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PeliSPOT^ä

human GRANZYME B kit 

288 tests

 An enzyme immuno spot assay for the quantitative enumeration

of human GRANZYME B secreting cells

 

 

PRODUCT INFORMATION

Cat.No. RDI-M2536clb, new cat#: 55R-M2536clb  $787.50/kit

 

 For Research Use Only

 

 

 

 

Sample Insert:see actual insert with each kit for batch specific information

 

I.   INTRODUCTION

Granzymes are exogenous serine proteinases released from cytoplasmic granules of cytotoxic lymphocytes (CTLs) and Natural killer cells (NK). Next to Granzymes these granules contain other proteins including a pore-forming protein (Perforin). Upon binding of the CTL to a target cell (by CTL-receptor and antigen-presenting MHC molecules on the target cell) the contents of the granules are released. The Granzymes enter the target cell by the granule excocytose pathway. Perforin enters the target cell by polymerization in the membrane and releases the Granzymes by perforating the Granzyme-endosomical membrane. In the cytoplasm Granzyme B can activate cell death by three different pathways, first by activating the cascade of caspases, secondly by cleaving cytoplasmic substrates and thirdly by cleaving directly in the nucleus. Granzyme A is also able to induce apoptosis in the target cell via a different pathway and with slower kinetics.

 

The ELISPOT or ELISA SPOT technique is an assay method that has demonstrated its significance in the last few years mainly in the field of anti-viral immunity and tumor-immunology. The ELISPOT assay for the detection of IFNg secreting T-cells was first described in 1983 by Prof. C.C. Czerkinski. A significant advantage compared to the ELISA technique is the increased sensitivity. Where ELISA requires at least 400 cells per well to produce sufficient Granzymes to give detectable levels, in contrast in ELISPOT only 1 positive cell per well can be detected resulting in a sensitivity of 1 x 10⁶ positive cells. Not only the high sensitivity, but also the possibility to enumerate cytokine or Granzyme B producing T cells at the single cell level, is an enormous advantage.

 

Using the ELISPOT assay, Granzyme B producing cells can now be enumerated on a single cell level facilitating a sensitive method to study vaccine efficiencies, anti-tumor effects, etc.

 

II.                 INTENDED USE

 

This PeliSPOT human GRANZYME B kit has been developed for reproducible and specific quantification of the frequency of Granzyme B secreting human CTL and NK cells. The kit is for research use only.

 

III.                PRINCIPLE OF THE TEST

 

Cells are overnight incubated with a stimulator in a well coated with a high affinity anti Granzyme B monoclonal antibody. Secreted Granzyme B will bind insitu to the coat antibody. Subsequently cells and unbound components are washed away and a biotin labelled anti Granzyme B antibody is added. Via streptavidin-poly-HRP and a precipitating substrate the presence of secreted Granzyme B are visualised by a blue/purple coloured spot.

Enumeration of spots can be reliably performed with the A.EL.VIS, or other automated plate reader system. The results can be expressed as the number of spot forming cells per million cells.

 

IV.               STORAGE AND STABILITY

 

The components of the humane Granzyme B PeliSPOT kit (except substrate solution) should be stored

at < -18°C. The substrate solution should be stored at 2-8 °C.

Performance of the kit is guaranteed until the expiration date shown on the case label.

 

V.              CONTENTS OF THE KIT

The PeliSPOT human GRANZYME B kit contains material sufficient for 288 tests.

The reagents provided are:

Kit component	Volume	Storage	Cap colour	Format
Coating antibody	190 µl	<-18 °C	Red	100-fold concentrated
Biotinylated antibody	375 µl	<-18 °C	Yellow	100-fold concentrated
Streptavidin-poly-HRP	20 µl	<-18 °C	Brown	6000-fold concentrated
Positive assay control	100 µl	<-18 °C	Black	8-fold concentrated
PeliSPOT buffer	50 ml	<-18 °C		5–fold concentrated
Substrate*	18 ml	2-8 °C		Ready for use

 

 

VI.               PRECAUTIONS FOR USE

 

1.      The PeliSPOTä human GRANZYME B kit is intended for research purposes only, ingredients are not for in vivo use.

2.      Only use the reagents supplied with the kit, do not mix reagents from different kit lots.

3.      Handle all blood, tissue and cell samples with care to prevent transmission of infections.

4.      Sodium azide inactivates HRP, do not use sodium azide-containing solutions, nor add sodium azide to the supplied materials.

