APPLICATION NOTEA CUSTOM, MULTIPLEX HUMAN CORONAVIRUS
SEROLOGICAL ASSAY

In less than a year, severe acute respiratory syndrome coronavirus
2 (SARS-CoV-2) swept globally, infecting over 30 million people.
Of those infected, more than 1 million people have been killed
by Coronavirus 2019 (COVID-19) disease. As such, assessing
the prevalence of SARS-CoV-2, along with SARS-CoV-2 vaccine
development have been top global priorities. COVID-19
screening assays can be broadly classified into diagnostic/
antigen tests1 that detect viral RNA or viral protein in the hostduring an active infection, and serological assays that detect theantibody response to the virus. Screening for active infections has
been challenging for two interrelated reasons. Although SARSCoV-2 has a lower mortality rate than the related SARS-CoV andmiddle east respiratory syndrome coronavirus (MERS-CoV), it has
a higher transmission rate2-4. Furthermore, it has been estimatedthat ~45% of those infected with the virus are asymptomatic5.
In the absence of symptoms to inspire diagnostic testing, it isapparent that the percentage of COVID-19 infected people may
be severely underestimated. Given the possibility of such a large
percentage of asymptomatic carriers, serological assays play acrucial role in the Center for Disease Control (CDC) COVID-19
serology surveillance strategy6.

SARS-CoV2 is a member of the coronaviridae family,
specifically the coronavirinae subfamily that includes 4 genera:
α-coronavirus, β-coronavirus, γ-coronavirus and δ-coronavirus2.
SARS-CoV-2, SARS-CoV and MERS-CoV are all RNA βcoronaviruses. SARS-CoV-2 shares 80% sequence identitywith SARS-CoV and 50% sequence identity with MERS-CoV3,7.
It has been estimated that members of the α-coronavirus andβ-coronavirus family are also responsible for 10-30% of common
colds8,9. Given the promiscuous nature of coronavirus, the needto evaluate the immune response to coronavirus subtypes
becomes apparent. To what degree does exposure to the common
cold virus, SARS-CoV or MERS-CoV enhance or diminish immunity
to SARS-CoV-2? To answer this question, investigators need tobe able to evaluate the specificity and cross-reactivity of theimmune response to coronaviruses. R&D Systems, a Bio-Technebrand, has developed a novel custom Luminex serological assaythat distinguishes between coronavirus subtypes. Using ourhigh-quality recombinant proteins, this antigen-down assay saves
time and precious sample by multiplexing many coronavirus
subtypes simultaneously in each sample.

CORONAVIRUS SPIKE PROTEINS USED IN THE LUMINEX ASSAY
CATALOG #PROTEIN

LUMINEX ASSAY STANDARD CURVE RANGE

10522-CV

SARS-CoV-2 Spike S141-7,478 pg/mL

10523-CV

SARS-CoV-2 Spike RBD49-7,608 pg/mL

10474-CV

SARS-CoV-2 Nucleocapsid

98-16,026 pg/mL

10521-CV

MERS-CoV Nucleocapsid

61-9,853 pg/mL

Coming Soon!

MERS-CoV Spike S1

964-21,912 pg/mL

10621-CV

MERS-CoV Spike RBD

58-8,380 pg/mL

10600-CV

HCoV-HKU1 Spike RBD

143.5-18,685.5 pg/mL

Coming Soon!

HCoV-OC43 Spike RBD

35-3,153 pg/mL

10605-CV

HCoV-NL63 Spike RBD Protein

22.5-2,286.3 pg/mL

10612-CV

HCoV-229E Spike RBD Protein

55.5-7,315.3 pg/mL

TABLE 1. Coronavirus Spike Proteins Used in the Luminex AssayAA

SARS-CoV-2 Standard Curves

Assay Linearity: SARS-CoV-2 Proteins

Mean Flourescence Intensity

100000

140%

10000

120%
100%

100080%60%

10040%20%

1010100

1000

10000

1000000%Concentration (pg/mL)

1:2

B

MERS-CoV Standard Curves
Mean Flourescence Intensity

1:8

1:16

B

Assay Linearity: MERS-CoV Proteins

100000

140%
120%

10000

100%
80%
1000

60%
40%
20%

100
100

1000

10000

1000000%Concentration (pg/mL)

1:2

C

1:4

1:8

1:16

C

Common Coronavirus Standard Curves

Assay Linearity: Common Coronavirus Proteins

100000Mean Flourescence Intensity

1:4140%
10000

120%
100%

1000

80%
60%

100

40%
20%

1010100

1000

10000

1000000%Concentration (pg/mL)

