TITLE: Quantitative Estimation of IgM and IgG Antibodies Against SARS-CoV-2
AUTHORS: Abhinay Gontu, Sreenidhi Srinivasan, Meera Surendran Nair, Scott E Lindner, Allen M Minns, Randall Rossi, Suresh Kuchipudi, Vivek Kapur

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. [ELISA procedure]
Coating of ELISA platesCoat the ELISA plates with 50 μL (per well) of the receptor-binding domain (RBD) of SARS-CoV-2 spike antigen at a concentration of 2 μg/mL diluted in phosphate-buffered saline (PBS, pH 7.3±0.1). A summary of recombinant RBD antigen production protocol is included in the Materials section.Seal the coated plates with adhesive plate sealers and stored at 5 ± 3°C. The coated plates can be stored at 5±3°C and can be used until 1 week post-coating for ELISA (Stadlbauer et al., 2020).
?. [Checking class specificity of the ELISAs]
The isotype specificity of the ELISA methods can be tested by treating the serum/plasma samples with 1,4-Dithiothreitol (DTT).Prepare 1M DTT in 10X PBS.Dilute the samples to be tested to 1:50 in 1x PBS.Add DTT to the diluted sample such that the final concentration of DTT is 0.005 M.Mix well and incubate at 37°C for 30 min.Dilute the treated samples three-fold in 3% non-fat milk.Test the samples as described in the ELISA method above.Expected result:DTT inactivates IgM rapidly (compared to IgG), and therefore a significant drop in ODs and/or titers is expected in the IgM titers post-treatment. The IgGs do also get inactivated. However, the drop in ODs and/or titers is not as significant as with IgM.
?. [ELISA procedure]
Sample addition to the ELISA platesAfter incubating with blocking buffer (step 2), wash the ELISA plates three times with wash buffer.Transfer 50 μL (per well) of the diluted samples and appropriate controls (positive controls: monoclonal antibodies; negative controls: dilution buffer) from the U-bottomed plate to the ELISA plate.Seal plates with adhesive plate sealers and incubate at 37°C for 1 h.
?. [ELISA procedure]
BlockingUse freshly prepared 1x PBS with 0.05% Polysorbate™ 20 as wash buffer.Use 5% non-fat dry milk powder in wash buffer as the blocking buffer. Vortex samples briefly to obtain a homogenous solution, and place in a 37°C incubator for 5-10 min.Note that all wash steps are performed with 200 μL/well wash volume; if using an ELISA washer, include program to aspirate well contents as the first step prior to dispensing of wash buffer to wellsAfter overnight incubation of coated plates, wash plates three times with wash buffer.Next, add 200 μL per well of the freshly prepared blocking buffer.Seal plates with adhesive plate sealers and incubate at 37°C for 1 h.
?. [ELISA procedure]
Sample preparationPlace serum/plasma samples at 56°C for 30 min in a dry/water bath to heat inactivate. The heat inactivated samples can be stored at 5±3°C until tested, or stored long-term at -80°C (samples can also be heat-inactivated earlier, stored appropriately, and then thawed on the day of the test).Prepare 3% non-fat milk in wash buffer as a dilution buffer.Dilute heat inactivated serum/plasma samples to 1:50 with dilution buffer in sterile U-bottom plates. Perform serial three-fold dilutions starting from 1:50 up to 1:4050.Prepare desired concentrations of commercially available monoclonal antibodies (typically 1 μg to 0.01 μg) in PBS. Use 50 μL of each dilution as a positive control.
?. [ELISA procedure]
Addition of secondary antibodyAfter incubation, wash ELISA plates three times with wash buffer.Add 50 μL (per well) of the following diluted secondary antibodies:For IgM ELISA: Peroxidase-conjugated anti-human IgM (Fc5μ) antibody, diluted 1:12,000 with 3% non-fat milk.For IgG ELISA: Peroxidase-conjugated anti-human IgG (Fc specific) antibody, diluted 1:10,000 with 3% non-fat milk.Seal plates with adhesive plate sealers and incubate at 37°C for 1 h.
?. [ELISA procedure]
Developing the reactionDilute chromogenic visualization solution with 1 mg (tablet) of 3,3′,5,5′-Tetramethylbenzidine (TMB) in 10 mL of phospho-citrate buffer with 3 μL of hydrogen peroxide prepared just before use.After incubation with secondary antibody (step 5), wash three times with wash buffer.Add 100 μL (per well) of the prepared TMB solution and incubate at room temperature (22±3°C) for 10 min in light-protected conditions.
?. [ELISA procedure]
Stopping the reaction and recording the resultsStop the reaction by adding of 50 μL per well 3 M hydrochloric acid.Read ELISA plates at 450 nm wavelength and record optical densities (OD).
?. [ELISA procedure]
Interpreting the results
?. [ELISA procedure]
Determining cut-off ODRun an appropriate number (≥75) of negative (preferably pre-COVID-19) serum/plasma samples.Determine a suitable cut-off OD by calculating the value representing three- or six-times the standard deviation over the mean ODs of the negative samples.