Comparisons of IgE, IgG, and IgG4 responsiveness to Dermatophagoides farinae in children by immunoblotting

Although asthmatic children are often sensitized to the house-dust mite (HDM) during early childhood, it is not clear what antigenic components are associated with the early immune response of these children. To investigate this problem, we evaluated IgE, IgG, and IgG4 antibody responses to Dermatophagoides farinae (Df) by immunoblotting among three groups of children: group I aged 0–4 years, group II aged 5–9 years, and group III aged 10–14 years. In the group I subjects, positive IgE-binding reactions to 15- and 25-kDa components were found in 76% and 44% of sera, respectively. Those to other components were generally low in frequency. In contrast, positive IgG-binding reactions to 15- and 25-kDa components were found in 44% and 24% of sera, and those to 30- and 110-kDa components in 48% and 88% of sera, respectively. Positive IgG4 reactions to 15- and 25-kDa components were found in 48% and 24%, respectively, and those to other components were very low. Positive IgE-binding reactions to these components gradually became more frequent with increasing age in groups II and III, while IgG and IgG4 reactivities were not markedly different in these age groups. These results suggest that the 15- and 25-kDa proteins of Df are important antigens associated with the initial IgE response to HDM in early childhood, while the 30- and 110-kDa proteins are associated with IgG and 15-kDa components with IgG4.     

IgG and IgE Antibody Patterns after Immunotherapy with Monomethoxy Polyethyleneglycol Modified Honey Bee Venom

Antibody responses to honey bee venom (HBV) were studied in 13 patients during a 4-month course of immunotherapy with monomethoxy polyethyleneglycol (mPEG) modified venom. There was a rise of HBV-specific IgG antibodies as measured by IgG-RAST in all patients and a slight decrease of IgE antibody in most of them. The IgG-antibody responses during mPEG-HBV treatment as examined by crossed radioimmunoelectrophoresis were directed to phospholipase A, hyaluronidase, acid phosphatase and to another allergen, antigen 1. Thus, despite a high degree of mPEG-modification of HBV, the immunogenicity of the most important HBV allergens was retained.     

粉尘螨变应原的分析

目的 对粉尘螨(Dermatophagoides farinae)变应原进行分离和鉴定其特异性变应原组分. 方法 采用ELISA检测粉尘螨变应原的生物活性.按常规方法制备粉尘螨浸出液,经SDS-PAGE分离,测定各组分的相对分子质量(M,);同时用对螨过敏的60例病人混合血清(建立血清库)作探针进行Westem blot,鉴定其特异性变应原组分. 结果 SDS-PAGE显示粉尘螨有15条蛋白带,Mr在14×103-109×103之间,其中主带有9条,M,分别为109×103、100×103、86×103、62×103、56×103、36×103、28 x103、19×103、14×103;Western blot结果表明,浸出液中共有5条致敏条带,其M,分别为109×103、100×103、36×103、28 x 103、14×103;经ELISA检测粉尘螨浸出液具有生物活性. 结论 粉尘螨的特异性变应原有5条,分别为109×103、100×103、36×103、28×103、14×103,其浸出液有稳定的生物活性.该研究为开发适合我国人群的粉尘螨标准化试剂提供了试验依据.     

Purification and Characterization of a Major Allergen from the House Dust Mite Dermatophagoides farinae

A major allergen from an extract of the house dust mite, Dermatophagoides farinae, was shown to be extremely heterogenic with respect to charge. A slightly basic component of this allergen with a pI of 8, was purified by isoelectric focusing in two steps. The purified component, denoted antigen 19/20 IIa, seemed to be representative for the allergenic activity of the major allergen. The amino acid analysis suggested that antigen 19/20 IIa had a molecular weight of 9400 and contained one residue of galactosamine. SDS polyacrylamide gel electrophoresis did indicate a somewhat higher molecular weight of 14,500. Antibodies against the purified component cross-reacted with a crude extract of the mite Dermatophagoides pteronyssinus.     

