ABSTRACT
OBJECTIVE To find out whether 2811 will cause toxic reactions in animals, and determine the safe dose of without any toxic reactions and the relationship between the dose, time of administration and results of the toxicity test. METHODS Thirty healthy rhesus monkeys were selected and randomly divided into 3 groups, 1 O in each group (half males and half females), namely, the solvent control group, 2811 100 and 400 mg* kg-1 groups, respectively. The solvent control group was iv given 0.9% sodium chloride injection, while the experimental groups were iv given 2811 100 and 400 mg* kg-1 , respectively, once every 6 days, 3 times in 2 weeks, and allowed to recover for 9 weeks after drug withdrawal. During the test, the body weight, food intakes, body temperatures, ophthalmology, blood pressure, electrocardiograms, blood routine, anticoagulation, blood biochemistry, electrolytes, urine, systemic anatomy, organ weight and coefficient were observed, while histopathology and immunology tests were performed. At the same time, the anti-drug antibody (ADA) and plasma concentrations were determined, and toxikinetic parameters were analyzed. RESULTS During the experiment, the observation of general symptoms, body mass, food intakes, body temperatures, ophthalmology, blood pressure, electrocardiograms, blood routine, anticoagulation, blood biochemistry, electrolytes, urine, organ weight and coefficient, histopathology and immunology of animals in each dose group showed no significant changes related to the tested animals. ADA was not detected in any of the groups. Plasma drug concentrations in 2B11 100 and 400 mg* kg-1 groups were basically consistent and proportional to the dose, so was the ratio of peak concentration and exposure. 2B11 injection showed linear kinetics in vivo. CONCLUSION Under the conditions set in this test, the 2-week repeated administration toxicity test of 2B11 in rhesus monkeys is safe, and no clinical adverse reactions are observed at the dose level of 400 mg*kg-1, which provides reference for the follow-up clinical study of 2B11. Copyright © 2022 Chinese Journal of Pharmacology and Toxicology. All rights reserved.
ABSTRACT
Objective: To establish and verify a competitive ELISA method for the detection of blocking activity of monoclonal antibody against SARS-CoV-2 RBD, and to compare the results by correlation analysis with that of live virus neutralization activity measured by the plaque reduction neutralization test (PRNT). Methods: Using RBD-Fc as coating antigen, ACE2-His and anti-SARS-CoV-2 RBD monoclonal antibodies were added to competitively bind to RBD. Anti-6×his antibody labeled with horseradish peroxidase was used as the secondary antibody. The competitive ELISA method detecting the ability of McAb to block the binding of RBD to ACE2 was established. The specificity, relative accuracy, precision, linearity and range of the method were verified. Seven monoclonal antibodies against SARS-CoV-2 RBD were detected by this method. The results were compared with PRNT method, and correlation analysis was performed. Results: The blocking activity of the relevant anti-SARS-CoV-2 RBD monoclonal antibody on RBD and ACE2 protein can be effectively detected using the established competitive ELISA method. The blocking ability of McAb was dose-dependent and conformed to the four-parameter equation. The samples with theoretical titers of 64%, 80%, 100%, 125% and 156% were determined for 10 times, and the relative bias was within ±20%. The logarithm (abscissa) of theoretical potency value was used for linear regression to the logarithm (ordinate) of the corresponding titer determination value. The regression equation was y=1.156x-0.021 3, in which the slope was between 0.8 and 1.25, meaning good relative accuracy. The geometric coefficient of variation (GCV%) of the relative titers of each titer level were 2.6%, 5.2%, 3.6%, 3.4% and 10.2%, respectively, all of which were less than 20% with good precision. The correlation coefficient of linear regression equation was 0.985, meeting the requirements. The relative accuracy, intermediate precision and linearity of the method all met the requirements of the titer level range was 64%~156%. The detection results of the blocking activity of the 7 RBD monoclonal antibodies showed good correlation with the results of the live virus neutralization activity measured by the PRNT method. Conclusion: A competitive ELISA method for the detection of anti-SARS-CoV-2 RBD monoclonal antibody has been successfully established. The method has satisfied specificity, accuracy, precision and linearity. The results had a good correlation with that by PRNT method. It can be used to indirectly evaluate the neutralizing activity of related SARS-CoV-2 monoclonal antibodies against the live viruses.