Overview
This video showcases an indirect enzyme-linked immunosorbent assay or ELISA sandwich immunocapture assay designed to assess immunogenic glycoprotein levels in vaccines. The assay plate, containing glycoprotein-specific antibodies, is incubated with serially diluted vaccine samples, including reference and test vaccines, followed by immunoassay to quantify glycoprotein content in the test vaccine.
Protocol
1. Security precautions
NOTE: This method is applicable to both live rabies virus (RABV) and inactivated vaccine.
- Use good Laboratory Practice and Safety procedures.
- Wear adequate Personal Protection Equipment (PPE) including disposable coat, gloves, masks, glasses, etc.
- When the live virus is titrated, use a class II biological safety cabinet.
- Consider any material in contact with the samples (reagents, washing solutions, etc.) as infectious material.
- Treat the contaminated material by immersing it in the bleach solution (2,5% of sodium hypochlorite) for 30 min for decontamination.
- Handle chemicals in accordance with good laboratory practices.
2. Preparation
- Use analytical grade reagents wherever possible.
- 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.
- 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).
- 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. - Cover the plate with an adhesive film and incubate the microplate for 3 h at 37 °C in a humidified atmosphere.
- Carefully aspirate and transfer the well content into a recipient containing 2,5% sodium hypochlorite solution.
- Invert the microplate and let it dry on an absorbent paper at room temperature for 5 min.
4. Microplate passivation
- To each well, add 300 µL of the passivation buffer.
- Cover the plate with an adhesive film and incubate for 30 min at 37 °C.
- Aspirate carefully and transfer the well content into a recipient containing 2,5% sodium hypochlorite solution.
- 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.
- Washing of the sensitized microplate
- To each well, add 300 µL of the washing buffer.
- Aspirate carefully and transfer the well content into a recipient containing 2,5% sodium hypochlorite solution.
- Repeat steps 5.1.1 and 5.1.2 five more times to wash the sensitized plate extensively.
- Invert the microplate and let it dry on an adsorbent paper at room temperature for 1 min.
- Dilutions of the reference vaccine for the control curve
- 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.
- Prepare a ten-fold dilution of the reconstituted reference vaccine in the diluent to reach 1 µg/mL of rabies virus glycoprotein.
- Prepare 6 serial two-fold dilutions of this reference vaccine in the diluent as indicated in Table 1.
- Distribute 200 µL of the diluent in duplicate (wells 1H/2H) to serve as a blank control.
- Distribute 200 µL per well of each reference vaccine dilution in duplicate (wells G1/G2 to A1/A2).
- Dilutions of the tested vaccine for its titration
- Prepare a ten-fold dilution of the tested vaccine in the diluent.
- Prepare 7 two-fold serial dilutions of the tested vaccine in diluent as indicated in Table 2.
- Distribute 200 µL per well of each tested vaccine dilution in duplicate (wells H3/H4 to A3/A4).
- Incubation/Washing of the ELISA plate
- Cover the microplate with an adhesive film and incubate for 1 h at 37 °C.
- Remove the film, aspirate carefully and transfer the content of each well into a recipient containing 2,5% sodium hypochlorite solution.
- To each well, add 300 µL of washing buffer.
- Aspirate carefully and transfer the content of each well into a recipient containing 2,5% sodium hypochlorite solution.
- 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).
- Invert the microplate and let it dry on an absorbent paper at room temperature for 1 min.
- Binding of the peroxidase conjugated mAb-D1
- 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.
- Cover the microplate with an adhesive film and incubate for 1 h at 37 °C.
- Remove the film, aspirate carefully and transfer the content of each well into a recipient containing 2,5% sodium hypochlorite solution.
- Add to each well, 300 µL of the washing buffer.
- Aspirate carefully and transfer the content of each well into a recipient containing 2,5% sodium hypochlorite solution.
- Repeat steps 5.5.4 and 5.5.5 five times to remove unbound peroxidase-labeled antibody (mAb D1).
- Invert the microplate and let it dry on an absorbent paper at room temperature for 1 min.
- Revelation using a substrate-chromogen
- Distribute 200 µL per well of substrate-chromogen solution.
- 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).
- Stop the reaction by adding 50 µL of stopping solution per well.
- 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.
- Collect OD data in .xls or .xlsx file format for analysis.
Table 1. Buffers used in the assay.
Buffers and reagents | Preparation |
Coating buffer (Carbonate buffer 50mM pH=9.6) |
Add Sodium carbonate 50 mM (Na2CO3-10H2O) to Sodium bicarbonate 50 mM (NaHCO3) until the desired pH (about 1/10 volume of sodium bicarbonate) |
Passivation buffer | 0.3% Bovine Serum Albumin (BSA, fraction V), 5% sucrose in carbonate buffer 50 mM pH 9.6 |
10x Phosphate buffered saline pH=7 (PBS 10x) | NaCl 80 g, KCl 2 g, Na2PO4-12H2O 11.33 g, KH2PO4 2g in 1L of distilled water. Adjust pH=7 with 4N NaOH |
Washing buffer | 0.05% Tween in 1x PBS |
Diluent | 0.5% Bovine Serum Albumin (Fraction V), 0.05% Tween in 1x PBS (adjust pH to 7 because of acidification by BSA) |
Citrate buffer pH-5.6 (for peroxidase substrate) |
11.67 g Tri-sodium citrate-2H2O (Na3C6H5O7-2H20), 2.17 g Citric acid-1H20 in 1L of distilled water |
Substrate-chromogen solution | 50 mg Ortho-phenylene diamine tablet, 0.1% Hydrogen peroxide 30% (110 vol) in 25 ml Citrate buffer pH 5.6 |
Stopping solution (4N sulfuric acid) |
10 ml H2SO4 36N in 80 ml cooled distilled water. Dilution must be carried out in an ice bath |
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Materials
Name | Company | Catalog Number | Comments |
Class II Biological Safety Cabinet | ThermoFisher Scientific | 10445753 | If titrating live virus |
Clear Flat-Bottom Immuno Nonsterile 96-Well Plates, 400 µL, MAXISORP | ThermoFisher Scientific | 439454 | Good for binding to the loaded antibody |
Equip Labo Polypropylene Laboratory Fume Hood | ThermoFisher Scientific | 12576606 | For the preparation of sulfuric acid |
Immunology Plate Strong Adsorption MAXISORP Flat Bottom Well F96 |
Dutscher | 55303 | Good for binding to the loaded antibody |
Microplate Sealing Tape(100 sheets) | ThermoFisher Scientific | 15036 | |
Microplate single mode reader Sunrise | TECAN | ||
Microplate shaker-incubator | Dutscher | 441504 | |
Microplate washer Wellwash | ThermoFisher Scientific | 5165000 | |
Multichannel pipette (30-300 µL) 12 channels | ThermoFisher Scientific | 4661180N | |
Single Channel pipettes (Kit 2 : Finnpipettes F2 0.2-2 μL micro, 2-20 μL, 20-200 μL & 100-1000 μL) | ThermoFisher Scientific | 4700880 |