INSTRUCTION FOR USE

Histamine ELISA Kit

Enzyme immunoassay for the detection of histamine
(Cat.nr. HU0030203)
Histamine ELISA (Cat.nr. HU0030203) is a kit prepared acylation reagent: histamine is converted into N-
for an immunoenzymatic assay for the detection of acylhistamine.
histamine. The kit contains the procedure and the The assay is performed in plastic microwells which have
materials sufficient for 96 determinations including been coated with histamine. Derivatized standard/control
standards. solutions, samples and the anti-histamine antibody are
For result evaluation a microtiter plate or a strip added to the microwells.
photometer is required (manual or automatic ELISA During the first incubation free N-acylhistamine molecules
reader). and histamine coated on the solid phase compete for the
anti-histamine antibodies binding sites. Any unbound
Type of samples that can be analyzed (matrices) molecule is removed in a washing step.
Fresh fish, fish meal, sausage, cheese, milk, wine and During the second incubation, the antibody anti-histamine
champagne. bound on the solid phase is detected by the addition of an
enzyme conjugate. After the incubation a second washing
Sample preparation step is performed.
- Fish meal: extraction with water, centrifugation, During the third incubation the enzyme activity is
dilution, acylation determined by addition of a fixed amount of a
- Fish, sausage, cheese: grinding, extraction with chromogenic substrate. The enzyme converts the
water, centrifugation, dilution, acylation colourless chromogen into a blue product. The addition of
- Milk: precipitation, acidification, centrifugation, the stop reagent leads to a colour change from blue to
acylation yellow. The absorbance is measured by a microplate
- Wine: dilution, acylation reader at 450 nm. The colour development is inversely
proportional to the histamine concentration in the
Assay time: 90 minutes (sample preparation not standard/control solution/sample.
included).
2. PROVIDED REAGENTS
Detection limit
- Fish meal: 100 ppm Acylation plate: 96 non-coated wells (12 strips x 8 wells).
- Fish, sausage, cheese: 2-2,5 ppm Microtiter plate: 96 wells (12 strips x 8 wells, breakable in
- Milk: 0.1 ppm single wells), coated with histamine.
- Wine: dilution, 0.25 ppm Histamine Std: 6 plastic vials, each containing 4 ml of the
following concentrations of histamine: 0 ng/ml; 0.5 ng/ml;
1.5 ng/ml; 5 ng/ml; 15 ng/ml; 50 ng/ml. Coloured caps.
Specificity 1-Control Histamine: 1 plastic vial containing 4 ml. Green
cap.
Compound Cross-reactivity % 2-Control Histamine: 1 plastic vial containing 4 ml. Red
Histamine 100 cap.
Antibody anti-histamine: 1 plastic bottle containing 12 ml.
3-methyl-histamine 0.01 Blue cap.
Tyramine < 0.001 Enzyme conjugate: 1 plastic bottle containing 12 ml. Red
cap.
L-phenylalanine < 0.001 Acylation Buffer: 1 plastic bottle containing 22 ml. Brown
L-histidine < 0.001 cap.
Acylation Reagent: 1 plastic vial containing 3 ml. Green
L-tyrosine < 0.001 cap.
Tryptamine < 0.001 Washing buffer 50X: 1 plastic bottle containing 20 ml.
Violet cap.
5-hydroxy-indole-acetic acid < 0.001 Developing solution: 1 dark plastic bottle containing 12
Serotonin < 0.001 ml. Black cap.
Stop solution: 1 plastic bottle containing 12 ml. Gray cap.
1. TEST PRINCIPLE 3. REQUIRED BUT NOT PROVIDED MATERIALS
In a non-coated microplate of acylation, standard/control - Distilled water.
solutions, and samples are derivatized by use of an - Hydrochloric acid (HCl) 0.1 N (milk).

