A subscription to JoVE is required to view this content. Sign in or start your free trial.

Method Article

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

13K views

⸱

DOI:

10.3791/59641

⸱

May 11th, 2020

 , 
1Unit Antiviral Strategies, OIE Reference Laboratories for Rift Valley Fever Virus & Crimean Congo Hemorrhagic Fever Virus, Institut Pasteur, 2Institut Pasteur de Guinée, Conakry

In This Article

Summary

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

Abstract

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.

The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Introduction

Since more than 50 years, the NIH test1 is used as a gold standard method to evaluate the rabies vaccine potency before the batch release. This test consists of an intraperitoneal immunization of groups of mice with the vaccine to be tested followed by an intra-cerebral (IC) challenge 14 days later with the Challenge Virus Standard (CVS) strain of rabies virus (RABV). The potency is evaluated from the proportion of mice surviving the IC challenge. Although WHO2 and European Pharmacopeia3 still require the NIH test for assessing the vaccine potency, this test suffers several hurdles: results are highly variable4; infectious RABV is used during the challenge and this requires both technical skill and strict biosafety measures; large numbers of animals are used, and the severity of the challenge raises serious ethical concerns5. A less severe variation of this test has been developed: two weeks after the intra-peritoneal immunization, mice are not challenged by IC but bled and tested for the presence of specific RABV neutralizing antibodies (VNAbs) in their serum using an in vitro neutralization test. However, this test still requires sacrificing a large number of laboratory mice although it is already in use for the veterinary vaccines6,7 and has been considered for human vaccines8.

As of now, both International9 and European10 recommendations encourage manufacturers and National Control Laboratories (Official Medicine Control Laboratories - OMCLs) to implement the Replacement, Reduction, and Refinement of laboratory animal testing, referred as the 3Rs strategy. European Directive 2010/63/EU (in force since 2013/01/01) related to the protection and welfare of animals has also reinforced the constraints for vaccine manufacturers and laboratories involved in the Quality Control of rabies vaccines as well as in rabies research11. As a result, the development, validation, and use of alternative in vitro approaches have now become a priority. These are not only ethically sound but can also reduce the batch testing costs and shorten the time for results to hours instead of weeks3.

At the surface of the RABV particle, the glycoprotein adopts a trimeric form12,13,14,15,16. In rabies vaccine, this native trimeric form constitutes the major immunogen inducing VNAbs17 while the monomeric, soluble or denatured glycoproteins are poorly immunogenic18,19. Thus, the preservation of trimers of the glycoprotein along the vaccine production process is a good indicator for the preservation of an optimal immunogenic potential. Several immunochemical methods, such as the antibody-binding-test20,21, the single radial immunodiffusion (SRD) test22 and the ELISA test23,24,25,26,27 are recommended by the WHO Technical Report Series2 and the European monograph3 to quantify the antigen content in rabies vaccines. These are used by manufacturers to monitor the consistency of vaccine production and by the OMCL to assess the consistent formulation of batches of human vaccines28, even if the NIH test is still considered for the potency.

However, all these immunochemical methods are not equivalent. The SRD test requires a pre-treatment which may alter the membrane-anchored trimers and result in a soluble or denatured form of the glycoprotein22,29. Hence, SRD is not much efficient in discriminating between immunogenic and non-immunogenic glycoproteins resulting in an imperfect appraisal of the immunogenicity of a vaccine lot. By contrast, the ELISA test is more sensitive22, preserves the native structure of the glycoprotein, and is more appropriate to determine the content of the natively folded trimers of glycoprotein. The ELISA test can use either rabbit polyclonal or mouse monoclonal anti-glycoprotein antibodies purified or concentrated with ammonium sulfate. Studies have demonstrated good concordance between the NIH test and the antigen content evaluated by ELISA in vaccines and concluded that ELISA methods are suitable for the in vitro potency test. This advocates that ELISA tests might at least supplement or even replace the NIH test4,26,27,30,31,32,33. Today, the European Pharmacopoeia recommends the use of validated serological or immunochemical assays as alternatives to the NIH test3. The complete avoidance of animal use for vaccine potency has become a realistic perspective.

