Thermo Fisher Scientific Dynabeads SARS-CoV-2 Spike IgG ELISA Kit User guide

Type
User guide
Dynabeads SARS-CoV-2 Spike IgG ELISA Kit
Catalog Numbers 18000D, 18000DFIVE
Pub. No. MAN0025845 Rev. A.0
WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,
clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support.
Product description
The Dynabeads SARS-CoV-2 Spike IgG ELISA Kit is a solidphase indirect EnzymeLinked Immunosorbent Assay (ELISA) designed to
detect and quantify the level of Human SARS-CoV-2 IgG from serum and plasma (EDTA, citrate, and heparin-treated) samples.
The spike protein is a patented recombinant trimeric structure combining the S1 subunit, including the receptor binding domain (RBD),
and the S2 subunit found on the surface of SARS-CoV-2 viruses where antibody binding occurs. The trimeric spike protein is coupled to
Dynabeads magnetic beads in contrast to traditional ELISAs where the spike protein is coupled to the bottom of microtiter plates. Using
spike-coupled Dynabeads magnetic beads dispersed in samples significantly reduces assay incubation time to only 45 minutes.
In addition, the use of magnetic beads facilitates automation, and running the assay on platforms such as the KingFisher Flex
instrument, drastically reduces hands-on time since all incubations and washing steps are automated. All that is required is to pre-dilute
reagents, fill the wells of the KingFisher plastic plates, and start the instrument.
This manual describes procedures for both an automated ELISA using the KingFisher Flex instrument (the KingFisher Apex instrument
can also be used), and a manual protocol using 96-well DynaMag magnets. The product can be used to perform both qualitative and
quantitative assays.
Contents and storage
Contents Cat. No. 18000D
(96 tests)
Cat. No. 18000DFIVE
(5 × 96 tests) Storage
Dynabeads SARS-CoV-2 Spike (1 mg beads/mL) [1] 3 mL 5 × 3 mL
2°C to 8°C.
Do not freeze.
Human SARS-CoV-2 IgG High Control, lyophilized 1 vial 5 vials
Human SARS-CoV-2 IgG Calibrator, lyophilized 1 vial 5 vials
Human SARS-CoV-2 IgG Low Control, lyophilized 1 vial 5 vials
Human SARS-CoV-2 IgG HRP Conjugate (100X) 0.12 mL 5 × 0.12 mL
Assay Buer (20X) 50 mL 5 × 50 mL
Wash Buer (20X) 50 mL 5 × 50 mL
Substrate Solution (Stabilized chromogen/Tetramethylbenzidine) 15 mL 5 × 15 mL
Stop Solution 15 mL 5 × 15 mL
[1] Dynabeads SARS-CoV-2 Spike beads can also be purchased as a separate unit.
USER GUIDE
For Research Use Only. Not for use in diagnostic procedures.
Guidelines for sample prepration
IMPORTANT! Reagents are lot specific. Do not mix or exchange
reagent between lots or from dierent kits.
Collect samples in pyrogen/endotoxin-free tubes.
Freeze samples at −20°C after collection if samples will not
be tested immediately. Avoid multiple freeze-thaw cycles of
frozen samples.
Thaw sample completely, keep on ice and mix well prior to
analysis.
Required materials not supplied
Distilled or deionized water
Calibrated adjustable precision pipettes
Mixer or shaker (e.g., HulaMixer Sample Mixer)
ELISA plate reader with software capable of measurement
at 450 nm, 490 nm, and 650 nm (e.g., Varioskan LUX
Multimode Microplate Reader)
See “Automated protocol using the KingFisher Flex instrument”,
or “Manual protocol using a DynaMag magnet” for additional
materials required for the respective procedures.
Before you begin
Allow Wash Buer (20X) and Assay Buer (20X) to reach
room temperature before use.
Dilute buers before use. Mix concentrated buers to re-
dissolve any precipitated salts.
Note: All buers are supplied in excess.
Do not allow the beads to settle to the bottom of the vial
when dispensing them into the plate wells. Resuspend the
beads with a pipette prior to transferring them into each well.
Prepare 1X Wash Buer
1. Dilute 25 mL of Wash Buer (20X) with 475 mL of deionized
or distilled water. Label as 1X Wash Buer.
2. Store 1X Wash Buer at 2–8. Use 1X Wash Buer within
30 days.
Prepare 1X Assay Buer
1. Dilute 5 mL of Assay Buer (20X) with 95 mL of deionized or
distilled water. Label as 1X Assay Buer.
