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>Treat the contaminated material by immersing in the bleach solution (2,5% of sodium hypochlorite) for 30 min for decontamination.</li>
	</ol>
	</li>
	<li>Handle chemicals in accordance with the Good Laboratory Practice.</li>
</ol>
2. Preparation
<ol>
	<li>Use analytical grade reagents where ever possible.</li>
	<li>Prepare fresh solutions of the coating buffer/carbonate buffer, passivation buffer, diluent and citrate buffer (Table 1), filter through 0.45 or 0.22 &#181;m filters and store at 4 &#176;C for one day prior to the use to preserve their analytical purity.</li>
	<li>Allow reagents to reach to the room temperature (+18 &#176;C to +25 &#176;C) 30 min before the use and homogenize by gentle mixing prior to the use.</li>
</ol>
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).
<ol>
	<li>To each well, add 200 &#181;L of the monoclonal antibody (mAb-D1) diluted in the carbonate buffer. 
	NOTE: An optimal concentration of about 1 &#181;g/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.</li>
	<li>Cover the plate with an adhesive film and incubate the microplate for 3 h at 37 &#176;C in a humidified atmosphere.</li>
	<li>Carefully aspirate and transfer the well content into a recipient containing 2,5% sodium hypochlorite solution.</li>
	<li>Invert the microplate and let it dry on an adsorbent paper at room temperature for 5 min.</li>
</ol>
4. Microplate passivation
<ol>
	<li>To each well, add 300 &#181;L of the passivation buffer.</li>
	<li>Cover the plate with an adhesive film and incubate for 30 min at 37 &#176;C.</li>
	<li>Aspirate carefully and transfer the well content into a recipient containing 2,5% sodium hypochlorite solution.</li>
	<li>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 &#176;C for up to 3 months until use.</li>
</ol>
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.
<ol>
	<li>Washing of the sensitized microplate
	<ol>
		<li>To each well, add 300 &#181;L of the washing buffer.</li>
		<li>Aspirate carefully and transfer the well content into a recipient containing 2,5% sodium hypochlorite solution.</li>
		<li>Repeat steps 5.1.1 and 5.1.2 five more times to extensively wash the sensitized plate.</li>
		<li>Invert the microplate and let it dry on an adsorbent paper at room temperature for 1 min.</li>
	</ol>
	</li>
	<li>Dilutions of the reference vaccine for the control curve
	<ol>
		<li>Reconstitute the reference vaccine (validation antigen Lot 09) in 1 mL of distilled water corresponding to a concentration of 10 &#181;g/mL of rabies virus glycoprotein.</li>
		<li>Prepare a ten-fold dilution of the reconstituted reference vaccine in the diluent to reach 1 &#181;g/mL of rabies virus glycoprotein.</li>
		<li>Prepare 6 serial two-fold dilutions of this reference vaccine in the diluent as indicated in Table 1.</li>
		<li>Distribute 200 &#181;L of the diluent in duplicate (wells 1H/2H) to serve as a blank control.</li>
		<li>Distribute 200 &#181;L per well of each reference vaccine dilution in duplicate (wells G1/G2 to A1/A2).</li>
	</ol>
	</li>
	<li>Dilutions of the tested vaccine for its titration
	<ol>
		<li>Prepare a ten-fold dilution of the tested vaccine in the diluent.</li>
		<li>Prepare 7 two-fold serial dilutions of the tested vaccine in diluent as indicated in Table 2.</li>
		<li>Distribute 200 &#181;L per well of each tested vaccine dilution in duplicate (wells H3/H4 to A3/A4).</li>
	</ol>
	</li>
	<li>Incubation/Washing of the ELISA plate
	<ol>
		<li>Cover the microplate with an adhesive film and incubate for 1 h at 37 &#176;C.</li>
		<li>Remove the film, aspirate carefully and transfer the content of each well into a recipient containing 2,5% sodium hypochlorite solution.</li>
		<li>To each well add 300 &#181;L of washing buffer.</li>
		<li>Aspirate carefully and transfer the content of each well into a recipient containing 2,5% sodium hypochlorite solution.</li>
		<li>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).</li>
		<li>Invert the microplate and let it dry on an adsorbent paper at room temperature for 1 min.</li>
	</ol>
	</li>
	<li>Binding of the peroxidase conjugated mAb-D1
	<ol>
		<li>Distribute 200 &#181;L per well of the recommended dilution (1/2000) of peroxidase-labeled mAb-D1 in diluent (approximate concentration of 1&#181;g/mL). A recommended concentration is indicated for each mAb-D1 batch and has to be periodically verified with the positive control.</li>
		<li>Cover the microplate with an adhesive film and incubate for 1 h at 37 &#176;C.</li>
		<li>Remove the film, aspirate carefully and transfer the content of each well into a recipient containing 2,5% sodium hypochlorite solution.</li>
		<li>Add to each well, 300 &#181;L of the washing buffer.</li>
		<li>Aspirate carefully and transfer the content of each well into a recipient containing 2,5% sodium hypochlorite solution.</li>
		<li>Repeat steps 5.5.4 and 5.5.5 five times to remove unbound peroxidase-labeled antibody (mAb D1).</li>
		<li>Invert the microplate and let it dry on an adsorbent paper at room temperature for 1 min.</li>
	</ol>
	</li>
	<li>Revelation using a substrate-chromogen
	<ol>
		<li>Distribute 200 &#181;L per well of substrate-chromogen solution.</li>
		<li>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).</li>
		<li>Stop the reaction by adding 50 &#181;L of stopping solution per well.</li>
		<li>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.</li>
		<li>Collect OD data as .xls or .xlsx file format for analysis.</li>
	</ol>
	</li>
	<li>Draw the reference vaccine curve, the trimeric glycoprotein content, as a function of optical density (492 nm)
	<ol>
		<li>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)</li>
		<li>Subtract the mean OD of the Blank (wells, H1/H2 in Table 2) from each calculated mean OD.</li>
		<li>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).</li>
		<li>Draw the reference curve by joining points (Figure 1).</li>
	</ol>
	</li>
</ol>

