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The optimum methanol concentration was 10% because Amax/IC50 had the highest value and IC50 had the lowest value. The result was also used to redissolve MT residue from the real sample. Increasing the concentration of PBS from 5 to 40 mM decreased Amax due to the PBS concentration increasing to cause the enhancement of ionic strength (Table S1, see Supplementary Materials). [http://www.selleckchem.com/products/Staurosporine.html Staurosporine in vitro] Ions bind to paratopes or charged groups of epitopes, inhibiting antigen-antibody reaction, and inducing decreased Amax [41]. Therefore, the optimum ionic strength of PBS was 10 mM. Five pH values (4.7, 6.0, 7.4, 8.6, and 9.6) were evaluated. The icELISA was sensitive at neutral (pH 7.4) and acidic conditions (pH 4.7) with IC50 values of 0.455 and 0.456 ng/mL, respectively (Table S1, see Supplementary Materials). However, low pH values contributed to low Amax and this phenomenon was attributed to the low reaction activities between antigen and antibody. Therefore, pH 7.4 was selected as the optimum pH value for icELISA. In conclusion, the MT standard was diluted with PBS (10 mM, pH 7.4) and analyzed by icELISA. The standard curve was established by plotting the B/B0 (B was the absorbance at each concentration of MT and B0 was [http://www.selleckchem.com/products/dorsomorphin-2hcl.html Dorsomorphin clinical trial] the absorbance in the absence of MT) against the concentration of MT (Figure 2). The standard curve equation was y = 0.081 + (0.999 ? 0.081)/(1 + (x/0.229)1.39), the linear regression correlation coefficient (R2) was 0.999, the IC50 value was 0.26 ng/mL, and the limit of detection (LOD, concentration calculated as IC10) was 0.045 ng/mL. Figure 2 Optimized inhibition standard curve for MT analysis by icELISA. Error bar was calculated according to three repeats at each concentration. 3.4. Specificity of mAb The specificity of mAb was assessed by CR. Eight of analogues were analyzed by icELISA (Table 1). Testosterone had a CR rate of approximately 2.17%, because testosterone without methyl group at the C-17 positon. The [http://en.wikipedia.org/wiki/Resminostat Resminostat] CR rate with nor-testosterone (1.04%) and estradiol (0.52%) was calculated. There were two extra methyl groups located in the C-17 and C-18 positions of these compounds, which were not present in MT. The results agreed with the immunogen synthesis scheme. Even though these analogues have CR with mAb, the rates were