2. Store 1X Assay Buer at 2–8. Use 1X Assay Buer within
30 days.
Resuspend Dynabeads magnetic beads
1. Resuspend the magnetic beads in the vial by vortexing
for at least 30 seconds or using a tilt-and-rotate mixer for
5 minutes.
2. Leave the vial on a mixer to keep the beads resuspended
until ready to use.
Reconstitute controls
High, Calibrator, and Low control antibodies are provided for
use in the assay.
The reconstitution volume for each control is indicated on the
respective label of each vial.
1. Centrifuge the tubes before use to collect the lyophilized
powder at the bottom of the tubes.
2. Add the volume of deionized or distilled water indicated on
the label of each vial to achieve the following concentrations:
Human SARS-CoV-2 IgG
High Control: 150,000 pg/mL
Human SARS-CoV-2 IgG
Calibrator: 37,500 pg/mL
Human SARS-CoV-2 IgG
Low Control: 9,375 pg/mL
3. Swirl or mix the tubes gently, then incubate for 10 minutes.
For optimal results, keep the vials on ice until ready to use.
Prepare 1X HRP Conjugate Solution
Note: Prepare 1X HRP Conjugate Solution within 15 minutes of
usage.
1. For each plate, dispense 120 μL of Human SARS-CoV-2 IgG
HRP Conjugate (100X) into a tube containing 11.88 mL of 1X
Assay Buer to make the 1X HRP Conjugate Solution.
2. Mix thoroughly. Keep away from light.
Predilute samples
For best results, predilute serum and plasma samples prior to
the experiment. High titers of immunoglobulins in the samples can
result in absorbance values outside of the assay range. Because
Human SARS-CoV-2 IgG titers can be very high, each investigator
should determine the optimal dilution appropriate for the type of
assay (qualitative or quantitative) being performed.
Qualitative assays
Note: See Figure 2 for recommended plate layout and “Determine
results for qualitative assay” on page 6 for how to interpret the
results.
Sample dilution
The results of the assay are determined by comparing the
signal in the sample to the signal from the Calibrator. Dilute
serum and plasma samples ~1:500 with 1X Assay Buer to
achieve the highest sensitivity, but the dilution factor should
be optimized depending on the immunoglobulin titer.
Antibody dilution
Dilute the resuspended High, Calibrator, and Low Controls
1:10 in 1X Assay Buer. If running 3 replicates, prepare
400 µL of solution (add 40 µL of control to 360 µL of 1X
Assay Buer).
2Dynabeads SARS-CoV-2 Spike IgG ELISA Kit User Guide
Quantitative assays
Note: See Figure 3 for recommended plate layout and “Determine
results for quantitative assay” on page 6 for how to interpret the
results.
Sample dilution
Make a serial dilution to determine the concentration of
antibody in the sample. Dilute serum and plasma samples
~1:1000 with 1X Assay Buer to achieve the highest
sensitivity, but the dilution factor should be optimized
depending upon the immunoglobulin titer.
Standard curve
Prepare a serial dilution of the High Control in 1X Assay
Buer to generate a standard curve. Make a 1:10 dilution
followed by six 2-fold dilutions as illustrated in Figure 1.
Figure 1 Dilution of standards
Prepare standard dilution series
1. Fill 8 microcentrifuge tubes with the volume of 1X Assay
Buer indicated in Figure 1.
2. Add 80 µL of the reconstituted High Control to the tube for
standard 1 (Std 1) to make a 1:10 dilution.
3. Transfer 400 µL from the Std 1 tube to the Std 2 tube to
make a 1:2 dilution.
4. Mix and transfer 400 µL from the Std 2 tube to the Std 3 and
continue the dilution process until the Std 7 tube is reached.
5. Discard 400 µL of Std 7.
6. Dispense standards into the assay plate (plate #2 for the
automated protocol). See Figure 3 for recommended plate
layout.
Recommended plate layouts
The following plate layouts can be used for the manual protocol,
or when setting up plate #2 for the automated protocol using the
KingFisher Flex instrument (see Table 1).