The method presented below is based on an indirect ELISA sandwich immunocapture using a mouse monoclonal antibody clone (mAb-D1) which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein15,34. This method was developed initially at the Institut Pasteur26,30 then optimized and validated by the Agence Nationale de Sécurité du Médicament et des produits de santé (ANSM) laboratory, i.e., the French OMCL4,33. The mAb-D1 is used both for sensitizing the plate and subsequently for detecting the captured antigen. This allows for the specific quantification of the glycoprotein trimers, i.e., the immunogenic RABV antigen. The mAb-D1 used for the detection is labeled with peroxidase, which is revealed in the presence of the substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content. It is of note that the same type of assay can be applied for different mAbs recognizing different antigenic sites of the RABV glycoprotein35. The method to obtain and purify or concentrate with ammonium sulfate anti-glycoprotein polyclonal rabbit immunoglobulins G (IgG) or monoclonal mouse globulins have been extensively described previously36 along with the method to conjugate antibodies with peroxidase37.

Access restricted. Please log in or start a trial to view this content.

Protocol

1. Security precautions

NOTE: This method is applicable to both live RABV and inactivated vaccine.

  1. Use good Laboratory Practice and Safety procedures.
  2. Wear adequate Personal Protection Equipment (PPE) including disposable coat, gloves, mask, glasses, etc.
  3. When the live virus is titrated, use a class II biological safety cabinet.
    1. Consider any material in contact with the samples (reagents, washing solutions, etc.) as infectious material.
    2. Treat the contaminated material by immersing in the bleach solution (2,5% of sodium hypochlorite) for 30 min for decontamination.
  4. Handle chemicals in accordance with the Good Laboratory Practice.

2. Preparation

  1. Use analytical grade reagents where ever possible.
  2. Prepare fresh solutions of the coating buffer/carbonate buffer, passivation buffer, diluent and citrate buffer (Table 1), filter through 0.45 or 0.22 µm filters and store at 4 °C for one day prior to the use to preserve their analytical purity.
  3. Allow reagents to reach to the room temperature (+18 °C to +25 °C) 30 min before the use and homogenize by gentle mixing prior to the use.

3. Microplate sensitization

NOTE: Use 96 well adsorption immunoassay plates which are optimized to bind high amounts of Immunoglobulins (e.g., see Table of Materials).

  1. To each well, add 200 µL of the monoclonal antibody (mAb-D1) diluted in the carbonate buffer.
    NOTE: An optimal concentration of about 1 µg/mL has been experimentally determined and corresponds to an approximate 1/2000 dilution of the purified mAb-D1. This recommended concentration is indicated for each mAb-D1 batch and must be periodically verified with the positive control.
  2. Cover the plate with an adhesive film and incubate the microplate for 3 h at 37 °C in a humidified atmosphere.
  3. Carefully aspirate and transfer the well content into a recipient containing 2,5% sodium hypochlorite solution.
  4. Invert the microplate and let it dry on an adsorbent paper at room temperature for 5 min.

4. Microplate passivation

  1. To each well, add 300 µL of the passivation buffer.
  2. Cover the plate with an adhesive film and incubate for 30 min at 37 °C.
  3. Aspirate carefully and transfer the well content into a recipient containing 2,5% sodium hypochlorite solution.
  4. Invert the microplate and let it dry on an adsorbent paper at room temperature for 1 min.
    NOTE: The microplate can be immediately used or stored sealed at -20 °C for up to 3 months until use.

5. ELISA assay

NOTE: For establishing the control curve of the reference vaccine, Step 5.3 is not required; to titrate the tested vaccine all Steps 5.1 to 5.6 are necessary.

