Instructions for use

Histamine ELISABA E-1000R

Histamine ELISA
1. Introduction
1.1 Intended use and principle of the test
Enzyme Immunoassay for the quantitative determination of Histamine in plasma and urine.
In combination with the supplementary kit Histamine Release (for details contact your local supplier), the assay
can be used for the measurement of histamine release in heparinized whole blood.
In the first part of the procedure, Histamine is quantitatively acylated. The subsequent competitive ELISA kit
uses the microtiter plate format. The antigen is bound to the solid phase of the microtiter plate. The acylated
analyte concentrations in the standards, controls and samples and the solid phase bound analyte compete
for a fixed number of antibody binding sites. After the system is in equilibrium, free antigen and free
antigen-antibody complexes are removed by washing. The antibody bound to the solid phase is detected byan anti-goat IgG-peroxidase conjugate using TMB as a substrate. The reaction is monitored at 450 nm.
Quantification of unknown samples is achieved by comparing their absorbance with a standard curve
prepared with known standard concentrations.
1.2 Background
Histamine belongs to the biogenic amines and is synthesized by decarboxylation from the amino acidhistidine. It is synthesized by mast cells, basophils, platelets, histaminergic neurons, and enterochromaffine
cells, where it is stored intracellularly in vesicles and released on stimulation.
Histamine acts by binding to its 4 receptors (H1R, H2R, H3R and H4R) on target cells in various tissues. Itcauses smooth muscle cell contraction, vasodilatation, increased vascular permeability and mucus secretion,tachycardia, alterations of blood pressure, and arrhythmias.
In humans, histamine is one of the most important mediators and takes part in the initial phase of ananaphylactic reaction (“immediate type” allergy).
2. Procedural cautions, guidelines, warnings and limitations
2.1 Precautions, guidelines and warnings(1) This kit is intended for professional use only. Users should have a thorough understanding of this protocol
for the successful use of this kit. Only the test instruction provided with the kit is valid and has to be usedto run the assay. Reliable performance will only be attained by strict and careful adherence to theinstructions provided.
(2) The principles of Good Laboratory Practice (GLP) have to be followed.
(3) In order to reduce exposure to potentially harmful substances, wear lab coats, disposable protective
gloves and protective glasses where necessary.
(4) All kit reagents and specimens should be brought to room temperature and mixed gently but thoroughly
before use. Avoid repeated freezing and thawing of reagents and specimens.
(5) For dilution or reconstitution purposes, use deionized, distilled, or ultra-pure water.
(6) The microplate contains snap-off strips. Unused wells must be stored at 2 °C to 8 °C in the sealed foil
pouch with desiccant and used in the frame provided.
(7) Duplicate determination of sample is highly recommended to be able to identify potential pipetting errors.
(8) Once the test has been started, all steps should be completed without interruption. Make sure that therequired reagents, materials and devices are prepared ready at the appropriate time.
(9) Incubation times do influence the results. All wells should be handled in the same order and timeintervals.
(10) To avoid cross-contamination of reagents, use new disposable pipette tips for dispensing each reagent,sample, standard and control.
(11) A standard curve must be established for each run.
(12) The controls should be included in each run and fall within established confidence limits. The confidence
limits are listed in the QC-Report provided with the kit.
(13) Do not mix kit components with different lot numbers within a test and do not use reagents beyond
expiry date as shown on the kit labels.
(14) Avoid contact with Stop Solution containing 0.25 M H2SO4. It may cause skin irritation and burns. In caseof contact with eyes or skin, rinse off immediately with water.
(15) TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with anabundant volume of water and skin with soap and abundant water. Wash contaminated objects before
reusing them.
(16) For information on hazardous substances included in the kit please refer to Safety Data Sheet (SDS). TheSafety Data Sheet for this product is made available directly on the website of the manufacturer or uponrequest.
(17) Kit reagents must be regarded as hazardous waste and disposed of according to national regulations.
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2.2 Limitations
Any inappropriate handling of samples or modification of this test might influence the results.
2.2.1 Interfering substances
PlasmaSamples containing precipitates or fibrin strands might cause inaccurate results. Hemolytic samples (up to1 mg/ml hemoglobin), icteric samples (up to 50 mg/dl bilirubin) and lipemic samples (up to 1600 mg/dl
triglycerides) have no influence on the assay results.
24-hour urine
Please note the sample preparation! If the percentage of the final concentration of acid is too high, this willlead to incorrect results for the urine samples.
2.2.2 Drug interferences
There are no known substances (drugs) which ingestion interferes with the measurement of histamine levelin the sample.
2.2.3 High-Dose-Hook effectNo hook effect was observed in this test.
3. Storage and stability
Store the unopened reagents at 2 - 8 C until expiration date. Do not use components beyond the expirydate 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 desiccantagain.
4. Materials
4.1 Contents of the kit
Adhesive Foil - Ready to use

