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PeliKine Compactä

human GRANZYME A ELISA kit

288 tests

An enzyme immunoassay for the quantitative determination

of human Granzyme A

PRODUCT INFORMATION

Central Laboratory of the

Cat.No.RDI- M1935clb  $937.25

Netherlands Red Cross

Blood transfusion Service

PO box 9190

1006 AD Amsterdam

The Netherlands

For other countries:

RDI Division of Fitzgerald Industries Intl

34 Junction Square Drive

Concord MA 01742-3049

USA

Ph (978) 371-6446 or (800) 370-2222

Fax (973) 584-0210

Web: http://www.researchd.com

For Research Use Only

2302011205

I. INTRODUCTION

Granzymes are exogenous serine proteinases (enzymes) that are released from cytoplasmic granules of cytotoxic lymphocytes (CTLs) and NK cells.

Granzymes; granules + enzymes. These granules contain next to granzymes 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 in the intercellular space where after perforine will "perforate" the target cell membrane by forming transmembrane pores. Through these pores the granzymes can now enter the cytosol of the target cell. Granzyme B activates the intracellular cascade of caspases finally resulting in the killing of the target cells. Also granzyme A is able to induce apoptosis in the target cell but the molecular mechanisms of the pathway involved need to be clarified.

Percentages of granzyme A and B positive CTL's can be determined by flow cytometry and immunocytochemical methods for many disorders.

Not all granzymes enter the target cell, part of them also "leak" in to the peripheral blood and other biological fluids. Detectable amounts of granzymes have been found to circulate in healthy volunteers. These soluble granzymes can now be measured by ELISA's, which were developed by CLB-researchers.

Viral infections

Increased levels of soluble granzymes have been found with patients suspected of an increased NK cell and CTL-response caused by systemic viral infections such as EBV, HIV, CMV, hepatitis A and Dengue fever.

Lymphoma's and carcinoma's

It is shown that the presence of a high percentage of granzyme B positive CTL's in glands of patients suffering from Hodgkin's disease correlate with a severe prognosis.

Rheumatoid arthritis

Soluble granzyme A and B is increased in synovial fluid from Rheumatoid Arthritis and significantly higher than levels in patients with osteo arthrosis.

Transplantation

Granzymes are likely involved in the acute rejection of kidney-transplants, as infiltrating lymphocytes in the rejected kidney strongly express granzymes. Increasing plasma levels of soluble granzymes in patients with a kidney transplants suggest a systemic viral infection, in particular an infection by CMV.

USE

This PeliKine compactä human GRANZYME A ELISA kit has been developed for fast, reproducible and specific quantification of human GRANZYME A (huGRANZYME A) in plasma and serum as well as in cell-culture supernatant. Also suitable for urine, synovium fluid and BAL fluid.

II. PRINCIPLE OF THE TEST

The PeliKine compactä human GRANZYME A ELISA kit is a "sandwich-type" of enzyme immunoassay in which a monoclonal anti-huGRANZYME A antibody is bound onto polystyrene microtiter wells. Human GRANZYME A, present in a measured volume of sample or standard is captured by the antibody on the microtiter well, and non-bound material is removed by washing. Subsequently, a biotinylated second monoclonal antibody to huGRANZYME A is added. This antibody binds to the huGRANZYME A-antibody complex present in the microtiter well. Excess biotinylated antibody is removed by washing, followed by the addition of horseradish peroxidase (HRP)-conjugated streptavidin, which binds to the biotinylated side of the huGRANZYME A sandwich. After removal of non-bound HRP conjugate by washing, a substrate solution is added to the wells. A coloured product is formed in proportion to the amount of huGRANZYME A present in the sample or standard. After the reaction has been terminated by the addition of a stop solution, absorbance is measured in a microtiter plate reader. From the absorbance of samples and those of a standard curve, the concentration of huGRANZYME A can be determined by interpolation with the standard curve.

III. STORAGE AND STABILITY

The PeliKine compactä human GRANZYME A ELISA kit should be stored at -18°C to -32°C. The performance of the kit is guaranteed until the expiration date shown on the case label.

IV. CONTENTS OF THE KIT

The PeliKine compactä human GRANZYME A ELISA kit contains material sufficient for 288 tests, including standard curve samples. The reagents provided are:

Quantity	Kit component		Volume	Cap colour
1 vial	Coating antibody	100-fold concentrated	375 µl	red
1 vial	Positive control	Range 550 to 850 units	200 µl	transparent
1 vial	GRANZYME A standard	22,750 units / ml	100 µl	black
1 vial	Biotinylated antibody	100-fold concentrated	375 µl	yellow
1 vials	Streptavidin-poly-HRP conjugate	10,000-fold concentrated	20 µl	brown
1 bottle	Kit buffer	5-fold concentrated	60 ml	-
3 pcs	Microtiter plates + lid	-	-	-
10 pcs	Plate seals	-	-	-

One unit approximately meets 1 pg / ml.

