Establishment and calibration of national reference for infective titer detection of coxsackievirus A16

Abstract

Objective To establish the national reference for detecting infective titer of coxsackievirus A16 (CA16), in order to provide the national reference for viral titer control during CA16 vaccine production and immune effect evaluation of CA16 vaccine. Methods The qualified CA16 culture was divided into 0.5 ml aliquots as candidate reference. Candidate reference was collaboratively calibrated by 3 independent laboratories. The infective titer of CA16 was detected by cell culture method, and 50% cell culture infective does (CCID50) of CA16 was calculated by Behrens-Karber method. Meanwhile, CA16 candidate reference was stored at -60 ℃ for 12 months, at -20 ℃ for 6 months, at 4 ℃ for 28 days, at 22-25 ℃ (room temperature) for 7 days and at 37 ℃ for 7 days or frozen and thawed repeatedly, and the stability under different circumstances was evaluated. Results The viral titer of CA16 candidate reference was (6.80±0.95) lgCCID50/ml. The viral titers of CA16 candidate reference had no significant reduction after keeping in storage at -60 ℃ for 12 months, -20 ℃ for 6 months, and 4 ℃ for 28 days, indicating high stability at these conditions. The viral titer loss rate of CA16 candidate reference at 22-25 ℃ (room temperature) and 37 ℃ were 3.4% and 4.4% per day respectively, and respective titer loss after 27 and 21 d storage was not significant. The viral titer loss rate of CA16 candidate reference was 4.8% per time under repeated freeze-thaw condition. Conclusion CA16 candidate reference meets the requirement as a national reference, and its valuation was (6.80±0.95) lgCCID50/ml. Key words: Enterovirus; Viral infective titer; National reference