Optimization and verification of a method for in vitro detection of bioactivity of recombinant human bone morphogenetic protein-2 based on ASTM F2131-02 standard test
10.13200/j.cnki.cjb.003934
- VernacularTitle:基于ASTM F2131-02标准试验的重组人骨形态发生蛋白-2体外生物活性检测方法的优化及验证
- Author:
XIE Qian
- Publication Type:Journal Article
- Keywords:
Recombinant human bone morphogenetic protein-2(rhBMP-2);
Bioactivity;
Specific activity
- From:
Chinese Journal of Biologicals
2024;37(9):1115-1121
- CountryChina
- Language:Chinese
-
Abstract:
Objective To optimize and verify an in vitro detection method for bioactivity of recombinant human bone morphogenetic protein-2(rhBMP-2)based on ASTM F2131-02 standard test.Methods Based on the method established by the American Society for Testing Material(ASTM),the detection cell lines(W-20-17 and C2C12 cells),dilution mode of rhBMP-2(2 and 3 times serial dilution),culture time(24,36,48 and 72 h),and freezing temperature(-20 and-80 ℃)were optimized,and the specificity,intermediate precision,linearity and durability of the optimized method were verified. The specific activity of rhBMP-2 from different manufacturers was detected using the optimized method.Results C2C12 cells were determined as detection cells;The best dilution method of rhBMP-2 was 2 times series dilution;The optimum cell culture timewas 48handtheoptimum freezingtemperatureofcellswas-80℃. C2C12 cells responded specifically to rhBMP-2 induction when adding heteroprotein(bovine serum albumin)to rhBMP-2;The CV of 12 detections by 2 experimenters at 2 time points was11. 7%. The theoretical titer of rhBMP-2 showed a good linear relationship with the measured titer in the range of 70%-142%,and the fitting linear regression equation was:y = 0. 983 1 x-0. 010 3,R~2= 0. 996 4;The CV of the detected specific of rhBMP-2 using 4,11 and 21 passages of C2C12 cells was 5. 2%. The CV of specific activity of rhBMP-2 samples from different manufacturers detected by the optimized method repeatedly for two times was 1. 3%-7. 5%.Conclusion The optimized method showed good specificity,intermediate precision,linearity and durability,and might be used to detect the bioactivity of rhBMP-2 in vitro,which provided a reliable analytical method for its research,production and application.
