Instructions for use

Histamine Research ELISA ™

BA E-5800

Histamine Research ELISA
1.

Intended use and principle of the test
Enzyme Immunoassay for the quantitative determination of Histamine in different animal species andbiological fluids.
During the sample preparation 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 standards, controls and samples and the solid phase bound analyte compete for a fixed numberof antiserum binding sites. After the system is in equilibrium, free antigen and free antigen-antiserum
complexes are removed by washing. The antibody bound to the solid phase is detected by an anti-rabbit
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 reference curve
prepared with known standard concentrations.

2.

Procedural Cautions, 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 used
to run the assay. Reliable performance will only be attained by strict and careful adherence to theinstructions provided.
(2) Reagents of this kit which contain human serum or plasma have been tested and confirmed negative for
HIV I/II, HBsAg and HCV by approved procedures. All reagents, however, should be treated as potential
biohazards in use and for disposal.
(3) The principles of Good Laboratory Practice (GLP) have to be followed.
(4) In order to reduce exposure to potentially harmful substances, wear lab coats, disposable latex gloves
and protective glasses where necessary.
(5) 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.
(6) For dilution or reconstitution purposes, use deionized, distilled, or ultra-pure water.
(7) 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.
(8) Duplicate determination of sample is highly recommended to be able to identify potential pipetting errors.
(9) 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.
(10) Incubation times do influence the results. All wells should be handled in the same order and timeintervals.
(11) To avoid cross-contamination of reagents, use new disposable pipette tips for dispensing each reagent,
sample, standard and control.
(12) A standard curve must be established for each run.
(13) The controls should be included in each run and fall within established confidence limits. The confidence
limits are listed in the QC-Report.
(14) 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.
(15) 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.
(16) Some reagents contain sodium azide (NaN3) as preservatives. In case of contact with eyes or skin, rinse
off immediately with water. NaN3 may react with lead and copper plumbing to form explosive metalazides. When disposing reagents, flush with a large volume of water to avoid azide build-up.
(17) 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.
(18) For information on hazardous substances included in the kit please refer to Material Safety Data Sheet(MSDS). The Material Safety Data Sheet for this product is made available directly on the website of themanufacturer or upon request.
(19) Kit reagents must be regarded as hazardous waste and disposed according to national regulations.
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 withdesiccant again.

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Effective: 2015-02-12

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4.

Materials

4.1

Content of the kit
BA D-0090

Adhesive Foil - Ready to use

Content:

Adhesive Foils in a resealable pouch

Volume:1 x 4 foils

BA E-0030

Wash Buffer Concentrate - Concentrated 50x

Content:

Buffer with a non-ionic detergent and physiological pH

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

BA D-0024
Content:

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

BA E-5840
Content:

Rabbit anti-goat immunoglobulins conjugated with peroxidase

Volume:1 x 12 ml/vial, red cap
Substrate - Ready to use

BA E-0055
Content:

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

Content:

0.25 M sulfuric acid

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

BA E-1031

Histamine Microtiter Strips - Ready to use

Content:

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

BA E-1010

Histamine Antiserum - Ready to use

Content:

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

orange

1.5

13.5

4 ml

BA E-1004

dark blue

5454 ml

BA E-1005

light grey

15135

4 ml

BA E-1006

50450

4 ml

BA E-1051

blacklight green

BA E-1052

dark red

Refer to QC-Report for expected value andacceptable range!

Conversion:

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

Acidic buffer spiked with defined quantity of Histamine

BA E-1011

TRIS-buffer containing a non-mercury preservative

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

Content:

Lyophilized acylation reagent

Volume:4 vials, purple cap

Version: 11.0

4 ml

Acylation Buffer - Ready to use

Content:

BA E-1012

4 ml

Effective: 2015-02-12

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BA E-0085

Acylation Solvent - Ready to use

Content:

Ethanol

Volume:1 x 10 ml/vial, brown cap

Hazards
identification:
H225 Highly flammable liquid and vapour.
BA E-0041

4.2

Diluent - Ready to use

Content:

Acidic buffer with non-mercury preservatives

Volume:1 x 22 ml/vial, white cap

Additional materials and equipment required but not provided in the kit− Calibrated precision pipettes to dispense volumes between 10 – 300 µl; 2 ml
− Microtiter plate washing device (manual, semi-automated or automated)− ELISA reader capable of reading absorbance at 450 nm and if possible 620 - 650 nm− 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
The kit was validated for EDTA –plasma from different animal species. In principle other sample types
than plasma are also suitable but have to be tested in advance. For cell culture supernatants the use ofthe ELISA BA E-1700 (Histamine ELISA Fast Track) is recommended. For more details please contact yourlocal supplier or the manufacturer directly.
In general haemolytic and lipemic samples should not be used with this assay.
Storage of plasma samples: up to 6 hours at 2 – 8 °C; for longer periods (up to 6 months) at -20 °C.
Repeated freezing and thawing should be avoided.

