ABSTRACT
OBJECTIVE:To establish a method for determining recombinant human calmodulin B subunit(rhCNB)in rat plas-ma,and study its pharmacokinetics characteristics. METHODS:ELISA double-antibody sandwich method was adopted. 1 μg/ml rhCNB monoclonal antibody mAb was wrapped,added to the to-be-test sample,rhCNB polyclonal antibody pAb(dilution ratio of 1∶5 000)and HRP-labeled conjugate of anti-IgG(dilution ratio of 1∶10 000)were added. Using tetramethylbenzidine for develop-ing,microplate reader was conducted in wavelength of 450 nm to determine the absorbance value(OD value)and plasma concen-tration of 6 rats after 2,15,30,60,120,240,480,720 min of iv 2.5 mg/kg rhCNB,and the pharmacokinetic parameters were calculated by BAPP 3.0 software. RESULTS:The linear range of rhCNB were 0.195-12.5 ng/ml(r2=0.995 0),lower limit of quan-titation was 0.195 ng/ml,accuracy were 97.300%-103.622%(RSD<7.5%,n=6);RSDs of within-batch,inter-batch,freezing and thawing 3 times were no higher than 8.5%(n=6,18,15). rhCNB pharmacokinetics characteristics in rat fitted to two-com-partment model,AUC0-720 min was 173.038 mg·min/L and t1/2 was 94.62 min. CONCLUSIONS:The established method has high specificity and sensitivity,good accuracy and precision,which can be used for rhCNB quantitative detection and pharmacokinetics study in biological samples.
ABSTRACT
Objective:To validate an enzyme linked immunosorbent assay (ELISA) method for the quantification of rhCNB in long-tailed macaque sera.Methods: The linear,sensitivity,accuracy,precision and recovery were determined using ELISA.Results:The present ELISA method had high linearity within 0.195 ng/ml-12.5 ng/ml,the working curve of rhCNB was Y=15.1X-0.26, R2=0.996 8 , the method showed good sensitivity of 0.195 ng/ml, the accuracy were in the range of 91.9%-108.8%, and the Coefficient of variation ( CV) for inter-assay were 3.55%,1.39%and 4.71%,the intra-assay were 1.59%,3.2%and 3.8%,all less than 10%, the recoveries were in the range of 88.5% -108.3%, <110% .Thus the method was coincidence with requirement.Conclusion:Double antibody sandwich ELISA assay of rhCNB in long-tailed macaque sera has good sensitivity ,accuracy, precision and recovery and it can be used to measure rhCNB concentration in biological samples .
ABSTRACT
<p><b>OBJECTIVE</b>To express N51 derived from the N-terminal heptad repeat (NHR) domain in gp41 of the HIV-1 CRF07_BC strain and analyze its molecular structure and antigenicity.</p><p><b>METHODS</b>Overlapping PCR was used to amplify the DNA fragment encoding N51Fd gene, which was then subcloned into the vector pFUSE-hIgG1-Fc2. The construct was confirmed by DNA sequencing. The structure and antigenicity of the recombinant protein N51FdFc-BC were analyzed using bioinformatic software, circular dichroism, and Western blotting.</p><p><b>RESULTS</b>A recombinant expression vector pFUSE/N51Fd-BC was successfully constructed. N51FdFc-BC recombinant protein with a relative molecular mass of about 35 000 was effectively expressed in mammalian 293T cells and could be recognized by rabbit antibodies against HIV-1 gp41 N/C peptides as shown by Western blotting. Bioinformatic analysis showed that the recombinant protein N51FdFc-BC, with a relative molecular mass of 34 315.1 and a PI of 7.59, formed a secondary structure of random coil to allow its interactions as an antigen with antibodies. Circular dichroism measurement confirmed the random coil structure of N51FdFc-BC protein.</p><p><b>CONCLUSION</b>The recombinant protein N51FdFc-BC has a random coil structure and can be used as an immunogen for development of HIV-1 subunit vaccine.</p>