  1. Washing of the sensitized microplate
    1. To each well, add 300 µL of the washing buffer.
    2. Aspirate carefully and transfer the well content into a recipient containing 2,5% sodium hypochlorite solution.
    3. Repeat steps 5.1.1 and 5.1.2 five more times to extensively wash the sensitized plate.
    4. Invert the microplate and let it dry on an adsorbent paper at room temperature for 1 min.
  2. Dilutions of the reference vaccine for the control curve
    1. Reconstitute the reference vaccine (validation antigen Lot 09) in 1 mL of distilled water corresponding to a concentration of 10 µg/mL of rabies virus glycoprotein.
    2. Prepare a ten-fold dilution of the reconstituted reference vaccine in the diluent to reach 1 µg/mL of rabies virus glycoprotein.
    3. Prepare 6 serial two-fold dilutions of this reference vaccine in the diluent as indicated in Table 1.
    4. Distribute 200 µL of the diluent in duplicate (wells 1H/2H) to serve as a blank control.
    5. Distribute 200 µL per well of each reference vaccine dilution in duplicate (wells G1/G2 to A1/A2).
  3. Dilutions of the tested vaccine for its titration
    1. Prepare a ten-fold dilution of the tested vaccine in the diluent.
    2. Prepare 7 two-fold serial dilutions of the tested vaccine in diluent as indicated in Table 2.
    3. Distribute 200 µL per well of each tested vaccine dilution in duplicate (wells H3/H4 to A3/A4).
  4. Incubation/Washing of the ELISA plate
    1. Cover the microplate with an adhesive film and incubate for 1 h at 37 °C.
    2. Remove the film, aspirate carefully and transfer the content of each well into a recipient containing 2,5% sodium hypochlorite solution.
    3. To each well add 300 µL of washing buffer.
    4. Aspirate carefully and transfer the content of each well into a recipient containing 2,5% sodium hypochlorite solution.
    5. Repeat steps 5.4.3 and 5.4.4 five times to remove the unbound antigen and conserve the G protein trimers bound to the coated antibody (mAb D1).
    6. Invert the microplate and let it dry on an adsorbent paper at room temperature for 1 min.
  5. Binding of the peroxidase conjugated mAb-D1
    1. Distribute 200 µL per well of the recommended dilution (1/2000) of peroxidase-labeled mAb-D1 in diluent (approximate concentration of 1µg/mL). A recommended concentration is indicated for each mAb-D1 batch and has to be periodically verified with the positive control.
    2. Cover the microplate with an adhesive film and incubate for 1 h at 37 °C.
    3. Remove the film, aspirate carefully and transfer the content of each well into a recipient containing 2,5% sodium hypochlorite solution.
    4. Add to each well, 300 µL of the washing buffer.
    5. Aspirate carefully and transfer the content of each well into a recipient containing 2,5% sodium hypochlorite solution.
    6. Repeat steps 5.5.4 and 5.5.5 five times to remove unbound peroxidase-labeled antibody (mAb D1).
    7. Invert the microplate and let it dry on an adsorbent paper at room temperature for 1 min.
  6. Revelation using a substrate-chromogen
    1. Distribute 200 µL per well of substrate-chromogen solution.
    2. Seal the microplate with a film and incubate in the dark at room temperature for 30 min. A yellow-orange color develops the intensity of which is proportional to the amount of bound peroxidase-labeled antibody (mAb D1).
    3. Stop the reaction by adding 50 µL of stopping solution per well.
    4. Carefully wipe the bottom of the microplate and place it in a spectrophotometer to determine the optical density (OD) at 492 nm of all used wells: negative control (blank), reference vaccine and tested vaccine.
    5. Collect OD data as .xls or .xlsx file format for analysis.
  7. Draw the reference vaccine curve, the trimeric glycoprotein content, as a function of optical density (492 nm)
    1. Calculate the mean OD at 492 nm for each duplicate at the different dilutions of the reference vaccine (wells G1/G2 to A1/A2 in Table 2)
    2. Subtract the mean OD of the Blank (wells, H1/H2 in Table 2) from each calculated mean OD.
    3. Plot the resulting OD values on the vertical axis (linear scale) and the corresponding concentration in glycoprotein trimers (ng/mL) on the horizontal axis (logarithmic scale).
    4. Draw the reference curve by joining points (Figure 1).

Access restricted. Please log in or start a trial to view this content.

Results

In the following example the reference vaccine Lot 09, consisting of purified inactivated rabies virus particles (PV vaccine strain), is used. The glycoprotein trimers (10 µg/mL) content in this has been established after the determination of the total amount of viral proteins (BCA test) and evaluation of the percentage of the glycoprotein by SDS-polyacrylamide gel electrophoresis. Alternatively, a calibrated reference vaccine, e.g., the WHO 6th International Standard (IS) for ...

