Jimeno L, Duffort O, Serrano C, Barber D, Polo F
Research and Development Department, ALK-Abelló, Madrid, Spain.
Allergy. 2004 Sep;59(9):995-1001. doi: 10.1111/j.1398-9995.2004.00464.x.
Pollen of Artemisia vulgaris (mugwort) is a relevant cause of pollinosis in temperate and humid regions. Recently, the major allergen of this pollen, Art v 1, has been characterized.
To develop a monoclonal antibody (mAb)-based enzyme-linked immunosorbent assay (ELISA) to quantify Art v 1, and to assess the correlation of Art v 1 content with the biological activity of mugwort pollen extracts.
Art v 1-specific mAbs were obtained from a BALB/c mouse immunized with high-performance liquid chromatography (HPLC)-purified Art v 1. One of these antibodies (Av 3.7), which recognizes the N-terminal defensin-like domain of Art v 1, was used as the capture antibody in an ELISA method for allergen quantitation. An anti-A. vulgaris rabbit serum was used as the second antibody. Art v 1 was purified by immunoaffinity chromatography and used as the standard in the assay.
The purity and identity of the affinity-purified Art v 1 was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometry, amino acid composition, and N-terminal amino acid sequencing. The prevalence of specific IgE against Art v 1, determined by radioallergosorbent test (RAST) in a population of 44 mugwort-allergic patients, was 79%. The Art v 1-ELISA developed displays a detection limit of 0.1 ng/ml, and a practical working range of 0.2-10 ng/ml. The concentration of Art v 1 was measured in 10 A. vulgaris pollen extracts, and a good correlation was observed between the Art v 1 content and the allergenic activity of the extracts.
The results prove the usefulness of the Art v 1-ELISA for the standardization of A. vulgaris pollen extracts intended for clinical use.
普通蒿（艾蒿）花粉是温带和湿润地区花粉症的一个相关病因。最近，这种花粉的主要变应原Art v 1已得到鉴定。
开发一种基于单克隆抗体（mAb）的酶联免疫吸附测定（ELISA）法来定量Art v 1，并评估Art v 1含量与艾蒿花粉提取物生物活性之间的相关性。
用高效液相色谱（HPLC）纯化的Art v 1免疫BALB/c小鼠，获得Art v 1特异性单克隆抗体。其中一种识别Art v 1 N端防御素样结构域的抗体（Av 3.7）用作ELISA法中变应原定量的捕获抗体。抗普通蒿兔血清用作二抗。通过免疫亲和色谱法纯化Art v 1并用作测定中的标准品。
通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、质谱分析、氨基酸组成分析和N端氨基酸测序，证实了亲和纯化的Art v 1的纯度和同一性。在44例艾蒿过敏患者群体中，通过放射变应原吸附试验（RAST）测定的针对Art v 1的特异性IgE患病率为百分之七十九。所开发的Art v 1-ELISA检测限为0.1 ng/ml，实际工作范围为0.2-10 ng/ml。测定了10种普通蒿花粉提取物中Art v 1的浓度，观察到Art v 1含量与提取物的变应原活性之间具有良好的相关性。
结果证明了Art v 1-ELISA对于临床用普通蒿花粉提取物标准化的实用性。
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