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><tbody><tr><td>Class II Biological Safety Cabinet</td><td>ThermoFisher Scientific</td><td>10445753</td><td>if titrating live virus</td></tr><tr><td>Clear Flat-Bottom Immuno Nonsterile 96-Well Plates, 400 &amp;micro;L, MAXISORP</td><td>ThermoFisher Scientific</td><td>439454</td><td>good for binding to the loaded antibody</td></tr><tr><td>Equip Labo Polypropylene Laboratory Fume Hood</td><td>ThermoFisher Scientific</td><td>12576606</td><td>for the preparation of sulfuric acid</td></tr><tr><td>Immunology Plate Strong&lt;br/&gt; Adsorption MAXISORP Flat Bottom&lt;br/&gt; Well F96</td><td>Dutscher</td><td>55303</td><td>good for binding to the loaded antibody</td></tr><tr><td>Microplate Sealing Tape(100 sheets)</td><td>ThermoFisher Scientific</td><td>15036</td><td></td></tr><tr><td>Microplate&amp;nbsp; single mode reader Sunrise</td><td>TECAN</td><td></td><td></td></tr><tr><td>Microplate shaker-incubator</td><td>Dutscher</td><td>441504</td><td></td></tr><tr><td>Microplate washer Wellwash</td><td>ThermoFisher Scientific</td><td>5165000</td><td></td></tr><tr><td>Multichannel pipette (30-300 &amp;micro;L) 12 channels</td><td>ThermoFisher Scientific</td><td>4661180N</td><td></td></tr><tr><td>Single Channel pipettes (Kit 2 : Finnpipettes F2 0.2-2 &amp;mu;L micro, 2-20 &amp;mu;L, 20-200 &amp;mu;L &amp;amp; 100-1000 &amp;mu;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-vitro-elisa-test-to-evaluate-rabies-vaccine-potency

Research

JoVE Journal

Immunology and Infection

12.3K Views.  Institut Pasteur. 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. 