Specific IgE and IgG antibody responses in children to timothy pollen components during immunotherapy

Fourteen children with timothy grass pollinosis were given immunotherapy (IT) for 3 years with a purified and characterized timothy grass pollen preparation or a crude aqueous timothy pollen extract. Crossed radioimmunoelectrophoresis (CRIE) showed that 75% of the children under 11 years of age developed new specificities of IgE antibodies against timothy antigens, in contrast to older children, where no development of IgE antibodies against new timothy antigens could be detected. IgE antibodies were only detected against antigens formerly known as allergens. Timothy-specific IgG antibodies increased in most children during hyposensitization against the major allergens Ag 19 and Ag 24/25 and several other IgE-binding timothy antigens.     

Protein sequence analysis and mapping of IgE and IgG epitopes of an allergenic 98-kDa Dermatophagoides farinae paramyosin, Der f 11

BACKGROUND: A 98-kDa mite paramyosin (Der f 11) from Dermatophagoides farinae (Df) is highly allergenic, and its cDNA (Df642) has been cloned. This paper describes the sequence characteristics and the mapping of the immunodominant human IgE and IgG epitopes of Der f 11. METHODS: The protein sequence analysis was performed with a combination of FASTA, GCG, and CLUSTAL W computing packages. The whole cDNA insert and its PCR-derived DNA fragments were generated and expressed in E. coli. These overlapping recombinant peptides (F1 to F5) were used for B-cell epitope mapping with 18 mite-allergic sera by dot immunoassays. RESULTS: Df642 cDNA encodes a partial sequence that contains the 2nd to 26th 28-residue repeats and lacks the N-terminus and the C-terminus. The sequence identity of Der f 11 with other known paramyosins is 34-60%. The dominant IgE epitopes are located in peptides F1 and F4, whereas the dominant IgG epitopes are located in peptides F1 and F2. These peptides are more reactive than whole rDf642. CONCLUSIONS: Mite paramyosin is very similar to other known paramyosins. The human IgE and IgG epitopes are scattered throughout the entire molecule. Data also indicate the presence of unique IgE and IgG epitopes in Der f 11.     

Characterization of Allergens in a Crude Extract of Dermatophagoides farinae and Their Identification in a New Purified Preparation

The allergens of a crude D. farinae extract were identified by means of crossed radioimmunoelectrophoresis (CRIE). A total of 11 allergens were identified. They were characterized as three major, three intermediate and five minor allergens, based on radiostaining in CRIE. In addition to the radiostaining due to allergens, some misleading radiostaining was observed associated with some of the other antigens. A purified commercially available preparation, Spectralgen Mite D. farinae, was compared with the crude extract by means of immunoelectrophoretic techniques and radioallergosorbent test (RAST) based techniques. The purified preparation was found to be fully representative of the allergens in the crude extract. All 11 allergens were identified in the purified preparation. The purification ratio of the allergens varied from 2 to 7, while the total specific allergenic activity was approximately 6 times higher in the purified preparation than in the crude extract, as judged from RAST-inhibition studies.     

粉尘螨抗细菌活性物质的分离与鉴定

目的从粉尘螨丙酮提取液中分离抗细菌活性成分,并对其抗菌活性进行初步鉴定。方法用丙酮抽提粉尘螨,并用Sephadex G50分子筛层析进行对粉尘螨提取液中的抗细菌活性进行初步分离、纯化。对各组分的抗细菌活性进行初步鉴定。结果经Sephadex G50分子筛层析分离得到Ⅰ及Ⅱ两个峰。粉尘螨丙酮粗提物对绿脓假单胞菌无抑制作用,但对金黄色葡萄球菌、枯草芽孢杆菌及短小芽孢杆菌均有抑制作用。峰Ⅰ及峰Ⅱ则对大肠埃希杆菌、枯草芽孢杆菌、短小芽孢杆菌及金黄色葡萄球菌都有抑制作用。粉尘螨粗提液及第二个峰在加热及蛋白酶K处理后仍然保留抗细菌活性,但是第一个峰在同样处理后失去了抗菌活性。结论首次从粉尘螨分离纯化得到抗细菌成分。     

