Sep 04, 2025
Neutralizing antibodies (NAbs) tests
- jeabchanida Ruchisrisarod1
- 1Thai Redcross
- Protocol for detection
Protocol Citation: jeabchanida Ruchisrisarod 2025. Neutralizing antibodies (NAbs) tests. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov112yyvr2/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 04, 2025
Last Modified: September 04, 2025
Protocol Integer ID: 226393
Keywords: respiratory syndrome coronavirus, coronavirus, pcr positive with antibody, antibody positive on day, antibody assays of igm, evaluation on antibody, antibody assay, antibody, neutralizing antibody, surrogate isotype independent virus, magnitude of viral load, virus, same day of symptom onset, quarantine among negative case, onset of symptom, severe acute, infection, symptom onset, sar, viral load, quarantine, state quarantine premise, days after infection
Funders Acknowledgements:
King Chulalongkorn Memorial Hospital’s Excellence Centre Program
Grant ID: EC-64-30101-15
Abstract
Antibody assays of IgM, IgG and surrogate isotype independent virus
neutralizing antibody (sVNT) targeting receptor binding domain of Severe Acute
Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) were employed in 97 real-time
Reverse Transcription Polymerase Chain Reaction (RT-PCR) confirmed Coronavirus
Disease 2019 (COVID-19) patients with varying severity admitted to King
Chulalongkorn Memorial Hospital. Concordance rate was 100% regardless of
severity, onset of symptoms and magnitude of viral load. Per available samples,
antibodies appeared on the same day of symptom onset in one patient; one day
after in 18 patients and two days after in 19 patients. In two patients,
antibodies appeared as early as 4 days after infection (exposure). IgM and IgG
were evident in all patients’ first assay (within two days of admission). sVNT
was also evident within two days of admission in all but 3 patients. IgM
usually remained positive during the entire course of hospital stay, where the
longest in this study was 32 days. Antibody assays were also applied to samples
collected at a State Quarantine premise from 77 asymptomatic Thais returning
from Sudan in October. Virus was detected by real-time RT-PCR in 15 cases (day
0=6, day 3=4, day 5=4 and day 9=1). Twenty-nine (including 11 RT-PCR positive
cases) were antibody positive on day 0, while 4 PCR positive with antibody
negative on day 0 became antibody positive on day 14. Evaluation on antibody
response at days 7 or 10 is needed to help build a case to shorten length of
quarantine among negative cases.
Guidelines
For human serum, blood was collected into a serum separator tube and allowed to clot for 30 minutes at room temperature. Samples were then centrifuged at 3,500 × rpm for 5 minutes to separate the serum. Assays were performed immediately after preparation; otherwise, serum was aliquoted and store at ≤ −20 °C until analysis. Repeated freeze–thaw cycles were avoided.
Materials
Materials
- 96 well capture plate (8 wells x 12 strips) pre-coated with recombinant ACE2 protein
- Adhesive plates sheet (for covering plate)
- Positive control 1 vial (0.05 ml)
- Negative control 1 vial (0.05 ml)
- HRP conjugated RBD 1 vial (0.02 mL)
- HRP dilution buffer 1 bottle (10 ml)
- Sample dilution buffer 1 bottle (30 ml)
- 20× wash solution 1 bottle (40 ml)
- TMB ELISA substrate solution 1 bottle (12 ml)
- Stop solution 1 bottle (6 ml)
- Plate washer-automated or manual (manifold dispenser)
- Microtiter plater reader with software capable of measuring at 450 nm
- Deionized water
Troubleshooting
Safety warnings
- 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.
All reagents were equilibrated to room temperature (20–25 °C) prior to use for 30 min. After handling reagents were promptly returned to refrigerated storage.
All samples and controls were mixed thoroughly by vortex prior to use.
HRP-conjugated RBD was diluted 1:1000 in HRP dilution buffer. For instance, to prepare sufficient working solution for a 96-well plate, 10 μl of HRP-conjugated RBD was mixed with 10 ml of HRP dilution buffer.
The 20× wash Solution was diluted 1:19 with deionized water to prepare a 1X working solution. Prepared solution was stored at 2–8 °C when not in use.
Dilute the test serum positive and negative controls 1:9 in dilution buffer, then mix each with HRP-RBD at a 1:1 ratio in a tube. Incubate at 37 °C for 30 minutes at room temperature. It is recommended that all positive and negative controls be prepared in duplicate.
Add 100 µl of the mixture to each well of the 96-well capture plate and incubate at 37 °C for 15 minutes.
Wash the plate three times with 1X wash buffer, removing 260 µl of excess solution each time using the ELISA washer.
Add TMB substrate at 100µl per well was added and incubated in a dark room at room temperature for 15 minutes.
Add 50 µL of stop solution to each well to stop the reaction.
Read the absorbance in the microtiter plate reader at 450 nm immediately.
Protocol references
Genescript SARS-CoV-2 Surrogate Virus Neutralization Test Kit