Jeoung B J, Reese G, Hauck P, Oliver J B, Daul C B, Lehrer S B
Tulane University Medical Center, Department of Medicine. New Orleans, La. 70112, USA.
J Allergy Clin Immunol. 1997 Aug;100(2):229-34. doi: 10.1016/s0091-6749(97)70229-x.
Among 13 allergens found in extracts of cooked brown shrimp (Penaeus aztecus) the 36 kd muscle protein tropomyosin has been identified as the only major shrimp allergen (Pen a 1). Cross-reacting molecules with similar molecular weights were detected in other crustacea species such as crab, lobster, and crawfish. Because Pen a 1 and Pen a 1-like allergens are important in crustacea allergy, the aim of this study was to develop a monoclonal antibody (mAb)-based sandwich ELISA to quantify Pen a 1 and to evaluate Pen a 1 levels in four commercial shrimp, crab, and lobster extracts.
Two Pen a 1-specific mAbs with different epitope specificities were selected. ELISA plates coated with captured mAb 3.2 were incubated with samples containing Pen a 1. Bound Pen a 1 was detected by a combination of biotinylated mAb 4.9.5 and alkaline phosphatase-labeled streptavidin.
The optimized sandwich ELISA could detect Pen a 1 concentrations ranging from 4 to 125 ng/ml. Four commercial shrimp extracts demonstrated a 40-fold difference in Pen a 1 levels (24 to 920 microg/ml). Crab and lobster extracts contained detectable levels of Pen a 1-like proteins. No reactivity to cockroach, house dust mite, oyster, codfish, or peanut extracts was detected, which indicates that the developed assay is crustacea-specific.
A sensitive sandwich assay was developed to quantify Pen a 1. This assay will be helpful to standardize shrimp extracts in regard to the content of the major allergen, Pen a 1, and to study cross-reactivities among and evaluate occupational exposure to different crustacea species.
在熟褐虾（墨西哥对虾）提取物中发现的13种变应原中，36kd肌肉蛋白原肌球蛋白已被确定为唯一主要的虾类变应原（Pen a 1）。在其他甲壳类物种如蟹、龙虾和小龙虾中检测到具有相似分子量的交叉反应分子。由于Pen a 1和Pen a 1样变应原在甲壳类过敏中很重要，本研究的目的是开发一种基于单克隆抗体（mAb）的夹心ELISA来定量Pen a 1，并评估四种市售虾、蟹和龙虾提取物中的Pen a 1水平。
选择两种具有不同表位特异性的Pen a 1特异性单克隆抗体。用捕获单克隆抗体3.2包被的ELISA板与含有Pen a 1的样品孵育。结合的Pen a 1通过生物素化单克隆抗体4.9.5和碱性磷酸酶标记的链霉亲和素的组合进行检测。
优化后的夹心ELISA可检测到4至125 ng/ml的Pen a 1浓度。四种市售虾提取物的Pen a 1水平相差40倍（24至920μg/ml）。蟹和龙虾提取物中含有可检测水平的Pen a 1样蛋白。未检测到对蟑螂、屋尘螨、牡蛎、鳕鱼或花生提取物的反应性，这表明所开发的检测方法具有甲壳类特异性。
开发了一种灵敏的夹心检测方法来定量Pen a 1。该检测方法将有助于在主要变应原Pen a 1的含量方面标准化虾提取物，并研究不同甲壳类物种之间的交叉反应性以及评估职业暴露情况。