BA D-0090
Contents:

Adhesive Foils in a resealable pouch

Volume:1 x 4 foils

BA E-0030

Wash Buffer Concentrate - Concentrated 50x

Contents:

Buffer with a non-ionic detergent and physiological pH

Volume:1 x 20 ml/vial, light purple cap

BA D-0024
Contents:BA E-1040

Reaction Plate - Ready to use
1 x 96 well plate, empty in a resealable pouch
Enzyme Conjugate - Ready to use

Contents:

Donkey anti-goat immunoglobulins conjugated with peroxidase

Volume:1 x 12 ml/vial, red cap

BA E-0055

Substrate - Ready to use

Contents:

Chromogenic substrate containing tetramethylbenzidine, substrate buffer and hydrogen
peroxide

Volume:1 x 12 ml/black vial, black cap

BA E-0080

Stop Solution - Ready to use

Contents:

0.25 M sulfuric acid

Volume:1 x 12 ml/vial, light grey cap

Hazards
identification:
H290 May be corrosive to metals.
BA E-1031

Histamine Microtiter Strips - Ready to use

Contents:

1 x 96 well (12x8) antigen precoated microwell plate in a resealable pouch withdesiccant.

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BA E-1010

Histamine Antiserum - Ready to use

Contents:

Goat Anti- Histamine antibody, blue coloured

Volume:1 x 12 ml/vial, blue cap

Standards and Controls - Ready to use
Cat. no.

Component

Colour/Cap

Concentration
ng/mlConcentration
nmol/lVolume /Vial

BA E-1001

white004 ml

BA E-1002

light yellow

0.5

4.5

4 ml

BA E-1003

orange1.513.54 ml

BA E-1004

dark blue

545

4 ml

BA E-1005

light grey

15135

4 ml

BA E-1006

blacklight green

50450

4 ml

BA E-1051
BA E-1052

dark red

Refer to QC-Report for expected value andacceptable range!

Conversion:

Histamine (ng/ml) x 9 = Histamine (nmol/l)Contents:

Acidic buffer spiked with defined quantity of Histamine

4 ml
4 ml

Acylation Buffer - Ready to use

BA E-1011
Contents:

TRIS-buffer containing a non-mercury preservative

Volume:1 x 4 ml/vial, pink cap
Acylation Reagent - Lyophilized

BA E-1012
Contents:

Lyophilized acylation reagent

Volume:2 vials, purple cap
Acylation Solvent - Ready to use

BA E-0085
Contents:

Organic solvent

Volume:1 x 5 ml/vial, brown cap

Hazards
identification:
H225 Highly flammable liquid and vapour.
4.2 Additional materials and equipment required but not provided in the kit







Calibrated precision pipettes to dispense volumes between 10 - 300 μl; 1.25 ml
Microtiter plate washing device (manual, semi-automated or automated)
ELISA reader capable of reading absorbance at 450 nm and if possible 620 - 650 nmMicrotiter plate shaker (shaking amplitude 3 mm; approx. 600 rpm)
Absorbent material (paper towel)
Water (deionized, distilled, or ultra-pure)
Vortex mixer

5. Sample collection and storage
In general the repeated freezing and thawing of samples should be avoided.
PlasmaWhole blood should be collected by venipuncture into centrifuge tubes containing EDTA as anti-coagulant(e.g. MonovetteTM or VacuetteTM for plasma) and centrifuged according to manufacturer’s instructions atroom temperature immediately after collection.
Haemolytic, icteric and lipemic samples should not be used for the assay.
Storage: up to 6 hours at 2 - 8 °C, for longer period (up to 6 month) at -20 °C.

