ELISpot Flex: Human IgM (ALP)
Product Code: 3880-2A

CONTENTS:
Vial 1 (red top)
Capture mAbs: MT11/12 (1.2 ml)Concentration: 0.5 mg/ml

Vial 2 (yellow top)
Detection mAb: MT22, biotin (100 μl)
Concentration: 0.5 mg/ml
Vial 3 (white top)
Streptavidin-ALP (50 μl)

Vial 4 (black top)
Polyclonal activator: R848 (100 μl)
Concentration: 1 mg/ml
Vial 5 (blue top)
Lyophilized recombinant human IL-2 (1 μg)
Antibodies are supplied in sterile filtered (0.2 μm) PBS with 0.02% sodium
azide. Streptavidin-ALP is supplied in 0.1 M Tris buffer with 0.002% Kathon
CG. R848 is supplied in sterile filtered (0.2 μm) PBS containing 1% DMSO. Vials
have been overfilled to ensure recovery of the specified amount.
STORAGE:

Shipped at ambient temperature. On arrival antibodies and Streptavidin-ALP
should be stored refrigerated at 4-8°C. R848 and IL-2 should be stored at-20°C. The expiry date indicates how long unopened products, stored according to instructions, are recommended for use.

Schematic illustration of the different protocolsPROTOCOL I

2PROTOCOL II

Kit description

Please read through before starting the assay
This kit contains reagents for detection and enumeration of B cells secreting IgM antibodies.
B cells secreting antigen-specific IgM as well as B cells secreting IgM irrespective of antigenspecificity (total IgM) can be measured.
Detection of antigen-specific B cells
Detection of antigen-specific IgM-secreting B cells, visualised as spots, can be made using
either of two protocols (Protocol I or II). The choice of protocol will depend on several factors
such as the available amount and molecular nature of the antigen. The use of biotinylated
antigen (Protocol I) may be more sensitive and result in spots of higher quality. In addition,
the antigen is used in smaller quantities and is not present during cultivation.
In Protocol I, antibodies from all secreting B cells are captured by anti-IgM antibodies coatedon the ELISpot plate. Cells producing specific antibody are then visualised by the addition ofbiotinylated antigen followed by Streptavidin-enzyme and substrate. Please note that antigen must be biotinylated prior to starting the experiment for this protocol.
In Protocol II, representing the conventional way of performing B cell ELISpot, the antigen
is coated on the ELISpot plate and antigen-specific antibodies secreted by the B cells bind to
the immobilised antigen. Spots are detected after addition of biotinylated anti-IgM followedby Streptavidin-enzyme and substrate.
Detection of total IgM-producing B cells
Both protocols allow determination of the total number of IgM-secreting B cells, used as apositive control or for establishing the proportion of antigen-specific B cells.

3Hints and comments

Please read through before starting the assay
These suggestions are based on the detection of antigen-specific immune responses using
PBMC. If using B-cell clones, mixtures of separated cell fractions etc., other protocols may
have to be considered.
PlatesWe recommend the use of PVDF-based membrane plates. Maximal antibody binding capacity of these plates is obtained by a brief treatment with ethanol. It is essential that themembrane is not allowed to dry after the treatment. If this occurs the treatment step (A2-A3)
needs to be repeated before adding the coating antibody.
The IgM antibodies may be polyreactive and have specificities that could cross-react withcomponents exposed in the ELISpot wells. Hence it is important to coat wells with PBS alone
to monitor the number of background spots.
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. Avoid getting liquid on the undersideof the membrane as this may cause leakage due to capillary drainage. If using MAIPSWU
plates, always remove the plate from the plate tray before manually emptying the plate.
Cells
Both fresh and cryopreserved cells may be used with good results. However it is recommended that the latter are rested for at least one hour to allow removal of cell debris before
addition to the plate. Triplicates or duplicates of 100,000-500,000 cells per well are often
used to assess antigen-specific responses. For detection of total IgM, less cells (e.g. 25,00050,000 cells per well) may be used to avoid confluent spot formation.
In vivo activated B cells
B cells that have been activated in vivo, for instance as a result of vaccination, may be analysed directly in the ELISpot wells without prior stimulation. Typically, cells secreting antigenspecific antibodies can be detected in the circulation 6 to 9 days after vaccination.
Memory B cells - in vitro activation
Memory B cells may require polyclonal stimulation before secreting detectable amounts ofantibody. Pre-stimulation can be made with a mixture of R848 (1 μg/ml) and rhIL-2 (10 ng/ml)(both included) in separate tubes/plates for 48-72 hours. After pre-stimulation, wash the cells
to ensure removal of any secreted antibodies. Resuspend the cells in medium and add thecell suspension to the ELISpot plate.
Reconstitution of rhIL-2: Add 1 ml PBS to obtain 1 μg/ml. Leave at room temperature for 15
minutes and then vortex. Use immediately or store in aliquots at -20°C.

