白茅花粉提取物的表征以及使用针对草地早熟禾的单克隆抗体对第1、4和5组同源物的初步分析。

Characterization of cogon grass (Imperata cylindrica) pollen extract and preliminary analysis of grass group 1, 4 and 5 homologues using monoclonal antibodies to Phleum pratense.

作者信息

Kumar L, Sridhara S, Singh B P, Gangal S V

机构信息

Centre for Biochemical Technology, Delhi, India.

出版信息

Int Arch Allergy Immunol. 1998 Nov;117(3):174-9. doi: 10.1159/000024007.

DOI:10.1159/000024007

PMID:9831804

Abstract

BACKGROUND

Previous studies have established the role of Imperata cylindrica (Ic) pollen in type I allergic disorders. However, no systematic information is available on the allergen composition of Ic pollen extract.

OBJECTIVES

To characterize the IgE-binding proteins of Ic pollen extract and to detect the presence of grass group 1, 4 and 5 allergen homologues, if any.

METHODS

Pollen extract of Ic was analyzed by in vivo and in vitro procedures such as intradermal tests (ID), enzyme-linked immunosorbent assay (ELISA), ELISA-inhibition, thin-layer isoelectric focusing (TLIEF), sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Dot blot assay was carried out to check the presence of well-known group 1, 4, and 5 allergen homologues in Ic pollen extract.

RESULTS

Out of 303 respiratory allergies patients skin-tested, 27 showed sensitivity to Ic pollen extract. Specific IgE levels were elevated in all 15 serum samples tested. The extract prepared for this study was found to be highly potent since it required only 400 ng of homologous proteins for 50% inhibition of binding in ELISA inhibition assays. TLIEF of Ic pollen extract showed 44 silver-stained bands (pI 3.5-7.0) while SDS-PAGE resolved it into 24 Coomassie-Brilliant-Blue-stained bands (MW 100-10 kD). Immunoblotting with individual patient sera recognized 7 major IgE-binding bands (MW 85, 62, 57, 43, 40, 28 and 16 kD) in Ic pollen extract. A panel of monoclonal antibodies, specific to group 1, 4 and 5 allergens from Phleum pratense pollen extract identified group 5 and group 4 homologues in Ic pollen extract.

CONCLUSION

Ic pollen extract was characterized for the protein profile by TLIEF and SDS-PAGE. IgE reactivity was determined by ELISA and immunoblot. Monoclonal antibodies to group 5 and group 4 allergens reacted weakly showing that this pollen contains group 5 and group 4 homologous allergens.

摘要

背景

先前的研究已证实白茅花粉在Ⅰ型过敏症中的作用。然而，关于白茅花粉提取物的过敏原组成尚无系统信息。

目的

鉴定白茅花粉提取物中的IgE结合蛋白，并检测是否存在禾本科1、4和5组过敏原同源物。

方法

采用体内和体外方法分析白茅花粉提取物，如皮内试验（ID）、酶联免疫吸附测定（ELISA）、ELISA抑制试验、薄层等电聚焦（TLIEF）、十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和免疫印迹。进行斑点印迹试验以检测白茅花粉提取物中是否存在已知的1、4和5组过敏原同源物。

结果

在303例接受皮肤试验的呼吸道过敏患者中，27例对白茅花粉提取物敏感。在所有检测的15份血清样本中，特异性IgE水平均升高。本研究制备的提取物被发现具有高效力，因为在ELISA抑制试验中仅需400 ng同源蛋白即可实现50%的结合抑制。白茅花粉提取物的TLIEF显示44条银染带（pI 3.5 - 7.0），而SDS-PAGE将其分离为条考马斯亮蓝染色带（分子量100 - 10 kD）。用个体患者血清进行免疫印迹可识别白茅花粉提取物中的7条主要IgE结合带（分子量85、62、57、43、40、28和16 kD）。一组针对早熟禾花粉提取物中1、4和5组过敏原的单克隆抗体在白茅花粉提取物中鉴定出5组和4组同源物。