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.

1. Security precautions
NOTE: This method is applicable to both live RABV and inactivated vaccine.
<ol>
	<li>Use good Laboratory Practice and Safety procedures.</li>
	<li>Wear adequate Personal Protection Equipment (PPE) including disposable coat, gloves, mask, glasses, etc.</li>
	<li>When the live virus is titrated, use a class II biological safety cabinet.
	<ol>
		<li>Consider any material in contact with the samples (reagents, washing solutions, etc.) as infectious material.</li>
		<li>Tr...

The authors have nothing to disclose.

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...

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&#160;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 S&#233;curit&#233; du M&#233;dicament et des produits de sant&#233; (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.

<table border="0" cellpadding="0" cellspacing="0" style="width:1523px;" width="1522">
	<tbody>
		<tr height="34">
			<td height="34" style="height:35px;width:465px;">Class II Biological Safety Cabinet</td>
			<td style="width:340px;">ThermoFisher Scientific</td>
			<td style="width:262px;">10445753</td>
			<td style="width:456px;">if titrating live virus</td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:465px;">Clear Flat-Bottom Immuno Nonsterile 96-Well Plates, 400 &#181;L, MAXISORP</td>
			<td>ThermoFisher Scientific</td>
			<td>439454</td>
			<td>good for binding to the loaded antibody</td>
		</tr>
		<tr height="86">
			<td height="86" style="height:85px;width:465px;">Equip Labo Polypropylene Laboratory Fume Hood</td>
			<td>ThermoFisher Scientific</td>
			<td>12576606</td>
			<td>for the preparation of sulfuric acid</td>
		</tr>
		<tr height="112">
			<td height="112" style="height:113px;width:465px;">Immunology Plate Strong 
			Adsorption MAXISORP Flat Bottom 
			Well F96</td>
			<td style="width:340px;">Dutscher</td>
			<td>55303</td>
			<td>good for binding to the loaded antibody</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:35px;">Microplate Sealing Tape(100 sheets)</td>
			<td>ThermoFisher Scientific</td>
			<td>15036</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:35px;width:465px;">Microplate&#160; single mode reader Sunrise</td>
			<td style="width:340px;">TECAN</td>
			<td style="width:262px;"></td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:35px;">Microplate shaker-incubator</td>
			<td style="width:340px;">Dutscher</td>
			<td>441504</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:35px;width:465px;">Microplate washer Wellwash</td>
			<td>ThermoFisher Scientific</td>
			<td>5165000</td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:465px;">Multichannel pipette (30-300 &#181;L) 12 channels</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:262px;">4661180N</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="103">
			<td height="103" style="height:104px;width:465px;">Single Channel pipettes (Kit 2 : Finnpipettes F2 0.2-2 &#956;L micro, 2-20 &#956;L, 20-200 &#956;L &#38; 100-1000 &#956;L)</td>
			<td>ThermoFisher Scientific</td>
			<td>4700880</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro elisa test to evaluate rabies vaccine potency

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.

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 &#181;g/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)...

Watch this Scientific Journal Video about In Vitro ELISA Test to Evaluate Rabies Vaccine Potency at JoVE.com

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

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.