1 2 34 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
S1 S1 S9 S9
S1 S9 S17 S17 S17 S25 S25 S25
S2 S2 S10 S10
S2 S10 S18 S18 S18 S26 S26 S26
S3 S3 S11 S11
S3 S11 S19 S19 S19 S27 S27 S27
S4 S4 S12 S12
S4 S12 S20 S20 S20 S28 S28 S28
S5 S5 S13 S13
S5 S13 S21 S21 S21 S29 S29 S29
S8 S8 S16 S16
S8 S16 S24 S24 S24
Low Low Low
Cal Cal Cal
S6 S6 S14 S14
S6 S14 S22 S22 S22
Hi Hi Hi
S7 S7 S15 S15
S7 S15 S23 S23 S23
Figure 2 Plate layout for qualitative assays (samples in
triplicate)
1 2 34 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
S1 S1 S9 S9
S1 S9 S17 S17 S17
Std
1
Std
1
Std
1
S2 S2 S10 S10
S2 S10 S18 S18 S18
S3 S3 S11 S11
S3 S11 S19 S19 S19
S4 S4 S12 S12
S4 S12 S20 S20 S20
S5 S5 S13 S13
S5 S13 S21 S21 S21
S8 S8 S16 S16
S8 S16 S24 S24 S24
S6 S6 S14 S14
S6 S14 S22 S22 S22
S7 S7 S15 S15
S7 S15 S23 S23 S23
Std
2
Std
2
Std
2
Std
3
Std
3
Std
3
Std
4
Std
4
Std
4
Std
5
Std
5
Std
5
Std
6
Std
6
Std
6
Std
7
Std
7
Std
7
Std
8
Std
8
Std
8
Figure 3 Plate layout for quantitative assays (samples in
triplicate)
Dynabeads SARS-CoV-2 Spike IgG ELISA Kit User Guide 3
Automated protocol using the KingFisher Flex
instrument
A video describing the automated procedure is available on the
product detail page and describes the set up of KingFisher
plates and the experimental procedure.
Each KingFisher Flex instrument has 7 positions that are used
for plates, and 1 position reserved for the tip comb. The basic
isolation process involves the automated movement of spike-
coated magnetic beads from plate to plate by the 96 -well
magnetic head to facilitate the antibody binding, incubation, and
washing steps, all with no hands-on work required until manual
addition of the stop solution at the end.
Required materials not supplied
• KingFisher Flex instrument
Suitable flat clearbottomed plates for ELISA readout
• KingFisher 96 microplate (200 µL)
• KingFisher 96 deep well (DW) plate
• KingFisher 96 tip comb for deepwell magnet
Plate covers (nontransparent plastic film)
Download automated protocol
To download the BindIt software and the script for the protocol,
follow the instructions at thermofisher.com/us/en/home/life-
science/dna-rna-purification-analysis/automated-purification-
extraction/automated-protocols-software.html?open=protein.
The script for the KingFisher Flex instrument is called: ELISA-
DB-SARS-CoV-2-Flex. The script can be run directly from the
instrument or from a computer connected to the KingFisher
instrument after the download.
Guidelines for plate preparation
A total of 7 plates are used per run for this protocol. Up to 96
wells in each plate can be used for the run, but if there are less
than 96 samples, make sure that reagents are added to the same
well numbers, and in the same order for all of the plates.
See Table 1 for a summary of the reagent and volumes for each of
the respective plates and the plate position when loaded into the
KingFisher Flex instrument.
See “Recommended plate layouts” on page 3 for examples of
quantitative and qualitative assay plate layouts (for plate #2).
Table 1 Plate set-up for the KingFisher Flex instrument
Plate# Name Prefill Plate type
#1 Dynabeads 70 µL 1X Assay Buer
+ 30 µl Dynabeads
96-well microplate
#2 Sample 100 µL of diluted
sample or 100 µL of
Antibody Controls
96-well microplate
#3 Wash I 600 µL 1X Wash
Buer
96 Deep-Well
#4 Detection
antibody
100 µL detection
antibody
96-well microplate
#5 Wash II 600 µL 1X Wash
Buer
96 Deep-Well
#6 Substrate 100 µL Substrate
Solution
96-well microplate
#7 Tip comb 96 Deep-Well
Run automated protocol
Dilute and resuspended all reagents before use as described in
“Before you begin” on page 2.
To make dispensing reagents into the plates easier, use of a multi-
channel pipette is recommended.
1. Fill the plates with the resuspended and pre-diluted reagents
according to the plate set-up described in Table 1.
Note: Gently resuspend the Dynabeads magnetic beads
after loading approximately every 8 wells to ensure
consistent bead concentrations.
Note: See page 3 for examples of qualitative (Figure 2) and
quantitative (Figure 3) assay plate layouts.