5.      All reagents contain merthiolate (0.001% w/v) and may be toxic upon ingestion, inhalation or skin contact. Avoid contact of skin, eyes or clothing with the solutions. In case of contact, wash skin or eyes with water and consult a physician.

 

VII.              ADDITIONAL MATERIALS REQUIRED

 

–          96-well microtiter PVDF-bottemed membrane plate : preferable MiIllipore multiscreen MAIP N45.

–          CO­₂ Incubator.

–          Preferably A.EL.VIS or other immunospot analyser.

–          Pipetting devices for accurate delivery of 1-10 µl, 50 µl, 100 µl and 1 ml volumes.

–          Beakers, flasks, cylinders necessary for preparation of reagents.

 

VIII.             ADDITIONAL REAGENTS REQUIRED

Note: in the concentrated buffer salt crystals may appear when stored at 2-8°C. Before preparing the working-strength buffer, first warm the concentrated buffer BRIEFLY to 37°C to dissolve the precipitate.

 

Cell culture medium: e.g. IMDM or RPMI

Supplements for cell culture medium : FCS,  5% (v/v), Glutamin, 2mM, Antibiotics: e.g. penicillin 100 U/ml; streptomycin 100 µg/ml

 ethanol: 70% or 96% (v/v)

  Ployclonal stimulator: e.g.

Dissolve in the cell culture medium; PMA (Sigma, P8139) 1 ng/ml and Ionomycine (Sigma I0634) 0.5 ug/ml.

 

 IX.           ASSAY PROCEDURE

 PLEASE READ CHAPTER X ON PAGE 7 FOR IMPORTANT INFORMATION FIRST!

 

For your convenience an easy-reference protocol summery with check list and plate plan are available on the last pages of this leaflet.

 

DAY 0

Bring all reagents required for this day to room temperature (18-25°C). Centrifuge vials before use (1 minute 3000 x g).

 

1. PeliSPOT BUFFER

The kit contains one bottle with 5-fold concentrated kit buffer.

 

 

2. MICROTITER PLATES

Make sure not to scrape the bottom of the wells during pipetting and do not let the membrane dry out, empty plate just before adding reagents.

 

3.a. Microtiterplate activation

Millipore MAIP N45 is recommended.

Activate the PVDF-bottemed-wells by adding 100 ul of 70% or 96% ethanol.

Cover the plate with the lid and incubate for10 minutes at room temperature.

 

3.b. Rince

Aspirate ethanol from wells and completely fill the wells > 300 ml with distilled water and aspirate, repeat this with PBS, after the final aspiration the wells should be dry (repeated tapping on absorbent paper).

 

3.c. Coating antibody

Prepare PBS as described on page 3 of the information leaflet.

Per microtiter plate, add 60 ml of coating antibody (red-capped vial) to 6 ml PBS.

Add 50 ml of the diluted coating antibody to all wells.

Cover the plate with the lid and incubate overnight at 2-8 °C.

 

 

DAY 1

Bring all required reagents for this day to room temperature (18-25°C). Centrifuge vials before use (1 minute 3000 x g).

 

4. Washing procedure

Prepare working-strength PBS (1:20 dilution of stock PBS as described on page 3 of the information leaflet) Ad Tween 20 to 0.005% as described on page 3.

Aspirate supernatants from wells or empty wells by flicking the plate over a sink, completely fill the wells > 300 ml with working-strength washing buffer (PBS/TWEEN) and aspirate. Repeat this five times, after the final aspiration the wells should be dry (repeated tapping on absorbent paper).

 

5. Blocking procedure

Prepare working-strength PeliSPOT buffer (1:5 dilution of stock PeliSPOT buffer as described in point 2. of this assay procedure).

Add 100 ml of working strength PeliSPOT buffer to all wells, gently agitate the microtiter plate by tapping the edge of the plate for a few seconds to mix contents of each well.

Cover the plate with the lid and incubate 1 hour at room temperature (18-25°C).

 

6.  INCUBATION WITH THE CELLS

Please read the chapter IMPORTANT INFORMATION on page 7 for additional recommendations regarding the best method for cell preparation!

 

6.a. Preparation of cells

Cells can be used either as freshly isolated peripheral blood mononuclear cells (PBMC) or frozen PBMC as well as cell lines and other cultured cells.

PBMC are isolated from venous blood by density centrifugation according to the manufacturers protocol, wash cells twice with culture medium.

After being drawn (both Heparin or EDTA-anti-coagulated blood can be used), blood is kept at room remperature for maximally 24 hours.

Optimal number of cells per well has to be determined experimentally by two fold serial dilution.

