extracts

Sera of 20 patients allergic to the house dust mite Dermatophagoides pteronyssinus were investigated by crossed radioimmunoelectrophoresis (CRIE) using extracts prepared from purified mite bodies (PMB) and whole mite culture (WMC). By CRIE, different allergen patterns and different numbers of allergens were detected in the two extracts. By summarizing the values of the CRIE patterns of the 20 patients' sera into allergograms, 6 out of 16 allergens in PMB and 5 out of 10 allergens in WMC extracts could be identified as major allergens. These diverging results, together with the published literature, emphasize the necessity to reevaluate the International Standard for D. pteronyssinus.     

Identification of major allergens from the house dust mites, Dermatophagoides farinae and Dermatophagoides pteronyssinus, by electroblotting

The allergens were separated from the extracts of house dust mites by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by autoradiography. Over 30 protein bands of the whole body extract of Dermatophagoides farinae were apparent on 10-20% gradient SDS-PAGE, and 13 bands with MW between 93KD and 12KD bound with specific IgE antibodies in patients' sera sensitive to house dust mites. The major allergenic component of the whole body extract of D. farinae was the protein of MW 14-15KD, which was detected in 95.7% of 47 patients' sera sensitive to house dust mites. The extract of Dermatophagoides pteronyssinus supplied by Bencard Company, England was thought to contain feces enriched material as noted in a few broad protein bands on SDS-PAGE. Seven allergenic components were shown by autoradiography. The protein band of MW 14-15KD was one of the most frequently revealed allergens on autoradiography, which has appeared in 32.5% of 40 patients' sera sensitive to house dust mites. The electrobotting technique used in the present study was fast, convenient and highly useful for both the identification of allergen components and the screening of specific IgE antibody. The individual variations of IgE immune responses to the allergenic components of the two house dust mites were discussed.     

A reference allergen preparation of the house dust mite D. pteronyssinus, produced from whole mite culture--a part of the DAS 76 study. Comparison with allergen preparations from other raw materials

A D. pteronyssinus whole culture allergen preparation contained 49 antigens as revealed by crossed immunoelectrophoresis (CIE), using polyspecific rabbit antibodies. Crossed radio-immunoelectrophoresis ( CRIE ) with sera from 30 patients revealed nine allergens, antigens 42, X, Y and 23 (in rank order) showing the most frequent and intense IgE-uptake. Nine antigens originated from the culture medium (human dander + yeast), but none of these gave rise to specific IgE-uptake. Extremely few and weak reactions were observed in radioallergosorbent (RAST) with 129 sera, using media extracts on the discs. Purified mite body extract (PMB) contained less ag 42 and more ag Y and ag 23 than whole mite culture extract ( WMC ), whereas an acetone-extracted mite excreta preparation (AML) contained 5 times more ag 42, but was devoid of ag Y and ag 23. Ag X was present in all preparations. The RAST-inhibitory potency of PMB was best correlated with the content of ag X. Preparations with properties similar to WMC and PMB were judged as suitable for clinical application.     

Immunoblot and radioallergosorbent test inhibition studies of allergenic cross-reactivity of the predatory mite Amblyseius cucumeris with the house dust mite Dermatophagoides pteronyssinus.

BACKGROUND: In 1999, an extensive study among bell pepper growers showed that a predatory mite, Amblyseius cucumeris, is a potentially relevant source of occupational allergens because 23% of the population had positive skin prick test reactions. OBJECTIVE: To investigate whether cross-reactivity between A. cucumeris and Dermatophagoides pteronyssinus is responsible for the cosensitization to both mite species found in 58.7% of A. cucumeris-sensitized greenhouse workers. METHODS: Fifteen serum samples from greenhouse workers with work-related inhalant allergy and a positive radioallergosorbent test (RAST) reaction to A. cucumeris or D. pteronyssinus were selected for immunoblot analysis using extracts of both mites. A subselection (n = 5) was used for RAST and immunoblot inhibition to investigate potential cross-reactivity. RESULTS: On immunoblot, 2 distinct patterns were observed: one pattern showed common protein bands in A. cucumeris and D. pteronyssinus blots suggestive of cross-reactivity between A. cucumeris and D. pteronyssinus and the other pattern showed no shared protein bands. Dermatophagoides pteronyssinus RAST inhibition with A. cucumeris extract was low in 4 serum samples (<25% inhibition) and nearly absent in 1 serum sample; A. cucumeris RAST inhibition with D. pteronyssinus extract was high in 1 serum sample (75% inhibition), low in 2 serum samples (35% and <15% inhibition), and absent in 2 serum samples. These results were confirmed by immunoblot inhibition experiments. CONCLUSIONS: Amblyseius cucumeris, a new occupational allergen, has species-specific antigens and common antigens that are cross-reactive with the house dust mite D. pteronyssinus.     

