改良抗体捕获ELISA方法检测流行性出血热特异性IgE,IgA,IgG抗体的初步研究

初步报道建立了一种检测流行性出血热(EHF)特异性IgE、IgA、IgG抗体的改良抗体捕获ELISA方法(EacELISA,AacELISA,GacELISA)。该法以抗人IgE、IgA或IgG单克隆抗体作包被抗体;酶标记物系两株组特异性较强的EHF·McAb(A_(35),A_(25-1)株);在实验中采用EHF病毒抗原与酶标记物混合后一次加入,而不是分别依次加入的方式,使操作步骤简化,实验时间缩短,又适当提高了试验敏感性。该法具有简便,特异性高,灵敏度较高(检测EHF·IgE,EHF·IgA>1:100;EHF·IgG>1:2560),重复性较好(CV:EHF·IgE 2.51％,EHF·IgA 11.80％,EHF·IgG 10.85％)等优点。检测39份EHF病人血清,急性期病人(3～7病日)血清三种抗体的检出率分别为84.21％(16/19,EHF·IgG),89.47％(17/19,EHF·IgA)和100％(19/19,EHF·IgG);而发病3～6月后患者血清三种抗体检出率分别为10.00％(2/20),45.00％(9/20)和100.00％。在急性期与恢复期病人之间,EHF·IgE和EHF·IgA两种抗体的检出率差异较显著(P<0.01)。70价其他人群血清三种抗体检出率均为阴性。     

岩梧桐属的花粉形态及其分类学意义

本文通过光学显微镜和扫描电镜对岩梧桐属Wightia Wall.花粉进行了深入的观察,并与紫葳科和玄参科部分属种作了详尽的比较。结果认为,从花粉的大小等级、萌发孔特征、外壁内外层比例等看,岩梧桐属花粉与玄参科的花粉更类似,而与紫葳科的相距较大。孢粉学支持把岩梧桐属置于玄参科。该属的国产种,根据花粉的大小和外壁纹饰特征的差异划分为两个种较为合理和自然,不该合并为一个种。     

沈阳地区气传致敏花粉探讨

本文报道在沈阳地区1965—1985年间于空气中间断收集大气花粉5年,共收花粉玻片1100张,花粉45780粒,属于24科37属或种。其中9种(或属)超过千粒,为优势种类花粉。每年沈阳市大气中花粉出现两个高峰,一个为春季(四、五月),另一个为夏秋季(七八月)。通过长期观察发现,花粉种类和数量每年虽有不同,伹差异不大,唯有豚草花粉消长明显。大气中花粉种类及数量的变化往往受到气温、国家有关绿化政策以及社会风气的影响。花粉与过敏症关系十分密切,我们采用17种花粉制成浸液为病人皮试和治疗。皮试结果阳性率最高的为蒿属花粉,其次为其它夏秋花粉,春季花粉阳性率不高,致敏性不强。用花粉浸液对过敏症者进行免疫治疗,收到一定的疗效。     

滇桐属的花粉形态及其系统位置的探讨

滇桐属(Craigia w.w.Smith ＆ W．E. Evans)植物的生殖器官与花粉的形态都比 较特殊,一直受到国内外学者的重视,究竟应将它归入梧桐科(sterculiaceae)还是椴树科 (Tiliaceae),长期以来都存在着不同的见解。为了帮助澄清这个问题,本文在过去研究中国 梧桐科花粉形态的基础上,从花粉形态的角度进行探讨,对滇桐属的花粉以及椴树科的椴属 (Tilia)、海南椴属(Hainania)、蚬木属(Excentrodendron)等4属16种花粉在扫描电子显微镜 和光学显微镜下进行观察。研究结果表明,滇桐属花粉与椴树科3个属的花粉在形状、极性、 萌发孔位置及其形状结构(包括孔室的形状)、表面纹饰等方面都有差异,但与梧桐科的苹婆 属(Sterculia)、梭罗树属(Reevesia)等的花粉形态比较接近,而且滇桐属的花的形态与苹婆属等的也很相似,因此我们认为把滇桐属置于梧桐科内是比较合适的。     

基于深度最大输出网络的抗体种型特异性B细胞表位预测

B细胞表位研究有助于肽段疫苗研制,抗体研制以及疾病诊断和治疗研究.不同的B细胞表位诱导免疫系统产生不同的抗体种型,探索研究能够诱导特异性抗体产生的B细胞表位具有重要意义.基于二肽组成特征,利用深度最大输出网络算法训练构建三个二类分类器,分别对应诱导三种不同特异性抗体的B细胞表位,即IgA表位,IgE表位以及IgG表位.通过五折交叉验证训练和测试这三个分类器,获得AUC的值分别为0.78,0.93以及0.78.IgA表位和IgE表位分类器的预测能力优于其它IgA表位和IgE表位分类器,IgG表位分类器和其它IgG表位分类器的预测能力相当.     

