My lab has been working in COVID-19 research, and we have recently developed a novel assay that allowed us to quickly and accurately detect SARS-CoV-2 antibodies. This protocol outlines a high throughput assay for detecting anti-SARS-CoV-2 antibodies in patient serum samples, which is critical given the current pandemic. The majority of current serological assays sacrifice speed for sensitivity or vice versa.
Our data shows that our assay is both sensitive and has a quick response time. In order to produce a sufficient quantity of high HiBiT-RBD bioreporter, begin by preparing for cell culture. First, prepare complete Dulbecco's modified eagle medium containing 10%fetal bovine serum and 1%penicillin and streptomycin.
Then warm the media in a 37 degree Celsius water bath. Turn the biological safety cabinet on and use 70%ethanol for sterilizing the cabinet surface. To culture the cell line, take it out from minus 80 degrees Celsius or liquid nitrogen and thaw it in a 37 degree Celsius water bath.
Mix the thawed cells with at least 10 milliliters of complete medium. Pipette the cell suspension into a 15 centimeter Petri dish and swirl the plate to distribute the cells uniformly. Place the dish in a cell culture incubator at 37 degrees Celsius, 5%carbon dioxide, and 85 to 95%humidity.
Observe the cells under the microscope until the confluency level reaches 80 to 90%At high confluency, remove the medium, wash the cells with warm PBS and add three to five milliliters of 0.25%trypsin-EDTA to detach the cells from the surface. After approximately five minutes, look at the cells under a microscope. If all cells are floating, add at least four milliliters of medium and transfer the cell suspension into a new sterile tube.
Count the cells using a hemocytometer and add one million cells into each well of a six-well plate for transfection. To perform transfection of the HiBiT-RBD plasmid, use one microgram of the HiBiT-RBD expression plasmid with a suitable transfection reagent. Incubate for 10 to 15 minutes at room temperature, then add the total volume to each well of the plate dropwise.
On the next day, replace the medium containing the transfection mixture with complete medium. Observe the transfection control well 48 hours after transfection. Collect the supernatant in 1.5 milliliter microtubes.
Add 500 microliters of 1X passive lysis buffer or PLB to the cells, then incubate and shake the plate for 15 minutes at room temperature for cell lysis. Next, to evaluate the luminescence signal from the bioreporter using a luciferase assay, first prepare the reaction components and use the supernatant as the source of the bioreporter. Dilute the large BiT and substrate to 1X before use.
Transfer 50 microliters of the supernatant from each well or tube to a 96-well plate and add 50 microliters of 1X large BiT to each well. Incubate for five minutes at room temperature. Open the luminometer software, add 50 microliters of 1X substrate to each well, place the plate in the luminometer and run the software.
In order to perform the HiBiT-RBD antibody detection assay, prepare the HiBiT-RBD bioreporter as previously described and combine 50 microliters of the HiBiT-RBD containing supernatant with one microgram of the commercial SARS-CoV-2 RBD antibody in a 1.5 milliliter microtube. Add 20 microliters of immunoglobulin binding protein or Protein G to the solution. Bring the total volume to 300 microliters by adding PBS.
Incubate the tubes on a tube shaker or rotator for 30 minutes. Centrifuge at 12, 000 G for 30 seconds. Then discard the supernatant and wash with PBS.
Repeat the process three times to remove free HiBiT-RBD. Resuspend the cells in 50 microliters of PBS and transfer it to a 96-well plate. Add 50 microliters of 1X large BiT and wait for five minutes.
Then add 50 microliters of 1X NanoLuc substrate. Immediately read the luminescence signal with a luminometer. Prepare larger quantities of the HiBiT-RBD bioreporter for a high throughput assay and combine 20 microliters of magnetic Protein G with 50 microliters of the HiBiT-RBD supernatant in each well of a 96-well plate for each sample.
Add 10 microliters of the serum sample to each well and bring the total volume to 150 microliters by adding PBS. Incubate for 30 minutes on a shaker at room temperature. Then place the plate on a magnetic washer to precipitate the Protein G antibody complex.
Discard the solution in the middle section of each well and add PBS for washing. Repeat the washing step at least three times to remove excess HiBiT-RBD. Add 50 microliters of 1X PBS and 50 microliters of 1X large BiT and incubate for at least five minutes at room temperature.
Prepare the luminometer software. Then add 50 microliters of 1X substrate. Place the plate in the machine, run software and record the signals.
Compare the signals from serum samples, control samples, and background signal from empty wells. The signals from both the HiBiT-RBD containing cell lysate and supernatant of the transfected cells were recorded to evaluate the appropriate protein source. The data showed low background compared to a strong signal when both parts were combined.
The addition of Protein G helps antibody precipitation. The assay was used to compare the signal from a commercial SARS-CoV-2 neutralizing antibody with a control IgG. The specific antibody signal was robust, while the control antibody had close to the luminescent background level.
Recombinant attenuated oncolytic vesicular stomatitis virus or VSV with a mutation at position 51 of the M protein expressing exogenous RBD was used to vaccinate mice. The serum collected from vaccinated mice produced a robust signal compared to no signal in mice injected with control VSV. This protocol can be used for detection of the SARS-CoV-2 antibodies in patient serum samples.
The wash steps following incubation with Protein G is critical for reducing background. In addition, it's important to add the substrate as quickly as possible to avoid signal loss. With a few additional optimization steps, the same assay can be used to analyze blood samples and could be an effective method for the determining anti-SARS-CoV-2 antibodies in patient blood samples.
