Scribd
Upload
9 views2 pages

ELISA for SARS-CoV-2 IgG Detection

The document outlines the procedure for conducting an Enzyme-Linked Immunosorbent Assay (ELISA) to qualitatively determine human serum IgG antibodies against the SARS-CoV-2 spike protein receptor-binding domain. It details the necessary reagents, sample preparation, incubation, washing, and measurement steps. Results interpretation is based on absorbance values compared to controls to determine the presence of antibodies.

Uploaded by

yyj7qnnr2p
Copyright
© All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
9 views2 pages

ELISA for SARS-CoV-2 IgG Detection

The document outlines the procedure for conducting an Enzyme-Linked Immunosorbent Assay (ELISA) to qualitatively determine human serum IgG antibodies against the SARS-CoV-2 spike protein receptor-binding domain. It details the necessary reagents, sample preparation, incubation, washing, and measurement steps. Results interpretation is based on absorbance values compared to controls to determine the presence of antibodies.

Uploaded by

yyj7qnnr2p
Copyright
© All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd

Enzyme-Linked Immunosorbent Assay (ELISA)

Assay applicability
For the qualitative determination of human serum IgG antibody that recognizes
SARS-CoV-2 spike protein receptor-binding domain (RBD)

Assay principle

Assay procedures

1. Work in groups of two classmates.


2. You need these reagents/samples:
a) PBS for blank control; b) Positive control sample; c) Negative sample; d) Unknown
sample for your examination; e) Detection solution; f) Substrate solution; g) Stop
solution
3. Label and orient the given strip of wells.

4. Distribute in the wells according to the following well arrangement (in duplicate)

1 Blank control (PBS) 100 l


2 100 l
3 Positive control 100 l
4 100 l
5 Negative control 100 l
6 100 l
7 Unknown sample 100 l
8 100 l

PBS, phosphate-buffered saline

1
5. Loading sample/buffer:
• Add 100 μl of relevant sample/control into the respective wells in duplicate
according to the above table.
• Mix by tapping the plate gently.

Note: Use a separate disposal pipette tip for each sample/control to avoid cross-
contamination.

6. Incubation: Cover the plate and incubate at room temperature for 45 minutes.

7. Washing:
• Discard the content and tap the plate on a clean paper towel to remove residual
solution in each well.
• Add 300 µl of wash buffer to each well and let it stand for about 10 seconds.
• Discard the wash buffer and tap the plate on a clean paper towel to remove
residual wash buffer.
• Repeat the wash step for a total of 3 washes.

8. Addition of secondary antibody: Add 100 µl Detection Solution (horseradish


peroxidase-conjugated mouse anti-human IgG antibody) to each well.

9. Incubation: Cover the plate and incubate at room temperature for 45 minutes.

10. Washing: Wash each well 4 times as described in step 7.

11. Colouring:

• Add 100 µl of Substrate Solution to each well.


• Incubate at room temperature for 15 minutes (protect the plate strip from
light).

12. Stopping reaction: Add 100 µl of Stop Solution to each well, gently tap the plate
frame for a few seconds to ensure thorough mixing.

13. Measurement: Pool your trips from different groups and place them on a 96-well
frame and measure absorbance of each well at 450 nm.

Results validity and interpretation


• The Absorbance value of the Blank Control should be < 0.17 at 450 nm.
• The Absorbance value of the Positive Control should be > 0.50 at 450 nm.
• Calculation of the cut-off value: Average of negative control OD values + 0.05
• If the OD value of the sample ≥ cut-off value, it is positive for the IgG antibody
against the spike protein of SARS-CoV-2.

You might also like