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 incubated to allow for any rabies-specific antibodies to attach to the virus antigen7. Following a washing procedure, a fluorescently labeled anti-human IgG or IgM antibody is applied and binds to any virus-bound antibody from the sample application. Labeled antibodies can then be visualized under a fluorescent microscope.
When preparing to perform this assay, it is important to determine the best preparation of patient samples, controls, and conjugates. Initial patient samples were screened at a low dilution factor or undiluted to test for any antibody presence. Paired samples or previously screened samples that demonstrated a high level of antibody were then further diluted to the end point. Initial patient sample dilutions for IgG testing were prepared in commercially available IFA diluent. IgM testing samples were initially prepared in a commercially available IgG blocking reagent. Subsequent serial dilutions for both tests were then prepared in PBS.
The controls used for testing were obtained from known recipients of rabies PrEP or from individuals with no history of rabies vaccination. All control samples were tested for antibody presence prior to use. The IgG control was obtained from a rabies vaccine recipient with an established rabies-neutralizing antibody titer confirmed by FAVN. The IgM-positive control sample was obtained from a rabies vaccine recipient approximately 2 weeks after completion of vaccination.
The use of appropriate conjugate is another vital aspect of performing the IFA test. The conjugate used for each test depends on the antibody isotype target for that assay. In this case, commercially available FITC-labeled anti-human IgG and anti-human IgM antibodies were utilized. Working antibody conjugate dilutions were determined prior to testing based on the manufacturer&#39;s recommendation.
There are several key steps in the procedure that will ensure successful execution of the IFA test, perhaps the most important being antigen slide preparation. Reaching approximately 50% infectivity provides abundant binding sites for available antibodies, while also creating clarity when reading and grading samples. The rabies-negative cells create a contrasting background to visualize labeled antibodies better. Non-specific staining can also present a problem when performing fluorescent staining using complex sample matrices, such as serum. The use of IgG inactivation reagents (e.g., Gullsorb) helps to cut down on non-specific staining due to interfering IgG antibodies when assessing the presence of IgM8. The proper preparation of materials and incorporation of special reagents into the procedure help decrease problematic staining while preserving high-quality results.
A multitude of testing methods have been developed that focus on rabies antibody detection, quantification, and identification. Each of these tests offers results that can be used in various ways depending on the information sought. Rabies IFA testing is a powerful tool for identifying virus-specific antibody isotypes, and the results can be ready in a relatively short period of time compared to other testing methods, such as the RFFIT and FAVN tests.
Although the IFA test can detect rabies antibodies in a sample, it does not provide a standardized quantification of the antibodies. The IFA test provides a serum dilution factor at which antibody detection ends. In comparison, the RFFIT and FAVN tests quantify the neutralizing capabilities of antibodies in a sample, resulting in a titer of international units (IU), providing a more detailed and standardized result. Results from a FAVN or an RFFIT test demonstrate the presence of neutralizing antibodies, which have been determined to be IgG antibodies9. The role of IgM in neutralizing activity is understood to be limited due to its structure10. Therefore, these tests do not specifically detect the presence of IgM.
The type of test and its result can be somewhat problematic when applying them to a specific problem. The concept of a protective level of antibody protection against rabies virus infection is no longer used to evaluate the immune response to rabies vaccination. Previously, a protective response was defined as &#8805;0.5 IU. However, current publications typically refer to the antibody response following immunization as acceptable (&#8805;0.5 IU) or unacceptable (&#60;0.5 IU). As stated, the IFA test does not provide an antibody titer in IU and should not be used to derive a level of protection against rabies. Using the IFA test while evaluating a patient who has developed rabies may not always result in a positive antibody presence due to variations in immune response throughout the disease. Due to the near 100% lethality of a rabies infection, it is important to consider the test and how much information can be safely derived from these results11. For these reasons, it is always best to evaluate all rabies antibody testing methods and determine which works best in a given scenario.

