Datasheet & Protocol

ELISA Flex:
Human Perforin (ALP)
3465-1A-6 ǀ 3465-1A-20
ELISA Flex kit for quantitative determination of human Perforin in solution, e.g. cellsupernatant and serum/plasma.

The kit includes

3465-1A-6

3465-1A-20

for 6 plates

for 20 plates

Capture mAbs:

Pf-80/164 (1 mg/ml)

300 μl1000 μl

Detection mAb:

Pf-344, biotin (1 mg/ml)

80 μl

250 μlStreptavidin-ALP

80 μl

250 μlHuman Perforin ELISA standard

1 vial1 vialStandard reconstitution buffer A51 ml

1 ml

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

Shipping and storage

Shipped at ambient temperature. All reagents should be stored at 4-8 °C upon receipt,
except the standard which should be stored at -20 °C. Antibodies are supplied insterile-filtered PBS with sodium azide (0.02%). Streptavidin-ALP is supplied in 0.1 M
Tris buffer with 0.002% Kathon CG. The expiry date indicates how long unopened
products, stored according to instructions, are recommended for use.

General and Preparations
Specificity

The kit contains a matched pair of monoclonal antibodies (mAbs) specific for humanperforin. The mAbs cross-react with perforin from non-human primates (NHP). Please
visit www.mabtech.com for reactivity on NHP species.

Standard range
50-5000 pg/ml

Calibration

No international standard exists for calibration.

Analysis of serum and plasma samples

Analysis of serum/plasma requires the use of ELISA diluent (product code: 3652-D2). The
ELISA diluent blocks heterophilic antibodies, commonly found in serum/plasma, fromcross-linking the assay antibodies, thereby preventing false positive read-outs. The ELISA
diluent should be used for dilution of standard, samples, and detection antibody.

Reconstitution of ELISA standard

Reconstitute the ELISA standard to a stock solution of 0.5 μg/ml by adding 0.5 ml of thestandard reconstitution buffer. Allow the standard to dissolve for 5 minutes and mix
thoroughly. The standard should be kept in aliquots at -20 °C. Avoid repeated freeze-thawcycles.

Preparation of standard curve

Prepare within 30 minutes of use. Volumes are sufficient for duplicates.

20 µl

300 µl 300 µl300 µl

300 µl 300 µl

300 µl300 µl300 µl

Standard stock0.5 µg/mlIncubation buffer
or ELISA diluent:
Concentration:

980 µl 300 µl

300 µl 300 µl

300 µl300 µl300 µl300 µl300 µl10000 5000
pg/ml pg/ml

2500
pg/ml

625pg/ml

312pg/ml

156pg/ml

78.1
pg/ml

39pg/ml

1250
pg/ml

Standard curve2

www.mabtech.com

Protocol
Day 1

1. Add 100 μl/well of capture mAb Pf-80/164 diluted to 4 μg/ml in PBS, pH 7.4. Use highprotein binding ELISA plates. Incubate overnight at 4-8 °C.

Day 2

2. Empty the plate and add 200 μl/well of PBS with 0.05% Tween 20 and 0.1% BSA(incubation buffer) to block the plate. Incubate for 1 hour at room temperature.

3. Wash the plate 5 times with PBS containing 0.05% Tween 20 (300 μl/well).
4. Add 100 μl/well of samples or standards diluted in incubation buffer or ELISA diluent.

Include assay background control, i.e. wells without standard. Incubate for 2 hours atroom temperature.

5. Wash as above.
6. Add 100 μl/well of detection mAb Pf-344-biotin diluted to 1 μg/ml in incubation bufferor ELISA diluent. Incubate for 1 hour at room temperature.

7. Wash as above.
8. Add 100 μl/well of Streptavidin-ALP diluted 1:1000 in incubation buffer. Incubate for
1 hour at room temperature.

9. Wash as above.
10. Add 100 μl/well of pNPP substrate (product code: 3652-P10) and incubate at room
temperature protected from direct light for approximately 60 minutes.

11. Measure the optical density in an ELISA reader at 405 nm. Preferably use a reader
capable of subtracting a reference wavelength of between 570 and 650 nm.
Representative standard curve shown below.

Human Perforin

OD (405-650 nm)

1010.1

0.01
10100

1000

10000

100000Standard concentration (pg/ml)

3

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Updated on 2022/12/01
Developed and manufactured by MABTECH AB,Sweden, whose quality management systemcomplies with the standards
ISO 9001:2015 & ISO 13485:2016.

CERTIFIED
ISO 9001

CERTIFIED
ISO 13485

Quality management systems

Medical devices
Quality management systems

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 00mabtech@mabtech.com

www.mabtech.com

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