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PeliSPOT(tm)

Human Granzyme B kit

288 Elispot determinations

cat#RDI-M2536clb  $750.00/kit  

Click here for data



  ANTIBODIES  

(anti-Human and others as indicated)

RDI Division of Fitzgerald Industries Intl  offers a wide line of  antibodies. Since no one antibody works best for all applications (neutralization, blotting, ELISA, etc), we offer many different types of antibodies to help solve this problem. Please inquire for other applications or types of antibodies not listed below.


GRANZYME A   &   GRANZYME   B  Monoclonals

Flow cytometry, Elisa, histochemistry , ELISPOT and more....

-See Also New Granzyme A Compact (tm) Kit   $812.00/1 kit  $750.00/kit 3+

-See Also New Granzyme B Compact (tm) Kit   $812.00/1 kit  $750.00/kit 3+ 


see below updated list of literature references on the Granzyme products
Mouse anti-Human Granzyme A & Granzyme B Monoclonals

GA=Granzyme A, GB-Granzyme B specific clones

Price:     $565.00/1 vial,    
Cat# Clone Format Unconjugated Especially suited for:
RDI-M1791clb GA6 200ul (1mg/ml) 200ug Immunochemistry/western blot
RDI-M1766clb GA28 100ul (2mg/ml) 200ug Elisa techniques
RDI-M1767clb GA29 100ul (2mg/ml) 200ug Elisa techniques
RDI-M1754clb GB7 200ul (1mg/ml) 200ug Immunochemistry/western blot
RDI-M1755clb GB10 100ul (2mg/ml) 200ug Elisa techniques Elispot (coat)
RDI-M1756clb GB11 100ul (2mg/ml) 200ug Elisa techniques,
RDI-M1757clb GB12 100ul (2mg/ml) 200ug Flow cytometry
Cat# Clone PE Conjugated Especially suited for:
RDI-M2289clb GB11 500ul (0.2ml/ml) 100ug Flow Cytometry
Cat# Clone Biotin-conjugated Especially suited for:
RDI-M1793clb GB10 100ul (1mg/ml) 100ug Elisa techniques
RDI-M1794clb GB11 100ul (1mg/ml) 100ug Flow cytometry (Elispot detection)
RDI-M1795clb GB12 100ul (1mg/ml) 100ug Flow cytometry
RDI-M1798clb GA28 100ul (1mg/ml) 100ug Elisa technologies
RDI-M2536clb Pelispot(tm)

Human Granzyme B kit

288 Elispot determinations

$688.00/1kit  $625.00/kit 5+

see sample protocol for intracellular staining below

see Sample protocol for paraffin sections below

see Sample Elisa protocol below

See sample ELISPOT Below

Click here for sample histograms

Click here for sample western blot data

For In Vitro Research Use Only-Not for use in Diagnostics


PeliCluster Granzyme A

Cat# RDI-M1791clb

Clone: CLB-GA6

This clone has been derived from hybridization of SP2?0 cells with spleen cells of a BALB/c mouse immunized with recombinant Granzyme A.

Isotype: Mouse IgG1

Source: Hybridoma supernatant

Purification: Protein A Chromatography

Packing: Each vial conatins 0.2ml with approx 1mg/ml monoclonal antibody in PBS pH7.5

Preservative: none

Storage & Stability: Monoclonal antibodies should be stored below -18 DEG C. The reagent is stable until the expiry date stated on the label

Major Reactivity: The monoclonal is directed against the Granzyme A antigen, which is expressed on human lymphocytes (CTL-and NK-cells)

Molecular Mass: 56-60Kda

Methods: This clone has been shown to be suitable for indirect staining on paraffin sections in immunohistochemistry

References: Kummer, J.A. et al., Production and characterization of monoclonal antibodies raised against recombinant human granzyme A and B and showing cross reactions with the natural proteins., J. Immunological methods 163. 77-83 (1993)

For Research Use only


PeliCluster Granzyme A

Cat# RDI-M1766clb

Clone: CLB-GA28

This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with human Granzyme A derived from IL-2 stimulated LAK cells.