The spectrum of low molecular weight house dust mite (Dermatophagoides pteronyssinus) allergens with emphasis on Der p II

Analysis by protein blotting of sera from 96 different house dust mite-allergic subjects revealed previously unrecognized complexity of low molecular weight (MW) (less than 20 kD) IgE-binding proteins in extracts of whole bodies of Dermatophagoides pteronyssinus. Of 11 different IgE-binding components of MW less than 20 kD identified, two (MW approximately 16 kD and approximately 15 kD), showed both a high frequency (88% and 49% respectively) and a high intensity of IgE-binding. The approximately 16 kD component, identified as allergen Der p II, showed the highest frequency of IgE antibody reactivity of any of the major D. pteronyssinus allergens including Der p I and Der p III.     

Measurement of IgG, IgA and IgE antibodies to Dermatophagoides pteronyssinus by antigen-binding assay, using a partially purified fraction of mite extract (F4P1).

An extract of Dermatophagoides pteronyssinus culture has been fractionated by chromatography on Sephadex G-100 and Pevikon block electrophoresis to obtain a partially purified allergen (F4P1). This preparation has a molecular weight of between 15--25,000 Dalton, migrates slowly on electrophoresis, and is colourless in solution. The skin-test reactivity of F1P1 was comparable to that of crude D. pteronyssinus extract. F4P1 was radio-labelled with 125I and used in an antigen-binding radioimmunoassay to measure IgG, IgA and IgE antibody (ab) to D. pteronyssinus. IgG, ab was detected in serum from 32/34 (94%) mite-allergic persons, and from 10/31 (30%) nonallergic persons. IgA ab and IgE ab were found in sera from 22/34 (65%) and 37/34 (79%) allergic persons respectively. Neither IgA nor IgE ab could be detected in sera from non-allergic persons. An excellent correlation was found between radioallergo-sorbent technique (RAST), using crude D. pteronyssinus extract and IgE-binding activity (BA) for F4P1, (r=0.94, P less than 0.001). The antigen-binding assay for IgE BA was as sensitive as RAST, but less sensitive than PK testing. There was a very good quantitative correlation between IgG BA and IgE BA (r = 0.84, P less than 0.001). IgG BA was shown to rise in the serum of three patients treated with injections of D. pteronyssinus extract.     

Production and proteomic characterization of pharmaceutical-grade Dermatophagoides pteronyssinus and Dermatophagoides farinae extracts for allergy vaccines

BACKGROUND: House dust mites (HDM) such as Dermatophagoides pteronyssinus and Dermatophagoides farinae represent a major cause of type 1 allergies worldwide. Hence large quantities of well-characterized HDM extracts are needed to prepare pharmaceutical-grade allergy vaccines. To this aim, the present study was undertaken to define optimal conditions for large-scale cultures. METHODS: D. pteronyssinus and D. farinae were grown on different media combining various proportions of wheat germ, yeast and synthetic amino acids (the latter resembling the composition of the human stratum corneum). Extracts thus obtained were analyzed for their total allergenic activity, as well as major allergen and protein contents, using immunosorbent assays, HPLC, immunoblotting, two-dimensional electrophoresis and peptide mass fingerprinting. RESULTS: An optimal culture medium (Stalmite APF) based on wheat germ, yeast and amino acids in defined proportion (42, 42 and 15% w/w, respectively) was selected to grow various HDM species with high yields. A detailed proteomic analysis revealed that D. pteronyssinus extracts generated under such conditions did not contain allergens originating from culture medium components and that major prevalent HDM allergens (i.e. groups 1, 2, 7, 10, 13 and 20) are found among the most abundant proteins in the D. pteronyssinus extract. Semiquantitative dot-blot assays confirmed the presence of Der p 3-10 as well as Der p 13 and 14 allergens within the extracts. CONCLUSIONS: We developed a well-defined medium allowing to grow various HDM species at an industrial scale in a highly reproducible manner. Extracts from mites produced under such pharmaceutical conditions contain all the relevant allergens for desensitization purposes and in vivo diagnosis.     

Studies on Dermatophagoides pteronyssinus allergens: measurement of the relative potencies of D. pteronyssinus purified extracts by in vitro and in vivo methods

To standardize the Dermatophagoides pteronyssinus extracts used in clinical allergy practice, the relative potencies of several purified extracts were estimated by radioallergosorbent test (RAST) inhibition, histamine release, and intracutaneous titration testing, and the results were compared. The potencies of nine independently prepared D. pteronyssinus extracts were assayed with respect to an extract adopted as a reference. In vivo the cutaneous wheal surface after intradermal injections was measured on a population of D. pteronyssinus-sensitive subjects. In vitro RAST inhibition was performed with the reference extract as allergosorbent and with a pool of human IgE-rich sera from patients sensitized to D. pteronyssinus but untreated. For histamine release human basophils from patients allergic to D. pteronyssinus were used. In the three method dose-response curves were plotted for the reference extract and the other extracts. A comparison of the measurement of in vitro and in vivo potencies is reported. Simialr results were obtained with the three methods: all the extracts but one were as potent as the reference preparation. However, for routine purposes RAST inhibition appears to be the most convenient and reliable procedure.