ELISpot Path:
SARS-CoV-2 (RBD) Human IgG (ALP)
3850-4APW-R1-1

CONTENTS:
1 pre-coated plate, mAbs MT91/145
For detection of RBD-specific IgG spots:
Antigen: RBD-WASP, lyophilized (1 vial)
Anti-WASP-ALP (60 μl)
For detection of total IgG spots:
Detection mAb:s MT78/145, biotin, 0.5 mg/ml (40 μl)
Streptavidin-ALP (40 μl)
Polyclonal activator: R848, 1 mg/ml, (100 μl)
Lyophilized recombinant human IL-2 (1 μg)
Standard reconstitution buffer A5 (1 ml)
BCIP/NBT-plus substrate (25 ml)
The biotinylated mAbs contain 0.02% sodium azide. AntiWASP-ALP and SA-ALP are supplied in PBS with 0.002%
Kathon CG. Vials have been overfilled to ensure recovery ofthe speci-fied amount.
STORAGE:
Shipped at ambient temperature. On arrival store RBD-WASP,
R848, and IL-2 at -20 °C. Store all other reagents at 4-8 °C.
Keep plate at room temp. The expiry date indicates how longunopened products, stored according to instructions, arerecommended for use.

Guidelines

PLEASE READ THROUGH BEFORE STARTING THE ASSAY

A Preparation of ELISpot plate (sterile conditions)
1. Remove the plate from the sealed package and wash 4 times with sterile PBS (200 μl/well).
2. Condition the plate with medium (200 μl/well) containing 10% of the same serum as
used for the cell suspensions. Incubate for at least 30 minutes at room temperature.
B Incubation of cells in plate (sterile conditions)
1. Remove the medium and add the cell suspension. The number of cells/well needs to beadapted dependent on the type of cells analyzed, e.g., RBD-specific IgG secreting cells or
all cells secreting IgG (total IgG). The included R848 and recombinant IL-2 can be used toinduce IgG secretion (see Hints and comments).
2. Put the plate in a 37°C humidified incubator with 5% CO2 and incubate for 18-24 hours.
Do not move the plate during this time and take measures to avoid evaporation (e.g. bywrapping the plate in aluminium foil).
C Detection of spots
1. Remove the cells by emptying the plate and wash 5 times with PBS, 200 μl/well.
2. RBD-specific IgG spots: Add 500 μl standard reconstitution buffer to the lyophilized
RBD-WASP, allow it to dissolve for 5 minutes and mix thoroughly. Dilute the RBD-WASP
solution 1:25, e.g. by adding 500 μl RBD-WASP to 12 ml PBS-0.5% FCS. Add 100 μl/well.
Total IgG spots: Dilute the detection mAbs MT78/145-biotin to 1 μg/ml in PBS-0.5% FCS.
Add 100 μl/well.
Incubate for 2 hours at room temperature.
3. Wash plate as above (step C1).
4. RBD-specific IgG spots: Dilute the anti-WASP-ALP (1:200) in PBS-0.5% FCS and add 100μl/well.
Total IgG spots: Dilute the Streptavidin-ALP (1:1000) in PBS-0.5% FCS and add 100 μl/well. Incubate for 1 hour at room temperature.
5. Wash plate as above (step C1).
6. Filter the ready-to-use substrate solution (BCIP/NBT-plus) through a 0.45 μm filter andadd 100 μl/well. Develop until distinct spots emerge.
7. Stop color development by washing extensively in tap water. If desirable, remove theunderdrain (the soft plastic under the plate) and rinse the underside of the membrane.
8. Leave the plate to dry. Inspect and count spots in an ELISpot reader or in a dissection
microscope.
9. Store plate in the dark at room temperature.

Hints and Comments
This protocol describes enumeration of B cells secreting IgG antibodies specific for the SARSCoV-2 Receptor Binding Domain (RBD) and enumeration of all cells secreting IgG (total IgG).
The two types of detection are set up in different wells in the plate and require different cellnumbers.
Plate washing
Washing of plates can be done using a multi-channel micropipette. In washing steps not
requiring sterile conditions (C1-C5), a regular ELISA plate washer can also be used, provided
that the washing head is adapted to the ELISpot plates.
Cells
In vivo activated B cells (e.g., during ongoing infection) may be analyzed directly in the ELISpot plate without prior stimulation. Memory B cells may require pre-stimulation in vitro witha mixture of R848 (1 μg/ml) and IL-2* (10 ng/ml) in tubes for 3-4 days to secrete detectableamount of antibody. After pre-stimulation, wash the cells extensively to remove secreted
antibodies. 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. For detection of RBD-specific IgG
spots add e.g.,100,000-500,000 cells/well and for total IgG spots e.g., 25,000-50,000 cells/well. Set up samples in triplicates or duplicates.
* 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.
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, i.e., IgG which
will interfere with the assay.
Assay controls
Preferentially include sample collected pre-SARS-CoV-2. Enumeration of all cells secreting IgG(total IgG) can be including as a control of cell viability.
Buffers
PBS for washing and dilution should be filtered (0.2 μm) for optimal results. Avoid the inclusion of Tween or other detergents in the washing and incubation buffers.
Substrate development
Development is made until distinct spots are visible in positive wells (usually 5-30 minutes). A
general darkening of the membrane may occur during development but disappears afterdrying.

Updated on 2023/05/31
Developed and manufactured by MABTECH AB,
Sweden, whose quality management systemcomplies with the standards ISO 9001:2015 & ISO
13485:2016.

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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 forconsequential, special, indirect or incidental damages there from.
Mabtech AB (Head Office)Sweden
Tel: +46 8 716 27 00
mabtech@mabtech.com

www.mabtech.com

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USA
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