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Immunology and Infection

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

Published: January 19th, 2024

DOI:

10.3791/65459

1Rabies Laboratory, Wadsworth Center, 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.

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.

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 ....

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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 mo.......

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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.......

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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 .......

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

....

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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 Teflon coating glass slides Any
PBS Any pH 7.6 
Penicillin Sigma P-3032 EGM
Rabies Direct Fluorescent Antibody Conjugate Millipore Sigma 5100, 5500 or 6500
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

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