FluoroSpot Path:
SARS-CoV-2 (Spike+RBD) Human IgG
FSP-05R-RS1-1

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The products are for research use only.
MABTECH shall not be liable for the use or handling of the product or for consequential, special, indirect or incidental damages therefrom.

INTRODUCTION
Intended use
For enumeration of B cells secreting IgG antibodies specific for the SARS-CoV-2
Spike protein and Receptor Binding Domain (RBD). Enumeration of all cells secreting
IgG (total IgG) can also be made.

Assay principle
Please read all instructions prior to starting the assay. Note that the reagents should not
be prepared long in advance. It is recommended to consult the section Tips for a
successful assay.

Cells are added to a FluoroSpot plate pre-coated with anti-IgG monoclonal capture
antibodies (mAbs). During incubation, IgG secreted by B cells will bind to the capturemAbs. After cell removal, Spike-specific IgG spots, RBD-specific IgG spots and total IgG
spots can be detected. Spike-specific IgG spots are detected using Spike-GAL, i.e.,
recombinant Spike protein with peptide tag GAL*, followed by fluorophore-conjugatedmAb anti-GAL-490. RBD-specific IgG spots are detected using RBD-WASP, i.e.,
recombinant RBD with peptide tag WASP**, followed by fluorophore-conjugated mAb antiWASP-640. Total IgG spots are detected by addition of fluorophore-conjugated anti-IgG
detection mAbs-550.
*Spike-GAL: Recombinant SARS-CoV-2 Spike protein prefusion ectodomain trimer basedon uniprot P0DTC2 (aa 1-1208) with proline substitutions at residues 986 and 987, aGSAS substitution at the furin cleavage site (residues 682-685), a C-terminal T4 fibritin
trimerization motif (Wrapp et al., Science, Feb 19, 2020), followed by GAL peptide tag
used for detection (YPGQAPPGAYPGQAPPGA), and a C-tag (EPEA) used for purification.
**RBD-WASP: Recombinant SARS-CoV-2 Receptor Binding Domain (RBD) of the Spikeprotein, based on uniprot P0DTC2 (aa 319-541) with a C-terminal WASP peptide tag(PDYRPYDWASPDYRD).
2CONTENTS AND STORAGE
Amount Storage

ContentsFor detection of Spike-specific
IgG spots

Antigen: Spike-GAL, lyophilized1 vial-20 °C

Anti-GAL mAb, 490

60 μl

4-8 °C

Antigen: RBD-WASP, lyophilized

1 vial-20 °C

Anti-WASP mAb, 640

60 μl

4-8 °CFor detection of total IgG spots

Anti-IgG mAbs MT78/145, 550

25 μl

4-8 °C

Polyclonal activators

R848, 1 mg/ml
Lyophilized recombinant human IL-2

100 μl1 μg-20 °C-20 °C

Buffers/Solutions

Standard reconstitution buffer A5

2 x 1 ml 4-8 °C

FluoroSpot enhancer

25 ml

Plate pre-coated with mAbs
MT91/145

1 plate Roomtemperature

For detection of RBD-specificIgG spots

Pre-coated plate

4-8 °C

Vials have been overfilled to ensure recovery of the specified amount.

Shipping
Shipped at ambient temperature. On arrival, store components as specified above.
Fluorophore conjugates and FluoroSpot enhancer contain 0.002% Kathon CG.

Expiry date
The expiry date indicates how long unopened products are recommended for use
when stored according to instructions.

3

ASSAY PROTOCOL
Cells
In vivo activatedBcells (e.g., during acute infection) may be analyzed directly in theFluoroSpot plate without prior stimulation.
Memory B cells may require pre-stimulation in vitro with a mixture of R848 (1 μg/ml) and IL-2*(10 ng/ml) in tubes for 3-4 days to secrete detectable amount of antibody. After pre-stimulation,
wash the cells extensively to remove secreted antibodies in the supernatant.
* Reconstitute IL-2 with 1 ml PBS to obtain 1 μg/ml. Leave for 15 min and then vortex. Use
directly or store in aliquots at -20°C.

A. Plate preparation (sterile conditions)1.

Remove the plate from the sealed package and wash the plate three times with sterile
PBS (200 µl/well).

2.

Block/condition the plate by adding cell incubation medium containing 10% fetal calf
serum (200 µl/well). Incubate for at least 30 minutes at room temperature.

B. Cell incubation (sterile conditions)1.

Remove the medium and add the cell suspension. Spike-specific IgG and RBD-specificIgG are set up and analyzed in the same well. Samples for total IgG should be set up inseparate wells. For detection of Spike-specific IgG spots and RBD-specific IgG spots
add 100,000-500,000 cells/well and for total IgG spots 25,000-50,000 cells/well.
Set up samples in triplicates or duplicates.

2.

Incubate the cells in the plate at 37 °C in a humidified incubator with 5% CO2 for

18-24 hours. Do not move the plate during incubation and avoid evaporation, forexample by wrapping the plate in aluminum foil.

4C. Detection
1.