For Granzyme B ELISPOT using PBMC a cell concentration of 1 ×10⁵ cells per well is recommended.

 

6.b. Preparation of antigen stimulators and controls

For antigen specific stimulation the optimal concentration of the antigen as well as the optimal number of cells per well has to be determined experimentally. Start with a cell concentration of e.g. 2 ×10⁵ cells per well and an antigen concentration between 1 and 20 µg/ml.

 

As positive secretion control polyclonal (mitogen) stimulation supplement of PMA/Iono (as described on page 3 of the information leaflet) is recommended.

 

As negative secretion control culture medium without extra addition is added the corresponding wells.

Add 50 ml of the diluted stimuli and controls in triplicate into the corresponding wells (see recommended plate plan).

 

6.c. Granzyme B assay control

HuGranzyme B positive assay control (positive control) is from natural origin.

The kit contains one black-capped vial human Granzyme B.

Dilute for one well 15 µl in 120 µl cell culture medium. If the assay is performed correctly it will result in a totally even blue/purple well.

add 100 µl of the diluted positive assay control into the corresponding wells.

Cover the plate with the lid and Incubate overnight (16 – 24 hours) at 37°C in a CO₂ incubator
During this period make sure that the plate is completely horizontal and do not agitate or move the plate.

 

 

Day 2

 

 Bring all required reagents for this day to room temperature (18-25°C), with the exception of the streptavidin-poly-HRP conjugate. Centrifuge all vials before use (1 minute 3000 x g).

 

7.    WASHING PROCEDURE

See point 4.

 

8. INCUBATION WITH BIOTINYLATED ANTIBODY

 

The kit contains one yellow-capped vial with 100-fold concentrated antibody-biotin conjugate.

Per microtiter plate, add 120 ml biotinylated antibody in 12 ml working-strength PeliSPOT buffer just before use.

8.    WASHING PROCEDURE

See point 4.

 

10. WASHING PROCEDURE

See point 4.

 

Note: The speed of the enzymatic colour development is influenced by many factors including temperature therefore monitor the development by eye.

 

12. Rince

Aspirate the substrate from the wells and completely fill the wells > 300 ml with distilled water and aspirate, after the final aspiration the wells should be dry (repeated tapping on absorbent paper).

 

Monocytes and spontaneous secretion of Granzyme B. 

Non-stimulated PBMC from healthy donors have shown to secrete Granzyme B when directly added to the assay plate. After depletion of the monocytes (adherend cells) this spontaneous release decreases dramatically.

 

We therefore strongly recommend to remove monocytes by magnetic cell separation or adherence.

 

Magnetic cell separation

Monocytes are depleted from the PBMC with CD14 beads (e.g. MACS, Miltenyi) according to the manufacturer‘s protocol. The CD14 negative fraction is collected and washed once with culture medium.

 

Adherence

A pre-incubation of the cells in a 24 wells culture plate for 20 to 44 hours at +37 °C in a CO₂ incubator (2 x 10⁶ cells /ml / well). After pre-incubation the non-adherent cell fraction is collected and washed once with pre-warmed culture medium.

 

Following data are results from initial experiments.

Cell preparation	Method	Spot forming cells per million cells	Reduction of spots
PBMC	Ficoll	1420	0%
Macs	CD14 depletion	420	70-90 %
Adherence	20 hours	624	35-65%
Adherence	44 hours	206	70-90%

 

 

Washing

Washing the plates thoroughly will result in a low background and a good spot development.

We strongly recommend to remove the plastic holder of the plate after the cell incubation and to rinse and dry the back of the membrane-bottom after each washing cycle.

Washing can be carried out manually or with an accurate automated washing device.

 

Pipetting

Make sure not to scrape the bottom of the wells during pipetting and do not let the membrane dry out, empty plate just before adding reagents.

 

PeliSPOT buffer

The provided PeliSPOT buffer can be used for the blocking step and as a dilution buffer for the biotinylated antibody and the streptavidine–poly-HRP. 

Working conditions

Sterile conditions are not necessary for coating and blocking if the cell incubation is not longer than 40 hours and the culture medium is supplemented with antibiotics. For working with human specimens working in a flow cabinet is recommended.

 

Interruption of the assay

After adding the biotinylated antibody it is possible to store the plate over night at 2-8 °C in the dark and finish the assay the next day. 

 

Specificity

No cross reactivity was observed with the following human proteins: Proteinase 2 (PR3), Tryptase, Cathepsin G (Cath G), Granzyme A, Human Neutrophil Elastase (HNE), Trypsin, Chymotrypsin.