Video: In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription polymerase chain reaction (nested RT-PCR) was developed to detect the conserved region of the nucleoprotein (N) gene of lyssaviruses. The method applies reverse transcription (RT) using viral RNA as template and oligo (dT)15 and random hexamers as primers to synthesize the viral complementary DNA (cDNA). Then, the viral cDNA is used as a template to amplify an 845 bp N gene fragment in first-round PCR using outer primers, followed by second-round nested PCR to amplify the final 371 bp fragment using inner primers. This method can detect different genetic clades of rabies viruses (RABV). The validation, using 9,624 brain specimens from eight domestic animal species in 10 years of clinical rabies diagnoses and surveillance in China, showed that the method has 100% sensitivity and 99.97% specificity in comparison with the direct fluorescent antibody test (FAT), the gold standard method recommended by the World Health Organization (WHO) and the World Organization for Animal Health (OIE). In addition, the method could also specifically amplify the targeted N gene fragment of 15 other approved and two novel lyssavirus species in the 10th Report of the International Committee on Taxonomy of Viruses (ICTV) as evaluated by a mimic detection of synthesized N gene plasmids of all lyssaviruses. The method provides a convenient alternative to FAT for rabies diagnosis and has been approved as a National Standard (GB/T36789-2018) of China.

A pan-lyssavirus nested reverse transcription polymerase chain reaction has been developed to detect specifically all known lyssaviruses. Validation using rabies brain samples of different animal species showed that this method has a sensitivity and specificity equivalent to the gold standard fluorescent antibody test and could be used for routine rabies diagnosis.

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

Genetics

Enhanced Rabies Surveillance Using a Direct Rapid Immunohistochemical Test

Laboratory-based surveillance is integral for rabies prevention, control and management efforts. While the DFA is the gold standard for rabies diagnosis, there is a need to validate additional diagnostic techniques to improve rabies surveillance, particularly in developing countries. Here, we present a standard protocol for the DRIT as an alternative, laboratory or field-based testing option that uses light microscopy as compared to the DFA. Touch impressions of brain tissue collected from suspect animals are fixed in 10% buffered formalin. The DRIT uses rabies virus-specific monoclonal or polyclonal antibodies (conjugated to biotin), a streptavidin-peroxidase enzyme, and a chromogen reporter (such as acetyl 3-amino-9-ethylcarbazole) to detect viral inclusions within infected tissue. In approximately 1 h, a brain tissue sample can be tested and interpreted by the DRIT. Evaluation of suspect animal brains tested from a variety of species in North America, Asia, Africa, and Europe have illustrated high sensitivity and specificity by the DRIT approaching 100% with results compared to DFA. Since 2005, the United States Department of Agriculture&#8217;s Wildlife Services (USDA WS) program has conducted large-scale enhanced rabies surveillance efforts using the DRIT to test &#62;94,000 samples collected from wildlife in strategic rabies management areas. The DRIT provides a powerful, economical tool for rabies diagnosis that can be used by laboratorians and field biologists to improve current rabies surveillance, prevention and control programs globally.

The direct rapid immunohistochemical test (DRIT) offers a World Organization for Animal Health and World Health Organization (OIE/WHO) recognized alternative to the direct fluorescent antibody (DFA) test for rabies diagnosis. This test allows for field-based applications that can be performed in approximately 1 h upon brain impressions using light microscopy.

Laboratory-based surveillance is integral for rabies prevention, control and management efforts. While the DFA is the gold standard for rabies diagnosis, ...

Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis

Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to confirm rabies diagnosis but have only recently been accepted by the OIE (World Organisation for Animal Health) as a primary method to detect rabies infection. Real-time RT-PCR assays provide real-time data, and are closed-tube systems, minimizing the risk of contamination during setup. DNA intercalating fluorochrome real-time RT-PCR assays do not require expensive probes, minimizing the cost per sample, and when the primers are designed in conserved regions, assays that are specific across virus genera rather than specific to just one virus species are possible. Here we describe a pan-lyssavirus SYBR real-time RT-PCR assay that detects lyssaviruses across the Lyssavirus genus, including the most divergent viruses IKOV, WCBV and LLEBV. In conjunction with dissociation curve analysis, this assay is sensitive and specific, with the advantage of detecting all lyssavirus species. The assay has been adopted in many diagnostic laboratories with quality assured environments, enabling robust, rapid, sensitive diagnosis of animal and human rabies cases.

This real-time RT-PCR using dsDNA intercalating dye is suitable to diagnose lyssavirus infections. The method begins with RNA extracted from rabies suspected ante-mortem or post-mortem samples, detailing master mix preparation, RNA addition, setup of the real-time machine and correct interpretation of results.

Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to ...

Field Postmortem Rabies Rapid Immunochromatographic Diagnostic Test for Resource-Limited Settings with Further Molecular Applications

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, animals suspected as rabies-positive must be submitted to a postmortem confirmation using classical or molecular laboratory methods. However, most endemic areas are in low- and middle-income countries where animal rabies diagnosis is restricted to central veterinary laboratories. Poor availability of surveillance infrastructure leads to serious disease underreporting from remote areas. Several diagnostic protocols requiring low technical expertise have been recently developed, providing opportunity to establish rabies diagnosis in decentralized laboratories. We present here a complete protocol for field postmortem diagnosis of animal rabies using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to the final interpretation. We complete the protocol by describing a further use of the device for molecular analysis and viral genotyping. RIDT easily detects rabies virus and other lyssaviruses in brain samples. The principle of such tests is simple: brain material is applied on a test strip where gold conjugated antibodies bind specifically to rabies antigens. The antigen-antibody complexes bind further to fixed antibodies on the test line, resulting in a clearly visible purple line. The virus is inactivated in the test strip, but viral RNA can be subsequently extracted. This allows the test strip, rather than the infectious brain sample, to be safely and easily sent to an equipped laboratory for confirmation and molecular typing. Based on a modification of the manufacturer&#39;s protocol, we found increased test sensitivity, reaching 98% compared to the gold standard reference method, the direct immunofluorescence antibody test. The advantages of the test are numerous: rapid, easy-to-use, low cost and no requirement for laboratory infrastructure, such as microscopy or cold-chain compliance. RIDTs represent a useful alternative for areas where reference diagnostic methods are not available.

We present a complete protocol for postmortem diagnosis of animal rabies under field conditions using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to final interpretation. We also describe further applications using the device for molecular analysis and viral genotyping.

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, ...

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. While the direct fluorescent antibody (DFA) test or the direct rapid immunohistochemical test (DRIT) are most commonly utilized for the antigen detection, both tests require fresh and/or frozen tissues for impressions on slides prior to the antigen detection using antibodies. If samples are collected and fixed in formalin, neither test is optimal for the antigen detection, however, testing can be performed by conventional immunohistochemistry (IHC) after embedding in paraffin blocks and sectioning. With this IHC method, tissues are stained with anti-rabies antibodies, sections are deparaffinized, antigen retrieved by partial proteolysis or other methods, and incubated with primary and secondary antibodies. Antigens are stained using horseradish peroxidase / amino ethyl carbazole and counterstained with hematoxylin for the visualization using a light microscope. In addition to the specific antigen detection, formalin fixation offers other advantages like the determination of histological changes, relaxed conditions for specimen storage and transport (under ambient temperatures), ability to test retrospective cases and improved biological safety through the inactivation of infectious agents.

Here, we present an immunohistochemistry test protocol for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues.

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. ...

