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

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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

1. Don personal protective equipment (PPE), at minimum eye protection (glasses or face shield), a surgical mask, and non-latex gloves.
2. Ensure personnel are vaccinated for rabies and that a titer of ≥0.5 IU/mL has been demonstrated within the past 6 months.

2. Antigen slide preparation

NOTE: Perform all virus, CSF, and serum manipulations in a biosafety cabinet (BSC) using universal precautions.

1. Prepare 20 mL of mouse neuroblastoma or BHK-21 cells to a concentration of 3.0 x 10⁵ cells/mL in Eagle's minimum essential media supplemented with 10% fetal bovine serum (EGM) and keep cold until use.
2. Prepare CVS-11 virus by diluting in EGM to a working dilution of 1.0 x 10^6.5 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.
3. Clean a humidity slide chamber and polytetrafluoroethylene (PTFE)-coated well-microscope slides with 70% ethanol and allow to air-dry in the BSC.
4. Add distilled water (dH₂O) to strips of absorbent paper in the slide chamber to ensure the humidity remains constant throughout the procedure.
5. 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).
6. Apply 50 µL of virus dilution to each well on the microscope slides with a repeating pipette. Then, apply 50 µL of cell dilution to each well, being careful not to contaminate the pipette tip with the virus already on the well.
7. Close the humidity slide chamber and place it in the humid incubator at 34-36 °C. Assess the cell infectivity after 24 h.
   NOTE: Perform steps 2.8-2.12 on only one slide.
8. 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.
9. Place the slide in the Coplin jar and fix the slide in cold acetone for a minimum of 1 h in a -20 °C freezer approved for flammable materials. Perform all acetone pouring and air-drying procedures in a fume hood.
10. Allow the acetone to flash off and slide to dry. Apply rabies direct fluorescent antibody (DFA) conjugate prepared according to the manufacturer's instructions to the wells of the slide and incubate for 30 min in a 34-36 °C humid incubator.
11. Wash the slide in Coplin jars of PBS twice for 2 min each. Air-dry the slide.
12. 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.
13. 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 °C until ready for use.

3. Sample preparation

1. 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.
   ​NOTE: Keep the conjugate in the dark to maintain the fluorophore integrity prior to application

4. IFA procedure

1. Remove the necessary number of prepared antigen slides needed for each assay and allow the slides to defrost and dry completely.
2. Place the slides in a Coplin jar(s) and fix the slides in cold acetone for between 2 h to overnight in a -20 °C freezer approved for flammable materials. Remove the slides from the acetone and allow to air-dry.
3. Place the slides in a humidity chamber box inside the BSC with dH₂O-soaked absorbent strips to maintain humidity. Apply 50 µL 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.
4. Place the closed humidity slide chamber in a 37 °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.
5. 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.
6. Repeat careful aspiration, then place each slide in a PBS-filled Coplin jar and wash twice for a total of 15 min.
7. Place the slides back in the humidity chamber box and apply 50 µL of appropriate anti-human antibody conjugate to each well. Repeat steps 4.4 to 4.6.
8. Allow the slides to air-dry, mount a coverslip with mounting media, and read the slides under fluorescent microscope.

5. Slide analysis

1. Grade samples based on the staining pattern and fluorescence intensity compared to positive control samples with confirmed high anti-rabies antibody titer.
2. 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.
3. 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.

Representative Results

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.

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 µm. Please click here to view a larger version of this figure.

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 µm. Please click here to view a larger version of this figure.

Figure 3: Rabies IFA flowchart. Flowchart showing execution stages of the major steps in the IFA procedure to aid in process visualization. Please click here to view a larger version of this figure.

Patient Sample	IgM	IgG
PS1-1	1:32	1:512
PS2-1	1:8	1:128
PS3-1	1:16	1:256
PS4-1	1:64	1:512
PS5-1	1:16	1:128

Table 1: Results 2 weeks post-vaccination. Patient sample results from serum samples collected approximately 2 weeks following completion of the PrEP vaccine series.

Patient Sample	IgM	IgG
PS1-2	1:2	1:128
PS2-2	1:1	1:128
PS3-2	1:1	1:64
PS4-2	1:1	1:256
PS5-2	1:1	1:32

Table 2: Results 6 months post-vaccination. Patient sample results from serum samples collected approximately 6 months following completion of the PrEP vaccine series.

Patient Sample	IgM	IgG
PS1-3	Not Detected	1:128
PS2-3	Not Detected	1:128
PS3-3	Not Detected	1:64
PS4-3	Not Detected	1:64
PS5-3	Not Detected	1:32

Table 3: Results 18 months post-vaccination. Patient sample results from serum samples collected approximately 18 months following completion of the PrEP vaccine series.

Discussion

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'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 ≥0.5 IU. However, current publications typically refer to the antibody response following immunization as acceptable (≥0.5 IU) or unacceptable (<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.

Materials

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