1:2

1:4

1:8

1:16

FIGURE 2. Natural Sample Assay Linearity. To show that the proteins used in themultiplex serological assay to capture anti-coronavirus antibodies bound specificallyto their intended target, linearity was measured by diluting high positive serum
samples with negative serum samples. Linearity plots were generated for: (A) SARSCoV-2 proteins (Nucleocapsid-Blue, Spike S1-Orange, Spike RBD-Gray), (B) MERSCoV proteins (Nucleocapsid-Blue, Spike S1-Orange, Spike RBD-Gray), and (C)common human coronavirus proteins (HKU1 RBD-Blue, OC43 RBD-Orange, NL63
RBD-Gray, 229E RBD-Green).

FIGURE 1. Bio-Techne Human Coronavirus Proteins in a Serological Immunoassay. A
multiplex serological assay using Luminex MagPlex® Microspheres was assembled
using human coronavirus proteins offered by R&D Systems, a Bio-Techne brand.
Using anti-coronavirus and anti-His-Tag antibodies, standard curves were generated
for: (A) SARS-CoV-2 proteins (Nucleocapsid-Blue, Spike S1-Orange, Spike RBDGray), (B) MERS-CoV proteins (Nucleocapsid-Blue, Spike S1-Orange, Spike RBDGray), and (C) common human coronavirus proteins (HKU1 RBD-Blue, OC43 RBDOrange, NL63 RBD-Gray, 229E RBD-Green).

2AB

SARS-CoV-2 Nucelocapsid Protein

SARS-CoV-2 Spike Protein Subunit 1
25,000Mean Flourescence Intensity

Mean Flourescence Intensity

30,00025,00020,00015,00010,0005,000-

20,00015,00010,0005,000-CSARS-CoV-2 Spike Protein Receptor Binding Domain
Mean Flourescence Intensity

25,00020,00015,00010,0005,000-DMultiplex Serology to Measure Immune Response to Coronavirus

Mean Flourescence Intensity

25,00020,00015,00010,0005,000-MERS NP

MERS S1

COVID19-Neg 1

MERS RBDSARS2 NP

COVID19-Neg 2

SARS2 S1

COVID19-Neg 3

SARS2 RBDHKU1 RBDCOVID19-Pos 1

OC43 RBDCOVID19-Pos 2

NL63 RBD

229 E RBDCOVID19-Pos 3

FIGURE 3. Patient Serum Sample Antibody Measurements. Forty pre-COVID-19 and COVID-19 PCR-negative samples (blue), and 24 COVID-19 PCR-positive samples(orange) were tested using the multiplex serological assay to determine the human IgG response to (A) the Nucleocapsid, (B) Spike Glycoprotein and (C) Spike Protein
Receptor Binding Protein. (D) Six representative COVID-19 patient serum samples show the variable IgG immune response each patient generates to coronavirus.

3PCR NEGATIVE (N=8)/PRE-COVID19 SAMPLES (N=32)
SARS2
NP

SARS2
S1SARS2
RBD

MERS
NP

MERS
S1

MERS
RBD

HKU1
RBD

OC43
RBD

NL63
RBD

229E
RBD

Negative

40

40

3740

40

40

0300

04

19

Positive

0000

03000000

3740

36

21Total Samples

40404040404040404040

Negative Agreement

100%

100%

93%100%100%100%8%0%

10%

48%

SARS2
NPSARS2S1SARS2
RBD(N=24)

MERS
NP

MERS
S1MERS
RBD

HKU1
RBD

OC43
RBD

NL63
RBD

229E
RBD

Negative

02

02

03

21

20

21

01

01

01

12

Positive

22

22

2103

04

03

23

23

23

12Total Samples

2424

24

24

24

24

24

2424

24

Positive Agreement

92%

92%

88%

13%

17%

13%

96%

96%

96%

50%

PCR POSITIVE COVID19 SAMPLESTABLE 2. Negative and Positive Agreement. To determine the clinical performance of the SARS-CoV-2 nucleocapsid and spike glycoprotein IgG assay, a positive percent
agreement between the IgG assay and a PCR-based diagnostic test was performed. The MERS and common coronaviruses sample agreements were calculated to show
little correlation between the SARS-CoV-2 and other coronaviruses.