粉尘螨变应原第5组分基因克隆及分子特征分析

目的 获得粉尘螨变应原第5组分的编码基因(Der f5)并了解其分子特征.方法 用RNAiso试剂盒提取粉尘螨总RNA,根据GenBank已公布的Der f5核酸序列设计引物,用RT-PCR扩增获得其编码基因,插入pMD19-T Simple载体进行序列测定和分析.结果 获得的Der f5基因与参考序列(GenBank AY283283)同源性达97.8%,含1个完整的开放阅读框架(ORF),由132个氨基酸组成,信号肽位于1～19AA、跨膜区域位于1～19AA,为细胞外疏水性蛋白.二级结构由延伸主链(1.52%)、无规则卷曲(7.58%)和α螺旋(90.91%)组成.具有酪蛋白激酶Ⅱ磷酸化位点2个.其氨基酸序列与屋尘螨变应原第5组分相似率为78%.结论 成功克隆了 Der f5,并初步预测得其分子特征.     

Detailed IgG and IgE antibody patterns during immunotherapy with honey bee venom

Fourteen patients with a known honey bee venom (HBV) allergy were followed during 1-2 years of immunotherapy. HBV-specific IgG antibody levels increased in all patients but one. HBV-specific IgE antibodies decreased slightly during the first year of therapy. The ratio HBV-specific IgG-/IgE showed a marked increase during the first year for most of the patients, and a further increase during the second year in the four patients followed that long. As could be expected an increased radiostaining was found after 1 year of treatment to all important allergens in IgG CRIE, but after 2 years a sustained or increased radiostaining was obtained to phospholipase (PLA) alone. A decreased radiostaining might more easily be seen with weaker immunogens.     

Localization of Der f 2 in the gut and fecal pellets of Dermatophagoides farinae

BACKGROUND: House dust mite derived materials are known to be the most potent agent inducing allergic diseases. Localization of Der f 2 was attempted to specify the sites and concentrations of Der f 2 within the mite, which may indicate the importance of secreted materials and nonexcreted body components as allergen sources. METHODS: Serial cryostat sections of embedded live mites and the fecal pellets, collected by brush, were immunoprobed using monoclonal antibody (mAb) 2F38 raised against recombinant (r) Der f 2. RESULTS: Highest concentrations were found in the anterior midgut, implying that this is the site of Der f 2 synthesis and secretion. Digestive material and defecated fecal pellets were also labeled with mAb. CONCLUSIONS: Our results show that the major allergen, Der f 2, found in the house dust mite D. farinae is derived from the digestive tract, and is concentrated in the feces.     

Lymphocyte proliferative responses to the purified Dermatophagoides farinae major allergen in untreated and hyposensitized atopic patients

We investigated the proliferative response of lymphocytes from mite-sensitive patients (RAST D.far greater than 3.5 PRU/ml) in the presence of the major allergen Der.f.I purified from Dermatophagoides farinae. Comparative studies were carried out with peripheral blood mononuclear cells from non-atopic donors (RAST = 0), and from patients undergoing hyposensitization treatment (5 to 24 months). According to Student's t-test, there was no significant difference in the Der.f.I-induced proliferation of peripheral blood mononuclear cells from normal donors, untreated atopic patients and hyposensitized patients. In conclusion, it was impossible to discriminate between normal donors, atopic patients and hyposensitized patients with regard to their circulating lymphocyte responses to the purified major allergen Der.f.I.     

Cross antigenic and allergenic properties of the house dust mite Dermatophagoides farinae and the storage mite Tyrophagus putrescentiae

The crossed antigenicity and allergenicity of the storage mite Tyrophagus putrescentiae (TP) and the house dust mite Dermatophagoides farinae (DF) were characterized by means of crossed immunoelectrophoresis and crossed radioimmunoelectrophoresis. DF extracts exhibited 32 antigens and as many as eight were demonstrated to be allergens. DF feces exhibited 20 antigens and six of these were allergens. Twenty antigens and two allergens were demonstrated for TP. Two antigenic and allergenic determinants were shared by DF and TP, and two determinants were also shared by DF feces and TP feces. TP feces and DF shared two antigenic and allergenic determinants. Our results demonstrated that the two mites and their feces extracts contain multiple antigens and allergens.     