6.

Test procedure
The following protocol for rat plasma samples should be used as a guideline and is suitable for animal
species where high Histamine concentrations are expected. In such cases, the samples have to beprediluted with the Diluent (BA E-0041). In cases, where low concentrations are expected, no sample
predilution will be necessary.
The following concentrations were detected with the Histamine Research ELISA in different animalspecies:
Animal species

Concentration (ng/ml)

Mouse

22.9

Rat

20Cat

1.1

Dog

0.3

Horse0.6

Allow all reagents and samples to reach room temperature and and mix thoroughly by gentle inversion
before use. Duplicate determinations are recommended.
The binding of the antisera and of the enzyme conjugate and the activity of the enzyme are temperaturedependent, and the absorption values may vary if a thermostat is not used. The higher the temperature,
the higher the extinction values will be. Varying incubation times will have similar influences on theabsorption. The optimal temperature during the Enzyme Immunoassay is between 20 – 25 °C.
In case of overflow, read the absorbance of the solution in the wells within 10 minutes, using a
microplate reader set to 405 nm

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6.1

Preparation of reagents
Wash Buffer
Dilute the 20 ml Wash Buffer Concentrate with water (deionized, distilled, or ultra-pure) to a final volumeof 1000 ml.
Storage: 1 month at 2 – 8 °C
Acylation Solution
The Acylation Solution has to be prepared freshly prior to the assay:
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 content of the individual vials and mix thoroughly.
Discard after use!

6.2

Sample predilution

1.

Pipette 10 µl of the sample into an Eppendorf tube or similar device.

2.

Add 200 µl of Diluent.

3.

Vortex for 1 min at RT (20 – 25 °C).

4.

25 µl of the prediluted sample are needed for the subsequent acylation step.

6.3

Sample preparation and acylation

1.

Pipette 25 µl of standards, controls and plasma samples 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).

7.

Take 25 µl of the prepared standards, controls and samples for the Histamine ELISA

6.4

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.

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

4.

Remove the foil. Discard or aspirate the content 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
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 content 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 absorbent
material.

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).

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7.

Calculation of results
The standard curve is obtained by plotting the absorbance readings (calculate the mean absorbance) of
the standards (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).
Controls
The concentrations of the controls can be read directly from the standard curve.
Samples
For this example (rat plasma) a sample pre-dilution of 1:21 was used. Therefore the concentrations readfrom the standard curve have to be multiplied by 21.
In general, if the samples have been pre-diluted, the concentrations read from the standard curve haveto be multiplied by the dilution factor to get the final results. If no pre-dilution was necessary the final
result could be read directly from the standard curve.

7.1

Quality control
It is recommended to use control samples according to national regulations. Use controls at both normal
and pathological levels. The kit, or other commercially available, controls should fall within establishedconfidence limits. The confidence limits of the kit controls are printed on the QC-Report.

7.2

Typical standard curve
Example, do not use for calculation!
Calibration Curve Histamine Research ELISA

10090

Extinction values (%)

80706050403020
1000,1110100

Histamineconcentration (ng/mL)

8.

Assay characteristics
Histamine

Analytical Sensitivity(Limit of Detection)

0.2 ng/ml

Substance

Analytical Specificity(Cross Reactivity)

Version: 11.0

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

Effective: 2015-02-12

Cross Reactivity (%)
Histamine
1000.10.01< 0.001< 0.001< 0.001< 0.001< 0.001< 0.001

6/7Recovery and Linearity for different animal species (plasma samples):Species

RecoveryLinearity

Mouse

Mean Recovery: 97%
Range Recovery: 86 – 104 %Mean Recovery: 86%
Range Recovery: 75 - 93 %Mean Recovery: 82%
Range Recovery: 70 – 93 %Mean Recovery: 82%
Range Recovery: 70 – 93 %Mean Recovery: 90%
Range Recovery: 72 – 94 %Mean Linearity: 115%
Range Linearity: 94 - 134 %Mean Linearity: 115%
Range Linearity: 88 - 131 %Mean Linearity: 115%
Range Linearity: 94-134 %Mean Linearity: 115%
Range Linearity: 94-134 %Mean Linearity: 115%
Range Linearity: 94-134 %RatCatDog
HorseFor updated literature or any other information please contact your local supplier.
Symbols:

Version: 11.0

Storagetemperature

Manufacturer

Contains sufficient for<n> tests

Expiry date

Batch code

For in-vitro diagnostic
use only!

Consult instructions
for useContentCE labelledCaution

Catalogue
numberFor research use
only!

Effective: 2015-02-12

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