ELISpot Pro:
Human IL-4 (ALP)
Product Code: 3410-2APW-2

CONTENTS:
Pre-coated plates, mAb IL4-I (2 white plates)
Detection mAb: IL4-II, ALP (150 μl)
BCIP/NBT-plus substrate (25 ml)
Positive control: Anti-CD3 mAb (CD3-2) (100 μl)
STORAGE:
Shipped at ambient temperature. On arrival all reagents should
be stored refrigerated 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 for use.
Detection mAb is supplied in 0.1M Tris buffer with 0.002% Kathon
CG. CD3-2 is supplied in sterile filtered (0.2 μm) PBS. Vials havebeen overfilled to ensure recovery of stated quantity.

Guidelines for ELISpot Pro: Human IL-4 (ALP)
Please read through before starting the assay

A Preparation and blocking of plate
1. Remove the plate from the sealed package and wash 4 times with sterile PBS (200μl/well).
2. Condition the plate with medium containing 10% of the same serum as used for the cellsuspensions (200 μl/well). Incubate for at least 30 minutes at room temperature.
B Incubation of cells in plate
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 in a dilution of
1:1000.
2. Put the plate in a 37°C humidified incubator with 5% CO2 and incubate for 12-48 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 IL4-II-ALP 1:300 in filtered PBS containing 0.5% fetal calf serum. Add 100 μl toeach well and incubate for 2 hours at room temperature.
3. Wash the plate 5 times with PBS, 200 μl/well.
4. Filter the ready-to-use substrate solution (BCIP/NBT-plus) through a 0.45 μm filter andadd 100 μl of substrate per well. Develop until distinct spots emerge. Stop the colour development by washing extensively in tap water. If desirable, remove the underdrain (thesoft plastic under the plate) and rinse the underside of the membrane.
5. Leave the plate to dry. Inspect and count spots in an ELISpot reader or in a dissection
microscope.
6. 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 T-cell clones, mixtures of separated cell fractions etc., other protocols may
have to be 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. Avoid getting liquid on the undersideof the membrane as this may cause leakage due to capillary drainage.
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.
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 heterophilic antibodies or intrinsic analyte which may interfere with the assay.
Conjugate
To reduce unspecific background it is recommended to filter the detection mAb-enzyme conjugate (0.2 μm).
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,CD3-2, or phytohemagglutinin (1-10 μg/ml) is often used as a control for cell viability andfunctionality of the test system. For stimulation of monkey cells we recommend mAb CD3-1
from Mabtech (product code 3610-1-50).
BuffersPBS 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
Develop until distinct spots are visible in positive wells (usually 5-30 minutes). A general darkening of the membrane may occur but disappears after drying.

Updated on 2022/11/22
Developed and manufactured by MABTECH AB,
Sweden, whose quality management systemcomplies with the standards ISO 9001:2015 & ISO
13485:2016.
The products are for research use only.

CERTIFIED
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CERTIFIEDISO 13485

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MABTECH shall not be liable for the use or handling of the product or for consequential, special, indirect or incidental damages there from.
Mabtech AB (Head Office)

Mabtech, Inc.

Tel: +46 8 716 27 00

Tel: +1 513 871-4500

Swedenmabtech@mabtech.com

www.mabtech.com

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