SARS-CoV-2 Spike S1 Protein ELISA Kit
Catalog No. 55030(Version A1)
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, California 92008, USA
Toll free:877 222 9543
Telephone:
760 431 1263
Fax:
760 431 1351
Active Motif Europe
Waterloo Atrium
Drève Richelle 167 – boîte 4
BE-1410 Waterloo, Belgium
UK Free Phone:		
0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone:		+32 (0)2 653 0001
Fax:		
+32 (0)2 653 0050
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone:+81 3 5225 3638
Fax:
+81 3 5261 8733
Active Motif China
787 Kangqiao Road
Building 10, Suite 202, Pudong District
Shanghai, 201315, China
Telephone:(86)-21-20926090
Hotline:400-018-8123
Copyright 2020 Active Motif, Inc.

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Information in this manual is subject to change without notice and does not constitute a commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of any
kind, either explicit or implied. Information may be changed or updated in this manual at any time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated, inwhole or in part, without the prior written consent of Active Motif, Inc. This documentation is
proprietary information and protected by the copyright laws of the United States and international treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2020 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All rights reserved.
All trademarks, trade names, service marks or logos referenced herein belong to their respective
companies.
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TABLE OF CONTENTS

Page

Overview  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Flowchart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Kit Performance and Benefits  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Protocol . .  .  .  .  .  .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Standard Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Sample Preparation  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
ELISA Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  10
Troubleshooting Guide  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Technical Services  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

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Overview
COVID-19, which is short for coronavirus disease 2019, is the official name of the respiratory disease caused by infection with the novel coronavirus SARS-CoV-2. The virus that causes COVID-19
was named SARS-CoV-2 because it is a coronavirus that is genetically similar to, yet distinct from,the virus that caused the severe acute respiratory syndrome (SARS) outbreak in 2003. Like many
viruses, the COVID-19 virus attaches to a cell that it is infecting through an interaction between aspike protein (S-protein) on the surface of the viral particle and a receptor protein on the surfaceof the cell.
Since the S-proteins are on the surface of the viral particles, antibodies that bind very specificallyand with high affinity to the S-protein can be used in immunological assays like ELISAs to detect
the presence of the virus in biological samples. Each S-protein, contains two subunits, S1 and S2,
with the S1 subunit mainly comprised of the receptor binding domain (RBD). It is the RBD which is
responsible for recognizing the cell surface receptor.
The SARS-CoV-2 Spike S1 Protein ELISA Kit is designed for the detection of SARS-CoV-2 Spike S1(RBD) protein in a high-throughput format. This kit contains enough reagents to be performed in a
96-stripwell plate, enabling you to simultaneously perform up to 96 assays in a single experiment.
For added convenience and a more quantitative interpretation of the results, the kit also includes
recombinant RBD which enables you to build a reference standard curve.
This ELISA kit is based on the sandwich enzyme immunoassay technique, which uses pre-coatedwell strips with a recombinant SARS-CoV-2 Spike S1 antibody. Both RBD protein standards andsamples are added to the pre-coated wells and the SARS-CoV-2 Spike S1 present in the sample
is captured by the antibody. A biotinylated recombinant SARS-CoV-2 antibody is then added,
producing the antibody-antigen-antibody “sandwich” immunocomplex. A streptavidin-horseradish
peroxidase (HRP) conjugate is then bound to the detection antibodies and developing solutions
enable a sensitive colorimetric readout that is easily quantified by spectrophotometry.

Product

FormatCatalog no.

SARS-CoV-2 Spike S1 Protein ELISA Kit

1 x 96 rxns

55030

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1Flowchart of Process

2

1

5

4

3

7

6

8

9

10

11

12

A
B
C
D
E
F

Sample containing
SARS-CoV-2 Spike S1 Protein

G
H

SARS-CoV-2 Spike S1 Antibodycoated Plate

Add sample
to the plate
21

11

01

9

8

7

6

5

4

3

2

1
A
B

C
D
E
F
G
H

1.5 hr.

