| The neutralization ability of tetanus antitoxin(TAT)is an important indicator for specific antibodies and evaluation of product quality,also called antibody titer.The level of potency determines the quality and effectiveness of the product.Up to now,the tetanus antitoxin titer test method is still in accordance with the provisions of the "People’s Republic of China veterinary pharmacopoeia"(The three part of 2015 version): using the mouse in vivo test method for determination.Because the animal law of the Pharmacopoeia has its own defects,it requires a large amount of animals,long detection time,high feeding requirements,high cost,and results are often unsatisfactory due to the differences in experimental animals.In recent years,international and domestic attention has been paid more and more to the welfare of experimental animals.Therefore,it is necessary to explore a rapid and simple method of vitro detection for the detection of plasma and intermediate drug titer of tetanus antitoxin raw materials.The floc unit assay is based on the immunological reaction of anti-toxin with pre-calibrated toxoid in the reactor to produce macroscopically visible flocs,which are a toxoid-dependent flocculent unit in vitro.To demonstrate the rapid method of anti-toxin titer,however,whether this method can replace the mouse test method prescribed by the pharmacopoeia,there is still no systematic analysis.In this study,the method of determination of tetanus antitoxin floc unit was carried out:Accuracy test results showed that the results of 6 floc determinations of the national standards for tetanus antitoxin were 950 Lf/mL,the average value was 950 Lf/mL,and the recovery rate was 100%,in the range of 90%-110%,meeting the standard requirements,indicating that the floc unit measurement method has good accuracy;the repeatability test results show that the results of 6 floc determinations are 2 000,1 950,2 000,2 000 respectively.1,950,2 000 Lf/mL,mean 1 983.33 Lf/mL,S is 25.82 Lf/mL,CV is 1.30%,less than 2%,meeting the requirements of the standard,indicating that the method has good repeatability;The precision test results showed that the average value of the flocculating results of the two testers on the test sample was 1 987.50 Lf/mL,S was 22.61 Lf/mL,CV was 1.13%,less than 2%,and the standard was met.The specified requirements indicate that the intermediate precision of the method is good.The above results show that the method meets the potency measurement requirements and has accurate and exclusive measurement capabilities for the test samples.The experiment selected 30 batches of tetanus antitoxin raw material plasma,30 batches of stock solution and 31 batches of finished products produced by the company from 2014 to 2016.A total of 91 samples were tested and passed the sterility test as the tested samples.The selected samples were tested by flocculation assay and mouse assay to test the titer,and the correlation between the two was statistically analyzed.The results showed that the correlation coefficients of raw material plasma,stock solution and finished product were determined by flocculation method and mouse test method as R=0.825、P<0.001,R=0.937、P<0.001 and R=0.945、P<0.001,both were significant.Positive correlation,can be used to determine the titer of tetanus antitoxin.On this basis,we selected 20 batches of raw materials plasma and stock solution of different batches in different months of 2017 for the determination of potency.The results showed that the floc unit method has good applicability in the detection of the intermediate semi-finished products.In summary,the floc assay is easy to operate,saves time and effort,and has good controllability.It can be used as an auxiliary rapid detection method for internal control of enterprises.It is a good method for measuring anti-toxin titer,which is cost-effective,convenient to use,operation and so on are all important. |
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