小鼠抗天花粉蛋白IgE单克隆抗体的制备、分离和鉴定(英文)

天花粉蛋白是中药天花粉的有效引产成份,在应用中它偶尔也引起过敏反应。我们用杂交瘤技术建立了一株分泌抗天花粉蛋白特异性IgE单克隆抗体的杂交瘤细胞系。在实验中,二次免疫的C57 BL/6 J小鼠的肠系膜淋巴结细胞和脾细胞分别被用来同NSI骨髓瘤细胞进行融合。虽然在融合前动物血清IgE抗体效价仅为40～160 PCA滴度,但用肠系膜淋巴结细胞进行的4次融合都产生了IgE杂交瘤。阳性率为1.0-6.7%。对比之下,用脾细胞进行的另外两次融合却没有观察到IgE杂交瘤产生,统计结果说明差异显著(P<0.05)。该IgE单克隆抗体可以在体内和体外诱发大鼠肥大细胞脱颗粒。56℃热处理2小时能使该抗体诱导PCA反应的能力完全丧失,然而却不影响其结合抗原的能力。对该单克隆抗体的特异性的鉴定表明,它可以特异性地识别精制天花粉蛋白和结晶天花粉蛋白上的抗原决定簇。     

干酪乳杆菌干预对花生过敏小鼠模型的免疫调节作用

【目的】研究干酪乳杆菌(Lactobacillus casei)Zhang在预防及治疗花生过敏方面的作用。【方法】构建小鼠花生过敏模型,饲喂L.casei Zhang,并检测血清中IgE、IgG1和IgG2a抗体,粪便中组胺和IgA抗体,细胞因子和CD4+Foxp3+Tregs细胞的含量。【结果】与模型组相比,口服L.casei Zhang的实验组小鼠血清过敏原特异性的IgG2a和粪便总IgA抗体水平有所上调,花生过敏症状及Th2型反应(血清总IgE、特异性IgE、粪便组胺和IL-4水平等指标)明显降低。L.casei Zhang的摄入对血清IgG1抗体和其他细胞因子(IL-10、IL-12及IFN-γ)的表达水平没有明显影响,而IFN-γ/IL-4的比例及实验组脾脏和肠系膜淋巴结中CD4~+Foxp3~+Tregs细胞的表达增加。【结论】L.casei Zhang能促使Th2优势反应向Th1方向转变,从而起到预防和治疗花生过敏的作用。     

乳杆菌对牛乳β-乳球蛋白致敏小鼠淋巴细胞分泌Th1/Th2型细胞因子和抗体的体外影响

为了分析乳杆菌对致敏小鼠脾淋巴细胞分泌Th1/Th2细胞因子及抗体的体外影响,用牛乳β-乳球蛋白腹腔注射BALB/c小鼠建立过敏症模型,造模成功后,分离致敏小鼠的脾淋巴细胞并与4种活/死乳杆菌(107 CFU/mL)体外共同孵育,ELISA法检测细胞上清液中细胞因子(IL-12、IFN-γ和IL-4)和抗体(总IgE、β-Lg特异性IgE和总IgG)含量。4种活/死乳杆菌均可体外调节致敏小鼠脾淋巴细胞分泌细胞因子和抗体的水平,特别是热致死的发酵乳杆菌和嗜酸乳杆菌可提高淋巴细胞IL-12和IFN-γ的分泌,抑制IL-4的分泌,使其IFN-γ/IL-4比值(代表Th1/Th2细胞平衡)高于活菌,与空白对照组比较差异显著(P<0.05)。同时,这两株热致死菌还可显著下调细胞上清液中总IgE、特异性IgE和总IgG抗体的浓度(P<0.05)。试验结果表明乳杆菌可提高牛乳β-乳球蛋白致敏小鼠脾淋巴细胞的IFN-γ/IL-4比值,进而纠正Th2占优势的Th1/Th2失衡,下调抗体分泌量,且具有菌株特异性。     

天花粉蛋白分子的抗原决定簇研究(英文)

