We are grateful to the New York State Department of Health Wadsworth Center for supporting this project.

The following protocol has been approved for the ethical use of human samples by the New York State Department of Health Wadsworth Center for assay development, protocol approval number #03-019.
1. Safety
<ol>
	<li>Don personal protective equipment (PPE), at minimum eye protection (glasses or face shield), a surgical mask, and non-latex gloves.</li>
	<li>Ensure personnel are vaccinated for rabies and that a titer of &#8805;0.5 IU/mL has been demonstrated within the past 6 months.</li>
</ol>
2. Antigen slide preparation
NOTE: Perform all virus, CSF, and serum manipulations in a biosafety cabinet (BSC) using universal precautions.
<ol>
	<li>Prepare 20 mL of mouse neuroblastoma or BHK-21 cells to a concentration of 3.0 x 105 cells/mL in Eagle&#39;s minimum essential media supplemented with 10% fetal bovine serum (EGM) and keep cold until use.</li>
	<li>Prepare CVS-11 virus by diluting in EGM to a working dilution of 1.0 x 106.5 50% tissue culture infectious dose (TCID50) per milliliter and keep cold until ready for use. 
	NOTE: TCID50 is determined by the Reed and Muench method published previously5.</li>
	<li>Clean a humidity slide chamber and polytetrafluoroethylene (PTFE)-coated well-microscope slides with 70% ethanol and allow to air-dry in the BSC.</li>
	<li>Add distilled water (dH2O) to strips of absorbent paper in the slide chamber to ensure the humidity remains constant throughout the procedure.</li>
	<li>Using a pencil, label each slide to be used with the lot number, date, cell type, and any other identifying information required for storage, and place in the slide chamber. 
	NOTE: Most markers, pens, or labels will not withstand the future acetone fixation step (step 2.9).</li>
	<li>Apply 50 &#181;L of virus dilution to each well on the microscope slides with a repeating pipette. Then, apply 50 &#181;L of cell dilution to each well, being careful not to contaminate the pipette tip with the virus already on the well.</li>
	<li>Close the humidity slide chamber and place it in the humid incubator at 34-36 &#176;C. Assess the cell infectivity after 24 h. 
	NOTE: Perform steps 2.8-2.12 on only one slide.</li>
	<li>Remove the slide from the humidity chamber and carefully aspirate the supernatant. Wash the slide in a phosphate-buffered saline (PBS)-filled Coplin jar for 2 min, then allow the slide to air-dry.</li>
	<li>Place the slide in the Coplin jar and fix the slide in cold acetone for a minimum of 1 h in a -20 &#176;C freezer approved for flammable materials. Perform all acetone pouring and air-drying procedures in a fume hood.</li>
	<li>Allow the acetone to flash off and slide to dry. Apply rabies direct fluorescent antibody (DFA) conjugate prepared according to the manufacturer&#39;s instructions to the wells of the slide and incubate for 30 min in a 34-36 &#176;C humid incubator.</li>
	<li>Wash the slide in Coplin jars of PBS twice for 2 min each. Air-dry the slide.</li>
	<li>Mount a coverslip with 0.05 M Tris, 0.15 M NaCl (pH 9.0), and 20% glycerol mountant, and read the slide using a fluorescent microscope at 200x magnification to assess the infectivity. If the cells are not approximately 50% infected, repeat steps 2.7-2.12 the following day until the desired infectivity is reached. 
	NOTE: Cell infectivity is approximated based on visually assessing the ratio of negative cells to rabies-positive cells on the slide well. Negative cells appear red in color, while positive cells show green fluorescent staining.</li>
	<li>Remove the remaining slides from the incubator and humidity chamber. Carefully aspirate the supernatant from each well, then place the slides in a Coplin jar(s) with PBS for 1-2 min. Air-dry the slides for approximately 30 min. Store the slides at -80 &#176;C until ready for use.</li>
</ol>
3. Sample preparation
<ol>
	<li>Prepare patient serum or CSF sample dilutions for testing. Prepare the necessary conjugate for a proper working concentration diluted in PBS with 0.05% Evans blue. 
	&#8203;NOTE: Keep the conjugate in the dark to maintain the fluorophore integrity prior to application</li>
</ol>
4. IFA procedure
<ol>
	<li>Remove the necessary number of prepared antigen slides needed for each assay and allow the slides to defrost and dry completely.</li>
	<li>Place the slides in a Coplin jar(s) and fix the slides in cold acetone for between 2 h to overnight in a -20 &#176;C freezer approved for flammable materials. Remove the slides from the acetone and allow to air-dry.</li>
	<li>Place the slides in a humidity chamber box inside the BSC with dH2O-soaked absorbent strips to maintain humidity. Apply 50 &#181;L of each sample dilution, control sample, or PBS to the pre-determined well. 
	NOTE: Each slide must contain an appropriate number of patient sample wells, positive control, negative control, and PBS cell control well.</li>
	<li>Place the closed humidity slide chamber in a 37 &#176;C, 5% CO2 humid incubator. Incubate the slides for 30 min, then remove the humidity slide chamber from the incubator and place it into a BSC.</li>
	<li>Use an aspirator tip to carefully aspirate the supernatant from each well, ensuring not to disturb the cell monolayer. Use a sterile dropper pipette to apply one drop of PBS to each well.</li>
	<li>Repeat careful aspiration, then place each slide in a PBS-filled Coplin jar and wash twice for a total of 15 min.</li>
	<li>Place the slides back in the humidity chamber box and apply 50 &#181;L of appropriate anti-human antibody conjugate to each well. Repeat steps 4.4 to 4.6.</li>
	<li>Allow the slides to air-dry, mount a coverslip with mounting media, and read the slides under fluorescent microscope.</li>
</ol>
5. Slide analysis
<ol>
	<li>Grade samples based on the staining pattern and fluorescence intensity compared to positive control samples with confirmed high anti-rabies antibody titer.</li>
	<li>Grade the samples on a scale of negative, 1+, 2+, 3+, and 4+, with a negative result showing no fluorescent staining and 4+ representing a bright green apple color fluorescence with a staining pattern similar to positive control samples1. 
	NOTE: The green apple color applies only to FITC-labeled antibodies; the color varies depending on fluorophore selection.</li>
	<li>Assign the samples an end point value represented by the dilution factor at which the sample displays a 1-2+ grade. Test the samples at a higher dilution factor if the end point is not reached in the initial assay.</li>
</ol>