Aspergillus fumigatus specific IgE and IgG antibodies for diagnosis of Aspergillus-related lung diseases

IgE and IgG antibodies against Aspergillus fumigatus were detected by crossed radio immunoelectrophoresis (CRIE) on the sera of seven patients with aspergilloma, six patients with allergic broncho-pulmonary aspergillosis (ABPA) and 25 patients with extrinsic asthma with Aspergillus allergy. IgE-CRIE analysis indicated the presence of A. fumigatus-specific IgE in sera of patients with ABPA and Aspergillus asthma but not of aspergilloma patients. IgG-CRIE showed that both aspergilloma and ABPA patient sera contained high levels of circulating specific IgG antibodies in contrast to sera of Aspergillus asthma patients, which did not show detectable amounts of Aspergillus-specific IgG antibodies. Specific IgE binding could be demonstrated for the major allergens Ag-10 and AG-40 in all ABPA patients, in 80% of Aspergillus asthma patients but not in sera from aspergilloma patients. Specific IgG antibodies directed towards the major allergens could be detected in most of the aspergilloma patients, between 30-70% of the ABPA patients but not in sera from patients with Aspergillus asthma.     

Sequence analysis and expression of a cDNA clone encoding a 98-kDa allergen in Dermatophagoides farinae

BACKGROUND: The important dust mite allergens identified to date are of molecular weights ranging from 14 to 60 kDa. Our previous protein study indicated that the 98-kDa native paramyosin in Dermatophagoides farinae mite showed IgE reactivity with 82% of the mite-sensitive asthmatic patients suggesting that it is a novel major mite allergen. This study described the isolation and characterization of the cDNA clone encoding the 98-kDa mite allergen. METHODS: A Dermatophagoides farinae cDNA library was constructed in lambda ZAPII vector and the library was immunoscreened with a monoclonal antibody 642. The cDNA insert was sub-cloned into M13 sequencing vector for single-stranded sequencing. The whole cDNA insert was expressed in pGEX-2T Escherichia coli expression system as a fusion protein with GST. The allergenicity of the recombinant peptides was tested by skin tests and IgE immunoassay. The IgE and IgG immunoassays were performed with sera from 20 mite-allergic patients. RESULTS: The cDNA clone Df642 was 2134 bp long, coding for a polypeptide of 711 amino acid residues. Protein sequence analysis and alignment confirmed that the deduced polypeptide is a mite paramyosin which is truncated slightly at the N- and C-terminuses. In vivo skin tests and in vitro IgE-binding study showed that 62% (13/21) and 50% (10/20) of the mite-sensitive asthmatic patients reacted positively with the recombinant Dermatophagoides farinae paramyosin, respectively. CONCLUSION: The study indicated that 98-kDa mite paramyosin is an important allergen.     

粉尘螨主要变应原Derf1基因的改造与可溶性表达

目的在大肠杆菌中提高粉尘螨1类变应原（Derf1）的可溶性表达。方法用RT-PCR方法扩增得到Derf1的全长序列与成熟肽序列（mDerf1）；以已知DerP5基因的前导序列替换Derf1前导序列和原酶序列，重新构建出rDerf1基因；将上述3个基因分别克隆入原核表达载体pGEX-4T-1中表达，经Western-blot对三者的表达产物进行分析鉴定。结果Western-blot表明，pGEX-Derf1与pGEX-mDerf1的表达产物分别为分子量约63000与51000的重组蛋白质，重组蛋白质主要存在于细胞裂解液的沉淀中。pGEX-rDerf1大量表达了可溶性目的蛋白质，分子量约53000，并被成功地分离纯化。以上三种表达产物均可被鼠抗GST抗体与螨过敏患者血清特异地识别。结论表达了含Derf1，mDerf1与rDerf1的三种GST融合蛋白质，它们均具免疫反应性，纯化获得了GST-rDerf1融合蛋白质，为后期的诊断及治疗奠定了基础。     