4

SerumThe 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 is not recommended as it may contain antibodies which
can interfere with the assay.
Buffers
PBS for washing and dilution should be filtered (0.2 μm) for optimal results. Although possible to use, we do not recommend the inclusion of Tween or other detergents in the washingand incubation buffers.
Substrate development
Develop until distinct spots are visible in positive wells (usually 10-30 minutes). A generaldarkening of the membrane may occur but disappears after drying.
Biotinylation of antigen
If biotinylated antigen will be used for detection, (Protocol I), this should be labeled before
starting the ELISpot experiment. A suitable reagent for biotinylation is e.g. Sulfo-NHS-LCBiotin (Pierce product code 21335). Further information on biotin and biotin conjugation may
be found at www.piercenet.com-The protein antigen to be biotinylated with Sulfo-NHS-LC-Biotin should be in a suitableamine-free buffer (e.g. PBS, pH 7.4). If solution contains sodium azide, this should first be removed e.g. by dialysis. For small volumes (< 200 μl) a MINI-dialysis unit Slide-A-Lyser (10.000MWCO, Pierce) can be used. The same dialysis unit can be used for removal of free biotin (seebelow). Conjugation is normally performed using a 20- to 50-fold molar excess of biotin toprotein where the higher ratio is for protein at low concentration.
-The Sulfo-NHS-LC-Biotin should be dissolved in pure H2O and immediately added to theantigen solution. After incubation for 30 minutes at room temperature, free biotin may beremoved by dialysis against PBS or other buffer.
The concentration of the biotinylated antigen in the assay is typically 0.01-1 μg/ml but depends on the size of the antigen and the degree of biotinylation and should be establishedseparately.

5

Protocol I(using biotinylated antigen)A

Preparation of ELISpot plate (sterile conditions)1.

Dilute the coating mAbs MT11/12 to 15 μg/ml in sterile PBS, pH 7.4.

2.

PVDF plates need to be treated (activated) with ethanol before coating. PVDF plates from Millipore,type MAIPSWU should be treated with 50 μl 70% ethanol/well for maximum 2 minutes. PVDFplates, type MSIP should be treated with 15 μl 35% ethanol/well for maximum 1 minute. After ethanol treatment, immediately proceed to plate washing.

3.

Wash plate 5 times with sterile water, 200 μl/well.

4.

Add 100 μl/well of the antibody solution and incubate overnight at 4-8°C. Include control wells withPBS only.

B

Incubation of cells in plate (sterile conditions)

1.

Wash plate 5 times with sterile PBS, 200 μl/well, to remove excess antibody.

2.

For blocking, add 200 μl/well of medium containing 10% of the same serum as used for the cellsuspensions. Incubate for at least 30 minutes at room temperature.

3.

For analysis of in vivo activated B cells, remove the medium and add the cell suspension to theELISpot plate. For analysis of memory B cells, the cells can be pre-activated separately in tubes prior to addition to the plates. See further Hints and Comments/In vivo activated B cells and Memory
B cells.