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

<ol>
	<li>Rupprecht, C. E., Fooks, A. R., Abela-Ridder, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+Techniques+in+Rabies.+Volume+1.">Laboratory Techniques in Rabies. Volume 1.</a> World Health Organization. , 232-245 (2018).</li><li>Moore, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+of+rabies+serology:+defining+context+of+interpretation.">Challenges of rabies serology: defining context of interpretation.</a> Viruses. 13 (8), 1516 (2021).</li><li>Zajac, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+a+rabies+enzyme-linked+immunosorbent+assay+(ELISA)+targeting+IgM+and+IgG+in+human+sera.">Development and evaluation of a rabies enzyme-linked immunosorbent assay (ELISA) targeting IgM and IgG in human sera.</a> Viruses. , 40-49 (2019).</li><li>Mills, D. J., Lau, C. L., Mills, C., Furuya-Kanamori, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+persistence+of+antibodies+and+boostability+after+rabies+intradermal+pre-exposure+prophylaxis.">Long-term persistence of antibodies and boostability after rabies intradermal pre-exposure prophylaxis.</a> Journal of Travel Medicine. 29 (2), (2022).</li><li>Ramakrishnan, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+50%+endpoint+titer+using+a+simple+formula.">Determination of 50% endpoint titer using a simple formula.</a> World Journal of Virology. 5 (2), 85-86 (2016).</li><li>Rudd, R. J., Appler, K. A., Wong, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+cross-reactions+with+other+viral+encephalitides+in+the+indirect+fluorescent-antibody+test+for+diagnosis+of+rabies.">Presence of cross-reactions with other viral encephalitides in the indirect fluorescent-antibody test for diagnosis of rabies.</a> Journal of Clinical Microbiology. 51 (12), 4079-4082 (2013).</li><li>Fooks, A. R., Jackson, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Rabies: scientific basis of the disease and its management. , (2020).</li><li>Paldanius, M., Bloigu, A., Leinonen, M., Saikku, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Chlamydia+pneumoniae-specific+immunoglobulin+A+(IgA)+antibodies+by+the+microimmunofluorescence+(MIF)+method:+comparison+of+seven+fluorescein-labeled+anti-human+IgA+conjugates+in+an+in-house+MIF+test+using+one+commercial+MIF+and+one+enzyme+immunoassay+kit.">Measurement of Chlamydia pneumoniae-specific immunoglobulin A (IgA) antibodies by the microimmunofluorescence (MIF) method: comparison of seven fluorescein-labeled anti-human IgA conjugates in an in-house MIF test using one commercial MIF and one enzyme immunoassay kit.</a> Clinical and Diagnostic Laboratory Immunology. 10 (1), 8-12 (2003).</li><li>Rodriguez, M. C., Fontana, D., Garay, E., Prieto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+quantification+of+anti-rabies+glycoprotein+antibodies:+current+state+and+perspectives.">Detection and quantification of anti-rabies glycoprotein antibodies: current state and perspectives.</a> Applied Microbiology and Biotechnology. 105 (18), 6547-6557 (2021).</li><li>Katz, I. S. S., Guedes, F., Fernandes, E. R., Dos Ramos Silva, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+aspects+of+rabies:+a+literature+review.">Immunological aspects of rabies: a literature review.</a> Archives of Virology. 162 (1), 3251-3268 (2017).</li><li>Moore, S. M., Hanlon, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabies-specific+antibodies:+measuring+surrogates+of+protection+against+a+fatal+disease.">Rabies-specific antibodies: measuring surrogates of protection against a fatal disease.</a> PLoS Neglected Tropical Diseases. 4 (3), 595 (2010).</li></ol>