Isotype: Mouse IgG1

Source: Hybridoma supernatant

Purification: ammonium sulphate precipitation followed by affinity chromatography

Packing: Each vial conatains 0.1ml with approx 2mg/ml monoclonal antibody in 20mM TRIS and 150mM NaCl, pH8.0.

Preservative: Merthiolate (0.001%)

Storage & Stability: Monoclonal antibodies should be stored below -18 DEG C. The reagent is stable until the expiry date stated on the label

Major Reactivity: The monoclonal is directed against the Granzyme A antigen, which is expressed in human, chimpanzee and Rhesus.

Molecular Mass: 56-60Kda

Methods: This clone has been shown to be suitable for Elisa techniques

References: 1. Spaeny-Dekking, E.H.A. et al, Extracellular Granzyme A and B in humans: detection of native species during CTL response in vitro and in vivo, J. Immunology, 160:3610-3616 (1998)

For Research Use only


PeliCluster Granzyme A

Cat# RDI-M1767clb

Clone: CLB-GA29

This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with human Granzyme A derived from IL-2 stimulated LAK cells.

Isotype: Mouse IgG1

Source: Hybridoma supernatant

Purification: ammonium sulphate precipitation followed by affinity chromatography

Packing: Each vial conatains 0.2ml with approx 1mg/ml monoclonal antibody in 20mM TRIS and 150mM NaCl, pH8.0.

Preservative: Merthiolate (0.001%)

Storage & Stability: Monoclonal antibodies should be stored below -18 DEG C. The reagent is stable until the expiry date stated on the label

Major Reactivity: The monoclonal is directed against the Granzyme A antigen, which is expressed in human, chimpanzee and Rhesus.

Molecular Mass: 56-60Kda

Methods: This clone has been shown to be suitable for Elisa techniques

References: 1. Spaeny-Dekking, E.H.A. et al, Extracellular Granzyme A and B in humans: detection of native species during CTL response in vitro and in vivo, J. Immunology, 160:3610-3616 (1998)

For Research Use only


PeliCluster Granzyme B

Cat# RDI-M1754clb

Clone: CLB-GB7

This clone has been derived from hybridization of SP2?0 cells with spleen cells of a BALB/c mouse immunized with recombinant Granzyme B.

Isotype: Mouse IgG2a

Source: Hybridoma supernatant

Purification: Protein A Chromatography

Packing: Each vial conatins 0.2ml with approx 1mg/ml monoclonal antibody in PBS pH7.5

Preservative: none

Storage & Stability: Monoclonal antibodies should be stored below -18 DEG C. The reagent is stable until the expiry date stated on the label

Major Reactivity: The monoclonal is directed against the Granzyme B antigen, which is expressed on human lymphocytes (CTL-and NK-cells)

Molecular Mass: 56-60Kda

Methods: This clone has been shown to be suitable for indirect staining on paraffin sections in immunohistochemistry

References: Kummer, J.A. et al., Production and characterization of monoclonal antibodies raised against recombinant human granzyme A and B and showing cross reactions with the natural proteins., J. Immunological methods 163. 77-83 (1993)

For Research Use only


PeliCluster Granzyme B

Cat# RDI-M1757clb

Clone: CLB-GB12

This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with human Granzyme B derived from NK cell line TY-INDY.

Isotype: Mouse IgG1

Source: Hybridoma supernatant

Purification: ammonium sulphate precipitation followed by affinity chromatography

Packing: Each vial contains 0.1ml with approx 2mg/ml monoclonal antibody in 20mM TRIS and 150mM NaCl, pH8.0.

Preservative: Merthiolate (0.001%)

Storage & Stability: Monoclonal antibodies should be stored below -18 DEG C. The reagent is stable until the expiry date stated on the label

Major Reactivity: The monoclonal is directed against the Granzyme B antigen, which is expressed in human, chimpanzee and Rhesus.

