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 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-vitro-elisa-test-to-evaluate-rabies-vaccine-potency

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JoVE Journal

Immunology and Infection

12.2K 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

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

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, ...

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. ...

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is requisite to phagocytosis and killing, the assay is an alternative method to&#160;opsono-phagocytic killing assays. An advantage of the opsono-adherence&#160;assay is the option of using inactivated pathogens and mammalian cell lines, which allows standardization across multiple experiments. The use of an inactivated pathogen in the assay also facilitates work with biosafety level 3 infectious agents and other virulent pathogens. In our work, the opsono-adherence assay was used to assess the functional ability of antibodies, from sera of animals immunized with an anthrax capsule-based vaccine, to induce adherence of fixed Bacillus anthracis to a mouse macrophage cell line, RAW 264.7. Automated fluorescence microscopy was used to capture images of bacilli adhering to macrophages. Increased adherence was correlated with the presence of anti-capsule antibodies in the serum. Non-human primates that exhibited high serum anti-capsule antibody concentrations were protected from anthrax challenge. Thus, the opsono-adherence assay can be used to elucidate the biological functions of antigen specific antibodies in sera, to evaluate the efficacy of vaccine candidates and other therapeutics, and to serve as a possible correlate of immunity.&#160;&#160;&#160;&#160;&#160;&#160;

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

The opsono-adherence assay is an alternative method to the opsono-phagocytic killing assay to evaluate the opsonic functions of antibodies in vaccine development.

The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...