Sep 03, 2025
Human ACE2 ELISA
- jeabchanida Ruchisrisarod1
- 1Thai Red Cross Emerging Infectious Diseases Health Science Centre, King Chulalongkorn Memorial Hospital, Bangkok, Thailand
- Protocol for detection
Protocol Citation: jeabchanida Ruchisrisarod 2025. Human ACE2 ELISA. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5k24nv1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 03, 2025
Last Modified: September 03, 2025
Protocol Integer ID: 226305
Keywords: human ace2 elisa, immune dysregulation, possible autoimmunity, specific antibody, antibody, inflammatory profiles of patient, cytokine profile, transient immune response, stable antibody level, underlying immune mechanism, cytokine, inflammatory profile, long covid, immunological heterogeneity, pandemic, elisa, infection patient, following sar, infection
Funders Acknowledgements:
National Research Council of Thailand
Grant ID: N35A650132
Abstract
The COVID-19
pandemic has led to a significant number of individuals experiencing persistent
post-acute sequelae, commonly referred to as long COVID, following SARS-CoV-2
infection. Similar symptoms, though less frequent, have also been observed
after COVID-19 vaccination.
This study investigated the immune and inflammatory profiles of patients with long COVID
symptoms, focusing on differences between post-infection and post-vaccination
cases. A total of 110 patients (61 post-infection and 49 post-vaccination) were
assessed at three and six months after symptom onset, alongside 40 controls.
Serological testing was performed for SARS-CoV-2-specific antibodies, including
neutralizing, non-neutralizing, and anti-ACE2 antibodies, and cytokine
profiling was conducted using a 19-marker panel.
Post-infection patients exhibited elevated and
increasing levels of anti-ACE2 antibodies and persistently high levels of
pro-inflammatory cytokines such as IL-1β, IFN-γ, and MCP-1, indicating ongoing
immune dysregulation and possible autoimmunity. In contrast, post-vaccination
patients demonstrated stable antibody levels and cytokine profiles comparable
to controls, suggesting a more regulated and transient immune response. These
findings highlight the immunological heterogeneity of long COVID and support
the need for targeted diagnostic and therapeutic approaches tailored to the
underlying immune mechanisms in post-infection and post-vaccination subgroups.
Attachments
Guidelines
Sample Preparation Guidelines:
- If samples will not be tested immediately, freeze them after collection.
- Avoid multiple freeze–thaw cycles of frozen samples.
- Thaw samples completely and mix gently (do not vortex) prior to analysis.
- Do not use hemolyzed or lipemic sera.
- Pre-dilute samples using 1X Assay Diluent for serum, plasma, and cell culture supernatant samples.
- Dilute serum 8-fold.
Materials
- Human ACE2 Antibody Coated wells, 96-well plate
- Human ACE2 Biotin Conjugate
- Human ACE2 Standard, recombinant human ACE2
- Assay Diluent (5X)
- Streptavidin-HRP (150X)
- TMB Substrate
- Stop Solution
- Wash Buffer Concentrate (20X)
- Adhesive Plate Covers
- Plate washer-automated or manual (manifold dispenser)
- Microtiter plater reader with software capable of measuring at 450 nm
- Deionized water
Troubleshooting
For the standard curve, add 100 µL of each standard to the appropriate wells for samples, add 100 µL
of diluted samples to the designated wells.
Cover the plate and incubate for 2.5 hours at room temperature or overnight at 4 °C with gentle shaking.
Discard the solution and wash the plate four times with 1× wash Buffer. Fill each well with 300 µL of wash Buffer using a multi-channel pipette or an auto washer. Complete removal of liquid at each wash is critical for optimal performance. After the final wash, remove any residual buffer by aspiration or decanting. Invert the plate and gently blot against clean paper towels.
Add 100 µL of prepared biotin conjugate to each well.
Incubate for 1 hour at room temperature with gentle shaking.
Discard the solution and repeat the wash
Add 100 µL of prepared Streptavidin-HRP solution to each well.
Incubate for 45 minutes at room temperature with gentle shaking.
Discard the solution and repeat the wash.
Add 100 µL of TMB Substrate to each well. The solution will start to turn blue.
Incubate for 30 minutes at room temperature in the dark with gentle shaking.
Add 50 µL of Stop Solution to each well and gently tap the plate to mix. The color will change from blue to yellow.
Read the absorbance at 450 nm. Read the plate within 30 minutes after adding the
Stop Solution.
Protocol references
Human ACE2 ELISA Kit
Catalog Number EH489RB (96 tests)