1

9

8

7

6

5

4

3

2

11

10

12

A
B
C
D
E
F
G
H

Add SARS-CoV-2 Spike S1
Detection Antibody
1 hr.

21

11

01

9

8

7

6

5

4

3

2

1
A

Add StreptavidinHRP conjugate

B
C
D
E
F
G

Add Developing
and Stop Solution

HHRP

30 min.
1

5

4

3

2

7

6

8

9

10

11

12

A
B
C
D
E
F
G
H

HRP

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2

Kit Performance and Benefits
The SARS-CoV-2 Spike S1 Protein ELISA Kit is for research use only. Not for use in diagnostic
procedures.

Standard Curve

Absorbance (450 nm)

3

2

1

0

1

10

100

SARS-CoV-2 Spike S1 (RBD) Protein [log10(ng/ml)]

Figure 1: Example Standard Curve

A standard curve was generated by plotting raw absorbance (450 nm) versus concentration of the SARSCoV-2 Spike S1 (RBD) Protein Standard in Dilution Buffer. The data shown are for four independent experiments with each standard in duplicate. Standard deviation is shown. The development time was 5 minutes.
The curve is a 5-Parameter Logistic (5PL) regression fit to the data determined using GraphPad Prism 8
software.

Sensitivity: Sensitivity is the lowest amount of analyte that can be detected. The sensitivity wasdetermined to be 0.58 ng/ml from an average of 4 duplicate standard curves.
Average Intra-assay Precision: Coefficient of variation (CV) = 2.92%
Six replicates of the standard curve were tested on one plate.
Average Inter-assay Precision: Coefficient of variation (CV) = 4.59%
Three independent experiments were performed of the standard curve. Every solution was
prepared independently for each run and a single-use aliquot of the SARS-CoV-2 Spike S1 (RBD)
Protein was used.
Spike & Recovery: The %recovery for three concentrations of recombinant SARS-CoV-2 Spike S1(RBD) Protein was evaluated in both plasma and saliva.
Sample Type

Range Recovery (%)

Average Recovery (%)

Plasma77-11689

Saliva91-12198

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3Linearity: High concentration of the recombinant SARS-CoV-2 Spike S1 (RBD) Protein was spiked
into plasma and saliva, diluted to the range of the standard curve, and evaluated for linearity.

1:2
1:4
1:8

PlasmaSalivaRange (%)

93-10992-101

Average (%)

98

95

Range (%)

90-101

83-95

Average (%)

94

87

Range (%)

99-105

87-95

Average (%)

102

92

Assay time: 3 hours.

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4Kit Components and Storage
The SARS-CoV-2 Spike S1 Protein ELISA Kit is shipped on dry ice. Upon receipt store the components at the temperatures shown below. Components are guaranteed stable for 6 months fromdate of receipt when stored properly.
Reagents

Quantity

Storage

SARS-CoV-2 Spike S1 (RBD) Protein Standard

15 µl

-80°C

SARS-CoV-2 Spike S1 Detection Antibody

15 µl

-20°C

Dilution Buffer

2 x 20 ml-20°C

Streptavidin-HRP Conjugate

12 ml

4°C

10X Wash Buffer

25 ml

4°C

Developing Solution

12 ml

4°C

SARS-CoV-2 Spike S1 Antibody-coated Plate

14°C

Stop Solution

12 ml

RT

Plate Sealer

2

RT

Aluminum Plate Sealer

1

RT

Additional materials required• Distilled water• Multi-channel pipettor• Multi-channel pipettor reservoirs• Rocking platform/orbital shaker• Microplate spectrophotometer capable of reading at 450 nm (655 nm as optional reference
wavelength)www.activemotif.com