Access restricted. Please log in or start a trial to view this content.

Discussion

The epitope recognized by the mAb-D1 is located in the antigenic site III of the RABV glycoprotein which is not only immuno-dominant for the induction of VNAbs but also involved in neurovirulence, pathogenicity40,41 and receptor recognition42. There is another important antigenic site along the glycoprotein, site II43, against which several MAbs have been isolated such as mAb-WI-111235. These...

Access restricted. Please log in or start a trial to view this content.

Disclosures

The authors have nothing to disclose.

Acknowledgements

Dr. Sylvie Morgeaux and Dr. Jean-Michel Chapsal must be acknowledged for their key participation to establish the ELISA assay on vaccine batches and to organize international Workshops and collaborative studies. We thank Sabrina Kali for critical reading of the manuscript. Dr. Pierre Perrin was responsible for the isolation and characterization of mAb-D1. This work has been mainly supported by Institut Pasteur funding.

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Class II Biological Safety CabinetThermoFisher Scientific10445753if titrating live virus
Clear Flat-Bottom Immuno Nonsterile 96-Well Plates, 400 µL, MAXISORPThermoFisher Scientific439454good for binding to the loaded antibody
Equip Labo Polypropylene Laboratory Fume HoodThermoFisher Scientific12576606for the preparation of sulfuric acid
Immunology Plate Strong
Adsorption MAXISORP Flat Bottom
Well F96		Dutscher55303good for binding to the loaded antibody
Microplate Sealing Tape(100 sheets)ThermoFisher Scientific15036
Microplate  single mode reader SunriseTECAN
Microplate shaker-incubatorDutscher441504
Microplate washer WellwashThermoFisher Scientific5165000
Multichannel pipette (30-300 µL) 12 channelsThermoFisher Scientific4661180N
Single Channel pipettes (Kit 2 : Finnpipettes F2 0.2-2 μL micro, 2-20 μL, 20-200 μL & 100-1000 μL)ThermoFisher Scientific4700880