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

In the aim of reducing animal testing and because of the variability of the NH test, WHO encourages to develop in vitro approaches for evaluating rabies vaccine potency. This ELISA test shows a good concordance with a classical NH test in recognizing the immunogenic form of the glycoprotein, shows a high sensitivity, and can be performed within only one day. Evaluation of rabies vaccine potency in vitro by ELISA is an alternative to the in vivo NH test.It is promoted by the manufacturers and national control laboratories. It's critical to distribute pre-sized volumes in duplicate for each dilution and to well dry the plate on absorbent paper after washes to avoid To begin this procedure, put on adequate personal protection equipment including disposable coat, gloves, mask, and glasses. Treat the contaminated materials by immersing it in a solution of 2.6%sodium hypochlorite for 30 minutes for decontamination.Next, set out a 96-well immunoassay plate and add 200 microliters of the monoclonal antibody diluted in the carbonate buffer to each well. Cover the plate with adhesive film and incubate the microplate at 37 degrees Celsius for three hours in a humidified environment. Then carefully aspirate and transfer the well content into a recipient containing a solution of 2.6%sodium hypochlorite.Invert the microplate and let it dry on absorbent paper at room temperature for five minutes. First, add 300 microliters of the passivation buffer to each well. Cover the plate with adhesive film and incubate at 37 degrees Celsius for 30 minutes.After this, transfer the contents of the well to one containing a solution of 2.6%sodium hypochlorite. To wash the plate, add 300 microliters of the washing buffer to each well. Then aspirate carefully and transfer the well content into a recipient containing a solution of 2.6%sodium hypochlorite.Repeat this washing process five more times to extensively wash the sensitized plate. After this, invert the microplate and let it dry on a piece of absorbent paper at room temperature for one minute. Reconstitute the reference vaccine in one milliliter of distilled water such that the final concentration of rabies virus glycoprotein is 10 micrograms per milliliter.Prepare a 10-fold dilution of the reconstituted reference vaccine in the diluent to reach a rabies virus glycoprotein concentration of one microgram per milliliter. Next, perform six serial two-fold dilutions of this reference vaccine in the diluent as outlined in table one of the text protocol. Distribute 200 microliters of the diluent in duplicate wells to serve as a blind control and 200 microliters per well for each reference vaccine dilution in duplicate.Then prepare a 10-fold dilution of the tested vaccine in the diluent and prepare seven two-fold serial dilutions of the tested vaccine in diluent as outlined in table two of the text protocol. Distribute 200 microliters of each tested vaccine dilution in duplicate to each well of the microplate. Cover the microplate with adhesive film and incubate at 37 degrees Celsius for one hour.After this, remove the film. Aspirate carefully and transfer the contents of each well into a recipient containing a 2.7%sodium hypochlorite solution. To wash the plate, add 300 microliters of washing buffer to each well, aspirate carefully and transfer the contents of each well into a recipient containing a 2.6%sodium hypochlorite solution.Repeat the washing process five more times to remove the unbound antigen and conserve the G-protein trimers bound to the coated antibody. Invert the washed microplate and let it dry on a piece of absorbent paper at room temperature for one minute. Then distribute 200 microliters of the recommended dilution of peroxidase labeled D1 monoclonal antibody in diluent to each well.Cover the microplate with adhesive film and incubate at 37 degrees Celsius for one hour. Next, remove the film, aspirate carefully, and transfer the contents of each well into a recipient containing a 2.6%sodium hypochlorite solution. To wash the microplate, add 300 microliters of washing buffer to each well.Aspirate carefully and transfer the contents of each well into a recipient containing 2.6%sodium hypochlorite solution. Repeat this washing process five more times to remove the unbound peroxidase labeled antibody. Invert the washed microplate and let it dry on a piece of absorbent paper at room temperature for one minute.Next, distribute 200 microliters of substrate-chromogen solution into each well. Seal the microplate with film and incubate at room temperature in the dark for 30 minutes. Then add 50 microliters of stopping solution into each well to stop the reaction.Carefully wipe the bottom of the microplate and place it in the spectrophotometer. Determine the optical density at 492 nanometers for all of the wells used. Collect this data in a spreadsheet file for analysis.After this, create plots for both the reference vaccine curve and the optical density values as outlined in the text protocol. In this study, the in vitro ELISA test is used to evaluate rabies vaccine potency. Representative optical density values from a typical experiment are shown here.These values are used to draw the reference vaccine curve by plotting the mean optical density for the different dilutions of the reference vaccine on the vertical axis and the concentration of the glycoprotein on the horizontal axis. The glycoprotein content of the tested vaccine is then estimated using this curve. The evaluation is precise for dilutions that have a mean optical density value in the liner area of the reference vaccine curve.Taking into account the dilution of one-to-40, the vertical projection of OD 1534 on the x-axis corresponds to 500 nanograms per milliliter. So the tested vaccine is estimated at 20 micrograms per milliliter of glycoprotein. When the in vitro potency is established, it is necessary to compare the tested vaccine to the reference six WHO international standard calibrated in international units.The same method can be used with other antibodies to enlarge detection of more vaccine strains including those used in veterinary vaccines which are classically more variable. Antibodies can also be used in competition for titration of antirabies antibodies in equine or human serum. Precautions must be taken with vaccines that are not inactivated or with viruses.Be sure to use personal protective equipment, wear gloves and decontaminate everything properly. Also, be careful with tablets of as they are carcinogenic and with the acidic sodium hypochlorite solution.

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Research

JoVE Journal

Immunology and Infection

12.1K visualizações.  Institut Pasteur. Com o objetivo de reduzir os testes em animais e por causa da variabilidade do teste de NH, a OMS incentiva a desenvolver abordagens in vitro para avaliar a potência da vacina antirrábica. Este teste ELISA mostra uma boa concordância com um teste clássico de NH no reconhecimento da forma imunogênica da glicoproteína, mostra uma alta sensibilidade, podendo ser realizado em apenas um dia. A avaliação da potência da vacina antirrábica in vitro pela ELISA é uma alternativa ao teste in ...