Yari Shamsi, Hadizadeh Tasbiti Alireza, Ghanei Mostafa, Siadat Seyed Davar, Yari Fatemeh, Bahrmand A
Tuberculosis Dept., Pasteur Institute of Iran, Tehran, Iran.
Tuberculosis Dept., Pasteur Institute of Iran, Tehran, Iran.
Int J Mycobacteriol. 2016 Dec;5 Suppl 1:S134-S135. doi: 10.1016/j.ijmyco.2016.10.011. Epub 2016 Oct 27.
Multidrug-resistant tuberculosis (MDR-TB) is caused by Mycobacterium tuberculosis strains that do not respond to isoniazid and rifampicin, the two most effective first-line anti-TB drugs. Here, we designed and produced antibodies based on biomarkers that exist only in MDR-TB.
Bacilli were cultured for 4weeks at 37°C, and protein extraction was performed by sequential extraction. Bacterial cells were sonicated, centrifuged at 5000rpm for 45min, and the supernatant was collected and subjected to multiple rounds of treatment to prior to protein isolation. Protein concentration was determined using the Bradford method, and extracted proteins (50μg) from each strain (drug-sensitive- and MDR-TB isolates) were visualized on polyacrylamide gels (5-15%) with Coomassie Brilliant Blue R-250 staining. Three extracts were mixed and dialyzed against 0.1M ammonium bicarbonate (pH 8.0), followed by mass spectrometry. Specific polyclonal antibodies against purified MDR-TB proteins were purified by affinity chromatography and prepared in rabbits using three booster injections. The ELIZA test was performed for evaluation the antibody production. The antibody was treated with normal oral flora to remove any non specificity and cross reactivity. Analyses of different protein patterns (drug-sensitive- and MDR-TB) were performed by western blot.
Our revealed that the MDR-TB strains contained specific antigens, and that the protein profiles of drug-sensitive TB strains differed from those of the MDR-TB isolates. Five bands from the MDR-TB fractions were detected as diagnostic antigens and were not observed in drug-sensitive-TB fractions. Western blot results showed that the MDR-TB antigenic fractions showed immunogenic bands at 50.0kDa and 70.0kDa, with the five antigenic MDR-TB-specific bands were identified as Rv3248c, Rv0350, Rv0440, Rv0475, and Rv3588c.
Western blot data revealed dynamic properties of antibody responses that led to actionable findings for further research. Moreover, specific anti-mycobacterial antibodies, such as MDR-TB antibodies, can be essential tools in the identification of species-restricted antigens, such as drug-resistant TB antigens. The MDR-TB antibodies described here might promote identification of mycobacterial antigens during the course of infection, which could be helpful for the development of newer TB-vaccine candidates or therapeutic agents for improved TB treatment or diagnosis.
耐多药结核病（MDR-TB）由结核分枝杆菌菌株引起，这些菌株对两种最有效的一线抗结核药物异烟肼和利福平无反应。在此，我们基于仅存在于耐多药结核病中的生物标志物设计并制备了抗体。
将杆菌在37°C培养4周，通过顺序提取进行蛋白质提取。对细菌细胞进行超声处理，以5000rpm离心45分钟，收集上清液并进行多轮处理以进行蛋白质分离。使用Bradford法测定蛋白质浓度，将来自每种菌株（药物敏感和耐多药结核分枝杆菌分离株）的提取蛋白质（50μg）在聚丙烯酰胺凝胶（5-15%）上用考马斯亮蓝R-250染色进行可视化。将三种提取物混合并对0.1M碳酸氢铵（pH 8.0）进行透析，然后进行质谱分析。针对纯化的耐多药结核分枝杆菌蛋白质的特异性多克隆抗体通过亲和色谱法纯化，并在兔中使用三次加强注射制备。进行酶联免疫吸附测定（ELISA）试验以评估抗体产生情况。用正常口腔菌群处理抗体以去除任何非特异性和交叉反应性。通过蛋白质印迹法对不同的蛋白质模式（药物敏感和耐多药结核分枝杆菌）进行分析。
我们的研究表明，耐多药结核分枝杆菌菌株含有特异性抗原，药物敏感结核分枝杆菌菌株的蛋白质谱与耐多药结核分枝杆菌分离株不同。在耐多药结核分枝杆菌组分中检测到五条带作为诊断抗原，而在药物敏感结核分枝杆菌组分中未观察到。蛋白质印迹结果表明，耐多药结核分枝杆菌抗原组分在50.0kDa和70.0kDa处显示免疫原性条带，耐多药结核分枝杆菌特异性的五条抗原性条带被鉴定为Rv3248c、Rv0350、Rv0440、Rv0475和Rv3588c。
蛋白质印迹数据揭示了抗体反应的动态特性，这为进一步研究带来了可操作的发现。此外，特异性抗分枝杆菌抗体，如耐多药结核分枝杆菌抗体，可能是鉴定物种限制性抗原（如耐药结核分枝杆菌抗原）的重要工具。本文所述的耐多药结核分枝杆菌抗体可能有助于在感染过程中鉴定分枝杆菌抗原，这可能有助于开发新型结核疫苗候选物或治疗剂，以改善结核病的治疗或诊断。