 

 

X.               Protocol summery and checklist for the PeliSPOT Granzyme B kit.

 

PLEASE READ CHAPTER X ON PAGE 7 FOR IMPORTANT INFORMATION FIRST!

 

Day 0:

¡      Bring coating antibody to room temperature.

¡      Prepare PBS.

¡      Dilute coating antibody 1:100 in PBS.

¡      Activate PVDF-bottomed-well microtiter plate with 70% or 96% ethanol for 10 minutes at room temperature.

¡      Rinse once with distilled water and wash once with PBS.

¡      Remove all residual buffer by repeated tapping on absorbent paper.

¡      Add 50 ml of diluted coating antibody to all wells, cover the plate with the lid and incubate overnight at +4°C.

 

Day 1:

¡          Bring all required reagents to room temperature.

¡          Prepare PeliSPOT buffer.

¡          Prepare washing buffer.

¡          Prepare culture medium.

¡          Wash the plate(s) five times with washing buffer.

¡          Empty wells by flicking the plate over a sink and tapping it on absorbent paper.

¡          Add 200 ml PeliSPOT buffer to all wells, cover the plate with the lid and incubate for one hour at room temperature.

¡          Prepare cell suspensions.

¡          Wash the plate(s) five times with washing buffer.

¡          Empty wells by flicking the plate over a sink and tapping it on absorbent paper.

¡          Add 50 ml of the cell suspension containing the appropriate number of cells according the plate plan, (see plate plan example).

¡          Subsequently add to the wells 50 ml of the specific antigens (peptides) of interest in the appropriate concentrations.

¡          Add for each sample 50 ml of a positive secretion control (polyclonal stimulator e.g. PMA / Ionomycin) as well as 50 ml culture medium as a negative secretion control (wells only containing the cell suspension).

¡          Add 100 ml of the positive Granzyme B assay control (per plate) to the wells.

¡          Cover the plate with the lid and Incubate overnight (16 – 24 hours) at 37°C in a CO2 incubator
During this period make sure that the plate is completely horizontal and do not agitate or move the plate.

 

Day2

¡          Bring all required reagents, with the exception of streptavidin-poly-HRP, to room temperature.

¡          Dilute biotinylated antibody 1:100 in PeliSPOT buffer.

¡          Wash the plate(s) five times with washing buffer.

¡          Empty wells by flicking the plate over a sink and tapping it on absorbent paper.

¡          Add 100 ml of the diluted biotinylated antibody to all wells, cover the plate(s) with the lid and incubate for one hour at room temperature.

¡          Dilute the streptavidin-poly-HRP conjugate 1:6,000 in PeliSPOT buffer.

¡          Wash the plate(s) five times with washing buffer.

¡          Empty wells by flicking the plate over a sink and tapping it on absorbent paper.

¡          Add 100 ml of the diluted streptavidin-poly-HRP conjugate to all wells, cover plate(s) with the lid and incubate for one hour at room temperature.

¡          Wash the plate(s) five times with washing buffer.

¡          Empty wells by flicking the plate over a sink and tapping it on absorbent paper.

¡          Add 50 ml substrate to all wells, cover the plate with the lid and incubate for 10-15 minutes at room temperature in the dark (monitor spot intensity by eye).

¡          Wash the plate(s) with an excess of distilled water.

¡          Remove all residual water by repeated tapping on absorbent paper

¡          Dry the plate overnight at room temperature (in dark) or place the plate in an air flow in front of a fan until the membranes are completely dry.

¡          Count the number of spots preferably using the AELVIS automated spot analyzer.

Plate plan proposed for the PeliSPOT Granzyme B kit:

 

 

1

2

3

4

5

6

7

8

9

10

11

12

A

neg

neg

neg

pos

pos

pos

Pept1

Pept1

Pept1

Pept2

Pept2

Pept2

B

C

D

E

F

G

H

Pos assay contr

Key:   

neg: negative secretion control control (wells only containing the cell suspension).

pos: positive secretion control (polyclonal stimulator e.g. PMA / Ionomycin).

Pept 1 and 2 : the specific antigens (peptides) of interest.

Pos assay contr: 100 ml of the positive Granzyme B assay control.

 

 

Research Diagnostics Inc

Pleasant Hill Road

Flanders NJ 07836

Phone (973) 584-7093

Fax (973) 584-0210

Email: antibodies@fitzgerald-fii.com

Web: http://www.researchd.com

 

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