Note: Use a plastic film seal to protect the substrate in
plate #6. Also wrap the plate in aluminum foil to protect the
substrate from light. Do not allow the substrate solution to
come in direct contact with aluminum foil or other metals.
2. Load the script called ELISA-DB-SARS-CoV-2-Flex on the
instrument, and push Start to initiate loading of each plate
(and the KingFisher DW 96 Tip Comb) to the correct
position.
Note: Remove any covering from the plate upon loading into
the instrument.
3. Immediately after the run (45 minutes) remove plate #6 from
the instrument (the color should now be blue), and add
100 µL of Stop Solution to each well.
4. Transfer 100 µL from each well into a clear flat-bottomed
ELISA plate and measure absorbance at 450 nm within
10 minutes after adding Stop Solution.
4Dynabeads SARS-CoV-2 Spike IgG ELISA Kit User Guide
Manual protocol using a DynaMag magnet
A manual protocol can be used to take advantage of the shorter bead-based ELISA assay time (45 minutes) if access to a KingFisher
instrument is unavailable. Note that the hands-on time will be longer due to the manual steps.
Required materials not supplied
Horizontal microplate shaker capable of 700 rpm
Mixer or shaker (e.g., HulaMixer Sample Mixer)
• DynaMag–96 Side Magnet or DynaMag–96 Side Skirted Magnet
Suitable white U-bottomed 96 well microplate
Suitable clear flat-bottomed ELISA plate
Plate covers
Perform manual ELISA
All reagents must be resuspended and diluted prior to use as described in “Before you begin” on page 2.
To make dispensing reagents into the plates easier, use of a multi-channel pipette is recommended.
See “Recommended plate layouts” on page 3 for examples of quantitative and qualitative assay plate layouts.
Note: U-bottomed plates are recommended for easier handling of magnetic beads, but flat-bottomed plates are required for ELISA
read-out.
1.1. Load 70 µL of 1X Assay Buer into a 96-well U-bottomed white ELISA plate according to your
plate layout.
1.2. Add 30 µL of resuspended Dynabeads magnetic beads to the same wells.
Note: Gently resuspend the vial of Dynabeads magnetic beads after loading approximately
every eight wells to ensure consistent bead concentrations.
1.3. Apply the plate to a magnet for 1–2 minutes. Invert the plate while still on the magnet to remove
the liquids.
1.4. Remove the plate from the magnet and add 100 µL of standards (quantitative) or controls
(qualitative), and samples to the appropriate wells. Cover the plate with a plastic film seal.
1.5. Incubate for 15 minutes at 37 with shaking to facilitate the binding of the antibodies to the
beads.
1Bind antibodies
2.1. Apply the plate to a magnet for 1–2 minutes. Invert the plate while still on the magnet to remove
the liquids.
2.2. Remove the plate from the magnet and add 200 µL of 1X Wash Buer.
2.3. Apply the plate to a magnet for 1–2 minutes. Invert the plate while still on the magnet to remove
the liquids.
2.4. Repeat steps step 2.2–step 2.3 twice to achieve a total of 3 wash steps.
2Wash magnetic beads
Dynabeads SARS-CoV-2 Spike IgG ELISA Kit User Guide 5
3.1. Add 100 µL of Human SARS-CoV-2 IgG HRP Conjugate to the wells, then cover the plate with a
plastic film seal.
3.2. Incubate for 15 minutes at 37 with shaking to bind the detection antibody to the beads.
3Add HRP Conjugate
Solution
4.1. Apply the plate to a magnet for 1–2 minutes. Invert the plate while still on the magnet to remove
the liquids.
4.2. Remove the plate from the magnet and add 200 µL of 1X Wash Buer.
4.3. Apply the plate to a magnet for 1–2 minutes. Invert the plate while still on the magnet to remove
the liquids.
4.4. Repeat steps step 4.2–step 4.3 twice to achieve a total of 3 wash steps.
4Wash magnetic beads
5.1. Add 100 µL of Substrate Solution and cover the plate with a plastic film seal to prevent spillage,
and protect the substrate. Cover the plate with aluminum foil to protect from light. Do not allow
the substrate to come into direct contact with the aluminum foil or other metals.
5.2. Incubate by shaking at 37 for 5 minutes, protect from light.
The solution should now be blue.
5Add Substrate Solution
6.1. Add 100 µL of Stop Solution to each well.
6.2. Apply the plate to a magnet for 2 minutes to collect the beads, protect from light.
6.3. Transfer 100 µL of all the samples from the wells to a clear flat-bottomed ELISA plate and
measure absorbance at 450 nm within 10 minutes.