Quantitation of Rabies Virus in Various Bovine Brain Structures

Bovine paralytic rabies (BPR) is a form of viral encephalitis that is of substantial economic importance throughout Latin America, where it poses a major zoonotic risk. Here, our objective was to utilize a laboratory protocol to determine the relative copy number of the rabies virus (RABV) genome in different bovine brain anatomical structures using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). qRT-PCR quantifies the specific number of gene copies present in a sample based on fluorescence emitted after amplification that is directly proportional to the amount of target nucleic acid present in the sample. This method is advantageous owing to its short duration, reduced risk of contamination, and potential to detect viral nucleic acids in different samples more easily compared to other techniques. The brains of six rabid animals were divided into six anatomical structures, namely the Ammon&#8217;s horn, cerebellum, cortex, medulla, pons, and thalamus. All brains were identified as positive for RABV antigens based on a direct immunofluorescence test. The same anatomical structures from the brains of four RABV-negative bovines were also assessed. RNA was extracted from each structure and used for qRT-PCR. An assay was performed to determine the copy numbers of RABV genes using an in vitro transcribed nucleoprotein gene. The standard curve used to quantify viral RNA exhibited an efficiency of 100% and linearity of 0.99. Analysis revealed that the cortex, medulla, and thalamus were the ideal CNS portions for use in RABV detection, based on the observation that these structures possessed the highest levels of RABV. The test specificity was 100%. All samples were positive, no false positives were detected. This method can be used to detect RABV in samples that contain low levels of RABV during diagnosis of BPR.

This protocol presents a qRT-PCR-based approach for determining the rabies virus nucleoprotein (N) gene copy number within various bovine brain anatomical structures using in vitro transcription.

Bovine paralytic rabies (BPR) is a form of viral encephalitis that is of substantial economic importance throughout Latin America, where it poses a major ...

Fabrication of Pulsatile Polymeric Microparticles Encapsulating Rabies Antigen

The current guidelines for rabies post-exposure prophylaxis require multiple injections administered over several weeks. This can be disproportionately burdensome to those living in low- and middle-income countries (LMICs), where the majority of deadly exposures to rabies occur. Different drug delivery strategies have been explored to condense vaccine regimens to a single injection by encapsulating antigens into polymeric particles. However, harsh stressors during the encapsulation process can cause denaturation of the encapsulated antigen. This article describes a method for encapsulating the rabies virus (RABV) antigen into polymeric microparticles that exhibit tunable pulsatile release. This method, termed Particles Uniformly Liquified and Sealed to Encapsulate Drugs (PULSED), generates microparticles using soft lithography to create inverse polydimethylsiloxane (PDMS) molds from a multi-photon, 3D-printed master mold. Poly(lactic-co-glycolic acid) (PLGA) films are then compression-molded into the PDMS molds to generate open-faced cylinders that are filled with concentrated RABV using a piezoelectric dispensing robot. These microstructures are then sealed by heating the top of the particles, allowing the material to flow and form a continuous, nonporous polymeric barrier. Post-fabrication, an enzyme-linked immunosorbent assay (ELISA) specific to the detection of intact trimeric rabies virus glycoprotein is used to confirm the high recovery of immunogenic antigen from the microparticles.

This method describes the encapsulation of the rabies antigen into biodegradable polymeric microparticles with structural and material properties that enable pulsatile release after a predetermined delay. Enzyme-linked immunosorbent assay (ELISA) assessment of the antigen retrieved from the particle core confirms the presence of intact trimeric rabies virus glycoprotein through particle fabrication.

The current guidelines for rabies post-exposure prophylaxis require multiple injections administered over several weeks. This can be disproportionately ...

Bioengineering

Quick and Early Detection of Rabies Antibodies to Evaluate the Immune Response in a Patient

Detection of Rabies IgG and IgM Antibodies Using the Rabies Indirect Fluorescent Antibody Test

The rabies indirect fluorescent antibody (IFA) test was developed to detect various rabies-specific antibody isotypes in sera or cerebral spinal fluid. This test provides rapid results and can be used to detect rabies antibodies in several different scenarios. The rabies IFA test is especially useful for the quick and early detection of antibodies to evaluate the immune response in a patient who has developed rabies. Although other methods for antemortem rabies diagnosis take precedence, this test may be utilized to demonstrate recent rabies virus exposure through antibody detection. The IFA test does not provide a virus-neutralizing antibody (VNA) titer, but the pre-exposure prophylaxis (PrEP) response can be evaluated through positive or negative antibody presence. This test can be utilized in various situations and can provide results for a number of different targets. In this study, we used several paired serum samples from individuals who received PrEP and demonstrated their rabies antibody presence over time using the IFA test.