4.

Put the plate in a 37°C humidified incubator with 5% CO2 and incubate for 16-24 hours. Do not
move the plate during this time and take measures to avoid evaporation (e.g. by wrapping theplate in aluminium foil).

CDetection of spots

2a.

Antigen-specific IgM
Dilute biotinylated antigen to a suitable concentration (0.01-1 μg/ml) in PBS containing
0.5% fetal calf serum (FCS). Add 100 μl/welland incubate for 2 hours at room temperature.

3.

Wash plate as above (step C1).

4.

Dilute the Streptavidin-ALP (1:1000) in PBS-0.5% FCS and add 100 μl/well. Incubate for 1 hour atroom temperature.

5.

Wash plate as above (step C1).

6.

Add 100 μl/well of substrate solution (e.g. BCIP/NBT) and develop until distinct spots emerge.

7.

Stop colour development by washing extensively in tap water. If desirable, remove the plate fromthe plate tray or the underdrain 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.

1.

6

See Hints and Comments section for details on cell stimulation.

To remove the cells, empty the plate and wash 5 times with PBS, 200 μl/well.
2b.

Total IgM
Dilute the detection mAb (MT22-biotin) to 1μg/ml in PBS-0.5% FCS. Add 100 μl/well andincubate for 2 hours at room temperature.

Protocol II(using coated antigen)A

Preparation of ELISpot plate (sterile conditions)

1a.

Antigen-specific IgM
Dilute the antigen to suitable concentration(1-50 μg/ml in sterile PBS, pH 7.4).

2.

PVDF plates need to be treated (activated) with ethanol before coating. PVDF plates from Millipore,type MAIPSWU should be treated with 50 μl 70% ethanol/well for maximum 2 minutes. PVDFplates, type MSIP should be treated with 15 μl 35% ethanol/well for maximum 1 minute. After ethanol treatment, immediately proceed to plate washing.

3.

Wash plate 5 times with sterile water, 200 μl/well.

4.

Add 100 μl/well of the antigen or antibody solution and incubate overnight at 4-8°C. Include control wells with PBS only.

B

Incubation of cells in plate (sterile conditions)

1.

Wash plate 5 times with sterile PBS, 200 μl/well, to remove excess antibody

2.

For blocking, add 200 μl/well of medium containing 10% of the same serum as used for the cellsuspensions. Incubate for at least 30 minutes at room temperature.

3.

For analysis of in vivo activated B cells, remove the medium and add the cell suspension to theELISpot plate. For analysis of memory B cells, the cells can be pre-activated separately in tubes prior to addition to the plates. See further Hints and Comments/In vivo activated B cells and Memory
B cells.

4.

Put the plate in a 37°C humidified incubator with 5% CO2 and incubate for 16-24 hours. Do notmove the plate during this time and take measures to avoid evaporation (e.g. by wrapping theplate in aluminium foil).

CDetection of spots

1.

To remove the cells, empty the plate and wash 5 times with PBS, 200 μl/well.

2.

Dilute the detection mAb (MT22-biotin) to 1 μg/ml in PBS-0.5% fetal calf serum (PBS-0.5% FCS). Add
100 μl/well and incubate for 2 hours at room temperature.

3.

Wash plate as above (step C1).

4.

Dilute the Streptavidin-ALP (1:1000) in PBS-0.5% FCS and add 100 μl/well. Incubate for 1 hour atroom temperature.

5.

Wash plate as above (step C1).

6.

Add 100 μl/well of substrate solution (e.g. BCIP/NBT) and develop until distinct spots emerge.

7.

Stop colour development by washing extensively in tap water. If desirable, remove the plate fromthe plate tray or the underdrain 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.

1b.

Total IgM
Dilute the coating mAbs MT11/12 to 15 μg/ml
in sterile PBS, pH 7.4.

See Hints and Comments section for details on cell stimulation.

7

Updated on 2022/11/23

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with the standards ISO 9001:2015 & ISO 13485:2016.

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