The Improvement And Evaluation Of The RFFIT System

Objective:1. To improve RFFIT detection system to meet the domestic needs of the laboratory condition and testing requirement;2. To evaluate the impaction of the improved RFFIT system;3. Fuse the RFFIT system which has been mature in our laboratory and the method for rabies vaccine antigen to form a modified antibody test—M-ABT.Methods:1. The BHK-21 (Baby hamster kidney cell line), BSR (the small clone of BHK-21), MNA (Mouse Neuroblastoma Cell) and CVS-11, CTN-181 were introduced to RFFIT system separately.Testing 10 specimens using different combinations of three cells and two strains of rabies virus for the initial identification of the consistency of test results, then testing 106 human serum samples, comparing the test results of different combinations RFFIT systematically.2. Detected 1011 human serum samples with improved RFFIT, evaluated the detection performance of the improved RFFIT system.3. Anti-RVNP-Mc1, one strain of monoclonal antibody against the nucleoprotein of rabies virus is prepared by Balb/c mice immunized with a piece of linear epitope peptide on the nucleoprotein of rabies virus linked with KLH molecular. Anti-RVNP-Mc1 is concentrated, purified and labeled by FITC. FITC-Anti-RVNP-Mc1 is used to RFFIT on 200 human sera paralleled with DFA5100 (Rabies Diagnostic Reagent, Millilore Company). The results are statistically analysized.4. Combined RFFIT with the antibody test (antibody binding test, ABT) program, improve it in dilution the vaccine sample, detection the remaining neutralizing antibody and the calculation of results, formed the M-ABT. Application of M-ABT on 10 different stocks inactivated rabies vaccine in time for testing their efficacy and then observed the change of situation of vaccine efficacy over time.Results:1. Cell culture: BHK-21, BSR, MNA cells grew well, established these three kinds of cell seed bank; 2. Virus culture: rabies virus CVS-11 and CTN-181 adapted BHK-21, BSR, MNA cells well, the virus titer of passages were higher than 10~6FFU/ml, consistent with the requirements of detection of RFFIT;3. Improvements of RFFIT detection system:10 low-volume specimens, detection with improved, which was introduced BHK-21 /the MNA cell line and the CTN-181 strain respectively, the test results have no significant difference compared with the results reference to a combination of BSR and CVS-11 test, p>0.05. 10~6 samples of large quantities of test results also show that there was no significant difference between the new inspection system established RFFIT test results and the results with international standards, they have a good relationship and the correlation coefficient is greater than 0.75;4. Evaluate the application of the improved RFFIT test system:(1) Different cells of the test results on the RFFIT: CVS-11 as an attack viruse, were applied BHK-21, BSR, MNA cells, detected the 1011 human serum samples with RFFIT, The positive detection system of different rates have no significant difference, P>0.05; Under different detection system, the geometric mean of the positive test results of samples (geometric mean titer, GMT) with no significant difference, P>0.05; Test results correlated well under different detection systems, r >0.75;(2) The impact of different operating environment for RFFIT: Operating in different laboratories, apply by the same operator with CVS-11 strain and BHK-21, BSR cells in human positive serum sample; Laboratory test results have no significant difference between the latter and with the original environment, P > 0.05.5. By logistic analysis, the results of RFFIT used Anti-RVNP-Mc1 and DFA5100 is correlated, the correlative coefficient (r) is 0.980. The consistent rate of qualitative results (negative or positive) is 96.83%. Byχ~2 test, the difference between the positive rates of RFFIT used two kinds of antibodies is not significant (χ~2 = 0.098, P>0.05). All 6 (3.00%) human sera with different qualitative results are detected with values≤1.0 IU/ml in RFFIT used two kinds of antibodies. 6 (8.82%) ones among 68 human sera with values≤1.0 IU/ml give out opposite qualitative results in RFFIT used two kinds of antibodies; the other samples rise consistent qualitative results.6. The established of M-ABT method and its preliminary application: 10 rabies vaccines tested by the method of M-ABT, the results have good correlation with the results of NIH method, r = 0.927, M-ABT method can be used to monitor the potency of rabies vaccine antigen for their changes over time. Conclusions:1. Achieved using different rabies virus strains and cell lines for RFFIT test, Use much weaker pathogenic strain of CTN-181 increased the security of the detection, Using BHK-21 cells which is much more common to more laboratory make it is easy for them to establish and apply the technology;2. Different operating environment on the impact of test results to RFFIT is not obvious, therefore, the same operator using the same detection system in different laboratory, the test results can be depended on;3. FITC-Anti-RVNP-Mc1 and DFA5100 have equivalent results in RFFIT, and so on Anti-RVNP-Mc1 can replace the use of DFA5100 in RFFIT.4. M-ABT method has been established, is highly correlated with the NIH method when was used to detect potency of the inactivated rabies vaccine, and it is a suitable technology for monitoring the effectiveness of inactivated rabies vaccine means.