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 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.
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 authors have nothing to disclose.
We are grateful to the New York State Department of Health Wadsworth Center for supporting this project.
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 |