Simple and rapid serological tests to characterize antibody responses are important in the current COVID-19 pandemic caused by SARS-CoV-2. Multiple immunoblot (IB) assays called COVID-19 IB assays were developed to detect IgG and IgM antibodies against SARS-CoV-2 virus proteins in COVID-19 patients.


The recombinant nucleocapsid protein and the S1, S2, and (RBD) receptor-binding domain of the SARS-CoV-2 spike protein were used as target antigens in COVID-19 IBs. The specificity of the IB assay was established with 231 sera from persons with allergies, unrelated viral infections, autoimmune conditions, and suspected tick-borne diseases, and 32 goat antisera for human influenza proteins. IgG and IgM COVID-19 IB assays were performed on 84 sera obtained at different times after a positive RT-qPCR test from 37 COVID-19 patients with mild symptoms.

  • Sera from patients with COVID-19

The study used decoded leftover sera received for routine testing at the Medical Art Center, Middletown, NJ and IGeneX, Milpitas, CA that would have otherwise been discarded. The 84 sera were from 37 patients who had tested positive on the FDA-cleared Quest Diagnostics RC SARS-CoV-2, LabCorp COVID-19, or ThermoFisher Scientific TaqPath COVID-19 RT-qPCR tests. All the patients had shown only mild COVID-19 symptoms and none had required hospitalization. The 84 serum samples used in the study were collected at times ranging from 0 to 154 days after patients tested positive for RT-qPCR. The patients were 19 men and 18 women with an age range of 21 to 76 years.

  • Antigen Strips for COVID-19 IB Assays

Antigen strips were prepared essentially as described for our previously developed IB assays for borreliosis. The purified antigens diluted to produce approximately 7-19 ng of protein as a line on each 3mm membrane strip were sprayed in straight lines on a nitrocellulose membrane (Amersham Protran, GE Healthcare Life Science, Chicago, IL) using a dispenser. of BioDot Liquid (BioDot, Irvine, CA). Human IgG and IgM (Sigma, St. Louis, MO) were applied as C1 and C4 controls respectively on all IB strips to establish the specificity of antibody class detection and to confirm the addition of alkaline phosphatase-conjugated anti-human antibodies.

Protein L (Sigma, St. Louis, MO) was used as a C2 control to detect the addition of human serum. A C3 calibration standard was applied to the test strip for use in all IB tests. The membranes were then blocked with 5% skimmed milk powder and cut into 3mm wide strips. Membrane strips containing antigens can be stored for at least 6 months at 2-8 ° C prior to using for IB assays.

  • Detection of IgG and IgM antibodies in sera from COVID-19 patients

IgG and IgM antibodies were detected in COVID-19 IBs essentially as described for borreliosis IBs. Before use, each 3 mm strip was soaked in 1 ml of diluent (100 mM Tris, 0.9% NaCl, 0.1% Tween-20 and 1% skimmed milk powder) for 5 min in a bucket. A 10 μL aliquot of the test or control serum for IgG IB and 20 μL for IgM IB was then added to an IB strip, incubated at room temperature for 1 hour, and then washed three times with wash buffer (KPL, Gaithersburg, MD).

After aspirating the final wash solution, IgG and IgM strips were incubated with alkaline phosphatase-conjugated goat anti-human IgG at a dilution of 1: 10,000 and goat anti-human IgM at a dilution of 1: 3000 respectively (KPL, Gaithersburg, MD) for 1 h at room temperature. After three washes, the bands were visualized by reaction with 5-Bromo-4-chloro-3-indolyl phosphate nitro-blue tetrazolium (KPL, Gaithersburg, MD). Reactions were terminated by washing with distilled water when the C3 calibration standard produced a visible band. Bands reactive to the antigen of less intensity than the calibration standard were considered negative.


Criteria for determining overall IgG and IgM antibody positivity using the four SARS-CoV-2 proteins were developed by optimizing specificity and sensitivity in the COVID-19 IgG and IgM IB assays. The estimated sensitivities and specificities of COVID-19 IgG and IgM IBs for IgG and IgM antibodies individually or for IgG or IgM antibodies meet US recommendations for laboratory serological diagnostic tests.

The proportion of positive IgM sera from COVID-19 patients after a positive RT-qPCR test peaked at 83% before 10 days and decreased to 0% after 100 days, while the proportions of positive IgG sera tended to stabilize between days. 11 and 65 to 78-100% and drop to 44% after 100 days. Detection of IgG or IgM antibodies was better than IgG or IgM alone in assessing seroconversion in COVID-19. Both IgG and IgM antibodies detected RBD less frequently than the S1, S2, and N proteins.


Multiplex COVID-19 IB assays offer many advantages for simultaneously evaluating antibody responses to different SARS-CoV-2 proteins in COVID-19 patients.

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