nAb ELISA Kit

SARS-CoV-2 Neutralizing Antibody Detection ELISA Kit



Catalog Number: ELK05D

Size: 96T/box

Type: Competitive ELISA

Specimen Type: Serum or plasma

Detects: Neutralizing antibodies of all Ig classes

Test Interpretation:

A positive result indicates that patient have had COVID-19 and have neutralizing antibodies against the RBD protein of SARS-CoV-2 and may have a level of immunity to COVID-19


1. BACKGROUND

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, or 2019-nCoV), an enveloped non-segmented positive-sense RNA virus, is the cause of contagious coronavirus disease 2019 (COVID-19) in humans. SARS-CoV-2 has several structural proteins including spike (S), envelope (E), membrane (M) and nucleocapsid (N). The spike protein (S) contains a receptor binding domain (RBD), which is responsible for recognizing the cell surface receptor, angiotensin converting enzyme-2 (ACE2). It is found that the RBD strongly interacts with the human ACE2 receptor leading to viral endocytosis into the host cells of the deep lung and viral replication.

Infection with the SARS-CoV-2 initiates an immune response to produce antibodies (including IgA, IgM and IgG) in the blood. Some antibodies cut off the interaction between the RBD with the ACE2 receptor and thus block viral infiltration and replication. These antibodies are named neutralizing antibodies.


2. INTENDED USE

The SARS-CoV-2 Neutralizing Antibody Detection ELISA Kit can detect circulating neutralizing antibodies against SARS-CoV-2 that block the interaction between the RBD with the ACE2 receptor. The assay detects any antibodies in serum and plasma that neutralize the RBD-ACE2 interaction. The test is both species and isotype independent.


3. ASSAY PRINCIPLE

The SARS-CoV-2 Neutralizing Antibody Detection ELISA Kit is a blocking ELISA detection tool, which mimics the virus neutralization process. The kit contains two key components: the Horseradish peroxidase (HRP) conjugated recombinant SARS-CoV-2 RBD fragment (HRP-RBD) and the recombinant ACE2-Fc fusion protein (human ACE2 fused to human Fc). The interaction between HRP-RBD and hACE2 can be blocked by neutralizing antibodies against SARS-CoV-2 RBD.

First, the samples and controls are pre-incubated with the HRP-RBD to allow the binding of the circulating neutralization antibodies to HRP-RBD. The mixture is then added to the capture plate which is pre-coated with the ACE2-Fc protein. The unbound HRP-RBD as well as any HRP-RBD bound to non-neutralizing antibody will be captured on the plate, while the circulating neutralization antibodies_HRP-RBD complexes remain in the supernatant and get removed during washing. After washing steps, TMB solution is added, making the color blue. By adding Stop Solution, the reaction is quenched and the color turns yellow. This final solution can be read at 450 nm in a microtiter plate reader. The absorbance of the sample is inversely dependent on the titer of the anti-SARS-CoV-2 neutralizing antibodies.


4. KIT CONTENTS

Part No.

Component

Quantity

1

Capture Plate

1 plate

2

Positive Control

1 vial (0.05 mL)

3

Negative Control

1 vial (0.05 mL)

4

HRP-RBD

1 vial (0.02 mL)

5

HRP Dilution Buffer

1 bottle (10 mL)

6

Sample Dilution Buffer

1 bottle (30 mL)

7

20× Wash Solution

1 bottle (40 mL)

8

Chromogen Solution A

1 bottle (6mL)

9

Chromogen Solution B

1 bottle (6mL)

10

Stop Solution

1 bottle (6 mL)

11

Plate Sealer

2 pieces


Capture Plate: 96 well microplates (12 wells x 8 strips) pre-coated with recombinant ACE2 protein; 8 strips configured in plate; Plate sealed in a foil pouch with a desiccant.



4. PROTOCOL

Reagent Preparation

1. All reagents must be taken out from refrigeration and allowed to return to room temperature before use (20°C to 25°C). Save all reagents in refrigerator promptly after use.

