Instructions for use

Histamine Release

BA E-1100R

Histamine Release (Supplementary kit)
1. Introduction
1.1 Intended use and principle of the test
Supplementary kit for the quantitative determination of the release of histamine from heparinized whole
blood (this kit has to be used in combination with the Histamine ELISA, for details contact your local
supplier).
In humans, histamine (ß-imidazole ethylamine) is the most important mediator and is mostly found in theinitial phase of an anaphylactic reaction ("immediate type" allergy). Histamine is produced by the enzymatic
decarboxylation of histidine. In the organism, histamine is present in nearly all tissues, and it is mainly
stored in the metachromatic granules of mast cells and the basophilic leukocytes. It is present in an inactive
bound form and is released only as required.
Histamine acts predominantly on smooth muscle and blood vessels. In humans, it is responsible for thebroncho-constriction occurring during the acute phase. In the vessels, its constrictive effect is limited to thevenula, whereas arterioles are dilated. Furthermore, histamine causes a contraction of the cells of thevascular endothelium and increases the vascular permeability, thereby allowing higher-molecular substances
to escape into the tissue.
Like several other mediators, histamine does not exclusively mediate various clinical symptoms ofanaphylaxis but also induces a series of effects which are directed towards a termination of the anaphylacticreaction. Histamine may inhibit the release of lysosomal enzymes from polymorphonuclear leukocytes, thedegranulation of mast cells and basophiles and the production of complement components throughmononuclear phagocytes. Furthermore, histamine can activate suppressor T cells and, thus, may inhibit theproduction of IgE. The biological action of histamine in tissue is guaranteed by three different surface
receptors, i.e. H1, H2 and H3 receptors.
Heparinized whole blood samples are incubated with different concentrations of the suspected allergen.
Release of histamine will occur upon stimulation of basophilic granulocytes depending on their sensibility to
the allergen. The released histamine in the supernatant is subsequently determined using a specific plasma
immunoassay, the Histamine ELISA purchased in connection with this kit. This histamine value is related to
the 100% control (= Total Histamine) and the blank value (= Spontaneous Release).
1.2 Background
Please refer to the instructions for use of the Histamine ELISA, chapter 1.2.
2. Procedural cautions, guidelines, warnings and limitations
Please refer to the instructions for use of the Histamine ELISA, chapter 2.
3. Storage and stability
Store the unopened reagents at 2 - 8 C until expiration date. Do not use components beyond the expiry
date indicated on the kit labels. Once opened the reagents are stable for 1 month when stored at 2 – 8 °C.
Once the resealable pouch has been opened, care should be taken to close it tightly with desiccant again.
4. Materials
4.1 Contents of the kit
BA E-1145

Anti-IgE-Antiserum - Concentrated

Contents:

Rabbit Anti-Human IgE antibody

Volume:1 x 25 µl/tube

BA E-1126

Release Buffer – Ready to Use

Contents:

Buffer with physiological pH

Volume:1 x 50 ml/vial, orange cap

4.2 Additional materials and equipment required but not provided in the kit- Calibrated precision pipettes to dispense volumes between 5 – 500 µl- Microtiter plate washing device (manual, semi-automated or automated)- ELISA reader capable of reading absorbance at 450 nm and if possible 620 - 650 nm- Centrifuge capable of at least 3.000 x g- Microtiter plate shaker (shaking amplitude 3 mm; approx. 600 rpm)
- Absorbent material (paper towel)- Temperature controlled water bath (37 °C and 90 °C) or similar heating device- Histamine ELISA, Vortex mixer, water (deionized, distilled, or ultra-plus)

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5. Sample collection and storageHeparinized Whole Blood
24 h before drawing blood samples the patient should not ingest any allergy causing drugs, antihistaminics,
oral corticosteroids and substances which block H2 receptors.
The Histamine Release is performed with heparinized whole blood. That means whole blood is collected into
a tube (e.g. MonovetteTM or VacuetteTM) according to the manufacturer’s instructions that contains heparinas anti-coagulant.
The samples can be stored for up to 24 hours at room temperature. Please do not keep the samples
refrigerated, this will lead to clotting of the leucocytes. Avoid direct sunlight.
The following quantities of whole blood are needed:
 40 µl
for the determination of total histamine
 200 µl
for the spontaneous release 200 µl
for positive control with anti- IgE
 200 µl
for each individual allergen concentration
Example: for a histamine release determination with 4 allergens in 3 different concentrations one would
need approx. 2.5 ml of heparinized blood.
Storage:

20 - 25 °C

Keep away from heat or direct sun light.

