The use of Toxoplasma gondii tachyzoites produced in HeLa cells adhered to Cytodex 1 microcarriers as antigen in serological assays: an application of microcarrier technology
- 35 Downloads
Toxoplasma gondii can infect nearly all warm-blooded animals, including humans. In the laboratory diagnosis of toxoplasmosis, serological tests have importance in detecting antibody response. Traditionally T. gondii tachyzoites grown in vivo are being used as an antigen source in serological assays. Currently, tachyzoites produced in vitro are being tested as an antigen source in order to decrease animal use. Microcarrier technology allowed us to grow anchorage-dependent host cells on microcarrier suspension in short time and approximately 10 times more than traditional flask technique. The ability of T. gondii tachyzoites to grow in host cells adhered to microcarriers has not been analyzed yet. In this study, we aimed to develop a novel in vitro culture method to produce T. gondii tachyzoites abundantly using HeLa cells adhered to Cytodex 1 microcarriers. Initially, the growth of HeLa cells adhered to Cytodex 1 was analyzed using RPMI 1640, DMEM, and EMEM. Next, HeLa cells with a concentration of 1 × 105 cells/ml and 2 × 105 cells/ml were adhered to Cytodex 1 and grown in spinner flasks. Then, T. gondii tachyzoites were inoculated with 1:1 and 2:1 cell:tachyzoite ratios to HeLa cells adhered to microcarriers in spinner flaks. During continuous production in spinner flasks, tachyzoites were harvested at the 2nd, 4th, and 7th day of culture and the quality of antigens produced from these tachyzoites were tested in ELISA and Western Blotting using sera of patients with toxoplasmosis. The optimization studies showed that finest HeLa inoculation value was 2 × 105 cells/ml using RPMI 1640, and the cell:tachyzoite ratio to obtain the highest tachyzoite yield (17.1 × 107) was 1:1 at the 4th day of inoculation. According to the results of ELISA comparing HeLa cell and mouse derived antigens, the highest correlation with mouse antigen was achieved at the 4th day of HeLa cell culture with 1:1 HeLa:tachyzoite ratio (P < 0.0001). The sensitivity and specificity ratios of ELISA were 100%. In addition, Western blotting banding patterns of the antigen derived at the 4th day of HeLa cell culture with 1:1 HeLa:tachyzoite ratio was comparable with mouse derived antigen. Overall, this novel methodology can be an alternative source of antigen in diagnostic assays, decrease animal use for antigen production, and contribute to the solution of ethical and economic problems.
KeywordsToxoplasma gondii HeLa cell Microcarriers Antigen
This study was supported by the grant given by the Scientific Research Projects Branch Directorate of Ege University, Turkey (Grant No: 2014-TIP-042) to M.D. In addition, access to the Animal Cell & Tissue Engineering laboratories of Ege University, Department of Bioengineering are highly appreciated.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Contini C, Fainardi E, Cultrera R, Canipari R, Peyron F, Delia S, Paolino E, Granieri E (1998) Advanced laboratory techniques for diagnosing Toxoplasma gondii encephalitis in AIDS patients: significance of intrathecal production and comparison with PCR and ECL-western blotting. J Neuroimmunol 92:29–37CrossRefGoogle Scholar
- Döşkaya M, Caner A, Can H, Gülçe İz S, Gedik Y, Değirmenci Döşkaya A, Kalantari-Dehaghi M, Gürüz Y (2014) Diagnostic value of a Rec-ELISA using Toxoplasma gondii recombinant SporoSAG, BAG1, and GRA1 proteins in murine models infected orally with tissue cysts and oocysts. PLoS ONE 9:e108329CrossRefGoogle Scholar
- Ho-Yen DO (2010) Annual reports, 2005–2009, Scottish Toxoplasma Reference Laboratory. http://www.hps.scot.nhs.uk/reflab/RefLabDetail.aspx?id=21. Active 2 Aug 2010
- Liang L, Döşkaya M, Juarez S, Caner A, Jasinskas A, Tan X, Hajagos BE, Bradley PJ, Korkmaz M, Gürüz Y, Felgner PL, Davies DH (2011) Identification of potential serodiagnostic and subunit vaccine antigens by antibody profiling of toxoplasmosis cases in Turkey. Mol Cell Proteom 10:M110.006916CrossRefGoogle Scholar
- Microcarrier Cell Culture Prenciples & Methods (2005) http://www.gelifesciences.com. Accessed 13 Dec 2016