نيلوفر صديقي

Foot-and-mouth disease (FMD) causes significant economic losses in the livestock industry. The immunogenic component of this virus is a ribonucleoprotein particle with the precipitation coefficient 146S, called the diantigen. The integrated virus, also known as 146S, is very important for eva‎luating the efficacy and potency of FMD vaccines and the amount of this particle must be quantified. There are various measurement methods for this. Although the sucrose density gradient (SDG) ultracentrifugation method is the gold standard for quantifying the 146S FMD virus particles or diantigen, it is not suitable due to its lower accuracy and precision, operator-friendliness and time-consuming nature. On the other hand, titration with an antibody against this diantigen is also an expensive method. For this purpose, size exclusion high-performance liquid chromatography (SE-HPLC) was chosen as an alternative instrumental method. Purification of inactivated foot-and-mouth disease virus with inactivated Asia serotype and activated O2016 in order to prepare a standard for drawing a calibration curve will be performed using preparative chromatography or fast protein liquid chromatography (FPLC) in two dimensions (ion exchange chromatography - size exclusion chromatography). In the first step, purification will be carried out from a High Scale_16 column filled with Q_Sepharose XL resin under a gradient (stepwise) method using buffers (Tris_HCl 20mM, pH=7.6) as the starting (binding) buffer and buffer (Tris_HCl 20mM, pH=7.6) containing NaCl (1M, pH=7.6) as the washing buffer under a flow rate of 8.7 ml/min in the equilibration, washing and washing steps. The flow rate for the step will be 2 ml/min. The sample was loaded and then a Superdex 200 increase column with a mobile phase consisting of (Na2HPO4 20mM, pH=7.4) NaCl, (150mM, pH=7.4) was used to continue the purification in the second dimension according to an isocratic method with a flow rate of 0.75 ml/min. Finally, to observe the sample profile and draw the calibration curve, analytical size exclusion chromatography (SE_HPLC) was used in an isocratic method with a flow rate of 0.5 ml/min using a Bio_SEC_3 column with a mobile phase containing (Na2HPO4 50mM, pH=7.0) NaCl, (150mM, pH=7.0) by ultraviolet and fluorescence detector.

واكسن غير فعال تب برفكي , كروماتوگرافي دو بعدي تبادل يوني(آنيوني)_ اندازه طردي , ، دي¬آنتي¬ژن (146S) , اندازه¬گيري كمي , كروماتوگرافي مايع پروتئيني سريع ـ كروماتوگرافي مايع با عملكرد بالا اندازه طردي

Inactivated foot-and-mouth disease (FMD) vaccine , two-dimensional ion exchange chromatography (anion)_size exclusion , D-antigen (146S) , Quantitative measurement , FPLC_SE_HPLC

Nioofar Sedighi

Dr. Afsaneh Molla Hosseini/Dr. Ali Eshaghi