PeliKine Compactä

human GRANZYME B ELISA kit

 

288 tests

 

An enzyme immunoassay for the quantitative determination

of human GRANZYME B

 

 

 

 

PRODUCT INFORMATION

 

 

 

 

 

 

 

2302011153

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 B ELISA kit has been developed for fast, reproducible and

specific quantification of human GRANZYME B (huGRANZYME B) in plasma and serum as well as in

cell-culture supernatant. Also suitable for synovium fluid and BAL fluid.

 

 

II. PRINCIPLE OF THE TEST

 

The PeliKine compactä human GRANZYME B ELISA kit is a "sandwich-type" of enzyme immunoassay in which a monoclonal anti-huGRANZYME B antibody is bound onto polystyrene microtiter wells. Human GRANZYME B, 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 B is added. This antibody binds to the huGRANZYME B-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 B 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 B 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 B can be determined by interpolation with the standard curve.

 

 

III. STORAGE AND STABILITY

 

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

 

One unit approximately meets 1 pg / ml.

 

 

V. PRECAUTIONS FOR USE

 

1)  The Pelikine compactä human GRANZYME B 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

 

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.