Datasheet & Protocol

ELISpot Flex:
Human IgE (HRP)
3810-2H

The kit is intended for enumeration of cells secreting human IgE using the ELISpot assay.

Components

Concentration

AmountCapture mAb 107

1 mg/ml

600 μlDetection mAbs 107/182/101, biotin

1 mg/ml

50 μl-

500 μl1 mg/ml

80 μl

-

1 μg

Streptavidin-HRP
Anti-CD40 mAb S2C6
Lyophilized recombinant human IL-4

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 IgE.

Shipping & Storage• Shipped at ambient temperature.
• Store antibodies and Streptavidin-conjugate at 4-8 °C upon receipt. IL-4 should be
stored at -20 °C.
• Antibodies are supplied in sterile filtered PBS with 0.02% sodium azide, except mAb
S2C6 that is supplied without azide. Streptavidin-ALP 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 guidancePlates

ELISpot 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-freemedium 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. If using frozen cells, leave them diluted in cell culture medium at 37 °C for at least
one hour after the thawing procedure. Remove cell debris and count the cells. The assay
can be used for analysis of in vivo- as well as in vitro-induced IgE-secreting cells. Note thatin vitro stimulation with anti-CD40 mAb and IL-4 may induce switching to IgE and IgG1, andthus not represent the situation in vivo.

Stimuli and assay controls

IgE secretion can be induced by pre-activation of cells in tubes, with anti-CD40 mAb S2C6 (1μg/ml) and IL-4* (10 ng/ml), for approximately 5 days. After pre-activation, wash the cells toremove secreted IgE. An assay setup with triplicate wells of 100,000-300,000 cells per wellis recommended. As negative controls, include wells with unstimulated cells and wells withonly medium.
*Reconstitute lyophilized recombinant human IL-4 with 1 ml PBS to obtain 1 μg/ml. Leavefor 5 minutes, then vortex. Store in aliquots at -20 °C or below.

Detection antibody

Diluted detection mAb should be filtered (0.2 μm) to reduce the risk of unspecificbackground.

Analysis

We recommend analyzing plates in an ELISpot reader (e.g., Mabtech ASTOR or MabtechIRIS).

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 (107) 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. Incubatefor at least 30 minutes at room temperature.
3. Empty the plate and add 100 μl cell suspension per well.
4. Place the plate in a 37 °C humidified incubator with 5% CO2 for 16-24 hours. Avoid
evaporation, e.g., by wrapping 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-HRP. The PBS should be filtered (0.2 μm) for optimal results. Buffers should notcontain sodium azide, as it inhibits HRP activity.
1. Wash the plate 5 times with PBS, 200 μl per well.
2. Dilute the detection mAbs (107/182/101-biotin) to 1 μg/ml in PBS-0.5% FCS and filter
the solution. Add 100 μl per well and 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 1hour 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 code3650-10) 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
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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