León-Janampa Nancy, Shinkaruk Svitlana, Gilman Robert H, Kirwan Daniela E, Fouquet Eric, Szlosek Magali, Sheen Patricia, Zimic Mirko
Laboratorio de Bioinformática y Biología Molecular, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru; Univ. Bordeaux, CNRS, Bordeaux INP, ISM, UMR 5255, 33400 Talence, France.
Univ. Bordeaux, CNRS, Bordeaux INP, ISM, UMR 5255, 33400 Talence, France.
J Pharm Biomed Anal. 2022 Jun 5;215:114749. doi: 10.1016/j.jpba.2022.114749. Epub 2022 Apr 1.
Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is one of the 10 leading causes of death worldwide, especially in low-income areas. A rapid, low-cost diagnostic assay for TB with high sensitivity and specificity is not currently available. Bio-functionalized magnetic nanoparticles (MNPs) which are able to efficiently detect and concentrate biomolecules from complex biological samples, allows improving the diagnostic immunoassays. In this way, a proof-of-concept of MNP-based sandwich immunoassay was developed to detect various MTB protein antigens. The superficial and secretory antigenic proteins considered in this research were: CFP10, ESAT6, MTC28, MPT64, 38 kDa protein, Ag85B, and MoeX. The proteins were cloned and expressed in an E. coli system. Polyclonal antibodies (ab) against the recombinant antigens were elicited in rabbits and mice. Antibodies were immobilized on the surface of amine-silanized nanoparticles (MNP@Si). The functionalized MNP@Si@ab were tested in a colorimetric sandwich enzyme-linked immunosorbent assay (sELISA-MNP@Si@ab) to recognize the selected antigens in sputum samples. The selected MTB antigens were successfully detected in sputum from TB patients in a shorter time (~ 4 h) using the sELISA-MNP@Si@ab, compared to the conventional sELISA (~15 h) standardized in home. Moreover, the sELISA-MNP@Si@ab showed the higher sensitivity in the real biological samples from infected patients.
由结核分枝杆菌（MTB）引起的结核病（TB）是全球十大主要死因之一，在低收入地区尤为如此。目前尚无一种快速、低成本且具有高灵敏度和特异性的结核病诊断检测方法。能够有效从复杂生物样本中检测和浓缩生物分子的生物功能化磁性纳米颗粒（MNPs）有助于改进诊断免疫测定。通过这种方式，开发了基于MNP的夹心免疫测定的概念验证方法，以检测各种MTB蛋白抗原。本研究中考虑的表面和分泌抗原蛋白包括：CFP10、ESAT6、MTC28、MPT64、38 kDa蛋白、Ag85B和MoeX。这些蛋白在大肠杆菌系统中进行克隆和表达。在兔和小鼠体内诱导产生针对重组抗原的多克隆抗体（ab）。将抗体固定在胺硅烷化纳米颗粒（MNP@Si）表面。在比色夹心酶联免疫吸附测定（sELISA-MNP@Si@ab）中测试功能化的MNP@Si@ab，以识别痰液样本中的选定抗原。与在国内标准化的传统sELISA（约15小时）相比，使用sELISA-MNP@Si@ab能在更短时间（约4小时）内成功检测出结核病患者痰液中的选定MTB抗原。此外，sELISA-MNP@Si@ab在感染患者的实际生物样本中显示出更高的灵敏度。