Molecular Mass: 31Kda

Methods: This clone has been shown to be suitable for flow cytometry

References: 1. Spaeny-Dekking, E.H.A. et al, Extracellular Granzyme A and B in humans: detection of native species during CTL response in vitro and in vivo, J. Immunology, 160:3610-3616 (1998)

2. Wever, P.C. et al., The CD8+ granzyme B+ T-cell subset in peripheral blood from healthy individuals contains activated and apoptosis-prone cells, Immunology, 93:383-389 (1998)

3. Hamann, D. et al, Phenotypic and functional separation of memory and effector human CD8+ T cells, J. Exp Med 186,9, 1407-1418 (1997)

For Research Use only


PeliCluster Granzyme B

Cat# RDI-M1756clb

Clone: CLB-GB11

This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with human Granzyme B derived from NK cell line TY-INDY.

Isotype: Mouse IgG1

Source: Hybridoma supernatant

Purification: ammonium sulphate precipitation followed by affinity chromatography

Packing: Each vial contains 0.1ml with approx 2mg/ml monoclonal antibody in 20mM TRIS and 150mM NaCl, pH8.0.

Preservative: Merthiolate (0.001%)

Storage & Stability: Monoclonal antibodies should be stored below -18 DEG C. The reagent is stable until the expiry date stated on the label

Major Reactivity: The monoclonal is directed against the Granzyme B antigen, which is expressed in human, chimpanzee and Rhesus.

Molecular Mass: 31Kda

Methods: This clone has been shown to be suitable for ELISA

References: 1. Spaeny-Dekking, E.H.A. et al, Extracellular Granzyme A and B in humans:detection of native species during CTL response in vitro and in vivo, J. Immunology, 160:3610-3616 (1998)

For Research Use only


PeliCluster Granzyme B

Cat# RDI-M1755clb

Clone: CLB-GB10

This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with human Granzyme B derived from NK cell line TY-INDY.

Isotype: Mouse IgG1

Source: Hybridoma supernatant

Purification: ammonium sulphate precipitation followed by affinity chromatography

Packing: Each vial contains 0.1ml with approx 2mg/ml monoclonal antibody in 20mM TRIS and 150mM NaCl, pH8.0.

Preservative: Merthiolate (0.001%)

Storage & Stability: Monoclonal antibodies should be stored below -18 DEG C. The reagent is stable until the expiry date stated on the label

Major Reactivity: The monoclonal is directed against the Granzyme B antigen, which is expressed in human, chimpanzee and Rhesus.

Molecular Mass: 31Kda

Methods: This clone has been shown to be suitable for ELISA

References: 1. Spaeny-Dekking, E.H.A. et al, Extracellular Granzyme A and B in humans:detection of native species during CTL response in vitro and in vivo, J. Immunology, 160:3610-3616 (1998)

For Research Use only


PeliCluster Granzyme B:PE conjugated

Cat# RDI-M2289clb

Clone: CLB-GB11

This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with human Granzyme B, derived from NK cell line YT-INDY.

Isotype: Mouse IgG1

Source: Hybridoma supernatant

Purification: ammonium sulphate precipitation followed by affinity chromatography

Conjugation: Conjugated with R-Phycoerythrin (PE)

Molecular F/P ratio between 1.0-2.0
Packing: Each vial contains 0.5ml with approx 0.2mg/ml monoclonal antibody in PBS with 1mg/ml BSA

Preservative: sodium azide (0.1%)

Storage & Stability: Monoclonal antibodies should be stored at 4 DEG C. The reagent is stable until the expiry date stated on the label

Major Reactivity: The monoclonal is directed against the Granzyme B antigen, which is expressed in human, chimpanzee and Rhesus.

Methods: This clone has been shown to be suitable for direct flow cytometry

Molecular Mass: 31kda


References: 1. Spaeny-Dekking, E.H.A. et al, Extracellular Granzyme A and B in humans:detection of native species during CTL response in vitro and in vivo, J. Immunology, 160:3610-3616 (1998)

2. Wever, P.C. et al., The CD8+ granzyme B+ T-cell subset in peripheral blood from healthy individuals contains activated and apoptosis-prone cells, Immunology, 93:383-389 (1998)

3. Hamann, D. et al, Phenotypic and functional separation of memory and effector human CD8+ T cells, J. Exp Med 186,9, 1407-1418 (1997)

For Research Use only


PeliCluster Granzyme A: Biotin Conjugated

Cat# RDI-M1798clb |

Clone: CLB-GA28

This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with human Granzyme A derived from IL-2 stimulated LAK cells.

