ELISpot Plus:
Human IFN-γ (HRP)
PRODUCT CODE: 3420-4HPW-2

CONTENTS:
2 pre-coated plates, mAb 1-D1K
Vial 1 (yellow top)
Detection mAb: 7-B6-1, biotin (40 μl)
Concentration 1 mg/ml
Vial 2 (white top)
Streptavidin-HRP (40 μl)
Vial 3
Positive control: Anti-CD3 mAb (CD3-2) (100 μl)
TMB substrate (25 ml)
The detection antibody is supplied in sterile filtered (0.2μm)
PBS with 0.02% sodium azide. Streptavidin-HRP is supplied in
PBS with 0.002% Kathon CG. Anti-CD3 mAb is supplied in sterile filtered (0.2 μm) PBS. Vials have been overfilled to ensure
recovery of the specified amount.
STORAGE:
Shipped at ambient temperature. On arrival all reagentsshould be stored at 4-8 °C. Plates may be kept at room temperature. The expiry date indicates how long unopened products, stored according to instructions, are recommended foruse.

Guidelines for ELISpot Plus: Human IFN-γ (HRP)
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 stimuli followed by the cell suspension. Alternatively
cells and stimuli can be mixed before addition to the plate. The mAb CD3-2, includedin the kit, is recommended as a positive control for cytokine production at a dilution of1:1000.
2. Put the plate in a 37°C humidified incubator with 5% CO2 and incubate for 18-48 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. Dilute the detection antibody (7-B6-1-biotin) to 1 μg/ml in PBS containing 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-HRP (1:1000) in PBS-0.5% FCS and add 100 μl/well. Incubate for 1
hour at room temperature.
Please note that sodium azide used in buffers will inhibit HRP activity.
5. Wash plate as above (step C1).
6. Add 100 μl/well of the ready-to-use TMB substrate solution and develop until distinct
spots emerge.
7. Stop color development by washing extensively in deionized water. If desirable, remove
the underdrain (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
These suggestions are based on the detection of antigen-specific immune responses using
PBMC. If using clones, mixtures of separated cell fractions etc., other protocols may have tobe considered.
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
Both freshly prepared and cryopreserved cells may be used in the assay. 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 250,000 cells per well are often used
to assess antigen-specific responses. For polyclonal activators, the cell number may have tobe reduced to avoid confluent spot formation. Protocols with other incubation times have tobe established by the user.
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 is not recommended as it may contain heterophilic antibodies or intrinsic analyte which may interfere with the assay.
Assay controls
The number of cells responding to stimulation is often compared to the number of cells
spontaneously producing the cytokine, which is determined by incubating the same numberof cells in the absence of stimuli. A polyclonal activator, such as the included anti-CD3 mAb or
phytohemagglutinin (1-10 μg/ml), is often used as a control for cell viability and functionalityof the test system.
Detection antibody
Diluted detection mAb can be filtered (0.2 μm) to reduce the risk of unspecific background.
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 2-20 minutes).
A general darkening of the membrane may occur during development but disappears afterdrying. Preferably use deionized water to stop the plates since some ions may cause fadingof TMB spots.

Updated on 2022/11/28
Developed and manufactured by MABTECH AB,
Sweden, whose quality management systemcomplies with the standards ISO 9001:2015 & ISO
13485:2016.

CERTIFIED
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CERTIFIED
ISO 13485

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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)SwedenTel: +46 8 716 27 00
mabtech@mabtech.com

www.mabtech.com

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