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

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.

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.

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

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