Author Spotlight: Rabies-Specific Antibody Isotypes Detection in Sera or Cerebral Spinal Fluid Using an IFA Test 

The aim of this manuscript is to examine the use of the rabies indirect fluorescent antibody test for the detection of rabies-specific IgG and IgM antibodies.

The rabies indirect fluorescent antibody (IFA) test was developed to detect various rabies-specific antibody isotypes in sera or cerebral spinal fluid. ...

Postmortem Diagnosis of Rabies in Animals by the Updated, Multiplexed LN34 Real-Time Reverse Transcription-Polymerase Chain Reaction Assay

Rabies is a fatal zoonotic disease caused by Lyssavirus rabies (RABV) and related negative strand RNA viruses from the Lyssavirus genus (family Rhabdoviridae). The LN34 assay targets the highly conserved leader region and nucleoprotein gene of the lyssavirus genome and utilizes degenerate primers and a TaqMan probe containing locked nucleotides to detect RNA across the diverse Lyssavirus genus. A negative finding for rabies should be made only if a full cross-section of the brain stem and three lobes of the cerebellum are examined; however, identification of lyssavirus RNA in any tissue is diagnostic of rabies infection. Tissue is collected and homogenized in TRIzol reagent, which also inactivates the virus. RNA extraction is performed using a spin column-based commercial extraction kit. Master mixes are prepared in a clean space and aliquoted into a 96 well plate before adding sample RNA. In clinical settings, each sample is tested by real-time RT-PCR for the presence of lyssavirus RNA in triplicate and singly for host &#946; -actin mRNA. Positive and negative controls are included at extraction and real-time RT-PCR steps of the protocol. Data analysis involves manual adjustment of the thresholds to standardize Ct values across instrument runs. Positive results are determined by the presence of typical amplification in the pan-lyssavirus assay (Ct &#8804; 35). Negative results are determined by the absence of typical amplification in the pan-lyssavirus assay and detection of host &#946;-actin mRNA (Ct &#8804; 33). Observation of values outside of these ranges or failure of the assay controls can invalidate the run or result in inconclusive results for a specimen. The protocol should be closely followed to ensure high assay sensitivity and specificity. Procedural modifications can affect assay performance and lead to false positive, false negative, or uninterpretable results.

This protocol demonstrates the pan-lyssavirus LN34 real-time reverse transcription-polymerase chain reaction (RT-PCR) assay from tissue collection to result interpretation, including updates to primer sequences and formulations to improve assay performance for some non-rabies lyssaviruses and lagomorphs. We also demonstrate the assay setup for a single-well LN34 multiplexed (LN34M) format.

Rabies is a fatal zoonotic disease caused by Lyssavirus rabies (RABV) and related negative strand RNA viruses from the Lyssavirus genus (family ...

Biology

Using a Sandwich ELISA to Determine the Immunogenic Glycoprotein Content in a Vaccine

Source: Jallet, C. et al., <a href="https://app.jove.com/v/59641">In Vitro ELISA Test to Evaluate Rabies Vaccine Potency.</a>&#160;J. Vis. Exp. (2020)
This video showcases an indirect enzyme-linked immunosorbent assay or ELISA sandwich immunocapture assay designed to assess immunogenic glycoprotein levels in vaccines. The assay plate, containing glycoprotein-specific antibodies, is incubated with serially diluted vaccine samples, including reference and test vaccines, followed by immunoassay to quantify glycoprotein content in the test vaccine.

1. Security precautions
NOTE: This method is applicable to both live rabies virus (RABV) and inactivated vaccine.

	Use good Laboratory Practice and ...