Duffort O, Calabozo B, González R, Carpizo J A, Barber D, Polo F
Research and Development Department, ALK-ABELLO, Madrid, Spain.
Int Arch Allergy Immunol. 2004 Dec;135(4):277-83. doi: 10.1159/000082320. Epub 2004 Nov 24.
Pollen of Bermuda grass (Cynodon dactylon) is an important cause of pollinosis in many areas of the world. Most patients show sensitivity to the major allergen Cyn d 1, a glycoprotein composed of a number of isoforms with a molecular mass of 31-32 kDa. The aim of this work was to develop a monoclonal antibody (mAb)-based ELISA to quantify Cyn d 1, and to assess the correlation of the allergen content with the biological activity of C. dactylon pollen extracts.
After fusion of myeloma cells with spleen cells from a BALB/c mouse immunized with C. dactylon pollen extract, Cyn d 1-specific mAbs secreting hybridomas were selected, and the antibodies characterized. One of them (4.4.1) was used as the capture antibody in an ELISA method for Cyn d 1 quantitation. An anti-Cyn d 1 rabbit serum was used as the second antibody. Cyn d 1 was purified by immunoaffinity chromatography with mAb 4.4.1, characterized, and used as the standard in the assay.
The identity, purity and isoallergen composition of affinity-purified Cyn d 1 was confirmed by N-terminal amino acid sequencing, SDS-PAGE, Western blot and 2D electrophoresis. The Cyn d 1 ELISA is highly specific and sensitive, with a detection limit of 0.24 ng/ml and a linear range of 1.1-9.2 ng/ml. An excellent correlation was found when the content of Cyn d 1, measured in 16 different extracts, was compared with the allergenic activity of the same extracts determined by RAST inhibition.
The results prove the usefulness of the Cyn d 1 ELISA for the standardization of C. dactylon-allergen products on the basis of major allergen content.
狗牙根（Cynodon dactylon）花粉是世界许多地区花粉症的重要病因。大多数患者对主要过敏原Cyn d 1敏感，Cyn d 1是一种糖蛋白，由多种分子量为31 - 32 kDa的亚型组成。本研究的目的是开发一种基于单克隆抗体（mAb）的ELISA方法来定量Cyn d 1，并评估过敏原含量与狗牙根花粉提取物生物活性之间的相关性。
将骨髓瘤细胞与用狗牙根花粉提取物免疫的BALB/c小鼠的脾细胞融合后，筛选出分泌Cyn d 1特异性mAb的杂交瘤，并对抗体进行表征。其中一种（4.4.1）用作ELISA方法中定量Cyn d 1的捕获抗体。抗Cyn d 1兔血清用作二抗。用mAb 4.4.1通过免疫亲和层析纯化Cyn d 1，对其进行表征，并用作测定中的标准品。
通过N端氨基酸测序、SDS - PAGE、Western印迹和二维电泳确认了亲和纯化的Cyn d 1的同一性、纯度和同种过敏原组成。Cyn d 1 ELISA具有高度特异性和敏感性，检测限为0.24 ng/ml，线性范围为1.1 - 9.2 ng/ml。当比较16种不同提取物中Cyn d 1的含量与通过RAST抑制测定的相同提取物的致敏活性时，发现两者具有极好的相关性。
结果证明了Cyn d 1 ELISA在基于主要过敏原含量对狗牙根过敏原产品进行标准化方面的实用性。