Remove the cells by emptying the plate and then wash the plate five times with PBS(200 µl/well).

2.

Spike-specific IgG spots and RBD-specific IgG spots: Prepare Spike-GAL and RBD-WASPby adding 500 μl standard reconstitution buffer to the lyophilized Spike-GAL and 500 μlstandard reconstitution buffer to the lyophilized RBD-WASP. Allow it to dissolve for 5
minutes and mix thoroughly. Dilute the Spike-GAL and RBD-WASP solutions 1:25 in thesame tube, e.g. by adding 500 μl Spike-GAL and 500 μl RBD-WASP to 11.5 ml PBS
containing 0.1% BSA (PBS-0.1% BSA). Add the dilution to the plate, 100 µl/well. Total
IgG spots: Add 100 µl/well of PBS-0.1% BSA.
Incubate for 2 hours at room temperature.

3.

Wash the plate five times with PBS (200 µl/well).

4.

Spike-specific IgG spotsand RBD-specific IgG spots: Dilute the anti-GAL-490 (1:200)
and anti-WASP-640 (1:200) in the same tube in PBS-0.1% BSA and add 100 µl/well.
Total IgG spots: Dilute MT78/145-550 (1:500) in PBS-0.1% BSA and add 100 µl/well.
Incubate for 1 hour at room temperature.

5.

Wash the plate five times with PBS (200 µl/well).

6.

Empty the plate, add FluoroSpot enhancer (50 µl/well), and leave for 5-15 minutes atroom temperature.

7.

Empty by flicking the plate to remove the FluoroSpot enhancer. Do not wash. It isimportant that the plate is not tapped against paper towels since dust particles may
interfere with the assay.

8.

Remove the underdrain (the soft plastic drain under the plate), for example using pliers.
Dry the plate protected from light. The plate should be completely dry before analysis.
Store plate in the dark at room temperature.

9.

Spot analysis is performed with an automated FluoroSpot reader equipped with filters
for the fluorophores used. Filters should have high specificity to avoid bleed-throughartifacts.
Analysis ofRequired filter

Spike-specific IgG spotsExcitation 490 nm/emission 510 nm (FITC)

RBD-specific IgG spots

Excitation 640 nm/emission 660 nm (Cy5)

Total IgG spots

Excitation 550 nm/emission 570 nm (Cy3)

5

TIPS FOR A SUCCESSFUL ASSAY
Buffers
PBS for washing and dilution should be filtered (0.2 μm) to remove any particles. We do notrecommend the inclusion of Tween or other detergents in the washing and incubation buffers.
WashingPlates can be washed using a multi-channel micropipette. A regular plate washer can be used in
washing steps when sterile conditions are not required, provided that the washing head is
adapted to ELISpot/FluoroSpot plates.
Cells
Freshly prepared and cryopreserved cells may be used. Let the latter rest for at least one hour
at 37 °C to allow removal of cell debris.
Assay controls
Preferably include sample collected pre-SARS-CoV-2 infection. Enumeration of all cells
secreting IgG (total IgG) can be included as a control of assay functionality.
Serum
The serum should be selected to support cell culture and give low background staining. We
recommend the use of fetal calf serum. Alternatively serum-free medium evaluated for cell
culture can be used. Human serum should not be used as it contains intrinsic IgG which willinterfere with the assay.
Analysis
An automated FluoroSpot reader with separate filters is required for analysis. The reader
should be equipped with filters for the fluorophores used. Filters should be selective for thespecific wavelengths to avoid bleed-through artifacts. Spots from cells secreting multiple
analytes are identified by coordinates, i.e. based on spot position. In order to obtain accurate
measurements of cells secreting multiple analytes, the automated reader must be able to create
an overlay analysis from the filters. To avoid fading, the plates should be kept dark and should
preferably be analyzed within 1 week of development.

6PROTOCOL SUMMARY
Start
Wash x3

Cellincubation

Block/condition plate for 30 min.
Add cells
Incubate at 37 °C, 18-24h

Sterileconditions

Remove cells
Wash x5

Detection

Add Spike-GAL and RBD-WASP
Incubate 2h at RT
Wash x5
Add fluorophore-conjugateddetection mAbs
Incubate 1h at RT
Wash x5
Add FluoroSpot enhancer
Incubate 5-15 min. at RT
Empty plate
Remove underdrain
and dry plate

Analysis

7

Updated on 2022/11/18

Developed and manufactured by MABTECH AB,
Sweden, whose quality management systemcomplies with the standards
ISO 9001:2015 & ISO 13485:2016.

CERTIFIED
ISO 9001

CERTIFIED
ISO 13485

Quality management systems

Medical devices
Quality management systems

The products are for research use only.
MABTECH shall not be liable for the use or handling of the product or forconsequential, special, indirect or incidental damages there from.
Mabtech AB (Head Office)SwedenTel: +46 8 716 27 00
mabtech@mabtech.com

www.mabtech.com

Mabtech, Inc.
USA
Tel: +1 513 871-4500
mabtech.usa@mabtech.com