Isotype: Mouse IgG1

Source: Hybridoma supernatant

Purification: ammonium sulphate precipitation followed by affinity chromatography

Conjugation: The monoclonal antibodies were conjugated to biotin using a modification of the procedure according to Hnatowich.

Packing: Each vial contains 0.1ml with approx 1mg/ml monoclonal antibody in 20mM TRIS and 150mM NaCl, pH8.0.

Preservative: Merthiolate (0.001%)

Storage & Stability: Monoclonal antibodies should be stored below -18 DEG C. The reagent is stable until the expiry date stated on the label

Major Reactivity: The monoclonal is directed against the Granzyme A antigen, which is expressed in human, chimpanzee and Rhesus.

Molecular Mass: 56-60Kda

Methods: This clone has been shown to be suitable for Elisa techniques

References: 1. Spaeny-Dekking, E.H.A. et al, Extracellular Granzyme A and B in humans:detection of native species during CTL response in vitro and in vivo, J. Immunology, 160:3610-3616 (1998)

For Research Use only


PeliCluster Granzyme B: Biotin Conjugated

Cat# RDI-M1795clb

Clone: CLB-GB12

This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with human Granzyme B derived from NK cell line YT-INDY.

Isotype: Mouse IgG1

Source: Hybridoma supernatant|

Purification: ammonium sulphate precipitation followed by affinity chromatography

Conjugation: The monoclonal antibodies were conjugated to biotin using a modification of the procedure according to Hnatowich.

Packing: Each vial contains 0.1ml with approx 1mg/ml monoclonal antibody in 20mM TRIS and 150mM NaCl, pH8.0.

Preservative: Merthiolate (0.001%)

Storage & Stability: Monoclonal antibodies should be stored below -18 DEG C. The reagent is stable until the expiry date stated on the label

Major Reactivity: The monoclonal is directed against the Granzyme B antigen, which is expressed in human, chimpanzee and Rhesus.|

Molecular Mass: 31Kda

Methods: This clone has been shown to be suitable for Elisa techniques

References: 1. Spaeny-Dekking, E.H.A. et al, Extracellular Granzyme A and B in humans:detection of native species during CTL response in vitro and in vivo, J. Immunology, 160:3610-3616 (1998)

2. Wever, P.C. et al., The CD8+ granzyme B+ T-cell subset in peripheral blood from healthy individuals contains activated and apoptosis-prone cells, Immunology, 93:383-389 (1998)

3. Hamann, D. et al, Phenotypic and functional separation of memory and effector human CD8+ T cells, J. Exp Med 186,9, 1407-1418 (1997)

For Research Use only


PeliCluster Granzyme B: Biotin Conjugated

Cat# RDI-M1794clb

Clone: CLB-GB11

This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with human Granzyme B derived from NK cell line YT-INDY.

Isotype: Mouse IgG1

Source: Hybridoma supernatant

Purification: ammonium sulphate precipitation followed by affinity chromatography

Conjugation: The monoclonal antibodies were conjugated to biotin using a modification of the procedure according to Hnatowich.

Packing: Each vial contains 0.1ml with approx 1mg/ml monoclonal antibody in 20mM TRIS and 150mM NaCl, pH8.0.

Preservative: Merthiolate (0.001%)

Storage & Stability: Monoclonal antibodies should be stored below -18 DEG C. The reagent is stable until the expiry date stated on the label|

Major Reactivity: The monoclonal is directed against the Granzyme B antigen, which is expressed in human, chimpanzee and Rhesus.