The authors have nothing to disclose.

The IFA test takes advantage of an antigen-antibody complex, allowing for a labeling site to visualize rabies-specific antibodies. Neuroblastoma or BHK cells are seeded on multi-well PTFE-coated microscope slides and inoculated with rabies virus lab strain CVS-11. Once the monolayer is confluent and the cells reach the desired infectivity of approximately 50%, the slides are stored until ready for use6.
Patient serum or CSF is applied to the infected cell monolayer and ...

The rabies indirect fluorescent antibody (IFA) test is used to detect various rabies-specific antibody isotypes in sera or cerebral spinal fluid. It is one of an arsenal of tests available for monitoring an antemortem rabies patient. It is especially useful for the early detection of antibodies to evaluate a patient's immune response to rabies infection. When used in conjunction with other tests, case history, and the patient's vaccination status, the IFA test can assist in determining exposure to rabies virus or a vaccine1. As the IFA test measures IgM and/or IgG, the values of the specific antibody can indicate an approximate time frame from exposure to the antigen1. This test may be useful in the listed applications or others not yet explored.
There are several rabies serological assays available. The rapid fluorescent focus inhibition test (RFFIT), fluorescent antibody virus neutralization (FAVN) test, or modifications of these are the primary methods for measuring rabies virus neutralizing antibodies (RVNAs)1. However, these tests do not differentiate IgM and IgG antibodies. When differentiating antibody isotype is important in monitoring the rabies immune response, the rabies IFA and the rabies enzyme-linked immunosorbent assay (ELISA) tests are used, but they do not measure RVNAs. Although the IFA and ELISA tests can be used to determine the presence of rabies-specific antibodies in a sample, there are some differences in how they are executed. The IFA test utilizes a cell-cultured live virus as its antigen substrate, whereas a typical ELISA for rabies detection uses one or more of the viral proteins. In a laboratory setting where the rabies virus can be cultured, the IFA test may be more easily performed instead of purchasing or cultivating individual viral proteins for the ELISA. The purpose of testing and the information garnered from the results of any rabies serological assay should be considered when determining which to choose2.
IgM is the first to respond, increasing until class switching is observed at around day 28, at which point IgG becomes the predominant circulating antibody3. Hence, IgM would only be expected for a limited amount of time following exposure to rabies virus or vaccination. Testing both serum and cerebrospinal fluid (CSF) can indicate if the exposure was through vaccination, in which antibodies would be seen only in sera, or from a viral infection, which would potentially show antibodies in CSF1.
It has been established that rabies antibodies persist for several years following pre-exposure prophylaxis (PrEP)4. The IFA test can be a useful tool to demonstrate this at different time points following vaccination or exposure.

