Datasheet & Protocol

ELISA Flex:
Human Granzyme B (HRP)
3486-1H-6 ǀ 3486-1H-20
ELISA Flex kit for quantitative determination of human Granzyme B in solution, e.g.
cell supernatant and serum/plasma.

The kit includes

3486-1H-6

3486-1H-20

for 6 plates

for 20 plates

Capture mAb:

MT34B6 (0.5 mg/ml)

300 μl1000 μl

Detection mAb:

MT28, biotin (0.5 mg/ml)

150 μl500 μlStreptavidin-HRP

80 μl

250 μlRecombinant human granzyme B 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-HRP is supplied in PBS with
0.002% Kathon CG. The expiry date indicates how long unopened products, storedaccording to instructions, are recommended for use.

General and Preparations
Specificity

The kit contains a matched pair of monoclonal antibodies (mAbs) specific for native andrecombinant human Granzyme B.

Standard range
10-1000 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 0.4 μg/ml by adding 0.8 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.

10 µl

120 µl 300 µl 300 µl

300 µl300 µl 300 µl

300 µl300 µl300 µlStandard stock
0.4 µg/ml

Incubation bufferor ELISA diluent: 390 µl
Concentration: 10 ng/ml

480 µl 300 µl300 µl 300 µl

20001000 500
pg/ml pg/ml pg/ml

250pg/ml

300 µl300 µl300 µl300 µl300 µl

125pg/ml

62.5pg/ml

31.2pg/ml

15.6pg/ml

7.8pg/mlStandard curve

2www.mabtech.com

Protocol
Day 1

1. Add 100 μl/well of capture mAb MT34B6 diluted to 2 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 MT28-biotin diluted to 1 μg/ml in incubation buffer or
ELISA diluent. Incubate for 1 hour at room temperature.

7. Wash as above.
8. Add 100 μl/well of Streptavidin-HRP diluted 1:1000 in incubation buffer. Incubate for

1 hour at room temperature. Please note that sodium azide used in buffers will inhibit
HRP activity.

9. Wash as above.
10. Add 100 μl/well of TMB substrate (product code: 3652-F10) and incubate at room
temperature, protected from direct light for 15 minutes.

11. Add 100 μl/well of 0.2 M H2SO4 to stop the reaction.

12. Measure the optical density in an ELISA reader at 450 nm within 15 min. Preferably use
a reader capable of subtracting a reference wavelength of between 570 and 650 nm.
Representative standard curve shown below.

Human Granzyme B

OD (450-650 nm)

1010.10.01
1101001000

10000

Standard concentration (pg/ml)

3

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Updated on 2024/03/19
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 00
mabtech@mabtech.com

www.mabtech.com

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