METHODSRESULTSR&D Systems developed human coronavirus proteins
were conjugated to distinct fluorescent Luminex MagPlex®
Microspheres to generate a multiplex serological assay.
Recombinant coronavirus spike protein (TABLE 1) conjugatedmicrospheres were pooled generating a 10 analyte multipleplex antibody capture serological assay. Assay positive control
samples were prepared using coronavirus-specific antibodies
SARS-CoV-2
NucleocapsidAntibody(MAB10474),
SARS-CoV-2 Spike S1 Subunit Antibody (MAB105403),
Anti-MERS Spike Protein (Absolute Antibody®), MERS-CoV
Nucleocapsid Antibody (Sino Biological®), Human Coronavirus
Spike HKU1 (Sino Biological®) or, when an appropriate antibody
is unavailable, an anti-histidine tag antibody (MAB050) was used.

Calibration curves were generated, using commerciallyavailable coronavirus spike and nucleocapsid protein antibodies(FIGURE 1). The standard curve ranges are indicated in TABLE
1. Linearity is an important measurement of assay accuracy
and specificity as non-specific binding can be identified witheach sample dilution if signal does not decrease. Seriallydiluted samples should have the same concentration once
back-calculated, giving you consistency in results and confidence
that the assay is measuring the correct analyte. The SARS-CoV-2,
MERS-CoV, and common cold coronavirus assay natural linearity
was 96-131% which is within the acceptable range for Luminexassays (FIGURE 2). To test for accuracy, COVID-19 positive samples
were compared to COVID-19 negative samples. Multiple serum
samples from COVID-19 PCR positive patients showed strong
assay signal for all three SARS-CoV-2 proteins, Nucleocapsid,
Spike S1 and Spike RBD, (FIGURE 3A-C). PCR-negative samples
had no signal. Representative COVID-19 negative and COVID-19
positive samples were run to show specificity of the assay tocapture SARS-CoV-2 IgG responses. The MERS-CoV proteins,which had nearly undetectable signal and a variable common
cold IgG response showed positivity across COVID-19 positiveand negative samples indicating no correlation with SARSCoV-2 IgG responses (FIGURE 3D). Finally, agreement between
this custom assay and commercially available ELISA tests wasevaluated (TABLE 2). The positive and negative agreement
showed the ability of a multiplexed R&D Systems coronavirus
proteins to accurately detect SARS-CoV-2 IgG responses.

A total of 64 archived serum samples were selected, including:pre-COVID-19 (n=32), SARS-CoV-2 PCR-confirmed negative(n=8), and PCR-confirmed positive (n=24) samples. Samples
were diluted 1:250 using the assay calibration buffer andincubated with microparticles for 2 hours at room temperature.
After washing away any unbound substances, Serotype-specific
IgG antibodies were detected with a biotinylated-anti-human
IgG antibody (Coming Soon!) or anti-mouse IgG (BAF018)
antibody (to detect positive control antibodies) and incubatedfor 1 hour at room temperature. Following a wash to remove
any unbound biotinylated antibody, streptavidin-phycoerythrin
conjugate (Streptavidin-PE), which binds to the biotinylated
antibody, is added to each well. Following washes to remove
unbound Streptavidin-PE, the microparticles were chemically
fixed by resuspending in 4% formaldehyde (FC004) and wereincubated for 1-hour at room temperature. The final washremoves fixation buffer and the microparticles were resuspended
in wash buffer and read using a Luminex® instrument.
4CONCLUSIONS
Taken together we have demonstrated that this custom multiplex
antibody capture assay allows for the simultaneous evaluationof SARS-CoV-2, MERS-CoV and common cold coronaviruses.
This assay can be used by investigators to determine if immune
responses to different coronaviruses has a positive (protective),negative (enhancement), or no effect on the clinical outcome of
an infection. Partner with R&D Systems, a Bio-Techne brand, tooptimize this and other future assays for your specific needs.

REFERENCES
1. https://www.fda.gov/consumers/consumer-updates/coronavirus-testing-basics
2. Fani, M. et al. (2020) Future Virol., [Epub ahead of print]
3. Hirano, T. & M. Murakami (2020) Immunity., 52:731
4. Perlman, S. (2020) N. Engl J. Med., 382:760
5. Oran, D. P. & E. J. Topal (2020) Ann Internal Med., [Epub ahead of print]
6. https://www.cdc.gov/coronavirus/2019-ncov/covid-data/serology-surveillance/
7. Coperchini, F. et al. (2020) Cytokine Growth Factor Rev., 53:25
8. Wat, D. (2004) Eur. J. Int. Med., 15:79
9. Mesel-Lemoine, M. et al. (2012) J. Virol., 86:7577

5

NOTES

67

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