Sep 02, 2025
Cytokines assay to analyze
- jeabchanida Ruchisrisarod1
- 1Thai Redcross
- Protocol for detection
Protocol Citation: jeabchanida Ruchisrisarod 2025. Cytokines assay to analyze . protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqpd1kvk5/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 02, 2025
Last Modified: September 02, 2025
Protocol Integer ID: 226218
Keywords: indicating ongoing immune dysregulation, ongoing immune dysregulation, long covidsymptom, cytokine profile, cytokine, possible autoimmunity, inflammatory profiles of patient, transient immune response, specific antibody, underlying immune mechanism, inflammatory profile, stable antibody level, immunological heterogeneity, long covid, following sar, infection, infection patient
Funders Acknowledgements:
National Research Council of Thailand
Grant ID: (N35A650132)
Abstract
Abstract
The COVID-19pandemic 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 studyinvestigated the immune and inflammatory profiles of patients with long COVIDsymptoms, 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
Materials
Materials
- Coupled magnetic beads
- Detection antibody
- Standard
- Quality control
- Detection antibody diluent HB
- Sample diluent HB
- Standard diluent HB
- Streptavidin PE
- Stop Solution
- Wash Buffer Concentrate (20X)
- Adhesive Plate Covers
- Bio-plex magnetic washer
- Bio-Plex‱ 200 Luminex
- Deionized water
Troubleshooting
Methods
Assay reagents: Prepare the assay diluent, standard
diluent HB, detection antibody diluent HB, and sample diluent by gentle mixing
until homogeneous. Leave the reagents at room temperature for 30 minutes prior
to use.
Wash buffer: Dilute the 10X wash buffer with
distilled water at a ratio of 1:9.
Samples: Dilute serum samples 1:4 with sample
diluent before analysis.
Standards and controls: Reconstitute the lyophilized
standard by adding 250 µL of standard diluent HB. Mix gently by vortexing for 5
seconds and keep the solution on ice for 30 minutes before use.
Standard dilutions: Prepare a 4-fold serial dilution
of the standard as illustrated. Mix each dilution by vortexing for 5 seconds to
ensure homogeneity.
Preparation of coupled beads
Mix the coupled beads by vortexing for 30 seconds, then dilute to 1× in assay
buffer. For a 96-well plate, prepare the working solution by mixing 570 µL of
10× coupled beads with 5130 µL of assay buffer (final volume: 5700 µL). Protect
the solution from light.
Bead addition
Add 50 µL of the prepared 1× coupled beads to each well of a 96-well plate.
Plate washing
Wash the plate twice with 100 µL of wash buffer per well.
Sample and standard addition
Add 50 µL of sample, standard, blank, or control to each well.
Incubation
Seal the plate with sealing tape and incubate at room temperature with shaking
at 850 rpm for 30 minutes.
Detection antibody preparation
Vortex the detection antibody for 5 seconds, then dilute to 1× in detection
antibody diluent. For a 96-well plate, prepare the solution by mixing 300 µL of
10× detection antibody with 2700 µL of detection antibody diluent (final
volume: 3000 µl).
Plate washing
Wash the plate twice with 100 µL of wash buffer per well.
Detection antibody incubation
Seal the plate with sealing tape and incubate at room temperature with shaking
at 850 rpm for 30 minutes.
Streptavidin-phycoerythrin (SA-PE) preparation
Vortex the 100× SA-PE for 5 seconds, then dilute to 1× in assay diluent. For a
96-well plate, prepare the solution by mixing 60 µL of 100× SA-PE with 5940 µL
of assay diluent buffer (final volume: 6000 µl). Protect the solution from
light.
Plate washing
Wash the plate three times with 100 µl of wash buffer per well.
SA-PE incubation
Add 50 µL of the prepared 1× SA-PE solution to each well. Seal the plate and
incubate at room temperature with shaking at 850 rpm for 10 minutes.
Final washing
Wash the plate three times with 100 µL of wash buffer per well.
Bead resuspension
Resuspend the beads in 125 µL of assay buffer per well. Seal the plate and
shake at 850 rpm for 30 seconds at room temperature.
Data acquisition
Read the plate using the Bio-Plex 3D system with DD Gates to select MagPlex
beads.
Protocol references
the Bio-Plex Pro‱ Human Cytokine Screening Panel from BIO-RAD, CA, USA