Mosavari N, Karimi A, Tadayon K, Shahhosseini Gh, Zavaran Hosseini A, Babaie M
. Reference Laboratory of Bovine Tuberculosis, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran.
Department of Molecular Medicine, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
Arch Razi Inst. 2021 Jan;75(4):439-449. doi: 10.22092/ari.2019.123082.1238. Epub 2021 Jan 1.
Tuberculin skin test, also known as the tuberculin or purified protein derivative (PPD) test, is an extensively applied diagnostic test for the detection of primary infection with Mycobacterium tuberculosis (Mtb). The production of PPD is accompanied by some difficulties that require a series of modifications in the production and purification processes. The present study aimed to determine the facilitation level of the manufacturing process by modifying evaluation methods for the production of PPD tuberculin. Mtb strains were cultured in Lowenstein-Jensen media, and the cultured strains were inoculated into the Dorset-Henley liquid medium by the biphasic medium of potato-Dorset-Henley. After incubation, flasks containing cultured strain were selected for bacterial inactivation, and the optimal gamma radiation dose(s) was determined. Tuberculoproteins were precipitated by ammonium sulfate (AS) and Trichloroacetic acid (TCA). Protein concentration was determined using the Bradford and Kjeldahl protein assay methods. Finally, the lymphocyte transformation test and potency test were performed. Based on the results, the Dorset-Henley liquid medium is suitable for the massive growth of the bacterium. The transferal of Mtb from solid to liquid medium was directly carried out without intermediate culture. It was found that during tuberculoprotein production, heating at 100°C for 3 h would be safe for killing mycobacterium. Furthermore, the simultaneous use of heating and gamma irradiation (8 kGgy) killed all of the mycobacteria, while doses of 1, 1.5, and 7 kGy decreased a significant number of bacterial cells. The results also indicated that the concentration of tuberculoprotein extracted by TCA precipitation method was higher than that obtained by AS precipitation. The tuberculoproteins which were produced by these two methods in the lymphocyte transformation test were not significantly different in terms of potency (P>0.05). Moreover, due to the high volume of produced protein, the protein measurement was more efficiently carried out by the Kjeldahl method, compared to the Bradford method. Finally, the results of the present study demonstrated that in addition to the novel approach of gamma irradiation, optimum methods are efficient and applicable in the production of PPD tuberculin.
结核菌素皮肤试验，也称为结核菌素或纯化蛋白衍生物（PPD）试验，是一种广泛应用于检测结核分枝杆菌（Mtb）原发性感染的诊断试验。PPD的生产存在一些困难，这需要在生产和纯化过程中进行一系列改进。本研究旨在通过修改PPD结核菌素生产的评估方法来确定制造过程的便利程度。将Mtb菌株在罗氏培养基中培养，然后通过马铃薯 - 多塞特 - 亨利双相培养基将培养的菌株接种到多塞特 - 亨利液体培养基中。培养后，选择含有培养菌株的烧瓶进行细菌灭活，并确定最佳γ辐射剂量。结核蛋白通过硫酸铵（AS）和三氯乙酸（TCA）沉淀。使用Bradford和凯氏定氮法测定蛋白质浓度。最后，进行淋巴细胞转化试验和效价试验。根据结果，多塞特 - 亨利液体培养基适合该细菌的大量生长。Mtb从固体培养基到液体培养基的转移直接进行，无需中间培养。发现在结核蛋白生产过程中，100℃加热3小时对杀死分枝杆菌是安全的。此外，加热和γ辐射（8 kGgy）同时使用可杀死所有分枝杆菌，而1、1.5和7 kGy的剂量可减少大量细菌细胞。结果还表明，通过TCA沉淀法提取的结核蛋白浓度高于AS沉淀法获得的浓度。这两种方法生产的结核蛋白在淋巴细胞转化试验中的效价没有显著差异（P>0.05）。此外，由于产生的蛋白量很大，与Bradford法相比，凯氏定氮法更有效地进行蛋白质测量。最后，本研究结果表明，除了γ辐射的新方法外，最佳方法在PPD结核菌素的生产中是有效且适用的。