<table><tbody><tr><td>25x55mm glass cover slips</td><td>Any</td><td></td><td></td></tr><tr><td>Acetone</td><td>Any</td><td></td><td></td></tr><tr><td>Anti-Human IgG Labeled Conjugate</td><td>Sigma-Aldrich</td><td>F9512</td><td></td></tr><tr><td>Anti-Human IgM Labeled Conjugate</td><td>SeraCare</td><td>5230-0286</td><td></td></tr><tr><td>Aspirating pipette tip</td><td>Any</td><td></td><td></td></tr><tr><td>BHK-21 Cells</td><td>ATCC</td><td>CCL-10</td><td></td></tr><tr><td>BION IFA Diluent</td><td>MBL BION</td><td>DIL-9993</td><td></td></tr><tr><td>Cell Culture water</td><td>Sigma-Aldrich</td><td>W3500</td><td>EGM</td></tr><tr><td>Coplin Jars</td><td>Any</td><td></td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F2442</td><td>EGM</td></tr><tr><td>Fluorescent microscope with FITC filter</td><td>Any</td><td></td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G7893</td><td>Mountant</td></tr><tr><td>Gullsorb IgM inactivation reagent</td><td>Fisher Scientific</td><td>23-043-158</td><td>IgG Inactivation Reagent</td></tr><tr><td>L-Glutamine</td><td>Sigma-Aldrich</td><td>G-7513</td><td>EGM</td></tr><tr><td>Minimum Essential Media Eagle &amp;ndash; w/Earle&amp;rsquo;s salts, L-glutamine, and non-essential amino acids, w/o sodium bicarbonate</td><td>Sigma-Aldrich</td><td>M0643</td><td>EGM</td></tr><tr><td>Mouse Neuroblastoma Cells</td><td>ATCC</td><td>CCL-131</td><td></td></tr><tr><td>Multi-well PTFE coating glass slides</td><td>Any</td><td></td><td></td></tr><tr><td>PBS</td><td>Any</td><td></td><td>pH 7.6&amp;nbsp;</td></tr><tr><td>Penicillin</td><td>Sigma</td><td>P-3032</td><td>EGM</td></tr><tr><td>Rabies Direct Fluorescent Antibody Conjugate</td><td>Millipore Sigma</td><td>5100, 5500, 6500, RU5100, or RU5500</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S-5761</td><td>EGM</td></tr><tr><td>Sodium Chloride crystals</td><td>Sigma-Aldrich</td><td>S5886</td><td>Mountant</td></tr><tr><td>Sterile dropper</td><td>Any</td><td></td><td></td></tr><tr><td>Streptomycin sulfate salt</td><td>Sigma</td><td>S9137</td><td>EGM</td></tr><tr><td>Trizma pre-set crystals pH 9.0</td><td>Sigma-Aldrich</td><td>S9693</td><td>Mountant</td></tr><tr><td>Tryptose Phosphate Broth</td><td>BD</td><td>260300</td><td>EGM</td></tr><tr><td>Vitamin mix</td><td>Sigma-Aldrich</td><td>M6895</td><td>EGM</td></tr></tbody></table>

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.

All serum samples were collected from the patients at approximately the same time frames following PrEP. The samples were tested from five different patients at the following time points: 2 weeks after the final rabies vaccine inoculation, 6 months after the rabies vaccine series, and 18 months after the rabies vaccine series. Each serum sample was diluted in series and graded for both IgM and IgG presence, as described in protocol steps 5.2 and 5.3. The antibody value assigned represents the dilution factor where the sa...

Watch this Scientific Journal Video about Rabies-Specific Antibody Isotypes Detection in Sera or Cerebral Spinal Fluid Using an IFA Test at JoVE.com

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.

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. ...

detection-rabies-igg-igm-antibodies-using-rabies-indirect-fluorescent

Research

JoVE Journal

Immunology and Infection

2.0K Views.  New York State Department of Health. De IFA-test beoordeelt de antilichaamrespons tijdens rabiësinfectie of evalueert de immuunrespons van vaccins. Het kan worden gebruikt om de aanwezigheid van antilichamen in een monster vast te stellen en te onderscheiden welke antilichaamisotypen aanwezig zijn. Neutraliserende antilichaamtesten zijn de gouden standaard voor de detectie van rabiësantilichamen om de antilichaamtiter te beoordelen bij een persoon die profylaxe voor of na blootstelling heeft gekregen.Hoewel een IFA-test...