Molecular Mass: 31Kda

Methods: This clone has been shown to be suitable for flow cytometry

References: 1. Spaeny-Dekking, E.H.A. et al, Extracellular Granzyme A and B in humans:detection of native species during CTL response in vitro and in vivo, J. Immunology, 160:3610-3616 (1998)

2. Wever, P.C. et al., The CD8+ granzyme B+ T-cell subset in peripheral blood from healthy individuals contains activated and apoptosis-prone cells, Immunology, 93:383-389 (1998)

3. Hamann, D. et al, Phenotypic and functional separation of memory and effector human CD8+ T cells, J. Exp Med 186,9, 1407-1418 (1997)

For Research Use only


PeliCluster Granzyme B: Biotin Conjugated

Cat# RDI-M1793clb

Clone: CLB-GB10

This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with human Granzyme B derived from NK cell line YT-INDY.

Isotype: Mouse IgG1

Source: Hybridoma supernatant

Purification: ammonium sulphate precipitation followed by affinity chromatography

Conjugation: The monoclonal antibodies were conjugated to biotin using a modification of the procedure according to Hnatowich.

Packing: Each vial contains 0.1ml with approx 1mg/ml monoclonal antibody in 20mM TRIS and 150mM NaCl, pH8.0.

Preservative: Merthiolate (0.001%)

Storage & Stability: Monoclonal antibodies should be stored below -18 DEG C. The reagent is stable until the expiry date stated on the label

Major Reactivity: The monoclonal is directed against the Granzyme B antigen, which is expressed in human, chimpanzee and Rhesus.

Molecular Mass: 31Kda

Methods: This clone has been shown to be suitable for Elisa techniques

References: 1. Spaeny-Dekking, E.H.A. et al, Extracellular Granzyme A and B in humans: detection of native species during CTL response in vitro and in vivo, J. Immunology, 160:3610-3616 (1998)

For Research Use only


Intracellular staining for Granzyme

Dr RA Robins, Division of Immunology, Nottingham University, Queens Medical Centre, Nottingham, UK

Cells to be analysed are washed and resuspended in RPMI + 1 O%FCS. Cells are stained in aliquots of 2x105 cells in FACS tubes (BD 2054). Washes use 1 ml of wash medium, centrifugation for 5 min at 600g, and removal of supernatant by decanting (followed by a shake to remove the last drop of wash medium)


Reagents

PBA Phosphate buffered saline containing 0.5% bovine serum albumin and 0. 1 %  sodium azide

PBA/Saponin PBA containing 50mM glucose and 0. 1 % saponin (S7900, Sigma)

Saponin/FCS PBA/Saponin containing 10% foetal calf serum

Note  Depending on the markers to be used together with granzyme B, surface labelling of cell population may be done before or after fixation and permeablization.

For surface staining before fixation, we use a completely standard staining protocol, which concludes with fixation in 1 ml of 0.5% formaldehyde in borate buffered saline (ie Cellpak sheath fluid). After mixing well, store at 4 DEG C overnight. We have used this method for most of our studies with biotin labelled anti-granzyme B antibody.

For combined cell surface and intracellular staining after fixation, aliquots of 2x 10 E05  (200K) cells are fixed by resuspending in 1 ml of 0.5% formaldehyde in borate buffered saline (ie Cellpak sheath fluid). After mixing well, store at 4 DEG C overnight. NB If cells cannot be stained the next day, they must be washed with PBA after 24hr fixation; cells can then be stored in PBA at 40C until stained.

Staining for Granzyme B

i) Fluorochrome labelled anti- Granzyme B antibody

Wash cells in fixative once with PBA. Cells are then washed once in PBA/Saponin, and once in Saponin/FCS.

After decanting and blotting, resuspend cells and add labelled antibodies to granzyme B, and cell surface markers if required. 2 ul of each antibody is added to the cells resuspended in residual saponin/FCS remaining after decanting and blotting.

After 30min incubation at 4 C wash 3 times in PBA/Saponin, finally resuspending cells in 0.5% formaldehyde in borate buffered saline.


ii) Biotin labelled anti-granzyme B

Cells are incubated with 5 ul appropriately diluted biotin labelled granzyme B after washing in PBA, PBA/Saponin and saponin/FCS as in the procedure above.