2. All samples and controls should be vortexed before use.

3. HRP-RBD Preparation: Dilute HRP conjugated RBD with HRP Dilution Buffer with a volume ratio of 1:1000. For example, for one 96 well plate testing, dilute 10 μL of HRP-RBD with 10 mL of HRP Dilution Buffer to make a HRP-RBD working solution.

4. 1× Wash Solution Preparation: Dilute the 20× Wash Solution with deionized or distilled water with a volume ratio of 1:19. For example, dilute 40 mL of 20× Wash Solution with 760 mL of deionized or distilled water to make 800 mL of 1× Wash Solution. Store the solution at 2°C to 8°C when not in use. 

Note: If any precipitate is observed in the 20× Wash Solution, incubate the bottle in a water bath (up to 50°C) with occasionally mixing until all the precipitate is dissolved.

Sample and Control Dilution

Dilute test samples, Positive, and Negative Controls with Sample Dilution Buffer with a volume ratio of 1:9. For example, dilute 10 μL of sample with 90 μL of Sample Dilution Buffer.

Capture Plate Preparation

1. It is recommended that all Positive Control and Negative Control should be prepared in duplicate.

2. Count the strips according to the number of test samples and install the strips. Make sure the strips are tightly snapped into the plate frame.

3. Leave the unused strips in the foil pouch and store at 2°C to 8°C. The strips must be stored in the closed foil pouch to prevent moisture from damaging the Capture Plate.

Test Procedure

Neutralization Reaction

1. In separate tubes, mix the diluted Positive Control, diluted Negative Control, and the samples with the diluted HRP-RBD solution with a volume ratio of 1:1. For example, mix 60 μL Positive Control with 60 μL HRP-RBD solution. Incubate the mixtures at 37°C for 30 minutes.

2. Add 100 µL each of the positive control mixture, the negative control mixture, and the sample mixture to the corresponding wells.

3. Cover the plate with Plate Sealer and incubate at 37°C for 15 minutes.

4. Remove the Plate Sealer and wash the plate with 280 µL of 1×Wash Solution for four times.

5. Pat the plate on paper towel to remove residual liquid in the wells after washing steps.

Substrate Reaction and Absorbance Measurement

6. Add 50 µL of Chromogen Solution A and 50 µL of Chromogen Solution B to each well and incubate the plate in the dark at 20 - 25°C for 15 minutes (start timing after the addition of TMB Solution to the first well).

7. Add 50 µL of Stop Solution to each well to quench the reaction.

8. Read the absorbance in the microtiter plate reader at 450 nm immediately.

Note: The substrate reaction time is determined by the temperature, the ideal reaction temperature is 25. If the temperature is lower than 25, extend the reaction time appropriately.


5. QUALITY CONTROL

To assure the validity of the results, each assay must include both Positive and Negative Controls. The net optical density (OD450) of each control must fall within the ranges listed in the following table. If OD450 values of controls do not meet the requirements in the following table, the test is invalid and must be repeated.

 OD450 values for quality control

Items

OD450 value

Control Result for Valid Assay

Quality Control

1.0

Negative Control

0.3

Positive Control

NoteThe standards in the table are only intended to evaluate the performance of the kit.


6. INTERPRETATION OF RESULTS

The positive cutoff and negative cutoff for SARS-CoV-2 neutralizing antibody detection can be used for interpretation of the inhibition rate. The operator can determine the result of the sample by comparing the inhibition rate to the following table.

Inhibition = (1 - OD value of Sample / OD value of Negative Control) × 100%


Cutoff Interpretation*

Items

Cutoff

Result

Interpretation

SARS-CoV-2

neutralizing

antibody test

≥ 20%

Positive

SARS-CoV-2 neutralizing antibody detected

20%

Negative

No detectable SARS-CoV-2 neutralizing antibody


*The cutoff value is based on validation with our panel of confirmed COVID-19 patient sera and healthy control sera. Users may want to set up their own cutoff based on different patient serum panels from different geographic locations or different ethnic backgrounds.