Using a Sandwich ELISA to Determine the Immunogenic Glycoprotein Content in a Vaccine

1. Security precautions

NOTE: This method is applicable to both live rabies virus (RABV) and inactivated vaccine.

1. Use good Laboratory Practice and Safety procedures.
2. Wear adequate Personal Protection Equipment (PPE) including disposable coat, gloves, masks, glasses, etc.
3. When the live virus is titrated, use a class II biological safety cabinet.
   1. Consider any material in contact with the samples (reagents, washing solutions, etc.) as infectious material.
   2. Treat the contaminated material by immersing it in the bleach solution (2,5% of sodium hypochlorite) for 30 min for decontamination.
4. Handle chemicals in accordance with good laboratory practices.

2. Preparation

1. Use analytical grade reagents wherever possible.
2. Prepare fresh solutions of the coating buffer/carbonate buffer, passivation buffer, diluent, and citrate buffer (Table 1), filter through 0.45 or 0.22 µm filters and store at 4 °C for one day prior to the use to preserve their analytical purity.
3. Allow reagents to reach room temperature (+18 °C to +25 °C) 30 min before the use and homogenize by gentle mixing prior to the use.

3. Microplate sensitization

NOTE: Use 96 well adsorption immunoassay plates which are optimized to bind high amounts of Immunoglobulins (e.g., see Table of Materials).

1. To each well, add 200 µL of the monoclonal antibody (mAb-D1) diluted in the carbonate buffer.  
   NOTE: An optimal concentration of about 1 µg/mL has been experimentally determined and corresponds to an approximate 1/2000 dilution of the purified mAb-D1. This recommended concentration is indicated for each mAb-D1 batch and must be periodically verified with the positive control.
2. Cover the plate with an adhesive film and incubate the microplate for 3 h at 37 °C in a humidified atmosphere.
3. Carefully aspirate and transfer the well content into a recipient containing 2,5% sodium hypochlorite solution.
4. Invert the microplate and let it dry on an absorbent paper at room temperature for 5 min.

4. Microplate passivation

1. To each well, add 300 µL of the passivation buffer.
2. Cover the plate with an adhesive film and incubate for 30 min at 37 °C.
3. Aspirate carefully and transfer the well content into a recipient containing 2,5% sodium hypochlorite solution.
4. Invert the microplate and let it dry on an absorbent paper at room temperature for 1 min.    
   NOTE: The microplate can be immediately used or stored sealed at -20 °C for up to 3 months until use.

5. ELISA assay

NOTE: For establishing the control curve of the reference vaccine, Step 5.3 is not required; to titrate the tested vaccine all Steps 5.1 to 5.6 are necessary.

1. Washing of the sensitized microplate
   1. To each well, add 300 µL of the washing buffer.
   2. Aspirate carefully and transfer the well content into a recipient containing 2,5% sodium hypochlorite solution.
   3. Repeat steps 5.1.1 and 5.1.2 five more times to wash the sensitized plate extensively.
   4. Invert the microplate and let it dry on an adsorbent paper at room temperature for 1 min.
2. Dilutions of the reference vaccine for the control curve
   1. Reconstitute the reference vaccine (validation antigen Lot 09) in 1 mL of distilled water corresponding to a concentration of 10 µg/mL of rabies virus glycoprotein.
   2. Prepare a ten-fold dilution of the reconstituted reference vaccine in the diluent to reach 1 µg/mL of rabies virus glycoprotein.
   3. Prepare 6 serial two-fold dilutions of this reference vaccine in the diluent as indicated in Table 1.
   4. Distribute 200 µL of the diluent in duplicate (wells 1H/2H) to serve as a blank control.
   5. Distribute 200 µL per well of each reference vaccine dilution in duplicate (wells G1/G2 to A1/A2).
3. Dilutions of the tested vaccine for its titration
   1. Prepare a ten-fold dilution of the tested vaccine in the diluent.
   2. Prepare 7 two-fold serial dilutions of the tested vaccine in diluent as indicated in Table 2.
   3. Distribute 200 µL per well of each tested vaccine dilution in duplicate (wells H3/H4 to A3/A4).
4. Incubation/Washing of the ELISA plate
   1. Cover the microplate with an adhesive film and incubate for 1 h at 37 °C.
   2. Remove the film, aspirate carefully and transfer the content of each well into a recipient containing 2,5% sodium hypochlorite solution.
   3. To each well, add 300 µL of washing buffer.
   4. Aspirate carefully and transfer the content of each well into a recipient containing 2,5% sodium hypochlorite solution.
   5. Repeat steps 5.4.3 and 5.4.4 five times to remove the unbound antigen and conserve the G protein trimers bound to the coated antibody (mAb D1).
   6. Invert the microplate and let it dry on an absorbent paper at room temperature for 1 min.
5. Binding of the peroxidase conjugated mAb-D1
   1. Distribute 200 µL per well of the recommended dilution (1/2000) of peroxidase-labeled mAb-D1 in diluent (approximate concentration of 1µg/mL). A recommended concentration is indicated for each mAb-D1 batch and has to be periodically verified with the positive control.
   2. Cover the microplate with an adhesive film and incubate for 1 h at 37 °C.
   3. Remove the film, aspirate carefully and transfer the content of each well into a recipient containing 2,5% sodium hypochlorite solution.
   4. Add to each well, 300 µL of the washing buffer.
   5. Aspirate carefully and transfer the content of each well into a recipient containing 2,5% sodium hypochlorite solution.
   6. Repeat steps 5.5.4 and 5.5.5 five times to remove unbound peroxidase-labeled antibody (mAb D1).
   7. Invert the microplate and let it dry on an absorbent paper at room temperature for 1 min.
6. Revelation using a substrate-chromogen
   1. Distribute 200 µL per well of substrate-chromogen solution.
   2. Seal the microplate with a film and incubate in the dark at room temperature for 30 min. A yellow-orange color develops the intensity of which is proportional to the amount of bound peroxidase-labeled antibodies (mAb D1).
   3. Stop the reaction by adding 50 µL of stopping solution per well.
   4. Carefully wipe the bottom of the microplate and place it in a spectrophotometer to determine the optical density (OD) at 492 nm of all used wells: negative control (blank), reference vaccine, and tested vaccine.
   5. Collect OD data in .xls or .xlsx file format for analysis.

Table 1. Buffers used in the assay.