Because the OD value of SQ 7 is showing an equivalent or more effective performance than ISA, it is expected that SQ7 will show comparable antibody formation like Montanide ISATM 201. HRP) from GeneTex (Hsinchu City, Taiwan) and goat anti-Mouse IgG (H+L) secondary antibody was purchased from Thermofisher (Waltham, MA, USA). The antibody test kit was purchased from IDEXX (Westbrook, ME, USA). Other used reagents were of analytical grade. 2.2. Preparation and Characterization of PCV2 Antigen 9 (Sf9) cells are produced in suspension for 2 to 3 3 days and it was inoculated with PCV2 ORF2 expression baculovirus in the range of 1 1 to 10 MOI (multiplicity of contamination) and incubated using a wave instrument at 27 C constant temperature room. When the CPE (cytopathic effect) was 80% or more at 8 to 9 days of inoculation, the supernatant was harvested by centrifugation at 250 Antigen The grasp seed in cryopreservation was main cultured using propagation medium and it was transferred to broth, and finally incubated at 37 C for ca. 7 days. Cultivation was terminated when the color of the medium changed to yellow, after which the incorporation of various bacteria was examined, and antigen titer was measured using the Color Change Unit (CCU) method. Briefly, after diluting the collected bacteria from 10?1 to 10?9 using sterile PBS, it was inoculated into each broth and the concentration checked using the CCU method. After that, cells were harvested by inactivation with formalin and stored at 4 C until vaccine preparation. 2.3.2. LY2801653 (Merestinib) Characterization Using Mycoplasma Selection Media A total of 1 1 mL of culture material (broth, followed by shaking and incubation at 37 C for 14 days. After 14 days of inoculation, cells were transplanted to agar, and incubated for 14 days at 37 C and 4%C6% CO2 condition, and it was observed using a low magnification microscope (100). was used as a positive control group, and the concentration was 100 colony-forming models (CFU). The un-inoculated group was used as the unfavorable control and boths were cultured under the same conditions. 2.3.3. Identification of Using Polymerase Chain Reaction (PCR) The DNA of the positive control (p36 gene forward and reverse primer (10 pmol/L, forward 5-GGG LY2801653 (Merestinib) CCG ATG AAA CCT ATT AAA ATA GCT-3; reverse 5-GCC GCG AAA TTA AAT ATT LY2801653 (Merestinib) TTT AAT TGC ATC CTG-3) were added to each PCR premix (iNtron Biotechnology, Seongnam, Korea) [16]. The PCR conditions used were as follow: One cycle of initial denaturation (for 15 min at 95 C), 35 cycles of amplification (for 1 min at 94 C, for 1 min at 50 C and for 1.5 min at 72 C) and one cycle of final extension (for 10 min at 72 C). The PCR products (948 bp) were electrophoresed in 1.5% agarose gels and visualized LY2801653 (Merestinib) with nucleic acid staining dye around the UV illuminator. 2.4. Construction of Pseudo-Ternary Phase Diagrams Pseudo-ternary phase diagrams were constructed following the method Rabbit polyclonal to beta defensin131 explained by Wang and Pal, with modifications [17]. Diagrams were prepared for different ratios of Span 80 to Cremophor ELP, including 1:4, 7:13 and 1:1. For each ratio, squalene oil and Smix (mixture of Span 80 and Cremophor ELP) were mixed in ratios (antigen to prepare vaccines. In 2 mL of vaccine, the concentration of PCV type 2 antigen was above 100 models, inactivated antigen was 1.0 108 CCU and the concentration of the preservative was less than 0.2%. 2.9. ELISA Analysis Method for PCV2 The plate coated with PCV2 ORF2 antibody was removed at room heat in advance and left for 1 h. After diluting the test sample and the standard sample 8000C64,000-fold with a dilution buffer, it was dispensed as 100 uL per well into the prepared plate, reacted at 37 C for 1 h and washed 4 occasions using washing buffer. After a 100-fold dilution of the anti-PCV2 conjugate, the resultant was developed on a TMB (3,3,5,5-Tetramethylbenzidine) substrate and absorbance was measured at 450 nm. A standard curve was prepared using the standard sample, and the final quantitative value was calculated by substituting the optical density (OD) value of the sample. 2.10. Toxicity Study A total of 120 BALB/c mice.