5Buffer Preparation and Recommendations
1X Wash Buffer
Prepare 1X Wash Buffer by diluting 10X Wash Buffer 1:10 with distilled water. For a half plate, dilute
12 ml 10X Wash Buffer with 108 ml distilled water. For a full plate dilute 24 ml 10X Wash Buffer with
216 ml distilled water. Mix gently by swirling to avoid foaming. 1X Wash Buffer can be stored at 4°C
for up to 1 month. Dispose of the 1X Wash Buffer if any particulates are observed.
Dilution Buffer
Supplied read-to-use. Two bottles of Dilution Buffer are supplied and should be stored frozen at-20°C until the day that the assay is conducted. On the day of the assay thaw Dilution Buffer atroom temperature for at least 1 hour before using. If crystals have formed in the buffer, invert
and mix gently until the crystals have completely dissolved. For a half plate one bottle is required.
For a full plate, two bottles are required.
Diluted Detection Antibody
Dilute SARS-CoV-2 Spike S1 Detection Antibody 1:1000 in Dilution Buffer for the number of wells
to be assayed. For a half plate, dilute 6 µl SARS-CoV-2 Spike S1 Detection Antibody into 6 ml fresh
Dilution Buffer. For a full plate dilute 12 µl SARS-CoV-2 Spike S1 Detection Antibody into 12 ml fresh
Dilution Buffer. Place on ice and use immediately. Any remaining undiluted SARS-CoV-2 Spike S1
Detection Antibody can be stored at -20°C and is guaranteed until six months after receipt.
Streptavidin-HRP Conjugate Solution
Supplied ready-to-use.
Developing Solution
Supplied ready-to-use. Allow to warm to room temperature for at least 30 minutes before using.
Note: The Developing Solution is light sensitive. Therefore, avoid direct exposure to light duringstorage. The Developing Solution should be colorless prior to addition to the wells. A green/bluecolor present in the Developing Solution indicates that it has been contaminated and must be
discarded.
Stop Solution
Supplied ready-to-use.

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6

Protocol
Read the entire protocol before use.
Determine the appropriate number of microwell strips required for testing samples, protein
standards and blanks in duplicate. Unused microwell strips may be stored in the aluminum pouchat 4°C for up to one month. Use the strip holder for the assay.
Prepare the 1X Wash Buffer and Dilution Buffer as described in the section Buffer Preparation andRecommendations. Multi-channel pipettor reservoirs may be used for dispensing the Dilution Buffer, Wash Buffer, Developing Solution and Stop Solution into the wells.

Standard Curve
For creating a standard curve, we suggest placing the serial dilutions of the SARS-CoV-2 Spike S1(RBD) Protein Standard in duplicate in the first two rows. Up to 40 samples in duplicate can beassayed in the remainder of the plate.
Example Plate Layout
SARS-CoV-2 Spike S1(RBD) Protein Standard
1

2

A

150 ng/ml

150 ng/ml

B

75 ng/ml

75 ng/ml

C

37.5 ng/ml

37.5 ng/ml

D

18.8 ng/ml

18.8 ng/ml

E

9.38 ng/ml

9.38 ng/ml

F

4.69 ng/ml

4.69 ng/ml

G

2.34 ng/ml

2.34 ng/ml

H

0 ng/ml

0 ng/ml

SAMPLES
3

4

5

6

7

8

9

10

11

12

The standard curve should be generated the same day and on the same plate as the samples.
SARS-CoV-2 Spike S1 (RBD) Protein Standard Preparation
1.

The recombinant SARS-CoV-2 Spike S1 (RBD) Protein Standard is provided at 1 mg/ml. Thaw
on ice. Before using, flick the bottom of the tube gently to mix and quick spin the contents
to the bottom of the tube.

2.

Make up a 1 µg/ml stock of diluted SARS-CoV-2 Spike S1 (RBD) Protein Standard by adding 2µl of the 1 mg/ml stock into 2 ml Dilution Buffer and mix by vortexing.

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7

Freeze the remaining undiluted SARS-CoV-2 Spike S1 (RBD) Protein Standard at -80°C. The protein
is stable for up to 5 freeze-thaws. The diluted 1 µg/ml stock of SARS-CoV-2 Spike S1 (RBD) ProteinStandard can be stored at 4°C for up to a week. Do not used after one week as the signal may be
reduced.
Standard Curve Preparation
1.

Label eight 1.5 ml microfuge tubes A to H.

2.