V. PRECAUTIONS FOR USE

1) The Pelikine compactä human GRANZYME A ELISA kit is intended for research purposes only, ingredients not for in vivo use.

2) Only use the reagents and microtiter plates supplied with the kit, do not mix reagents from different kit lots

3) Handle all plasma and serum samples with care to prevent transmission of blood-borne 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 thiomersal (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.

6) Centrifuge all vials before use (1 minute 3000 x g).

7) With the exception of the substrate blank wells, do not allow wells to stand uncovered or dry for extended periods between incubation steps.

VI. ADDITIONAL BUFFERS & SOLUTIONS REQUIRED

Coating buffer and substrate buffer : 0.11 M acetate buffer pH 5.5

Dissolve 15.0 g sodium-acetate ( CH₃COONa.3H₂O ) in 800 ml distilled water.

Adjust pH to 5.5 with glacial acetic acid, add distilled water to a volume of 1 liter.

Do not add any preservative (e.g. merthiolate, sodium azide), since this may affect the quality of the

The buffer must be prepared fresh.

PBS stock solution [20 x]: 0.2 M Phosphate Buffered Saline (PBS)

Dissolve 32 g Na₂HPO₄.2H₂O

6 g NaH₂PO₄.2H₂O

164 g NaCl

in 900 ml distilled water

(intensive stirring and some heating will speed dissolution).

Bring the temperature of the solution back to room temperature (18-25°C) and check pH; if necessary

adjust pH to 6.8 - 6.9 with concentrated HCl or NaOH, and add distilled water to a volume of 1 liter (when

diluted 20 times the obtained buffer will have a pH of 7.2 - 7.4).

Add 20 mg thiomersal as preservative. Do not use sodium azide (NaN₃) since this preservative reduces the

quality of the enzymatic label.

The prepared buffer can be stored up to three months at 2-8°C.

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.

Washing buffer: PBS with 0.02 % TWEEN 20

Make 1 liter of working-strength PBS by diluting the PBS stock solution (see above)

20-fold with distilled water.

Add 200 ml TWEEN 20.

The prepared buffer can be stored up to one month at 2-8°C.

Substrate buffer: 0.11 M acetate buffer pH 5.5

Dissolve 15.0 g sodium-acetate ( CH₃COONa.3H₂O ) in 800 ml distilled water.

Adjust pH to 5.5 with glacial acetic acid, add distilled water to a volume of 1 liter.

Do not add any preservative (e.g. merthiolate, sodium azide) since this may affect the quality of the

enzymatic colour development.

The buffer must be prepared fresh.

3,5,3',5'-tetramethylbenzidine (TMB) stock solution: 6 mg/ml TMB in DMSO

Dissolve 30 mg 3,5,3',5'-tetramethylbenzidine (TMB) in 5 ml dimethylsulfoxide (DMSO).

The prepared stock solution can be stored up to 1 month at room temperature (1825°C) and if protected

against light

Hydrogen peroxide stock solution: 3% H₂O₂ solution in distilled water.

The prepared stock solution can be stored up to one month at 2-8°C.

Substrate solution:

For each plate mix the following reagents:

12 ml substrate

200 µl TMB stock solution

12 µl H₂O₂

The substrate solution should be prepared just before use and has to be at room temperature (18-25°C) for optimal reproducible results

Stop solution: 1.8 M H₂SO₄ solution in distilled water.

VII. ADDITIONAL INFORMATION

Additional materials required

- 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.

- Device for delivery of washing buffer (wash bottle / automated plate washer).

- Microtiter plate reader.

Sensitivity

2 x MEAN calculated zero signal (shaken incubation): < 20 units/ml.

Note: the sensitivity is dependent upon the type and quality of enzymatic substrate.

Expected values

Granzyme A values in fresh serum and plasma samples of healthy individuals are below

60 units/ml.

Specificity

No crossreactivity was observed with the following human proteins: Proteinase 3 (PR3), Tryptase, Cathepsin G (Cath G), Granzyme B, Human Neutrophil Elastase (HNE), Trypsin, Chymotrypsin.

REFERENCES

1. Spaenij-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).