[Determination of optimized MTT colorimetric assay as the identification method for the activity of B type CpG ODN BW006].
OBJECTIVE
BW006, B type CpG ODN, will be approved as vaccine adjuvant for human use. So it is necessary to determine a stable, safety and easy-to-operate method for activity identification of BW006 with different lots.


METHODS
According to the characteristic of BW006 to stimulate the proliferation of PBMC or mice splenocytes, MTT assay was preferentially selected to test the characteristic of BW006. The splenocytes of female BALB/c mice was stimulated by BW006, and different experiment parameters including splenocyte numbers, the shape of plate, BW006 concentration, culture medium, culture time and optimized MTT conditions were determined in these courses.


RESULTS
The whole experiment scheme was defined as following: 6 x 10(5) splenocytes were co-cultured with 3 mg/L BW006 in 200 microliter RPMI1640(without phenol red) supplemented with 100 mL/L FBS in a 96 well square plate. PBS, the solvent of BW006 was used as negative control, and culture medium without cells was used as blank. After being co-cultured for 36 hours at 37 degrees C in humidified incubator with 50 mL/L CO(2), 100 microliter supernatants was aspired out and 10 microliter MTT (5 g/L) was added into each well. The plate was further incubated in dark at 37 degrees C for 4 hours that is sufficient for the formation of formazan. Afterward, 150 microliter DMSO was directly added into each well. The plate was shaken on plate shaker for nearly 20 minutes until the formazan was completely dissolved and detected A(578) value at at ELISA reader.


CONCLUSIONS
Optimized MTT assay, which is non-radioactive contamination, low-price and simple operation, could be used as activity identification of BW006 during large-scale production.
Wang
Xue-ju
XJ
Department of Molecular Biology, College of Basic Medicine, Jilin University, Changchun 130021, China. wxj20032005@yahoo.com.cn
Liu
Jing
J
Wan
Min
M
Wang
Li
L
Wu
Xiu-li
XL
Wei
Hong-fei
HF
Yu
Yong-li
YL
Wang
Li-ying
LY
D000276:Adjuvants, Immunologic; D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D003124:Colorimetry; D005260:Female; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects*; D051379:Mice; D008807:Mice, Inbred BALB C; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D011818:Rabies / Q000473:pathology*; D013778:Tetrazolium Salts / Q000737:chemistry*
D000276:Adjuvants, Immunologic; C408982:CPG-oligonucleotide; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D013778:Tetrazolium Salts