MULTIPLEX COVID-19 BIOMARKERS ASSAY

The SARS-CoV-2 Human IgG (4-Plex) Q-Plex multiplex assay is a qualitative enzyme-linked immunosorbent assay that detects IgG antibodies to SARS-CoV-2 in human serum or plasma. SARS-CoV-2 is the novel coronavirus that causes COVID-19. The SARS-CoV-2 virus has several structural proteins including two spike proteins, S1 and S2. When an individual is infected with the SARS-CoV-2 virus, their immune system produces antibodies to these viral proteins. The typical immune response produces detectable antibody levels ~8-10 days following the onset of symptoms.

The SARS-CoV-2 Human IgG (4-Plex) Q-Plex Multiplex ELISA detects IgG antibodies to both the S1 and S2 spike proteins present in a blood sample. If antibodies are detected, the result will be considered positive. If antibodies are not detected, the result will be considered negative. The assay only detects IgG antibodies, not other classes of antibodies such as IgM or IgA.

Samples or controls are pipetted into wells of an arrayed microplate thereby immobilizing antibodies to SARS-CoV-2 S1, SARS-CoV-2 S2, Sheep Fc (negative control), and Human IgGs (positive control) to their locations in the array. After washing away any unbound IgG, a mixture that contains biotinylated anti-human IgG is added. After washing away unbound biotinylated antibody, streptavidin-horseradish peroxidase (SHRP) is added. Following an additional wash, the amount of SHRP remaining on each location of the array is proportional to the amount of Human IgG antibody reactive to SARS-Cov2 S1, SARS-Cov2 S2, Sheep Fc (negative control), anti-Human IgG (positive control) initially captured.

The amount of conjugated enzyme on each location of the array is measured with the addition of a chemiluminescent substrate.

The multiplex assay allows for simultaneous indirect ELISA on the following four printed spots:

1. SARS-CoV-2 Spike Glycoprotein (S1), a recombinant antigen which contains amino acids 1-674 of subunit 1. Spike S1 is expressed in mammalian HEK293 cells with a Sheep Fc-Tag.
2. SARS-CoV-2 Spike Glycoprotein (S2) is a recombinant antigen which contains the Spike subunit 2 protein, amino acids 685-1211. Spike S2 is expressed in mammalian HEK293 cells with a Sheep Fc-Tag.
3. Sheep Fc is a negative control to ensure no cross-reactivity occurs between human IgGs in the sample and the Fc-Tag on the SARS-Cov-2 Spike proteins.
4. Anti-Human IgG is a positive control to ensure the kit performs and the IFU was followed correctly.

The validation testing of this assay consisted of testing known negative samples, which were collected prior to August 2019, and known positive samples that were collected from individuals who tested positive for COVID-19 on a molecular test. The negative samples included sample from a patient that tested positive for another human coronavirus, OC43. The negative samples also included samples from patients with Rheumatoid Arthritis, an immune disorder that causes changes to the immune system, whose samples are more likely to interfere with an antibody test and HBV and HCV seropositive samples.

SARS-CoV-2 Spike 1 Protein IgG Assay vs. Molecular COVID-19 Test
N = 206	Confirmed Positive	Confirmed Negative
IgG Test Positive	14	0
IgG Test Negative	0	192

SARS-CoV-2 Spike 2 Protein IgG Assay vs. Molecular COVID-19 Test
N = 206	Confirmed Positive	Confirmed Negative
IgG Test Positive	10	1
IgG Test Negative	3	191

As of April 17, 2020, the assay for antibodies to the Spike 1 protein demonstrates: Estimated Sensitivity (PPA)= 100%, Estimated Specificity (NPA) = 100%, PPV = 100%, NPV =100%. The assay for antibodies to the Spike 2 protein demonstrates: Estimated Sensitivity (PPA) = 72%, Estimated Specificity (NPA) = 99%, PPV = 91%, NPV =98%.

We will continue to update these validation studies as more samples become available.

SARS-CoV-2 IgG Assay Characteristics
Principle	Indirect ELISA
Sample Type	Serum, Plasma, Whole Blood
Sample Volume	2 µL
Assay Incubation	2 hours, RT
Total Wash Steps	3
Within Plate Reproducibility	8% CV
Between Plate Reproducibility	10% CV

Kit Components

Each kit contains a 96-well plate, featuring the relevant biomarker panel in each well, and all reagents required to perform testing.

Reagents include:

• Detection Mix
• Substrate
• Sample Diluents
• Streptavidin HRP
• Wash Buffer

Kit Description

The Q-Plex™ SARS-CoV-2 Human IgG (4-plex) is a qualitative chemiluminescent assay (ELISA) allowing concurrent measurement of human IgG antibodies to SARS-CoV-2 S1 and SARS-CoV-2 S2 proteins in serum and plasma samples.

• This test is for use by research or clinical laboratories.
• This test has not been reviewed by the FDA.
• Negative results do not rule out SARS-CoV-2 infection, particularly in those who have been in contact with the virus. Follow-up testing with a molecular diagnostic should be considered to rule out infection in these individuals.
• Results from antibody testing should not be used as the sole basis to diagnose or exclude SARS-CoV-2 infection or to inform infection status.
• Positive results may be due to past or present infection with non-SARSCoV-2 coronavirus strains, such as coronavirus HKU1, NL63, OC43, or 229E.
• Not for the screening of donated blood.

Kit Processing

The Q-Plex™ Multiplex assays require the use of the Q-View™ software to read and interpret the test results. A fully-functional, free trial version of the software is available to download, install, and use to analyze your first kit(s). At the end of the trial period, a purchased license is required to continue the use of the Q-View software.

Q-Plex arrays are developed and optimized to work with Q-View imagers. We do not guarantee the results obtained from other imagers because not all imagers are compatible with Q-Plex arrays.