Stability:

24 h

Avoid cooling. At 2 - 8 °C, the leucocytes will clot.

Storage of Supernatants: see chapter 6.4
6. Test procedure
6.1 Preparation of reagents
Anti-IgE-Antiserum Concentrate:
Prepare the working solution of the Anti-IgE-Antiserum by adding 5 µl of the Anti-IgE-Antiserum
Concentrate to 5 ml of Release Buffer.
Storage of diluted Anti-IgE-Antiserum:
2 – 8 °C
- 20 °C
Stability of diluted Anti-IgE-Antiserum:
3d1 month
6.2 Release of the allergen induced histamine
Allergen dilutions
Starting with a stock solution of 1 mg allergen per ml water (deionized, distilled, or ultra-pure), a set of 10x
dilutions is prepared according to the following pipetting scheme (example for 3 dilution steps):
No.
Dilution
Allergene solution
Release-Buffer
110-150 µl stock- solution
450 µl
210-250 µl solution No. 1450 µl
310-350 µl solution No. 2450 µl
6.3 Histamine Release protocol (example for 3 dilution steps)
For each allergen tested with a patient the following pipetting scheme has to be followed:
Dilution
Allergen Heparinized
Release Buffer
dilutionwhole blood
10-1200 µl
200 µl10-2200 µl
200 µl10-3200 µl
200 µlSpontaneous - Release
200 µl200 µlPositive - Control
200 µlAnti-IgE
Antiserum

200 µl

6.4 Pipetting scheme Releaseallergen induced
release
Release-Buffer
allergene dilution
anti-IgE
Heparinized Whole Blood

positivecontrol

spontaneous
release200 µltotal Histamine
360 µl200 µl200 µl200 µl200 µl200 µlmix carefully
incubate for 60 min at 37 °C

40 µl
mix carefully
incubate for 10 min
at 90 °C

Incubate for 10 min in an ice bath
Centrifuge for 10 min at 700 x g (brake switched-off)Take 50 µl for the Acylation
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Storage of Supernatants

2 – 8 °C

-20 °C

1d1 week

6.5 Acylation
The Acylation has to be done with the Histamine ELISA test kit, starting with Chapter 6 Test procedure ofthe test instructions.
6.6 Histamine ELISA
The quantification of the Histamine has to be done with the Histamine ELISA test kit.
7. Calculation of results
Histamine
0.12 - 50 ng/ml
108 – 450 nmol/ml

Measuring range

The histamine concentrations of the samples from the release test can be read directly from the standard
curve of the Histamine ELISA.
The Histamine ELISA is a competitive assay. This means: the OD-values are decreasing with increasing
concentrations of the analyte. OD-values found below the standard curve correspond to high concentrationsof the analyte in the sample and have to be reported as being positive.
The results for the total histamine have to be multiplied by factor 5.
The histamine concentration from spontaneous release has to be subtracted from the allergen-inducedhistamine.
Expected reference values
It is strongly recommended that each laboratory should determine its own reference values.
Total Histamine< 60 ng/mlSpontaneous Release< 5 % of total histamine value

Positive Control> 5 % of total histamine value

Allergen Induced Release> 5 % of total histamine value

7.1 Quality control
The valid confidence limits for the kit controls are listed in the QC-Report included in the kit.
8. Assay characteristics
Please refer to the instructions for use of the Histamine ELISA, chapter 8.
9. References/Literature
Please refer to the instructions for use of the Histamine ELISA, chapter 9.

For updated literature or any other information please contact your local supplier.
Symbols:

Version: 13.0-r

Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContentCaution

Catalogue
numberEffective: 2018-07-26

Contains sufficient for<n> tests

For research use
only!
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