This article describes a method for quantifying rabies virus glycoprotein content in vaccines using a microplate assay. The process involves coating plates with specific antibodies, introducing vaccine samples, and measuring absorbance to determine glycoprotein concentration.
Begin with a passivated assay plate coated with rabies immunogenic glycoprotein-specific monoclonal antibodies and a blocking agent to prevent non-specific interactions.
Introduce serially diluted reference and test rabies vaccines with decreasing glycoprotein content. Seal the plate to prevent contamination.
During incubation, immunogenic glycoproteins interact with coated monoclonal antibodies via a specific antigenic site.
Post-incubation, remove the film, aspirate the supernatant, and wash the plate.
Overlay with enzyme-conjugated glycoprotein-specific monoclonal antibodies that bind to a different antigenic site on the glycoprotein, forming an immunocomplex.
Add a solution containing a substrate and a chromogen. When the substrate interacts with the enzyme, it causes the oxidation of the chromogen, changing the solution color.
Add a stopping solution to inactivate the enzymes.
Measure the absorbance and generate a reference curve using the reference vaccine values.
Identify the region where there is a linear relationship between absorbance and the glycoprotein content of the reference vaccine dilutions. Then, extrapolate the glycoprotein content of the test vaccine dilutions based on the absorbance that falls within this region.
To wash the blade, 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 twofold 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 blank 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 twofold 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 a 2.6% sodium hypochlorite solution. Repeat this washing process five more times to remove the unbound peroxidase-labeled antibody. Invert the wash 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 microblade, and place it in a 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.
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