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UrineSpontaneous urine or 24-hour urine, collected in a bottle containing 10 - 15 ml of 6 M HCl, can be used.
If 24-hour urine is used please record the total volume of the collected urine.
Storage: up to 6 hours at 2 - 8 °C, for longer periods (up to 6 month) at -20 °C.
Avoid exposure to direct sunlight.
Whole Blood
The release of histamine is performed with heparinized whole blood. For further information please refer tothe instructions for use of the add-on kit Histamine Release (for details contact your local supplier).
6. Test procedure
Allow all reagents and samples to reach room temperature and mix thoroughly by gentle inversion before
use. Duplicate determinations are recommended. It is recommended to number the strips of the microwellplate before usage to avoid any mix-up.
The binding of the antibody and the enzyme conjugates and the activity of the enzyme used aretemperature dependent, and the absorbance values may vary if a thermostat is not used. The higher thetemperature, the higher the absorption values will be. Varying incubation times have similar influences on
the absorbance. The optimal temperature during the Enzyme Immunoassay is between 20 - 25 °C.
6.1 Preparation of reagents
Wash Buffer
Dilute the 20 ml Wash Buffer Concentrate with water (deionized, distilled or ultra-pure) to a final volume of1000 ml.
Storage: 1 month at 2 – 8 °C
Acylation Solution
Reconstitute each vial of the Acylation Reagent (BA E-1012) with 2 ml Acylation Solvent (BA E-0085). Please
make sure that it is completely dissolved before use.
If more than 2 ml are needed, pool the contents of the individual vials and mix thoroughly.
Storage: 1 month at 2 – 8 °C
Histamine Microtiter Strips
In rare cases residues of the blocking and stabilizing reagent can be seen in the wells as small, white dots orlines. These residues do not influence the quality of the product.
6.2 Sample preparation and acylation
1.

Pipette 25 µl of standards, controls and plasma samples, 10 µl of urine samples, or 50 µl ofsupernatant from the release test* into the respective wells of the Reaction Plate.

2.

Add 25 µl of Acylation Buffer to all wells.

3.

Add 25 µl of Acylation Solution (refer to 6.1) to all wells.

4.

Incubate for 45 min at RT (20 - 25 °C) on a shaker (approx. 600 rpm).

5.

Add 100 µl of water (deionized, distilled, or ultra-pure) to all wells.

6.

Incubate for 15 min at RT (20 - 25 °C) on a shaker (approx. 600 rpm).
Take 25 µl of the prepared standards, controls and samples for the Histamine ELISA* For the release test the Histamine Release supplementary kit (for details contact your local supplier) has tobe used.

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6.3 Histamine ELISA
1.

Pipette 25 µl of the acylated standards, controls and samples into the appropriate wells of theHistamine Microtiter Strips.

2.

Pipette 100 µl of the Histamine Antiserum into all wells and cover plate with Adhesive Foil.

3.

Incubate for 3 h at RT (20 - 25 °C) on a shaker (approx. 600 rpm).
Alternatively: shake the Histamine Microtiter Strips briefly by hand and incubate for 20 - 25 h at
2 - 8 °C.

4.

Remove the foil. Discard or aspirate the contents of the wells. Wash the plate 4 x by adding 300 µl ofWash Buffer, discarding the content and blotting dry each time by tapping the inverted plate on
absorbent material.

5.

Pipette 100 µl of the Enzyme Conjugate into all wells.

6.

Incubate for 30 min at RT (20 - 25 °C) on a shaker (approx. 600 rpm).

7.

Discard or aspirate the contents of the wells. Wash the plate 4 x by adding 300 µl of Wash Buffer,discarding the content and blotting dry each time by tapping the inverted plate on absorbentmaterial.

8.

Pipette 100 µl of the Substrate into all wells and incubate for 20 - 30 min at RT (20 - 25 °C) on ashaker (approx. 600 rpm). Avoid exposure to direct sunlight!

9.

Add 100 µl of the Stop Solution to each well and shake the microtiter plate to ensure a homogeneous
distribution of the solution.

10.

Read the absorbance of the solution in the wells within 10 minutes, using a microplate reader set to
450 nm (if available a reference wavelength between 620 nm and 650 nm is recommended).

7. Calculation of results

Measuring range

Histamine
0.12 - 50 ng/ml
0.3 – 125 ng/ml

PlasmaUrine

The standard curve is obtained by plotting the absorbance readings (calculate the mean absorbance) of thestandards (linear, y-axis) against the corresponding standard concentrations (logarithmic, x-axis).
Use a non-linear regression for curve fitting (e.g. spline, 4- parameter, akima).
This assay 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 highconcentrations of the analyte in the sample and have to be reported as being positive.
Plasma samples and controls:
The concentrations of the plasma samples and the controls can be read directly from the standard curve.
Urine samples:
The read concentrations of histamine in urine have to be multiplied by 2.5
The total amount of Histamine excreted in urine during 24 h is calculated as following:µg/24h = µg/l x l/24h
Conversion
Histamine (ng/ml) x 9 = Histamine (nmol/l)
Expected reference values
It is strongly recommended that each laboratory should determine its own reference values.
Plasma0.2 – 1.0 ng/ml

24 hour-urine
5 – 53 µg/d

Spontaneous urine
10 – 50 µg/g creatinine

7.1 Quality control
The confidence limits of the kit controls are indicated in the QC-Report.

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7.2 Typical standard curve
Example, do not use for calculation!