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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 &#8211; w/Earle&#8217;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 Teflon 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&#160;</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 or 6500</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 sample reached an end point grade of 1-2+.
The results from testing each time point following PrEP demonstrate the ability of the assay to detect varying levels of antibody presence. Shortly after initial vaccination, high levels of both IgM and IgG were present in the patient samples, as seen in Figure 1. Areas of bright green fluorescent cell staining indicate positive antibody presence; red cells are rabies-negative cells where no antibody could bind. Approximately 6 months after vaccination, significantly lower levels of both IgM and IgG were present in the patient samples, but IgM had dropped out almost completely. At the final time point, 18 months following vaccination, IgM antibodies were not detected in any patient samples, as demonstrated in Figure 2 where no green fluorescent cell staining is observed. However, IgG levels persisted and stayed similar to the levels detected at the 6 month time point following the initial decrease from the 2 week time point. The results for IgG and IgM detection in samples from 2 weeks following vaccination, 6 months following vaccination, and 18 months following vaccination are listed in Table 1, Table 2, and Table 3, respectively. Figure 3 shows the flow chart of the execution stages.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65459/65459fig01.jpg" /> 
Figure 1: Rabies IgM IFA positive staining. Patient serum at a 1:8 dilution demonstrated a positive staining pattern shortly following completion of the PrEP vaccine series. The scale bar on the image represents 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65459/65459fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65459/65459fig02.jpg" /> 
Figure 2: Rabies IgM IFA negative staining. Patient serum at a 1:2 dilution demonstrated no positive staining approximately 18 months following completion of the PrEP vaccine series. The scale bar on image represents 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65459/65459fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65459/65459fig03.jpg" /> 
Figure 3: Rabies IFA flowchart. Flowchart showing execution stages of the major steps in the IFA procedure to aid in process visualization. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65459/65459fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Patient Sample</td>
			<td>IgM</td>
			<td>IgG</td>
		</tr>
		<tr>
			<td>PS1-1</td>
			<td>1:32</td>
			<td>1:512</td>
		</tr>
		<tr>
			<td>PS2-1</td>
			<td>1:8</td>
			<td>1:128</td>
		</tr>
		<tr>
			<td>PS3-1</td>
			<td>1:16</td>
			<td>1:256</td>
		</tr>
		<tr>
			<td>PS4-1</td>
			<td>1:64</td>
			<td>1:512</td>
		</tr>
		<tr>
			<td>PS5-1</td>
			<td>1:16</td>
			<td>1:128</td>
		</tr>
	</tbody>
</table>
Table 1:&#160;Results 2 weeks post-vaccination. Patient sample results from serum samples collected approximately 2 weeks following completion of the PrEP vaccine series.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Patient Sample</td>
			<td>IgM</td>
			<td>IgG</td>
		</tr>
		<tr>
			<td>PS1-2</td>
			<td>1:2</td>
			<td>1:128</td>
		</tr>
		<tr>
			<td>PS2-2</td>
			<td>1:1</td>
			<td>1:128</td>
		</tr>
		<tr>
			<td>PS3-2</td>
			<td>1:1</td>
			<td>1:64</td>
		</tr>
		<tr>
			<td>PS4-2</td>
			<td>1:1</td>
			<td>1:256</td>
		</tr>
		<tr>
			<td>PS5-2</td>
			<td>1:1</td>
			<td>1:32</td>
		</tr>
	</tbody>
</table>
Table 2:&#160;Results 6 months post-vaccination. Patient sample results from serum samples collected approximately 6 months following completion of the PrEP vaccine series.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Patient Sample</td>
			<td>IgM</td>
			<td>IgG</td>
		</tr>
		<tr>
			<td>PS1-3</td>
			<td>Not Detected</td>
			<td>1:128</td>
		</tr>
		<tr>
			<td>PS2-3</td>
			<td>Not Detected</td>
			<td>1:128</td>
		</tr>
		<tr>
			<td>PS3-3</td>
			<td>Not Detected</td>
			<td>1:64</td>
		</tr>
		<tr>
			<td>PS4-3</td>
			<td>Not Detected</td>
			<td>1:64</td>
		</tr>
		<tr>
			<td>PS5-3</td>
			<td>Not Detected</td>
			<td>1:32</td>
		</tr>
	</tbody>
</table>
Table 3:&#160;Results 18 months post-vaccination. Patient sample results from serum samples collected approximately 18 months following completion of the PrEP vaccine series.

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

1.8K Views.  New York State Department of Health. 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.

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

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 ...

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, immunodiagnostics and more recently as therapeutic agents. Traditional methods for labeling antibodies with small molecules require purified antibodies at relatively high concentration, involve multiple dialysis steps and have limited throughput. However, several applications, including the field of Antibody Drug Conjugates (ADCs), will benefit from new methods that will allow labeling of antibodies directly from cell media. Such methods may allow antibodies to be screened in biologically relevant assays, for example, the receptor-mediated antibody internalization assay in the case of ADCs. Here, we describe a method (on-bead method) that enables labeling of small amounts of antibodies directly from cell media. This approach utilizes high capacity magnetic Protein A and Protein G affinity beads to capture antibodies from the cell media followed by labeling with small molecules using either amine or thiol chemistry and subsequent elution of the labeled antibodies. Taking fluorescent dyes as surrogates for small molecules, we demonstrate the on-bead labeling of three different mouse antibodies directly from cell media using both amine and thiol labeling chemistry. The high binding affinity of antibodies to Protein A and Protein G ensures high recoveries as well as high purity of the labeled antibodies. In addition, use of magnetic beads allows multiple samples to be handled manually, thereby significantly improving labeling throughput.

The on-bead method for labeling antibodies with small molecules enables labeling of a small amount of antibodies directly from cell media. This method is compatible with amine and thiol chemistry, and can handle multiple samples in parallel, manually or using automated platforms.

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, ...

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 ...

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. ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.