After 30min incubation at 4 C, cells are washed once in PBA/Saponin, and once in Saponin/FCS.

5 ul of an appropriate dilution of fluorochrome labelled avidin is then added. (We find that Sigma Extra-avidin-fitc can be diluted 1/100). Incubate for 20min at 4 C wash 3 times in PBA/Saponin, and resuspend in PBA.


PeliCluster Granzyme antibody protocol

protocol for immuno-cyto and histo-chemistry, including paraffin-embeded tissue sections.

1. Deparaffinise and rehydrate the slides

2. Incubate for 15 minutes in methanol containing 0.3% hydrogen peroxide to block endogenous activity

3. Wash subsequently in tap water and distilled water

4. Enhance immunoreactivity by treatment with 10 mM sodium citrate pH 6.0 at 100 C for 10 minutes

5. Let the slides cool for 30 minutes in the sodium citrate buffer

6. Wash subsequently in tap water, distilled water and phosphate buffered saline (PBS)

7. Incubate for 15 minutes with normal goat serum (diluted 1:10 in PBS)

8. Tap off serum and wipe away excess

9. Incubate for 60 minutes with GA6 or GB7 (diluted in PBS)

10. Tap off liquid and wash in PBS

11. Incubate for 30 minutes with biotinylated rabbit anti mouse immunoglobulins diluted (1:200) in PBS

12. Tap off liquid and wash in PBS

13. Incubate for 30 minutes with straptavidine-biotin horseradish peroxidase complex

14. Tap off liquid end wash in PBS

15. Incubate for 5-15 minutes with 1 mg/ml 3.3-diaminobenzidine tetrahydrochloride and 0.03% hydrogen peroxide in 30 mM Tris-HCL, pH 7. 6

16. Wash subsequently in tap water and distilled water

17. Counterstain with Mayer's hamatoxylin

18. Wash subsequently in tap water and distilled water

19. Dehydrate the slides and mount in media containing organic solvents

This protocol was used for following publication:

Kummer JA, Kamp AM, Tadema TM, Vos W, Meijer CJ, Hack CE, Localization and identification of granzymes A and B-expressing cells in normalhuman lymphoid tissue and peripheral blood.Clin Exp Immunol1995 Apr;100(1):164-72


Granzyme ELISA

Materials

Coat antibody Granzyme A: clone GA29 (CLB product M1767, purified 200 ug)

Granzyme B:                         clone GB11 (CLB product M1756, purified 200 ug)

Detection antibody Granzyme A: clone GA28 (CLB product M1798, biotinilated 100 ug)

Granzyme B: clone GB10 (CLB product M1793, biotinilated 100 ug)

HPE dilution buffer High Performance ELISA (HPE) dilution buffer (CLB product M1940)

HRP-conjugate streptavidin-polymerized horseradish peroxidase (cat#RDI-PHRP20-SA - $410.00/mg)

Coat buffer*: 0.1 M sodium carbonate/bicarbonate buffer, pH 9.6

PBS*: Phosphate buffered saline

Blocking buffer: PBS 2% (v/v) cow milk

Washing buffer*: PBS-0.02% (w/v) Tween-20

Substrate solution*: 100 ug/ml 3,31,5,51-tetramethyl-benzidine (Merck, Darmstadt, Germany) and 0.003% (v/v) H202 in 0.11 M sodium acetate buffer, pH 5.5.

Stop solution*: 2 M H2S04

Microtiterplates: Nunc Maxisorb Immunoplate

* these components are also available as PeliKine ELISA tool set, product cat#RDI-M1980clb  $188.00/set  (sufficient for 3 microtiter plates)


Assay

1.  Dilute purified mAb GA29 or GB11 to 2 ug/ml (1: 100) in coat buffer.

2.  Add 100 ul to all wells and incubate for 16 hours at 4 C.

3.  Wash the plates 5 times with PBS.

4.  Block residual binding sites by a 45 minutes-incubation with blocking buffer.

5.  Wash the plates 5 times with washing buffer.

6.  Samples and standards (preverable purified native GrA or B at different concentrations) are pretreated with 40 ug/ml hyauronidase (Sigma) for 30 minutes at room temperature in washing buffer.

