Datasheet & Protocol

ELISpot Flex:
Human IL-4 (ALP)
3410-2A

The kit is intended for enumeration of cells secreting human IL-4 using the ELISpot assay.

Components

Concentration

AmountCapture mAb IL4-I

1 mg/ml

600 μlDetection mAb IL4-II, biotin

1 mg/ml

50 μl-50 μlStreptavidin-ALP

To ensure total recovery of the stated quantity, vials have been overfilled.

Specificity

The kit is based on a matched pair of mAbs specific for human IL-4. The mAbs crossreact with IL-4 from non-human primates. Please visit www.mabtech.com for reactivity
on NHP species.

Shipping & Storage• Shipped at ambient temperature.
• Store at 4-8 °C upon receipt.
• Antibodies are supplied in sterile filtered PBS with 0.02% sodium azide. StreptavidinALP is supplied in in 0.1 M Tris buffer with 0.002% Kathon CG.
• The expiry date indicates how long unopened products, stored according toinstructions, are recommended for use.

Tips and guidance
PlatesELISpot plates with a PVDF membrane are recommended. An initial ethanol treatment of themembrane gives optimal binding of capture mAb, ensuring a sensitive assay. It is importantthat the plate membrane is not allowed to dry after treatment. If drying occurs, repeat
protocol steps A2-A3 before adding capture mAb.

Plate washing

Plates can be washed using a multi-channel micropipette. When sterile conditions are notrequired (protocol steps C1-C5), an ELISA plate washer can be used if the washer head
position has been adapted to ELISpot plates. After each wash cycle, empty the plate and tap
it gently against absorbent paper.

Serum and cell culture medium

For blocking and cell incubation, we recommend cell culture medium containing 10%
fetal calf serum (FCS). The serum should be selected to support cell culture and give low
background staining. Homologous serum is not recommended. If required, serum-free
medium evaluated for cell culture can be used.

Cells

This assay has been validated with peripheral blood mononuclear cells (PBMCs). For othercell types, the protocol may need adjustment. Freshly prepared or cryopreserved cells canbe used. After thawing and washing of cryopreserved cells, leave diluted in cell culture
medium at 37 °C for at least one hour. Remove cell debris and count the cells.

Stimuli and assay controls

An assay setup with triplicate wells is recommended. To measure antigen-specific responses,
add 200,000-500,000 cells per well. Fewer cells are typically needed for polyclonal activators(used as positive controls for assay functionality), e.g., PHA (1-10 μg/ml) or mAb CD3-2(product code 3605-1-50). As negative controls, include wells with unstimulated cells andwells with only medium. For stimulation of monkey cells, we recommend mAb CD3-1(product code 3610-1-50).

Detection antibody

Diluted detection mAb can be filtered (0.2 μm) to reduce the risk of unspecific background.

Development and analysis

A general darkening of the membrane can occur during development but disappears afterdrying. We recommend analyzing plates in an ELISpot reader (e.g., Mabtech ASTOR orMabtech IRIS).

Please note

• Do not allow the plate membrane to dry during the assay• Do not touch the membrane with pipette tips
• Avoid getting liquid on the underside of the membrane as it may cause leakage

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Protocol
A. Coating (sterile conditions)

1. Dilute the capture mAb (IL4-I) to 15 μg/ml in sterile PBS.
2. Add ethanol (EtOH) to PVDF plates as follows:- MSIP plates (Millipore): 20 μl 35% EtOH per well for maximum 1 minute.
- MAIPSWU plates (Millipore): 50 μl 70% EtOH per well for maximum 2 minutes.
3. Wash the plate 5 times with sterile water, 200 μl per well.
4. Add 100 μl per well of the diluted capture mAb. Cover the plate and incubate overnightat 4-8 °C.

B. Cell Incubation (sterile conditions)

1. Wash the plate 5 times with sterile PBS, 200 μl per well.
2. Add 200 μl per well of cell culture medium to block and condition the plate. Incubate
for at least 30 minutes at room temperature.
3. Empty the plate and add 50 μl stimuli and controls, followed by 50 μl cell suspension
per well. Alternatively, mix stimuli and cells before adding 100 μl per well.
4. Place the plate in a 37 °C humidified incubator with 5% CO2 for approximately 48 hours,adapt incubation time to cell type and antigen/stimuli. Avoid evaporation, e.g., bywrapping the plate in aluminum foil. Do not stack or move plates during incubation.

C. Detection
Use PBS containing 0.5% FCS (PBS-0.5% FCS) for dilution of the detection mAb andStreptavidin-ALP. The PBS should be filtered (0.2 μm) for optimal results.
1. Wash the plate 5 times with PBS, 200 μl per well.
2. Add 100 μl per well of the detection mAb (IL4-II-biotin) diluted to 1 μg/ml in PBS-0.5%FCS. Incubate for 2 hours at room temperature.
3. Wash as in step C1.
4. Add 100 μl per well of Streptavidin-ALP diluted 1:1000 in PBS-0.5% FCS. Incubate for
1 hour at room temperature.
5. Wash as in step C1.
6. Add 100 μl per well of substrate (e.g., BCIP/NBT-plus, 0.45 μm-filtered, product code
3650-10) and develop until distinct spots emerge (5-30 minutes).
7. Stop color development by washing thoroughly in tap water.
8. Leave the plate to dry. The underdrain or tray can be removed for quicker drying.
Plates should be completely dry before analysis. For the option to re-analyze later, store
plates protected from light at room temperature.

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Updated on 2022/11/25

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

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