粉尘螨变应原第10组分基因克隆表达及生物信息学分析

目的:克隆粉尘螨变应原第10组分(Der f 10)基因并建立其原核表达体系。方法:提取粉尘螨总RNA,反转录后经套式PCR扩增出目的基因并克隆至pMD19-T载体测序,将测序正确的目的基因插入表达质粒pET28a,转化E.coliBL21(DE3)用IPTG诱导表达,用SDS-PAGE和Western blot鉴定重组蛋白。结果:RT-PCR扩增获得Der f 10,琼脂糖凝胶电泳见一条约888 bp的条带,与参考序列(GenBank No.EU 106617)同源性高达99.8%。将pET28a(+)-Der f 10质粒转化宿主菌E.coli BL21,SDS-PAGE电泳时出现特异性条带,Western blot表明重组蛋白可与抗His-Tag Mab抗体结合。生物信息学软件预测获得的Der f 10重组体由295个氨基酸组成的疏水性蛋白,相对分子质量为34 233.1 Da,二级结构包括α-螺旋(91.86%)、延伸主链(1.36%)和无规卷曲(6.78%),含有5个原肌球蛋白模序分别位于84～101、120～140、145～173、175～198和231～256氨基酸处。结论:获得了Der f 10编码基因,成功建立其原核表达体系,为生产重组变应原用于临床诊断与治疗奠定基础。     

粉尘螨Ⅰ类抗原cDNA的克隆表达和初步鉴定

目的　构建粉尘螨Ⅰ类抗原 (Derf1)cDNA基因的重组表达质粒 ,并于E .coli表达。方法　用BamHⅠ和SacⅠ从重组质粒pMD 18T Derf 1上切下Derf1基因 ,插入表达载体pET32a( )质粒 ,转化大肠杆菌BL2 1,在氨苄青霉素阳性的LB平板上筛选阳性重组子 ,并经双酶切及PCR扩增鉴定。重组质粒pET32a( ) Derf 1转化大肠杆菌 ,IPTG诱导表达后进行SDS PAGE电泳和薄层凝胶扫描定量分析。结果　对重组质粒进行酶切和PCR鉴定 ,与预期结果相符 ,证明已成功构建携带Derf1基因的重组原核表达质粒pET32a( ) Derf 1。核酸序列测定及同源性分析证实所构建的原核表达质粒pET32a( ) Derf1中所含的Derf1基因与GenBank中的Derf1序列同源性达到 99.5 %。Derf 1基因在大肠杆菌诱导表达后获得Mr 约4 5 0 0 0的蛋白 ,蛋白含量占全菌体蛋白含量的 15 %。结论　成功构建了粉尘螨Ⅰ类抗原cDNA基因的重组表达质粒pET32a( ) Derf 1,并在大肠杆菌中获得高效表达 ,为获得重组纯化Derf 1变应原并用于尘螨变应性疾病的诊治奠定基础     

A mouse model of the atopic eczema/dermatitis syndrome by repeated application of a crude extract of house-dust mite Dermatophagoides farinae

BACKGROUND: We cultured Dermatophagoides farinae (Df), one of the most common mites in house dust and the most important allergen among natural allergens. With this material, we attempted to produce an animal model of the atopic eczema/dermatitis syndrome (AEDS). METHODS: We cultured Df mites in high density and prepared a crude extract of Df (DfE) together with the culture medium. We applied the extract to the back skin of NC/Nga and BALB/c mice three times per week for 8 weeks. RESULTS: In the NC/Nga group, dryness or scaling appeared on the skin, and scratching behavior increased at the second week in the DfE-treated group. Skin erosion and hemorrhage occurred at the fourth week. The epidermis thickened and deepened into the upper dermis, in which mast cells were highly accumulated, corresponding with the skin lesion of AEDS patients. Specific IgE and IgG to DfE and total IgE were elevated in the sera. Mice treated with an extract of mite culture medium did not develop skin lesions. In the BALB/c group, mice developed specific IgE and IgG to DfE, however, no typical skin lesions appeared. Mast cells in the upper dermis did not increase. CONCLUSIONS: Repeated painting of Dermatophagoides extract produced IgE-associated AEDS-like lesions on the skin of NC mice.