为了研究抗原结构和功能之间的关系,我们用天花粉蛋白作抗原,制备了16株分泌抗天花粉蛋白特异性单克隆抗体的杂交瘤细胞系,其中包括5株IgE杂交瘤细胞系。用这些单克隆抗体同天花粉蛋白进行抗体竞争结合试验,结果表明:16种单抗之间可产生不同程度的竞争,竞争率从0—100%不等。按照竞争的情况,16种单抗可以分成4个不同的组,即TE 1,1B 1,3A 12,4B 5组,分别识别4个可区分开的抗原决定簇区。值得注意的是TE1组包括了所有5种IgE单克隆抗体(以及另外两种IgG 1单克隆抗体),这一结果提示TE1组单克隆抗体识别的抗原决定簇区可能以某种方式介入了小鼠IgE抗体应答的过程。     

066 IL-8/RANTES作为DNA疫苗佐剂可增强对单纯疱疹病毒2型的特异性细胞免疫

为探讨趋化蛋白能否在小鼠体内调节对单纯疱疹病毒2型(HSV-2)的免疫反应以及在HSV-2侵犯机体时可否发挥保护反应,作者选择了5种趋化蛋白:IL-8、IP-10、RANTES、MIP-1α和MCP-1,以含编码相应趋化蛋白cDNA的pCDNA3和含编码HSV-2包膜糖蛋白gD的质粒(pCDNA3-gD)分别肌内免疫BALB/c小鼠,用ELISA检测鼠体内IgG的含量.结果仅pgD+IL-8或pgD+MIP-1α共免疫小鼠后引起gD特异性IgG的量稍增多.现已知IgG1和IgE是Th2细胞相关性抗体,而IgG2a是Th1细胞相关性抗体.     

Biological characterization of a formulated allergoid from Dermatophagoides pteronyssinus.

A comparative study between the biological properties of the allergen Dermatophagoides pteronyssinus (Dp) and its formulated allergoid (DpHCHO) was performed. After immunizing rats with Dp or DpHCHO the serum level time course of IgE and IgG was determined by passive cutaneous anaphylaxis (PCA) and indirect-ELISA, respectively. The avidity for IgE (PCA and PCA-inhibition) or IgG (indirect ELISA and ELISA inhibition) and the ability to induce anaphylactic shock were also determined. Serum IgE pattern differs between Dp and DpHCHO while IgG levels were greater after DpHCHO. The allergoid has lost the ability to bind IgE while its avidity for IgG falls 5-6 times. The allergoid, but not the allergen, fails to induce anaphylactic shock. A schedule for assaying the biological characteristics of allergoids is presented.     

Immunomodulation by liposome entrapped allergen

Liposomes are non-toxic, biodegradable and feebly immunogenic lipid vesicles made from natural and synthetic lipids. They are known to act as immunopotentiating agents and can be used to formulate sustained release preparation by encapsulation. In the present study, liposome entrapped allergen and free allergen were used to inject in Balb/C mice at different time intervals and their immune response in terms of specific IgG and specific IgE levels was quantitated by ELISA (Enzyme Linked Immuno sorbent Assay). The results indicated that specific IgE response was significantly higher in mice injected free allergen as compared to that of mice given liposome entrapped allergen. However, the specific IgG response was not statistically significant. Experiments carried out with liposome entrapped allergen and liposome coupled allergen showed no statistically significant difference in specific IgE and specific IgG titre between the two groups of mice. This type of immunomodulatory effect of liposomes in reducing IgE levels and without affecting IgG levels may be useful in Type I allergic disorders.     

Induction of allergen-specific IgE and IgG responses by anti-idiotypic antibodies

In order to explore idiotypic, anti-idiotypic, and anti-anti-idiotypic responses to allergens, BALB/c mice were immunized with affinity-purified human idiotypic antibodies directed against a highly purified shrimp allergen. This resulted in the production of anti-idiotypic antibodies which were quantitated by using rabbit idiotypic antibodies raised against the same purified allergen. The mouse anti-idiotypic antibodies recognized shrimp-specific human idiotypic antibodies of the IgE isotype from 18 of 20 individuals, and IgG antibodies from 14 of 20 shrimp-sensitive patients. Immunization of BALB/c mice with affinity-purified, allergen-specific anti-idiotypic antibodies induced anti-allergen IgE and IgG responses in the absence of the allergen. This paper thus presents evidence that anti-idiotypic antibodies raised against allergen-specific idiotypic antibodies may substitute for the original allergen in the induction of allergen-specific idiotypic antibodies. The demonstration of shared idiotopes on IgG and IgE antibodies in the sera of shrimp-sensitive patients supports the use of allergen-specific anti-idiotypic antibodies as surrogate allergens.     