Into tube A, transfer 15 µl of the diluted 1 µg/ml SARS-CoV-2 Spike S1 (RBD) Protein Standard
and 85 µl Dilution Buffer to make a 150 ng/ml solution and mix well.

3.

Add 30 µl Dilution Buffer to tubes B through H.

4.

Perform a 1:2 serial dilution by transferring 30 µl of the contents in tube A to the tube B.

5.

Mix the contents of tube B by pipetting up and down or vortexing.

6.

Transfer 30 µl of the contents in tube B to tube C and mix, as previous.

7.

Repeat serial dilution in tube C-G. Do not add anything to tube H.

Sample Preparation
Because levels of the SARS-CoV-2 Spike S1 protein may vary between different samples, optimal
dilution factors for each sample must be determined by the end-user. It is suggested to dilute saliva at least 1:10 with Dilution Buffer (10 µl saliva: 90 ul Dilution Buffer) as diluting less than 1:10 may
result in a matrix affect on the assay. Saliva not used immediately may be stored at -20°C. For longterm storage (>2 months) samples should be stored at -80°C. Avoid multiple freeze/thaw cycles.

ELISA Assay
Step 1: Binding of Samples/Controls to the Capture Plate
1.

Take out the required number of microwell strips and the tray from the protective pouchcontaining the SARS-CoV-2 Spike S1 Antibody-coated Plate.

2.

Wash the plate/wells/strips 3 times with 200 µl 1x Wash Buffer. Following each wash step,
remove the solution from the plate by decanting and tapping the plate on an absorbent
material (e.g. paper tissue or towel).

3.

For the standard curve wells, dispense 90 µl of Dilution Buffer into each well.

4.

Add 10 µl of each diluted standard from tubes A-H to the plate wells as outlined in the platelayout (range: 1.5 ng – 0.023 ng total input). Row H will serve as the blank wells with 10 µl of
Dilution Buffer.

5.

For the sample wells, add 100 µl of sample diluted in Dilution Buffer.

6.

Cover used wells with Plate Sealer and incubate plate for 1.5 hour at room temperature withagitation on an orbital shaker or rocking platform at approximately 700 rpm.
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8

Step 2: Binding of Detection Antibody
7.

Before the incubation in Step 1 ends, prepare Diluted Detection Antibody as described in the
Buffer Preparation and Recommendations and mix thoroughly. Keep diluted antibody solution on ice until ready to use.

8.

After the incubation, remove the adhesive film. Wash the wells 3 times with 200 µl 1x Wash
Buffer, tapping the plate dry on paper towels after each wash.

9.

Add 100 µl of diluted detection antibody solution to each well.

10. Cover used wells with Plate Sealer and incubate at room temperature for 1 hour with agitation on orbital shaker or rocking platform at approximately 700 rpm.

Step 3: Binding of Streptavidin-HRP
11.

After the incubation, wash the wells 3 times with 200 µl of 1x Wash Buffer, tapping the plate
dry on paper towels after each wash.

12.

Add 100 µl of the Streptavidin-HRP Conjugate solution to each well.

13.

Cover used wells with Aluminum Plate Sealer tightly and incubate at room temperature for
30 minutes with agitation on orbital shaker or rocking platform at approximately 700 rpm.

Step 4: Colorimetric Reaction
14.

During the incubation, set Developing Solution out on bench to warm to room temperature.

15.

After the incubation, wash the wells 3 times with 200 µl of 1x Wash Buffer. Remove as muchof the final wash as possible by blotting the plate on paper towels.

16.

Add 100 µl of room temperature Developing Solution to each well. Incubate for 5-10 minutesat room temperature in the dark with gentle shaking.

17.

Incubate under low light conditions. Monitor the blue color development in the standard
curve wells. The Blank wells should remain clear.

18. Following the incubation, stop the reaction by adding 100 μl of the Stop Solution.
19.

Read absorbance on a microplate reader at 450 nm within 15 minutes.
Reading the reference wavelength is optional. Most microtiter plate readers are equippedto perform dual wavelength analysis and with the appropriate software, will automaticallysubtract the reference wavelength absorbance from the test wavelength absorbance. If yourplate reader does not have this capability, you may read the plate twice, first at 450 nm andthen at 655 nm and manually subtract the 655 nm OD from the 450 nm OD values.

