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首页> 美国卫生研究院文献>Clinical and Experimental Immunology >A novel luminescence-based method for the detection of functionally active antibodies to muscarinic acetylcholine receptors of the M3 type (mAchR3) in patients sera
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A novel luminescence-based method for the detection of functionally active antibodies to muscarinic acetylcholine receptors of the M3 type (mAchR3) in patients sera

机译：一种基于发光的新颖方法可检测患者血清中M3型毒蕈碱型乙酰胆碱受体（mAchR3）的功能活性抗体

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In different bioassays, functional antibodies reacting with the human muscarinic acetylcholine receptor M3(mAchR3) have been detected in sera from patients with Sjögren's syndrome (SS), and there is strong evidence that those antibodies may have pathogenetic relevance. However, depending on the method of detection, their prevalence varied. Furthermore, those bioassays are difficult to standardize. We report on the development and optimization of a novel test system based on a luminometric method to determine downstream signalling of mAchR3 which produces specific and reproducible results. Chinese hamster ovarian (CHO) cells were transfected with plasmids encoding mAchR3 and a green fluorescence protein (GFP)/aequorin fusion protein. Incubation of cells with carbachol resulted in an increase in intracellular [Ca2+], which was detected by measuring light emission with a luminometer, and the effect of incubation with patients' immunoglobulins (Ig) was evaluated. Optimal cell density, Ig preparation and time of incubation with patients' sera were determined. Sera from patients with primary Sjögren's syndrome (pSS; n = 40), systemic sclerosis (SSc; n = 47), myasthenia gravis (MG; n = 133) and 50 blood donors were analysed. Optimal assay conditions were obtained with a cell density of 100 000 cells/ml, isolation of Ig by ammonium sulphate precipitation and short-term incubation. Based on this highly reliable assay, 50% of the pSS patients had antibodies which inhibited carbachol-induced activation of mAchR3; none of the SSc patients, 6% of the patients with MG and 12% of the blood donors had antibodies which reacted with the mAchR3. This method facilitates the determination of functional anti-mAchR3 antibodies in patients' sera, confirmed their high prevalence in pSS patients and may, therefore, help to analyse their pathogenetic and clinical relevance in more detail.
机译：在不同的生物测定中，已从患有干燥综合征的患者的血清中检测到与人毒蕈碱型乙酰胆碱受体M3（mAchR3）反应的功能抗体，并且有力的证据表明这些抗体可能与病原学相关。但是，根据检测方法的不同，其流行程度也有所不同。此外，这些生物测定法难以标准化。我们报告开发和优化的新型测试系统，基于发光法确定mAchR3的下游信号传导，该信号产生特异性和可重复的结果。用编码mAchR3的质粒和绿色荧光蛋白（GFP）/水母发光蛋白融合蛋白转染中国仓鼠卵巢（CHO）细胞。用卡巴胆碱孵育细胞会导致细胞内[Ca 2 + ]的增加，这是通过用发光计测量光发射来检测的，并评估了与患者免疫球蛋白（Ig）一起孵育的效果。确定了最佳的细胞密度，Ig制备和与患者血清的孵育时间。分析患有原发性干燥综合征（pSS； n = 40），系统性硬化症（SSc； n = 47），重症肌无力（MG； n = 133）和50名献血者的血清。获得了最佳的测定条件，细胞密度为100000个细胞/ ml，通过硫酸铵沉淀分离Ig和短期孵育。基于这种高度可靠的检测方法，有50％的pSS患者具有抑制卡巴胆碱诱导的mAchR3激活的抗体。没有SSc患者，6％的MG患者和12％的献血者具有与mAchR3反应的抗体。该方法有助于确定患者血清中的功能性抗mAchR3抗体，证实了其在pSS患者中的高患病率，因此可能有助于更详细地分析其致病性和临床相关性。

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