8. Assay characteristics
Histamine

Analytical Sensitivity
Limit of Detection

Sensitivity Plasma
Sensitivity Urine

0.18 ng/ml
0.22 ng/ml
Histamine

Analytical Sensitivity
Limit of Quantitation

Sensitivity Plasma
Sensitivity Urine

0.29 ng/ml
0.96 ng/ml
Cross Reactivity (%)

SubstanceHistamine
1000.10.01< 0.001< 0.001< 0.001< 0.001< 0.001< 0.001

Histamine
3-Methyl-Histamine
Tyramine
L-Phenylalanine
L-Histidine
L-Tyrosine
Tryptamine
5-Hydroxy-Indole-Acetic Acid
Serotonin

Analytical Specificity(Cross Reactivity)

Precision
Intra-Assay
SampleRange (ng/ml)

CV (%)

SampleRange(ng/ml)

CV (%)

Histamine1

9.7 ± 1.5

15

Histamine1

1.2 ± 0.2

16Urine218.6 ± 2.4

13

Plasma25.0 ± 0.6

12Sample

Range (ng/ml)

CV (%)

SampleRange (ng/ml)

CV (%)

18.2 ± 0.91110.29 ± 0.07

252

12.8 ± 1.7

132

3.1 ± 0.283

42.2 ± 6.0

143

5.5 ± 0.8

15Inter-Assay

Histamine
Urine

Linearity

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Urine
PlasmaHistamineRange
4.3 – 69.8 ng/ml
0.7 – 7.9 ng/ml

Effective: 2019-06-28

PlasmaSerial dilution up to
1:21
1:10

Range (%)
90 - 124
85 -106

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Recovery

Urine
PlasmaMethod comparison
versus other RIA

Range

Mean (%)

Range (%)

14.0 – 105 ng/ml
0.4 – 6.4 ng/ml

10984

101 - 11978 - 89

Urine
PlasmaRIA = 0.9 ELISA – 3.1

r = 0.97; n = 29

RIA = 1.0 ELISA – 0.4

r = 0.99; n = 49

9. References/Literature(1) Yagci et al. TCTP/HRF pathway and angiogenesis: A feasible intercourse in chronic lymphocytic
leukaemia. Leukemia Research, 37:665-670 (2013)(2) Coulson et al. Paracetamol (acetaminophen) attenuates in vitro mast cell and peripheral bloodmononucleocyte cell histamine release induced by N-acetylcysteine. Clinical Toxicology, 48(2):111-114(2010)(3) Rovere et al. Histamine and Selenium in Lung Cancer. Anticancer Research, 26: 2937-2942 (2006)(4) Miyazaki et al. Macrophage inflammatory protein-1alpha as a costimulatory signal for mast cell-mediatedimmediate hypersensitivity rections. J Clin Invest, 115(2): 434-442 (2005)(5) Brown, S. G. et al. Ant venom immunotherapy: a double-blind, placeb-controlled, crossover trial. Lancet,361(9362): 1001-1006 (2003)(6) Matsumoto, J. Adverse effects of praziquantel treatment of Schistosoma japonicum infection:
involvement of host anaphylactic reactions induced by parasite antigen release. Int J Parasitol, 32(4):461-471 (2002)(7) Matsumoto, J. and H. Matsuda. Mast-cell-dependent histamine release after praziquantel treatmet of
Schistosoma japonicum infection: implications for chemotherapy-related adverse effects. Parasitol Res,88(10): 888-893(8) Hsu, C. H. et al. Immunoprophylaxis of allergen-induced immunoglobulin E synthesis and airway
hyperresponsiveness in vivoby genetic immunization. Nat Med, 2(5): 540-544(9) Demoly P. et al. Predictive capacity of histamine release for the diagnosis of drug allergy. Allergy, 54(5):500-506 (1999)

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

Version: 17.0-r

Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContentCaution

Catalogue
numberEffective: 2019-06-28

Contains sufficient for<n> tests

For research use
only!

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