Video: Auteur in de schijnwerpers: detectie van rabiësspecifieke antilichaamisotypen in sera of cerebrale ruggenmergvloeistof met behulp van een IFA-test 

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

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Antibody Binding Specificity for Kappa (V&#954;) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents in standard laboratory techniques. In contrast, several human IgMs demonstrated proof of efficacy in cancer models and models of CNS disorders including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Reasons for their lack of consideration include difficulties to express, purify and stabilize IgM antibodies, challenge to identify (non-protein) antigens, low affinity binding and fundamental knowledge gaps in carbohydrate and lipid research. 
This manuscript uses HIgM12 as an example to provide a detailed protocol to detect antigens by Western blotting, immunoprecipitations and immunocytochemistry. HIgM12 targets polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM). Early postnatal mouse brain tissue from wild type (WT) and NCAM knockout (KO) mice lacking the three major central nervous system (CNS) splice variants NCAM180, 140 and 120 was used to evaluate the importance of NCAM for binding to HIgM12. Further enzymatic digestion of CNS tissue and cultured CNS cells using endoneuraminidases led us to identify PSA as the specific binding epitope for HIgM12.

We present a protocol to identify protein moieties containing antigens for human and mouse IgM antibodies with specific kappa (V&#954;) light chains. This protocol is not limited to IgM class antibodies but applies to all immunoglobulin isotypes that target their antigen with sufficiently high affinity during immunoprecipitations.

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents ...

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels of autoantibodies to specific antigens are typically detected with the enzyme-linked immunosorbent assay (ELISA) technique. However, screening for multiple autoantibodies with ELISA can be time-consuming and requires a large quantity of patient sample. The antigen microarray technique is an alternative method that can be used to screen for autoantibodies in a multiplex fashion. In this technique, antigens are arrayed onto specially coated microscope slides with a robotic microarrayer. The slides are probed with patient serum samples and subsequently fluorescent-labeled secondary antibodies are added to detect binding of serum autoantibodies to the antigens. The autoantibody reactivities are revealed and quantified by scanning the slides with a scanner that can detect fluorescent signals. Here we describe methods to generate custom antigen microarrays. Our current arrays are printed with 9 solid pins and can include up to 162 antigens spotted in duplicate. The arrays can be easily customized by changing the antigens in the source plate that is used by the microarrayer. We have developed a two-color secondary antibody detection scheme that can distinguish IgG and IgM reactivities on the same slide surface. The detection system has been optimized to study binding of human and murine autoantibodies.

We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest.

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels ...

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, ...

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.

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 ...

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 ...

An Immunohistochemistry Staining for the Detection of Rabies Virus Antigens

Using a microtome, make a 5-micrometer paraffin section. Float the section on a water bath at 38 degrees Celsius, and collect it onto glass slides. Label the slides with a reagent-resistant pen. Place the slides onto a tray, and melt in an oven at 55 to 60 degrees Celsius for one hour.
Then, remove the slides from the oven, and immediately deparaffinize them using three consecutive xylene rinses of five minutes each. After this, rehydrate the sections on the slide by sequential immersions in decreasing dilutions of ethanol to deionized water, as outlined in the text protocol.
Treat the slides with the protease for 30 minutes for proteolytic antigen retrieval, then, rinse the slides in PBST for 10 minutes, and treat them with 3% hydrogen peroxide for 10 minutes. After this, wash the slides again with PBST for 10 minutes.
To begin, remove one slide from the buffer, making sure to only handle one slide at a time, while the others remain submerged, and use a paper towel to blot off excess buffer from around the tissue section. Place the slides into a humidity chamber, and incubate with normal goat serum at room temperature for 15 minutes.
Next, incubate the slides in the humidity chamber with the optimal predetermined dilution primary anti-rabies antibody and negative control antibodies at room temperature for 60 minutes with no washes in between. After this, wash the slides with PBST for 10 minutes, and incubate in a humidity chamber with the biotinylated antibody at room temperature for 15 minutes.
Wash the slides again with PBST for 10 minutes. Incubate the slides in a humidity chamber with streptavidin-HRP complex at room temperature for 15 minutes, and wash the PBST for 10 minutes. Then, incubate with AEC in a humidity chamber at room temperature for 10 minutes. Wash the slides in deionized water for 10 minutes and counterstain with Gill&#39;s hematoxylin diluted 1 to 2 with deionized water for two minutes.
After this, rinse off the excess hematoxylin by dip-rinsing the slides in deionized water. Rinse the slides in Scott&#39;s tap water for 30 seconds, and wash in deionized water for 10 minutes. Remove the slides one at a time and mount them with water-soluble mounting medium. Then, use a light microscope to read the slides.

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

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