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

Brand: Thermo Fisher Scientific

Model: Dynabeads SARS-CoV-2 Spike Ig Total ELISA Kit

Type: User guide

Dynabeads™ SARS-CoV-2 Spike Ig Total ELISA Kit

Catalog Numbers 18020D, 18020DFIVE

Pub. No. MAN0025847 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 Ig Total ELISA Kit is a solid‑phase indirect Enzyme‑Linked Immunosorbent Assay (ELISA) designed

to detect and quantify the level of Human SARS-CoV-2 Ig Total 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. 18020D

(96 tests)

Cat. No. 18020DFIVE

(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 Ig Total High Control, lyophilized 1 vial 5 vials

Human SARS-CoV-2 Ig Total Calibrator, lyophilized 1 vial 5 vials

Human SARS-CoV-2 Ig Total Low Control, lyophilized 1 vial 5 vials

Human SARS-CoV-2 Ig Total HRP Conjugate (100X) [2] 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.

[2] The Ig Total antibody recognizes IgA, IgG, and IgM.

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 Ig Total

High Control: 40,000 pg/mL

• Human SARS-CoV-2 Ig Total

Calibrator: 10,000 pg/mL

• Human SARS-CoV-2 Ig Total

Low Control: 2,500 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 Ig

Total 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.

Pre‑dilute samples

For best results, pre‑dilute 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 Ig Total 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 Ig Total 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

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

S1 S1 S9 S9

S1 S9 S17 S17 S17

Std

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

Figure 3 Plate layout for quantitative assays (samples in

triplicate)

Dynabeads™ SARS-CoV-2 Spike Ig Total 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 clear‑bottomed plates for ELISA read‑out

• KingFisher™ 96 microplate (200 µL)

• KingFisher™ 96 deep well (DW) plate

• KingFisher™ 96 tip comb for deep‑well magnet

• Plate covers (non‑transparent 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 Ig Total 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 Ig Total ELISA Kit User Guide 5

3.1. Add 100 µL of Human SARS-CoV-2 Ig Total 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 Ig Total 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 62.5–125 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 160–475 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 2.8% to 3.2%. 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 3.9% to 9.3%. Higher %CV can be expected

in low target concentration ranges.

Recovery

Table 2 Recovery of Ig total in serum and plasma samples

Sample % Recovery after subtraction of endogenous

signal

Matrix (1:1000

dilution)

High

(4,000 pg/mL)

Mediium

(1,000 pg/mL)

Low

(250 pg/mL)

Serum 102 96 97

Heparin plasma 102 101 118

Citrate plasma 101 102 106

EDTA plasma 94 97 119

Dynabeads™ SARS-CoV-2 Spike Ig Total ELISA Kit User Guide 7

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

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

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

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