References

  1. Seligmann, E. B. The NIH test for potency. Laboratory Techniques in Rabies. , 3rd Edition, WHO Geneva. 279-286 (1973).
  2. Annex 2. Recommendations for inactivated rabies vaccine for human use produced in cell substrates and embryonated eggs. WHO Technical Report Series. 941, WHO Geneva. 83-132 (2007).
  3. Rabies vaccine for human use prepared in cell cultures. European Pharmacopoeia. , (2008).
  4. Gibert, R., Alberti, M., Poirier, B., Jallet, C., Tordo, N., Morgeaux, S. A relevant in vitro ELISA test in alternative to the in vivo NIH test for human rabies vaccine batch release. Vaccine. 31, 6022-6029 (2013).
  5. Stokes, W., et al. Report on the international workshop on alternative methods for human and veterinary rabies vaccine testing: state of the science and planning the way forward. Biologicals. 40 (5), 369-381 (2012).
  6. Krämer, B., Bruckner, L., Daas, A., Milne, C. Collaborative study for validation of a serological potency assay for rabies vaccine (inactivated) for veterinary use. Pharmeuropa Bio & Scientific Notes. 2, 37-55 (2010).
  7. Krämer, B., Kamphuis, E., Hanschmann, K. M., Milne, C., Daas, A., Duchow, K. A multi-dose serological assay suitable to quantify the potency of inactivated rabies vaccines for veterinary use. Biologicals. 41, 400-406 (2013).
  8. Fitzgerald, E. A., Gallagher, M., Hunter, W. S., Seligmann, E. B. Jr Use of the antibody assay in immunized mice for the determination of rabies vaccine potency. Developments in Biological Standardization. 40, 183-186 (1978).
  9. Brown, F., Cussler, K., Hendriksen, C. WHO activities towards the three Rs in the development and control of biological products. Replacement, reduction and refinement of animal experiments in the development and control of biological products. , Karger. 31-39 (1996).
  10. Behr-Gross, M. E., Spieser, J. M. Contributions of the European OMCL Network and Biological Standardisation Programme to animal Welfare. ALTEX-Alternatives to Animal Experimentation. 23, 21-28 (2006).
  11. Directive 2010/63/EU of the European Parliament and the Council of 22 September 2010 on the protection of animals used for scientific purposes. Official Journal of European Union. L276, 33-79 (2010).
  12. Whitt, M. A., Buonocore, L., Prehaud, C., Rose, J. K. Membrane fusion activity, oligomerization and assembly of the rabies virus glycoprotein. Virology. 185 (2), 681-688 (1991).
  13. Gaudin, Y., Ruigrok, R. W., Tuffereau, C., Knossow, M., Flamand, A. Rabies virus glycoprotein is a trimer. Virology. 187, 627-632 (1992).
  14. Roche, S., Gaudin, Y. Characterization of the equilibrium between the native and fusion-inactive conformation of rabies virus glycoprotein indicates that the fusion complex is made of several trimers. Virology. 297 (1), 128-135 (2002).
  15. Desmézières, E., Maillard, A. P., Gaudin, Y., Tordo, N., Perrin, P. Differential stability and fusion activity of Lyssavirus glycoprotein trimers. Virus Research. 91 (2), 181-187 (2003).
  16. Koraka, P., et al. A recombinant rabies vaccine expressing the trimeric form of the glycoprotein confers enhanced immunogenicity and protection in outbred mice. Vaccine. 32 (36), 4644-4650 (2014).
  17. Wiktor, T., Gyorgy, E., Schlumberger, D., Sokol, F., Koprowski, H. Antigenic properties of rabies virus components. Journal of Immunology. 110, 269-276 (1973).
  18. Gamoh, K., Senda, M., Itoh, O., Muramatsu, M., Hirayama, N., Koike, R., et al. Use of ELISA for in vitro potency test of rabies vaccines for animal use. Biologicals. 24, 95-101 (1996).
  19. Dietzschold, B., Wiktor, T., Wunner, W., Varrichio, A. Chemical and immunological analysis of the rabies soluble glycoprotein. Virology. 124, 330-337 (1983).
  20. Fitzgerald, E., Green, O., Seligmann, E. Rabies vaccine potency testing: a comparison between the antibody-binding test and the NIH test. Symposia Series in Immunobiological Standard. 21, 300-307 (1974).
  21. Barth, R., Groβ-Albenhausen, E., Jaeger, O., Milcke, L. The antibody-binding test, a useful method for quantitative determination of inactivated rabies virus antigen. Journal of Biological Standardization. 9, 81-89 (1981).
  22. Ferguson, M., Schild, G. A single-radial-immunodiffusion technique for the assay of rabies glycoprotein antigen: application for the potency tests of vaccines against rabies. Journal of General Virology. 59, 197-201 (1982).
  23. Atanasiu, P., Perrin, P., Delagneau, J. F. Use of an enzyme immunoassay with protein A for rabies antigen and antibody determination. Developments in Biological Standardization. 46, 207-215 (1980).
