基于抗元型抗体的半抗原非竞争免疫检测系统。

IF 7.1 2区 医学 Q1 MEDICAL LABORATORY TECHNOLOGY
Clinical chemistry Pub Date : 2015-04-01 Epub Date: 2015-02-18 DOI:10.1373/clinchem.2014.232728
Kazuya Omi, Tsuyoshi Ando, Takuya Sakyu, Takashi Shirakawa, Yoshiaki Uchida, Asako Oka, Nobuyuki Ise, Katsumi Aoyagi, Katsutoshi Goishi
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引用次数: 25

摘要

背景:半抗原类小分子通常采用竞争性免疫分析法进行检测,理论上在灵敏度和特异性方面都不如非竞争性三明治免疫分析法。我们创建了一种基于抗型抗体的三明治免疫测定方法来测量半抗原。方法:利用体外抗体开发系统——自主多样化文库(ADLib)系统生成针对半抗原抗体免疫复合物的抗元型单克隆抗体。我们选择了2种半抗原,雌二醇(E2)和25-羟基维生素D [25(OH)D]作为分析物。采用96孔微滴板和全自动化学发光分析仪对这2种半抗原进行夹心免疫分析,并对其免疫性能进行了研究。结果:所建立的检测方法具有足够高的灵敏度,可以检测血清样品中的目标半抗原。ELISA对E2的检出限为3.13 pg/mL,全自动化学发光酶免疫分析(CLEIA)系统对25(OH)D的检出限为2.1 ng/mL。与竞争性实验相比,有效地提高了与免疫反应性衍生物的交叉反应性。夹心ELISA法检测E2的CVs为4.2% ~ 12.6%(测定内)和6.2% ~ 21.8%(总不精确度)。25(OH)D的夹心CLEIA的CVs为1.0% ~ 2.3%(法内)和1.9% ~ 3.5%(总不精确度)。特别是,25(OH)D的夹心CLEIA与LC-MS/MS和市售(125)I RIA均显示r = 0.99的相关性。结论:我们的方法代表了一种潜在的简单实用的半抗原常规检测方法,包括维生素、激素、药物和毒素。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Noncompetitive immunoassay detection system for haptens on the basis of antimetatype antibodies.

Background: Small molecules classified as haptens are generally measured by competitive immunoassay, which is theoretically inferior to noncompetitive sandwich immunoassay in terms of sensitivity and specificity. We created a method for developing sandwich immunoassays to measure haptens on the basis of antimetatype antibodies.

Methods: We generated antimetatype monoclonal antibodies against a hapten-antibody immunocomplex using an ex vivo antibody development system, the Autonomously Diversifying Library (ADLib) system. We selected 2 haptens, estradiol (E2) and 25-hydroxyvitamin D [25(OH)D], as analytes. Sandwich immunoassays for these 2 haptens were developed by use of a 96-well microtiter plate and a fully automated chemiluminescence analyzer, and the performances of these immunoassays were investigated.

Results: The developed assays exhibited sensitivity high enough to detect target haptens in serum samples. The limit of detection of the ELISA for E2 was 3.13 pg/mL, and that of the fully automated chemiluminescent enzyme immunoassay (CLEIA) system was 2.1 ng/mL for 25(OH)D. The cross-reactivity with immunoreactive derivatives was effectively improved compared with the competitive assay. The CVs for the sandwich ELISA for E2 were 4.2%-12.6% (intraassay) and 6.2%-21.8% (total imprecision). The CVs for the sandwich CLEIA for 25(OH)D were 1.0%-2.3% (intraassay) and 1.9%-3.5% (total imprecision). In particular, the sandwich CLEIA for 25(OH)D showed correlations of r = 0.99 with both LC-MS/MS and a commercially available (125)I RIA.

Conclusions: Our method represents a potentially simple and practical approach for routine assays of haptens, including vitamins, hormones, drugs, and toxins.

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来源期刊
Clinical chemistry
Clinical chemistry 医学-医学实验技术
CiteScore
11.30
自引率
4.30%
发文量
212
审稿时长
1.7 months
期刊介绍: Clinical Chemistry is a peer-reviewed scientific journal that is the premier publication for the science and practice of clinical laboratory medicine. It was established in 1955 and is associated with the Association for Diagnostics & Laboratory Medicine (ADLM). The journal focuses on laboratory diagnosis and management of patients, and has expanded to include other clinical laboratory disciplines such as genomics, hematology, microbiology, and toxicology. It also publishes articles relevant to clinical specialties including cardiology, endocrinology, gastroenterology, genetics, immunology, infectious diseases, maternal-fetal medicine, neurology, nutrition, oncology, and pediatrics. In addition to original research, editorials, and reviews, Clinical Chemistry features recurring sections such as clinical case studies, perspectives, podcasts, and Q&A articles. It has the highest impact factor among journals of clinical chemistry, laboratory medicine, pathology, analytical chemistry, transfusion medicine, and clinical microbiology. The journal is indexed in databases such as MEDLINE and Web of Science.
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