Development and Clinical Application of Double Antibody Sandwich ELISA for WuTac Concentration in Serum

Objective To develop a double antibody sandwich ELISA for WuTac concentration in serum and preliminarily apply in clinic. Methods The WuTac concentration in sera was determined quantitatively by double antibody sandwich ELISA, based on which the concentration of coating antibody and the dilution of enzyme-labeled antibody were optimized and a standard curve was plotted. The developed ELISA was verified and used for the determination of serum specimens from 36 healthy volunteers at 14 time points before and after injection with WuTac at various dosages(0. 05, 0. 1 and 0. 2 mg / kg). Results The optimal concentration of coating antibody was 0. 2 μg / ml, and the optimal dilution of enzyme-labeled antibody was 1 ∶ 15 000. The correlation coefficient (r value)of standard curve was not less than 0. 99. The linear determination range of developed double antibody sandwich ELISA was 3. 9 ～ 125 ng / ml. The variation coefficient(CV)of determination results of high concentration WuTac standard by the developed ELISA was less than 15%. The developed ELISA showed a accuracy of 99. 05% ± 5. 00%(92. 43% ～ 110. 02%)and good specificity. The determination results of clinical serum specimens proved that WuTac showed a characteristic of linear pharmacokinetics within a concentration range of 0. 05 ～ 0. 2 mg / kg. Conclusion A double antibody sandwich ELISA for WuTac concentration in serum was developed, with sensitivity, precision and accuracy meeting the requirements of pharmacokinetic study on biologics.