FluoroSpot Path:
SARS-CoV-2 (S1scan+SNMO)
Human IFN-γ/Granzyme B
FSP-0136-P1-1

www.mabtech.com
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
FluoroSpot – General description
This dual FluoroSpot kit enables convenient enumeration of cells secreting either one or bothanalytes in response to SARS-CoV-2 peptides.
FluoroSpot combines the sensitivity of ELISpot with the capacity to analyze secretion ofseveral analytes simultaneously. This highly sensitive cellular assay is robust, easy to perform,and suitable both for testing single samples and for large-scale screening.

Anti-tag mAb-490
SA-550TAG
Detection mAb-tag
Analyte 1
Capture mAb

B
Detection mAb-biotin
Analyte 2
Capture mAb

Our FluoroSpot path kits include pre-coated plates ready to use. Monoclonal antibodies (mAbs)
are coated onto a PVDF membrane in a 96-well plate. If several analytes are analyzed, mAbs
with different specificities are coated. Detection of cells secreting single or multiple analytes ismade possible using biotinylated and/or tag-labeled detection mAbs. The detection step isvisualized and amplified by specific fluorophore-conjugated reagents and the resulting spots
are analyzed in an automated reader.

2CONTENTS AND STORAGE

The kit is shipped at ambient temperature. All reagents, except the peptide pools, should be
stored at 4-8°C upon arrival. Peptide pools should be stored at -20°C or below. Plates should
be stored at room temperature. The expiry date indicates how long unopened products, storedaccording to instructions, are recommended to remain in use.
Component
Detection mAbs

Anti-IFN-γ mAb 7-B6-1, BAM
Anti-Granzyme B mAb MT28, biotin

Fluorophore conjugates

Concentration

1-plate kit
120 μl

0.5 mg/ml

50 μl

Anti-BAM mAb, 490

120 μl

SA-550120 μl

Co-stimulus

Anti-CD28 mAb (CD28-A)

Stimuli

SARS-CoV-2 SNMO defined peptide pool
SARS-CoV-2 S1 scanning pool (pool 1 and 2)
anti-CD3 mAb CD3-2 (positive control)

0.1 mg/ml

100 μl0.1 mg/ml

1 vial2 vials
100 μlBuffer/Solution

FluoroSpot enhancer

25 ml

Pre-coated plates

Plate pre-coated with:mAbs 1-D1K and MT34B61Anti-CD28 and anti-CD3 mAbs are supplied in sterile filtered (0.2 μm) PBS. The biotinylated
antibody contains 0.02% sodium azide. The BAM-conjugated antibody, fluorophore-conjugatedreagents, and the FluoroSpot enhancer contain 0.002 % Kathon CG. Vials have been overfilled toensure recovery of stated volume.

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ASSAY PROTOCOL
Please consult the table below for recommendations on dilutions and cell incubation time. The reagentsshould not be diluted in advance. For additional information, read the Tips For A Successful Assaysection.
Component

Dilution

Step

Anti-IFN-γ mAb 7-B6-1, BAM

1:200

C2

Anti-Granzyme B mAb MT28, biotin

1:1000C2

Anti-BAM, 490

1:200

C4SA-550

1:200

C4Co-stimulus

Anti-CD28 mAb (CD28-A)

1:1000

B1

Stimuli

SARS-CoV-2 peptide pools

see below

B1

Anti-CD3 mAb (CD3-2) (positive control)

1:1000Detection mAbs

Fluorophore conjugates

Cell incubation time
Approximately 48 hours

B2A. 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.

The peptide pools can be used separately or mixed. If used separately, dissolve thepeptide pools by addition of 40 μl DMSO to each vial, then add 85 μl PBS. If usedmixed, add 40 μl DMSO to one vial and transfer the solution to the next vials serially.
Add 85 μl PBS to the last vial. The concentrations of these stock solutions are 200 μg/ml of each peptide. Aliquot the pools and store at -20°C or below.
Empty the plate and add 50 μl/well of peptides or controls, followed by 50 μl cellsuspension/well. Alternatively, mix cells and stimuli before addition of 100 μl/well. The
final peptide concentration should be 2 μg/ml of each peptide. Anti-CD28 can be
included at a final concentration of 0.1 µg/ml. Anti-CD3 is recommended as a positive
control at a dilution of 1:1000.

2.

Incubate the cells in the plate at 37°C in a humidified incubator with 5% CO2.
Approximate incubation time is indicated above and can be further optimized by theuser. Do not move the plate during incubation and avoid evaporation, for example by4wrapping the plate in aluminum foil.

C. Detection
1.

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

2.

Dilute the detection antibodies in PBS containing 0.1% BSA (PBS-0.1% BSA) according
to the table above. If more than one analyte is analyzed, dilute the detection antibodies
in the same tube. Add 100 µl/well and incubate for 2 hours at room temperature.

3.

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

4.

Dilute the fluorophore-conjugates in PBS-0.1% BSA to the concentrations as shown in
the table above. If more than one analyte is analyzed, dilute the conjugates in the sametube. Add 100 µl/well and incubate for 1 hour at room temperature. Protect the platefrom light throughout the assay.

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 at room temperature.

7.

Empty by flicking the plate to remove the FluoroSpot enhancer. Do not wash. It is
important 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.

AnalyteRequired filter

IFN-γ

Excitation 490 nm/emission 510 nm (FITC)

Granzyme B

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 usedin washing steps when sterile conditions are not required, provided that the washing head isadapted to ELISpot/FluoroSpot plates.
Serum
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 beused.
Cells
Both freshly prepared and cryopreserved cells may be used in FluoroSpot. We recommend thatcryopreserved cells are rested for at least 1 hour after thawing to allow removal of cell debris
before addition to the plate. Triplicates or duplicates of 250,000 cells/well are usually recommended to assess antigen-specific responses. For polyclonal activators, the cell number should
be reduced in order to avoid confluent spot formation.
Assay controls
The number of cells responding to antigen stimulation is often compared to the number of cells
spontaneously secreting cytokine which is determined by incubating the same number of cells
in the absence of stimuli. It can be valuable to include cells from a SARS-CoV-2 negative
individual (never infected and unvaccinated). A polyclonal activator such as anti-CD3 mAb(included in the kit) or phytohemagglutinin (1-10 μg/ml) is often included as a control for cell
viability and functionality of the assay.
Co-stimulation with anti-CD28
Anti-CD28 mAb provides a co-stimulatory signal to antigen-specific responses by binding toCD28 on T cells. Addition of an anti-CD28 mAb together with antigen (step B1) can be used
to enhance antigen-specific responses. However, if the concentration of anti-CD28 mAb is toohigh, non-specific cytokine secretion may be elevated. Anti-CD28 can be used to circumvent
capture effects, which can occur when different capture antibodies are coated in the same well,where absorption of one cytokine may negatively affect the secretion of another cytokine. See
www.mabtech.com for more information about capture effects.
Analysis
An automated FluoroSpot reader with separate filters is required for analysis. The reader shouldbe equipped with filters for the fluorophores used. Filters should be selective for the specificwavelengths to avoid bleed-through artifacts. Spots from cells secreting multiple analytes areidentified by coordinates, i.e. based on spot position. In order to obtain accurate measurementsof 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 stimuli and cells
Incubate at 37°C

Sterileconditions

Remove cells
Wash x5Detection

Add detection antibody mix
Incubate 2h at RT
Wash x5
Add conjugate mix
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 Off ce)
SwedenTel: +46 8 716 27 00
mabtech@mabtech.com

www.mabtech.com

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