Essential role of macrophages in the initiation of allergic rhinitis in mice sensitized intranasally once with cedar pollen: regulation of class switching of immunoglobulin in B cells by controlling interleukin-4 production in T cells of submandibular lymph nodes

The production of allergen-specific IgE antibodies (Abs) in allergen-sensitized patients or animals has a mutual relationship with the immunologic response leading to allergic rhinitis. We recently reported that, after an intranasal injection of cedar pollen into mice, an interleukin-4 (IL-4)-dependent increase in serum nonspecific IgE Abs was a prerequisite for the production of serum allergen-specific IgE Abs. Here, we explored which lymphoid organs were responsive to the intranasally injected allergen and how IL-4 and IgE Abs were produced in the lymphocytes. Time-dependent changes in the total cell numbers and in in vitro IgE Ab production in various lymphoid organs revealed that the submandibular lymph nodes were the main responsible organ. After treatment with allergen (for IgE production) or allergen and complete Freund's adjuvant (for IgG production), we separated submandibular lymph node cells into macrophage-, lymphocyte-, and granulocyte-rich populations by discontinuous Percoll density-gradient centrifugation. Unexpectedly, bulk cells, but not the lymphocyte- or macrophage-rich populations, produced significant amounts of IL-4, IgE, and IgG; whereas production was restored by addition of Mac-1(+) cells from the macrophage-rich to the lymphocyte-rich fraction. Furthermore, a combination of the lymphocyte-rich population (for IgG [or IgE]) production) and the macrophage-rich population (for IgE [or IgG]) production) produced a large amount of IgE (or IgG). These results indicate that, in the initiation of allergic rhinitis, macrophages in the submandibular lymph nodes are essential not only for IL-4 or immunoglobulin production, but also for class switching of immunoglobulin in lymphocytes.     

Antigen-specific inhibition of ongoing murine IgE responses. II. Inhibition of IgE responses induced by treatment with glutaraldehyde-modified allergens is paralleled by reciprocal increases in IgG2a synthesis.

Administration of high m.w. glutaraldehyde-polymerized OVA (termed OVA-POL) before OVA-[A1(OH)3] immunization of C57BL/6 mice markedly impairs their capacity to generate OVA-specific IgE responses, while simultaneously resulting in striking enhancement of Ag-specific IgG2a responses. We demonstrate here that treatment with this class of chemically modified allergen also results in pronounced inhibition of ongoing IgE responses in vivo. The abrogation of well established murine IgE responses that is elicited after treatment with OVA-POL (i) is potent (97%), (ii) is long lived, and (iii) reflects reciprocal regulation of Ag-specific IgE and IgG2a responses in vivo. Moreover, the capacity of OVA-POL-treated mice to generate secondary IgE responses remains strongly decreased for at least 260 days and six subsequent immunizations with native allergen, despite there being no further treatment with modified allergen. These changes in IgE and IgG2a responsiveness are Ag specific and T cell dependent.     

The localization of allergens of Paragonimus westermani by pleural exudates from patients.

The allergens of the lung fluke Paragonimus westermani were localized by indirect immunostaining in adult fluke sections using pleural exudates from 3 patients with P. westermani. Immunostaining performed by using pleural exudate with the highest level of specific IgE revealed that the P. westermani major allergen (or allergens) was located in the gut epithelium and luminal contents and that minor allergens were in the tegument and parenchyma. The antigens recognized by specific IgG were located at various sites including those recognized by specific IgE. Paragonimus westermani-specific IgE cross-reacted with only the gut of 2 other Paragonimus species, Paragonimus miyazakii and Paragonimus ohirai. The major allergen in the gut also was recognized by the other 2 pleural exudates. These results indicate that the substance present in and secreted from the gut is not only a major allergen but is also a common allergen among Paragonimus species.     

Disruption of allergenic activity of the major grass pollen allergen Phl p 2 by reassembly as a mosaic protein

The recognition of conformational epitopes on respiratory allergens by IgE Abs is a key event in allergic inflammation. We report a molecular strategy for the conversion of allergens into vaccines with reduced allergenic activity, which is based on the reassembly of non-IgE-reactive fragments in the form of mosaic proteins. This evolution process is exemplified for timothy grass pollen-derived Phl p 2, a major allergen for more than 200 million allergic patients. In a first step, the allergen was disrupted into peptide fragments lacking IgE reactivity. cDNAs coding for these peptides were reassembled in altered order and expressed as a recombinant mosaic molecule. The mosaic molecule had lost the three-dimensional structure, the IgE reactivity, and allergenic activity of the wild-type allergen, but it induced high levels of allergen-specific IgG Abs upon immunization. These IgG Abs crossreacted with group 2 allergens from other grass species and inhibited allergic patients' IgE binding to the wild-type allergen. The mosaic strategy is a general strategy for the reduction of allergenic activity of protein allergens and can be used to convert harmful allergens into safe vaccines.     