  24. van der Marel, P., van Wezel, A. Quantitative determination of rabies antigen by ELISA. Developments in Biological Standardization. 50, 267-275 (1981).
  25. Adamovicz, P., Aguillon, F., David, A., Le Fur, R., Mazert, M. C., Perrin, P., et al. The use of various immunochemical, biochemical and biological methods for the analysis of rabies virus production in tissue cultures. Developments in Biological Standardization. 55, 191-197 (1984).
  26. Lafon, M., Perrin, P., Versmisse, P., Sureau, P. Use of monoclonal antibody for quantification of rabies vaccine glycoprotein by enzyme immunoassay. Journal of Biological Standardization. 13, 295-301 (1985).
  27. Thraenhart, O., Ramakrishnan, K. Standardization of an enzyme immunoassay for the in vitro potency assay of inactivated tissue culture rabies vaccines: determination of the rabies virus glycoprotein with polyclonal antisera. Journal of Biological Standardization. 17, 291-309 (1989).
  28. Council of Europe. Official Authority Batch release of Rabies vaccines Guideline. , PA/PH/OMCL (11) 173 DEF (2019).
  29. Ferguson, M., Seagroatt, V., Schild, G. A collaborative study on the use of single radial immunodiffusion for the assay of rabies virus glycoprotein. Developments in Biological Standards. 12, 283-294 (1984).
  30. Perrin, P., Morgeaux, S., Sureau, P. In vitro rabies vaccine potency appraisal by ELISA: advantages of the immunocapture method with a neutralizing anti-glycoprotein monoclonal antibody. Biologicals. 18, 321-330 (1990).
  31. Rooijakkers, E. J., Uittenbogaard, J. P., Groen, J., Osterhaus, A. D. Rabies vaccine potency control: comparison of ELISA systems for antigenicity testing. Journal of Virological Methods. 58, 111-119 (1996).
  32. Rooijakkers, E. J., Uittenbogaard, J. P., Groen, J., van Herwijnen, J., Osterhaus, A. D. Development and evaluation of alternative testing methods for the in vivo NIH potency test used for the quality control of inactivated rabies vaccines. Brown, F., Cussler, K., Hendriksen, C. , Karger. 137-145 (1996).
  33. Fournier-Caruana, J., et al. Inactivated rabies vaccine control and release: use of an ELISA method. Biologicals. 31, 9-16 (2003).
  34. Sissoëff, L., Mousli, M., England, P., Tuffereau, C. Stable trimerization of recombinant rabies virus glycoprotein ectodomain is required for interaction with the p75NTR receptor. Journal of General Virology. 86, 2543-2552 (2005).
  35. Morgeaux, S., et al. Replacement of in vivo human rabies vaccine potency testing by in vitro glycoprotein quantification using ELISA - Results of an international collaborative study. Vaccine. 35 (6), 966-971 (2017).
  36. Lafon, M. Techniques for the production, screening and characterisation of monoclonal antibodies. Laboratory techniques in rabies, 4th Edition. Meslin, F. X., Kaplan, M. N., Kopowski, H. , WHO. Geneva. 133-144 (1996).
  37. Perrin, P. Techniques for the preparation of rabies confugates. Laboratory techniques in rabies, 4th Edition. Meslin, F. X., Kaplan, M. N., Kopowski, H. , WHO. Geneva. 433-444 (1996).
  38. Barth, R., Diderrich, G., Weinmann, E. NIH test, a problematic method for testing potency of inactivated rabies vaccine. Vaccine. 6, 369-377 (1988).
  39. Jallet, C., et al. Chimeric lyssavirus glycoproteins with increased immunological potential. Journal of Virology. 73, 225-233 (1999).
  40. Dietzschold, B., et al. Characterization of an antigenic determinant of the glycoprotein that correlates with pathogenicity of rabies virus. Proceedings of the National Academy of Science U.S.A. 80, 70-74 (1983).
  41. Seif, L., Coulon, P., Rollin, P., Flamand, A. Rabies virulence: effect on pathogenicity and sequence characterization of rabies virus mutations affecting antigenic site III of the glycoprotein. Journal of Virology. 53, 926-934 (1985).
  42. Lafon, M. Rabies virus receptors. Journal of Neurovirology. 11, 82-87 (2005).
  43. Benmansour, A., Leblois, H., Coulon, P., Tuffereau, C., Gaudin, Y., Flamand, Y., Lafay, A. Antigenicity of the rabies virus glycoprotein. Journal of Virology. 65, 4198-4203 (1991).
  44. Rabies Vaccine Workshop Summary. , http://ntp.niehs.nih.gov/iccvam/meetings/rabiesvaccwksp-2011/rabiesvaccinewkspsumm-30nov11.pdf (2011).
  45. De Mattia, F., et al. The Vaccines Consistency Approach Project: an EPAA initiative. Pharmeuropa Bio & Scientific Notes. 2015, 30-56 (2015).

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Tags

In Vitro TestNeutralizing AntibodiesGlycoprotein TrimerMonoclonal AntibodySerial DilutionOptical DensityReference VaccineVaccine Batch
In Vitro ELISA Test to Evaluate Rabies Vaccine Potency
Contact Us
Recommend to Library

Privacy

Terms of Use

Policies

Copyright © 2026 MyJoVE Corporation. All rights reserved.