7.  Dilute standard and samples in HPE-dilution buffer RANGES minimum dilution 1:2 add 100 ul to the wells and incubated for 1 hour.

8.  Wash plate 5 times with washing buffer.

9.  Dilute biotinylated mAb GA 28 or GB10 to 1 ug/ml (1:100) together with 1% (v/v) normal mouse serum (CLB product M1250)

10.  Add 100 ul to all wells and incubate for 1 hour.

11.  Wash the plates 5 times with washing buffer.

12.  Dilute streptavidin-polyHRP (1:5000 -10.000) in HPE-dilution buffer.

13.  Add 100 ul to all wells and incubate for 30 minutes.

14.  Wash 5 times with washing buffer.

15.  Add 100 ul substrate solution to all wells and incubate for 30 minutes at room temperature in dark.

16.  Add 100 ul stop solution to all wells.

17.  Read the absorbance at 450 nm.

18.  Results can be calculated using the logit-computer program developed by Ed Niewenhuis from CLB, this program can be down loaded for free from : http://www.xs4all.nl/~ednieuw/logit.htm


Sample Elispot protocol using CLB Granzyme B antibodies:

Granzyme B elispot

1. coat 96-well nylon membrane-coated plate with 50 µl 15 µg/ml GB10 antibody

leave 4 DEG C overnight

2. wash plate with RPMI and block for 1 hr with 50µl RPMI/10%FCS, wash plate

3. incubate cells in different concentrations (5x105-1x105) in triplicate overnight at 37 DEGC with and without stimuli (2µmol peptide/ 50µg/ml PHA) in 100 µl RPMI/10%FCS (do not disturb)

4. discard cells and wash plate with PBS/Tween

5. incubate for 3 hrs with 50µl/well 1 µg/ml detecting antibody GB11biotin conjugate

6. wash plate (PBS/Tween) and incubate wells for 1-2 hrs with 50 µl 1 µg/ml streptavidin-AP|

7. wash plate and add 50 µl chromogen (NBT/BCIP) for +/- 15 minutes and stop reaction with tap water when purple spots become visible

Antibody concnetartions must be optimized for each use

Clone GB10 is available as 200 ug purified mcab with order no. RDI-M1755clb

Clone GB11 biotin is available as 100 ug biotinilated mcab with order  RDI-M1794clb


Select Literature References:

Costain DJ, Guha AK, Liwski RS, et al.

Murine hypodense eosinophils induce tumour cell apoptosis by a granzyme B-dependent mechanism. Cancer Immunol Immunother (Germany), Aug 2001, 50(6) p293-9

Kim GE, Yang WI, Lee S, et al.

The significance of granzyme B expression in patients with angiocentric lymphoma of the head and neck. Cancer (United States), Jun 15 2001, 91(12) p2343-52

Vermijlen D, Froelich CJ, Luo D, et al.

Perforin and granzyme B induce apoptosis in FasL-resistant colon carcinoma cells. Cancer Immunol Immunother (Germany), Jun 2001, 50(4) p212-7

Yawalkar N, Schmid S, Braathen LR, et al.

Perforin and granzyme B may contribute to skin inflammation in atopic dermatitis and psoriasis. Br J Dermatol (England), Jun 2001, 144(6) p1133-9

Andrade F, Bull HG, Thornberry NA, et al.

Adenovirus L4-100K assembly protein is a granzyme B substrate that potently inhibits granzyme B-mediated cell death. Immunity (United States), Jun 2001, 14(6) p751-61

Buzza MS, Hirst CE, Bird CH, et al.

The granzyme B inhibitor, PI-9, is present in endothelial and mesothelial cells, suggesting that it protects bystander cells during immune responses. Cell Immunol (United States), May 25 2001, 210(1) p21-9

Sun J, Whisstock JC, Harriott P, et al.