Influence of sensitization and allergen provocation procedures on the development of allergen-induced bronchial hyperreactivity in conscious, unrestrained guinea-pigs

The effects of different sensitization and allergen provocation regimens on the development of allergen-induced bronchial hyperreactivity (BHR) to histamine were investigated in conscious, unrestrained guinea-pigs. Similar early and late phase asthmatic reactions, BHR for inhaled histamine after the early (6 h) as well as after the late reaction (24 h), and airway inflammation were observed after a single allergen provocation in animals sensitized to produce mainly IgG or IgE antibodies, respectively. Repeating the allergen provocation in the IgE-sensitized animals after 7 days, using identical provocation conditions, resulted in a similar development of BHR to histamine inhalation. Repetition of the allergen provocation during 4 subsequent days resulted in a decreased development of BHR after each provocation, despite a significant increase in the allergen provocation dose necessary to obtain similar airway obstruction. The number of inflammatory cells in the bronchoalveolar lavage was not significantly changed after repeated provocation, when compared with a single allergen provocation. Finally, we investigated allergen-induced bronchial hyperreactivity by repetition of the sensitization procedure at day 7 and 14 (booster), followed by repeated allergen provocation twice a week for 5 weeks. Surprisingly, no BHR to histamine could be observed after either provocation, while the number of inflammatory cells in the bronchoalveolar lavage fluid after 5 weeks was enhanced compared with controls. These data indicate that both IgE and IgG sensitized guinea-pigs may develop bronchial hyperreactivity after a single allergen provocation. Repeated allergen exposure of IgE sensitized animals causes a gradual fading of the induced hyperreactivity despite the on-going presence of inflammatory cells in the airways, indicating a mechanism of reduced cellular activation.     

Genetic engineering of the major timothy grass pollen allergen, Phl p 6, to reduce allergenic activity and preserve immunogenicity

On the basis of IgE epitope mapping data, we have produced three allergen fragments comprising aa 1-33, 1-57, and 31-110 of the major timothy grass pollen allergen Phl p 6 aa 1-110 by expression in Escherichia coli and chemical synthesis. Circular dichroism analysis showed that the purified fragments lack the typical alpha-helical fold of the complete allergen. Superposition of the sequences of the fragments onto the three-dimensional allergen structure indicated that the removal of only one of the four helices had led to the destabilization of the alpha helical structure of Phl p 6. The lack of structural fold was accompanied by a strong reduction of IgE reactivity and allergenic activity of the three fragments as determined by basophil histamine release in allergic patients. Each of the three Phl p 6 fragments adsorbed to CFA induced Phl p 6-specific IgG Abs in rabbits. However, immunization of mice with fragments adsorbed to an adjuvant allowed for human use (AluGel-S) showed that only the Phl p 6 aa 31-110 induced Phl p 6-specific IgG Abs. Anti-Phl p 6 IgG Abs induced by vaccination with Phl p 6 aa 31-110 inhibited patients' IgE reactivity to the wild-type allergen as well as Phl p 6-induced basophil degranulation. Our results are of importance for the design of hypoallergenic allergy vaccines. They show that it has to be demonstrated that the hypoallergenic derivative induces a robust IgG response in a formulation that can be used in allergic patients.     

盐城地区未成年过敏性疾病患者吸入性过敏原IgE检测结果分析

摘要 目的：通过研究分析盐城地区未成年过敏性疾病患者吸入性过敏原特异性IgE检测结果分布变化特点,为过敏性疾病预防和临床诊疗提供科学依据。方法：自2020年1月至2021年3月期间,选择430例未成年(<18岁)过敏性疾病患者,血清检测方法采用欧蒙公司生产的过敏原特异性IgE检测试剂盒。结果：430例患者中,血清过敏原IgE阳性166例(38.60%),其中尘螨和屋尘是主要的吸入性过敏原。血清IgE阳性率男性39%,女性36% (P>0.05),中学组女性阳性率（61.11 %）高于男性（45.83 %）（P<0.05）。不同年龄组间IgE阳性率有统计学差异（P<0.05）,蟑螂、尘螨、霉菌、豚草、屋尘和年龄组间有统计学差异（P<0.05）。不同临床症状与IgE阳性率有统计学差异(P<0.05),其中呼吸道过敏症状组IgE阳性率最高（48.30 %）,不同症状组间主要过敏原都是尘螨。屋尘阳性患者均合并尘螨阳性,其中70.93 %的患者表现出了呼吸道过敏症状。结论：尘螨、屋尘是盐城地区未成年过敏性疾病患者最主要的吸入性过敏原,研究血清过敏原分布,对未成年人过敏性疾病的预防、诊疗具有重要意义。