Semisynthetic Bioluminescent Sensor Proteins for Direct Detection of Antibodies and Small Molecules in Solution.
Semisynthetic Bioluminescent Sensor Proteins for Direct Detection of Antibodies and Small Molecules in Solution.
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DOI：
10.1021/acssensors.7b00695
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发表时间：
2017-11-22
期刊：
影响因子：
8.9
通讯作者：
Merkx M
中科院分区：
文献类型：
--
作者：
Arts R;Ludwig SKJ;van Gerven BCB;Estirado EM;Milroy LG;Merkx M
可以直接在溶液中进行的单步免疫分析非常适合于医疗保健诊断。我们团队最近开发了一种新的生物发光传感器蛋白平台(LUMABS；发光抗体传感器)，可以检测血浆中的抗体。到目前为止，LUMABS仅限于检测识别天然多肽表位的抗体。在这里，我们报告了识别非肽表位的半合成LUMABS传感器的发展。非天然氨基酸对叠氮苯丙氨酸被引入到原始抗体识别位点的位置作为化学手柄，使合成的表位分子能够通过菌株促进的点击化学连接到二苯并辛基辅基上。半合成的LUMABS传感器成功地检测了针对小分子二硝基苯酚和肌酐的抗体(DNP-LUMABS和CR-LUMABS)，亲和力分别为5.8 pm和1.3 nM。这些半合成的LUMABS的一个重要应用是使用竞争性分析格式来检测小分子，这里展示了对肌酐的检测。使用CR-LUMABS和抗肌酐抗体的预组装复合体，可以在缓冲液和1：1稀释的血浆中检测到高微摩尔浓度的肌酐。半合成LUMABS传感器的使用大大扩大了抗体靶标的范围，并使LUMABS传感器能够应用于使用竞争免疫分析形式的比率生物发光检测小分子。
Single-step immunoassays that can be performed directly in solution are ideally suited for point-of-care diagnostics. Our group recently developed a new platform of bioluminescent sensor proteins (LUMABS; LUMinescent AntiBody Sensor) that allow antibody detection in blood plasma. Thus far, LUMABS has been limited to the detection of antibodies recognizing natural peptide epitopes. Here, we report the development of semisynthetic LUMABS sensors that recognize nonpeptide epitopes. The non-natural amino acid para-azidophenylalanine was introduced at the position of the original antibody-recognition sites as a chemical handle to enable site-specific conjugation of synthetic epitope molecules coupled to a dibenzocylcooctyne moiety via strain-promoted click chemistry. The approach was successfully demonstrated by developing semisynthetic LUMABS sensors for antibodies targeting the small molecules dinitrophenol and creatinine (DNP-LUMABS and CR-LUMABS) with affinities of 5.8 pM and 1.3 nM, respectively. An important application of these semisynthetic LUMABS is the detection of small molecules using a competitive assay format, which is demonstrated here for the detection of creatinine. Using a preassembled complex of CR-LUMABS and an anti-creatinine antibody, the detection of high micromolar concentrations of creatinine was possible both in buffer and in 1:1 diluted blood plasma. The use of semisynthetic LUMABS sensors significantly expands the range of antibody targets and enables the application of LUMABS sensors for the ratiometric bioluminescent detection of small molecules using a competitive immunoassay format.
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