Importance of the P4' residue in human granzyme B inhibitors and substrates revealed by scanning mutagenesis of the proteinase inhibitor 9 reactive center loop.

J Biol Chem (United States), May 4 2001, 276(18) p15177-84

Pinkoski MJ, Waterhouse NJ, Heibein JA, et al.

Granzyme B-mediated apoptosis proceeds predominantly through a Bcl-2-inhibitable mitochondrial pathway. J Biol Chem (United States), Apr 13 2001, 276(15) p12060-7

Rotonda J, Garcia-Calvo M, Bull HG, et al.

The three-dimensional structure of human granzyme B compared to caspase-3, key mediators of cell death with cleavage specificity for aspartic acid in P1. Chem Biol (England), Apr 2001, 8(4) p357-68

Li B, Hartono C, Ding R, et al.

Noninvasive diagnosis of renal-allograft rejection by measurement of messenger RNA for perforin and granzyme B in urine. N Engl J Med (United States), Mar 29 2001, 344(13) p947-54

Espersen C, Pakkenberg B, Harder E, et al.

High levels of CD8-positive lymphocytes expressing CD45R0, granzyme B, and Ki-67 in lymph nodes of HIV-infected individuals are not associated with increased mortality. AIDS Res Hum Retroviruses (United States), Mar 1 2001, 17(4) p287-93

Kontani K, Sawai S, Hanaoka J, et al.

Involvement of granzyme B and perforin in suppressing nodal metastasis of cancer cells in breast and lung cancers.

Eur J Surg Oncol (England), Mar 2001, 27(2) p180-6

Yawalkar N, Hunger RE, Buri C, et al.

A comparative study of the expression of cytotoxic proteins in allergic contact dermatitis and psoriasis: spongiotic skin lesions in allergic contact dermatitis are highly infiltrated by T cells expressing perforin and granzyme B. Am J Pathol (United States), Mar 2001, 158(3) p803-8

Zhang D, Pasternack MS, Beresford PJ, et al.

Induction of rapid histone degradation by the cytotoxic T lymphocyte protease Granzyme A.

J Biol Chem (United States), Feb 2 2001, 276(5) p3683-90

Matter-Reissmann UB, Forte P, Seebach JD

Human NK cells lyse porcine endothelial cells via the perforin/granzyme B pathway. Transplant Proc (United States), Feb-Mar 2001, 33(1-2) p439-40

Davis JE, Smyth MJ, Trapani JA

Granzyme A and B-deficient killer lymphocytes are defective in eliciting DNA fragmentation but retain potent in vivo anti-tumor capacity. Eur J Immunol (Germany), Jan 2001, 31(1) p39-47

Ronday HK, van der Laan WH, Tak PP, et al.

Human granzyme B mediates cartilage proteoglycan degradation and is expressed at the invasive front of the synovium in rheumatoid arthritis. Rheumatology (Oxford) (England), Jan 2001, 40(1) p55-61

Higaki Y, Yamada O, Okamura T, et al.

Granzyme-B-containing lymphocyte involvement in epidermal injury in graft-versus-host disease. Dermatology (Switzerland), 2001, 202(2) p94-8

Mackewicz CE, Lieberman J, Froelich C, et al.

HIV virions and HIV infection in vitro are unaffected by human granzymes A and B. AIDS Res Hum Retroviruses (United States), Mar 1 2000, 16(4) p367-72

Pereira RA, Simon MM, Simmons A

Granzyme A, a noncytolytic component of CD8(+) cell granules, restricts the spread of herpes simplex virus in the peripheral nervous systems of experimentally infected mice. J Virol (United States), Jan 2000, 74(2) p1029-32

Shresta S, Graubert TA, Thomas DA, et al.

Granzyme A initiates an alternative pathway for granule-mediated apoptosis. Immunity (United States), May 1999, 10(5) p595-605

Wever PC, Spaeny LH, van der Vliet HJ, et al.

Expression of granzyme B during primary cytomegalovirus infection after renal transplantation. J Infect Dis (United States), Mar 1999, 179(3) p693-6


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