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9Data Analysis
Average the optical density (OD) for the standards, controls and samples and subtract the
average OD obtained from the zero standard. If samples generate values higher than thehighest standard, repeat the assay with an appropriate dilution.
Generate a standard curve by plotting the mean absorbance for each standard on the y-axis
against the concentration on the x-axis. For a best fit curve use graphing software with
4-Parameter Logistic (4PL) or 5-Parameter Logistic (5PL) regression analysis.
The concentration of SARS-CoV-2 Spike S1 (RBD) Protein in each sample well can be determined from the equation for the standard curve. If the samples have been diluted, the actual
concentration of SARS-CoV-2 Spike S1 (RBD) Protein determined from the standard curve
must be multiplied by the dilution factor. For example, if the samples were diluted 1:10 in thewells, the actual amount in the original sample would be determined by multiplying by 10.

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10Troubleshooting Guide
Problem/question

Possible cause

Recommendation

No signal or weaksignalOmission of key
reagent

Check that all reagents have been added in all
wells in the correct order.

Substrate or con- Test conjugate and substrate for activity by mixing
jugate is no longer a small aliquot of HRP and Developing Solution
activetogether.
Enzyme inhibitor
present

Sodium azide will inhibit the peroxidase reaction.
Follow our recommendations to prepare buffers.

Plate reader settings not optimal

Verify the wavelength and filter settings in theplate reader.

Incorrect assay
temperature

Bring Developing Solution and Stop Solution toroom temperature before using.

Inadequate volume of Developing Solution

Check to make sure that correct volume is delivered by pipette.

Developing time
too long

Stop enzymatic reaction as soon as the specified
wells turn medium-dark blue.

Concentration of
SARS-CoV-2 SpikeS1 Detection Antibody is too highIncrease antibody dilutions.

Inadequate washing

Ensure all wells are filled with Wash Buffer andfollow washing recommendations .

Incomplete washing of wells

Ensure all wells are filled with Wash Buffer andfollow washing recommendations .

Well cross-contamination

Follow washing recommendations.

Too much sample
per wellDecrease amount of sample per well.

Concentration of
SARS-CoV-2 SpikeS1 Detection Antibody is too high

Perform antibody titration to determine optimal
working concentration. The sensitivity of the assay
may decrease.

No signal or weaksignalin sample wells

Not enoughsample per well

Increase amount of sample if necessary.

No signal or weaksignal in SARS-CoV-2
Spike S1 (RBD) ProteinStandard

Improper storage.
Too many freeze/
thaw cycles ofprotein

Do not use the diluted SARS-CoV-2 Spike S1 (RBD)
Protein Standard after one week of storage at 4°C.

High background in
all wells

Uneven color
development

High background insample wells

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Store the undiluted SARS-CoV-2 Spike S1 (RBD)
Protein Standard at -80°C and do not freeze-thaw
more than 5 times..

11

Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of thenumbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008, USA
E-mail: tech_service@activemotif.com
Active Motif Europe
Waterloo Atrium
Drève Richelle 167 – boîte 4
BE-1410 Waterloo, Belgium
E-mail: eurotech@activemotif.com

Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Active Motif China
787 Kangqiao Road
Building 10, Suite 202
Pudong District
Shanghai, 201315, China

Toll Free: 877 222 9543
Direct: 760 431 1263
Fax: 760 431 1351

Direct: +32 (0)2 653 0001
Germany Free Phone: 0800 181 99 10
France Free Phone: 0800 90 99 79
UK Free Phone: 0800 169 31 47
Fax: +32 (0)2 653 0050

Direct: +81 3 5225 3638
Fax: +81 3 5261 8733
E-mail: japantech@activemotif.com

Direct: (86)-21-20926090
Hotline: 400-018-8123
E-mail: techchina@activemotif.com

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