The IFA test assesses antibody response during rabies infection or evaluates the immune response from vaccines. It can be used to establish antibody presence in a sample and distinguish which antibody isotypes are present. Neutralizing antibody assays are the gold standard for rabies antibody detection to assess antibody titer in a person who has received pre-or post-exposure prophylaxis.
Although an IFA test may yield different results than a neutralizing antibody assay, it can still provide valuable information when used as an alternative. Typically, assays that measure rabies neutralizing antibodies evaluate immune response. However, these tests take multiple days requiring virus and live cell culture.
The IFA test allows advanced preparation of antigen slides for testing with results in a few hours compared to days. Using the IFA test in human rabies cases may lead to more answers about how an individual's immune system responds to rabies infection. There is a great variation amongst cases, and this test can provide results in a real-time manner during the disease.
To begin, wear personal protective equipment including eye protection, a surgical mask, and non-latex gloves. Prepare 20 milliliters of mouse neuroblastoma or BHK-21 cells to a concentration of 3.0 times 10 to the fifth cells per milliliter in EGM. Keep the prepared cells cold until they are ready to use.
Next, prepare the CVS-11 virus by diluting it in EGM. Ensure to keep the prepared virus cold until used. Clean the humidity slide chamber and polytetrafluoroethylene-coated well microscope slides with 70%ethanol, and allow them to air dry in the biosafety cabinet.
After cleaning, add distilled water to the strips of absorbent paper in the slide chamber to ensure constant humidity throughout the procedure. Using a pencil, label each slide with the required identification information such as the lot number, date, and cell type. Place the labeled slides in the slide chamber for storage.
Then apply 50 microliters of virus dilution to the wells on the microscope slides using a repeating pipette. Apply 50 microliters of cell dilution to each well, being cautious not to contaminate the pipette tip with the virus already in the well. Afterward, close the humidity slide chamber and place it in the humid incubator between 34 and 36 degrees Celsius.
Next, remove the slide from the humidity chamber and meticulously aspirate the supernatant. Wash the slide for two minutes in a PBS-filled Coplin jar, then allow it to air dry. Put the slide in a cold acetone-filled Coplin jar to fix the sample.
Place the jar in a 20 degrees Celsius freezer approved for flammable materials for at least one hour. Once the acetone evaporates and the slide dries, apply rabies direct fluorescent antibody conjugate to the slide's wells and incubate the slide for 30 minutes in a 34 to 36 degree Celsius humid incubator. Then wash the slide twice in a Coplin jar of PBS for two minutes each.
Air dry the slide and mount the cover slip with the 0.05 molar Tris, 0.15 molar sodium chloride, and 20%glycerol mountant. Use a fluorescent microscope at 200 times magnification to evaluate the cell infectivity. Next, remove the remaining slides from the incubator and humidity chamber.
Aspirate the supernatant from each well carefully, then place the slides in a Coplin jar with PBS from one to two minutes. Allow the slides to air dry for about 30 minutes and then store them at 80 degrees Celsius until use. To begin, prepare dilution to the patient's serum or cerebral spinal fluid samples for testing.
Prepare the required conjugate at the appropriate working concentration by diluting it in PBS with 0.05%Evans blue. For indirect fluorescent antibody tests, retrieve the appropriate number of prepared antigen slides required for the assay. Allow the slides to defrost and dry completely.
Next, place the slides in an acetone-filled Coplin jar and transfer them to a 20 degree Celsius freezer approved for flammable materials for two hours to overnight. Afterward, remove the slides from the acetone and allow them to air dry. Now, place the slides in the humidity chamber box inside the biosafety cabinet.
To maintain the humidity, add distilled water-soaked absorbent strips to the chamber. Apply 50 microliters of each control sample, sample dilution, or PBS to the predetermined well. Place the closed humidity slide chamber at 37 degrees Celsius, 5%carbon dioxide humidity incubator for 30 minutes.
Once done, remove the humidity slide chamber from the incubator and transfer it into a biosafety cabinet. Using an aspirator tip, carefully aspirate the supernatant from each well without disturbing the cell monolayer. Afterward, apply one drop of PBS to each well using a sterile dropper pipette.
Carefully aspirate the PBS and transfer each slide into a Coplin jar filled with PBS. Next, place the slides back into the humidity chamber box. Apply 50 microliters of the appropriate anti-human antibody conjugate to each well and incubate for 30 minutes in a humid incubator.
At the end of the incubation, transfer the humidity chamber into a biosafety cabinet. Aspirate the supernatant using an aspirator tip. Afterward, apply one drop of PBS to each well using a sterile dropper pipette.
After removing the PBS, wash the slide twice in a Coplin jar of PBS for a total of 15 minutes. Once washed, allow the slides to air dry, then mount a cover slip with mounting media. Take the slides and read them under fluorescent microscopy.
Grade the samples from negative to 4+with negative samples showing no fluorescence and 4+samples showing a bright green fluorescence. Assign the samples and endpoint value represented by the dilution factor at which the sample displays a 1 to 2+grade. After initial vaccination, high levels of both immunoglobulin M and G were observed in the patient samples.
Approximately six months after vaccination, significantly lower Levels of both antibodies were present in the patient samples, but immunoglobulin M levels had dropped almost completely. 18 months after vaccination, immunoglobulin M antibodies were not detected in patient samples. Immunoglobulin G levels persisted and stayed like those detected at the six-month time point following the initial decrease from the two-week time point.