6Add Stop Solution
Analyze the results
Determine results for qualitative assay
Calculate the ratio of values using the equation: Ratio =
Absorbance sample/Absorbance calibrator
If the ratio is <1, the target is absent/not detectable.
If the ratio lies between 1.1–1.3, the result is intermediate.
If the ratio is >1.3 the target is present
As a negative control, divide the Low Control by the
Calibrator (ratio=<1).
As a positive control, divide the High Control by the
Calibrator Control (ratio =>1.3).
Determine results for quantitative assay
If the High Control was used to generate a standard
curve, use a curve-fitting software capable of a 4-parameter
logistic (4PL) algorithm for optimal results. The background
absorbance may be subtracted from all data points; including
standards, unknowns, and controls prior to plotting.
Read the concentrations for unknown samples and controls
from the standard curve. Multiply the value(s) obtained for
sample(s) by the dilution factor to correct for dilutions.
Note: Samples producing signals greater than the upper limit
of the standard curve should be further diluted in Assay
Buer and re-analyzed. Multiply the concentration by the
appropriate dilution factor.
6Dynabeads SARS-CoV-2 Spike IgG ELISA Kit User Guide
Performance characteristics
Expected values
These values are given only for guidance and it is recommended
that each laboratory establishes its own normal values.
Control Ratio
High Control 1.81
Calibrator 0.48
Low Control 0.14
Standard curve example
The following data obtained for the standard curve are for
illustration only and should never be used in place of a real time
standard curve.
Sensitivity
Limit of Detection
Limit of Detection (LOD) is defined as the analyte concentration
giving an absorbance significantly higher than that of the dilution
medium (blank). LOD is determined from the mean signal of the
blanks (from 3 independent assays) plus 3 standard deviations,
and falls between standard 7 and standard 6 at 234–469 pg/mL
(some inter-assay variation is to be expected).
Limit of Quantification
Limit of Quantification (LOQ) is defined as the lowest level of
analyte that can be quantified with acceptable precision and
accuracy. LOQ is determined from the mean signal from matrix
blanks (from 3 independent assays) plus 10 standard deviations,
and lies between 300–650 pg/mL (some inter-assay and matrix
variation is to be expected).
Reproducibility
Intra-assay precision
Assay results showed that the intra-assay precision had typical
values ranging from 1.4% to 4.3%. Higher %CV can be expected
in low target concentration ranges.
Inter-assay precision
Assay results showed that the inter-assay precision had typical
values ranging from 3.8% to 8.1%. Higher %CV can be expected
in low target concentration ranges.
Recovery
Table 2 Recovery of IgG in serum and plasma samples
Sample % Recovery after subtraction of endogenous
signal
Matrix (1:1000
dilution)
High
(15,000 pg/mL)
Medium
(3,750 pg/mL)
Low
(938 pg/mL)
Serum 102 113 125
Heparin plasma 107 112 121
Citrate plasma 96 114 123
EDTA plasma 103 119 133
Dynabeads SARS-CoV-2 Spike IgG ELISA Kit User Guide 7
Related products
Unless otherwise indicated, all materials are available through thermofisher.com.
Product Catalog No.
KingFisher Flex Instrument 24074420
Varioskan LUX Multimode Microplate Reader VLBLATD2
Clear Flat-Bottom Immuno Nonsterile 96-Well Plates 439454
White U-bottomed microplate Greiner 650207
HulaMixer Sample Mixer 15920D
KingFisher 96 microplate (200 µL) 97002540
KingFisher deep-well plate, v-bottom 95040450
KingFisher Tip Comb for deep-well magnets 97002534
Magnetic 96-Well Separator A14179
DynaMag–96 Side Skirted Magnet 12027
Dynabeads SARS-CoV-2 Spike Ig Total ELISA Kit 18020D
Dynabeads SARS-CoV-2 Spike IgM ELISA Kit 18010D
Dynabeads SARS-CoV-2 Spike 18100D
Bender MedSystems GmbH | Campus Vienna Biocenter 2 | 1030 Vienna, Austria
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,
PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0025845
Revision Date Description
A.0 2 November 2021 New manual for Dynabeads SARS-CoV-2 Spike IgG ELISA Kit.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all
applicable Limited Use Label Licenses.
©2021 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
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Thermo Fisher Scientific Dynabeads SARS-CoV-2 Spike IgG ELISA Kit User guide

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