In the aim of reducing animal testing and because of the variability of the NH test, WHO encourages to develop in vitro approaches for evaluating rabies vaccine potency. This ELISA test shows a good concordance with a classical NH test in recognizing the immunogenic form of the glycoprotein, shows a high sensitivity, and can be performed within only one day. Evaluation of rabies vaccine potency in vitro by ELISA is an alternative to the in vivo NH test.
It is promoted by the manufacturers and national control laboratories. It's critical to distribute pre-sized volumes in duplicate for each dilution and to well dry the plate on absorbent paper after washes to avoid To begin this procedure, put on adequate personal protection equipment including disposable coat, gloves, mask, and glasses. Treat the contaminated materials by immersing it in a solution of 2.6%sodium hypochlorite for 30 minutes for decontamination.
Next, set out a 96-well immunoassay plate and add 200 microliters of the monoclonal antibody diluted in the carbonate buffer to each well. Cover the plate with adhesive film and incubate the microplate at 37 degrees Celsius for three hours in a humidified environment. Then carefully aspirate and transfer the well content into a recipient containing a solution of 2.6%sodium hypochlorite.
Invert the microplate and let it dry on absorbent paper at room temperature for five minutes. First, add 300 microliters of the passivation buffer to each well. Cover the plate with adhesive film and incubate at 37 degrees Celsius for 30 minutes.
After this, transfer the contents of the well to one containing a solution of 2.6%sodium hypochlorite. To wash the plate, add 300 microliters of the washing buffer to each well. Then aspirate carefully and transfer the well content into a recipient containing a solution of 2.6%sodium hypochlorite.
Repeat this washing process five more times to extensively wash the sensitized plate. After this, invert the microplate and let it dry on a piece of absorbent paper at room temperature for one minute. Reconstitute the reference vaccine in one milliliter of distilled water such that the final concentration of rabies virus glycoprotein is 10 micrograms per milliliter.
Prepare a 10-fold dilution of the reconstituted reference vaccine in the diluent to reach a rabies virus glycoprotein concentration of one microgram per milliliter. Next, perform six serial two-fold dilutions of this reference vaccine in the diluent as outlined in table one of the text protocol. Distribute 200 microliters of the diluent in duplicate wells to serve as a blind control and 200 microliters per well for each reference vaccine dilution in duplicate.
Then prepare a 10-fold dilution of the tested vaccine in the diluent and prepare seven two-fold serial dilutions of the tested vaccine in diluent as outlined in table two of the text protocol. Distribute 200 microliters of each tested vaccine dilution in duplicate to each well of the microplate. Cover the microplate with adhesive film and incubate at 37 degrees Celsius for one hour.
After this, remove the film. Aspirate carefully and transfer the contents of each well into a recipient containing a 2.7%sodium hypochlorite solution. To wash the plate, add 300 microliters of washing buffer to each well, aspirate carefully and transfer the contents of each well into a recipient containing a 2.6%sodium hypochlorite solution.
Repeat the washing process five more times to remove the unbound antigen and conserve the G-protein trimers bound to the coated antibody. Invert the washed microplate and let it dry on a piece of absorbent paper at room temperature for one minute. Then distribute 200 microliters of the recommended dilution of peroxidase labeled D1 monoclonal antibody in diluent to each well.
Cover the microplate with adhesive film and incubate at 37 degrees Celsius for one hour. Next, remove the film, aspirate carefully, and transfer the contents of each well into a recipient containing a 2.6%sodium hypochlorite solution. To wash the microplate, add 300 microliters of washing buffer to each well.
Aspirate carefully and transfer the contents of each well into a recipient containing 2.6%sodium hypochlorite solution. Repeat this washing process five more times to remove the unbound peroxidase labeled antibody. Invert the washed microplate and let it dry on a piece of absorbent paper at room temperature for one minute.
Next, distribute 200 microliters of substrate-chromogen solution into each well. Seal the microplate with film and incubate at room temperature in the dark for 30 minutes. Then add 50 microliters of stopping solution into each well to stop the reaction.
Carefully wipe the bottom of the microplate and place it in the spectrophotometer. Determine the optical density at 492 nanometers for all of the wells used. Collect this data in a spreadsheet file for analysis.
After this, create plots for both the reference vaccine curve and the optical density values as outlined in the text protocol. In this study, the in vitro ELISA test is used to evaluate rabies vaccine potency. Representative optical density values from a typical experiment are shown here.
These values are used to draw the reference vaccine curve by plotting the mean optical density for the different dilutions of the reference vaccine on the vertical axis and the concentration of the glycoprotein on the horizontal axis. The glycoprotein content of the tested vaccine is then estimated using this curve. The evaluation is precise for dilutions that have a mean optical density value in the liner area of the reference vaccine curve.
Taking into account the dilution of one-to-40, the vertical projection of OD 1534 on the x-axis corresponds to 500 nanograms per milliliter. So the tested vaccine is estimated at 20 micrograms per milliliter of glycoprotein. When the in vitro potency is established, it is necessary to compare the tested vaccine to the reference six WHO international standard calibrated in international units.
The same method can be used with other antibodies to enlarge detection of more vaccine strains including those used in veterinary vaccines which are classically more variable. Antibodies can also be used in competition for titration of antirabies antibodies in equine or human serum. Precautions must be taken with vaccines that are not inactivated or with viruses.
Be sure to use personal protective equipment, wear gloves and decontaminate everything properly. Also, be careful with tablets of as they are carcinogenic and with the acidic sodium hypochlorite solution.