IFUHU0030203 _v01_EN Eurofins Technologies Hungary Kft., Fóti út 56., 1047 Budapest, Hungary 1/4
09 NOV 2020 E-mail: technologies.hu@eurofins.com
Website: www.eurofins-technologies.com
- Precipitator (milk). 6. SAMPLES PREPARATION
Equipment
- Balance (fish, sausage, cheese). 6.1 Fish meal
- Blender (fresh fish, sausage, cheese) 1) Weigh 1 g of homogenized sample and add 200 ml
- Disposable 2 ml polyropylene centrifuge tubes. of distilled water.
- Disposable 15 ml polypropylene tubes (fish sausage, 2) Stir for 15 minutes.
cheese, wine). 3) Transfer 1 ml of the suspension into a 2 ml tube.
- Disposable 100 ml polypropylene or glass tubes. 4) Centrifuge for 10 minutes at 2000xg at room
- Vortex (milk). temperature (RT = 18 - 25 oC).
- Microcentrifuge (fish, sausage, cheese, milk). 5) Dilute 20 μl of the supernatant with 20 ml of distilled
- Optional: Microplate shaker. water.
- 2-20 μl, micropipettes, tips optional for fish) 6) Use 100 μl of the extract for the acylation step.
- 20-200 μl, micropipettes, tips. ATTENTION: Do not use any glassware.
- 200-1000 μl, micropipettes, tips. 7) The dilution factor is 200000.
- 50-200 μl, multichannel micropipette, tips.
- Microplate reader, filter 450 nm. 6.2 Fresh fish, sausage, cheese
1) Homogenize the sample.
2) Weigh 10 g of homogenized sample and add 90 ml
4. WARNING AND PRECAUTIONS FOR THE USERS of distilled water.
3) Blend for 1-2 minutes.
- The test is for in vitro diagnostic use only. 4) Transfer 1 ml of the suspension into a 2 ml tube.
- Some reagents contain solutions that may be 5) Centrifuge for 10 minutes at 2000xg at room
identified as dangerous substance by the Regulation temperature (RT= 18 - 25 oC).
(EC) N° 1272/2008. Please refer to Material Safety 6) Remove lipid layer.
Data Sheet available on both the Eurofins 7) Dilute 20 μl of the supernatant with 10 ml of distilled
Technologies and Eurofins Tecna (tecna.eurofins- water.
technologies.com) web site. 8) Use 100 μl of the extract for the acylation step.
- Handle the reagents with caution, avoiding contact ATTENTION: Do not use any glassware.
with skin, eyes and mucous membranes. 9) The dilution factor is 5000.
An alternative sample preparation for fresh fish is
5. HANDLING AND STORAGE INSTRUCTIONS available upon request, limit of detection is 2 ppm.
For more information, please, contact the Technical
- Store the kit at +2/+8 °C and never freeze. Assistance at Support.ET.trieste@eurofins.com
- Bring all reagents to room temperature before
use (at least 1 hour). ATTENTION: Do not unseal 6.3 Milk
the microplate until it reaches the room temperature. 1) Dispense 10 l of sample into a 2 ml tube and add 50
- Reseal the unused strips of the microtiter plate in the μl of precipitator.
bag together with the desiccant bag provided. 2) Vortex mix.
- Return all reagents to +2/+8 °C immediately after use. 3) Incubate for 5 minutes.
- Do not use components after the expiration date. 4) Add 2 ml of 0,1 N hydrochloric acid (HCl).
- Do not intermix components from different kit lots. 5) Centrifuge for 5 minutes at 3000xg at room
- Do not use photocopies of the instruction booklet. temperature (RT= 18 - 25 oC).
Follow the original instruction booklet that is included 6) Remove the lipid layer.
with the kit. 7) Use 100 μl of the extract for the acylation step.
- Do not change the assay procedure, in particular: ATTENTION: Do not use any glassware.
- do not prolong the incubation times, 8) The dilution factor is 200.
- do not incubate the plate at temperatures higher
than 25°C
- Use accurate and precise micropipettes with suitable 6.4 Wine, champagne
tips for dispensing. 1) Dilute 20 μl of the supernatant with 10 ml of distilled
- Once started, complete all the steps without water.
interruption. 2) Use 100 μl of the extract for the acylation step.
- The reproducibility of ELISA results depends largely ATTENTION: Do not use any glassware.
upon the efficiency and uniformity of microwells 3) The dilution factor is 500.
washing; always keep to the described procedure.
- Use a single disposable tip for each standard solution 7. WORKING SOLUTIONS PREPARATION
and sample to avoid cross-contamination.
- Do not allow tips to contact the liquid already in the Histamine Standards: ready to use.
microwells. Histamine Control 1: ready to use.
- Avoid exposure to direct light during all incubations. It Histamine Control 2: ready to use.
is recommended to cover the microtiter plate without Anti-histamine antibody: ready to use.
using plate sealers. Enzyme conjugate: ready to use.

IFUHU0030203 _v01_EN Eurofins Technologies Hungary Kft., Fóti út 56., 1047 Budapest, Hungary 2/4
09 NOV 2020 E-mail: technologies.hu@eurofins.com
Website: www.eurofins-technologies.com
Acylation buffer: ready to use.
Acylation reagent: ready to use. ATTENTION: In
presence of crystals, bring the solution at room
temperature and stir in order to solve them completely
The solution must be completely liquid and clear before
use
Washing buffer: dilute the concentrate 1:50 (1+49) with
distilled water. ATTENTION: In presence of crystals,
bring the solution at room temperature and stir in order to
solve them completely.
Developing solution: ready to use. The solution is light
sensitive and must be stored away from direct light.
Stop solution: ready to use. ATTENTION: Handle with
care; in case of contact flush immediately with plenty of
water.
8. ASSAY PROCEDURE
1) Predispose an assay layout, recording the
standards/controls and samples positions, taking
into account that all have to be run in duplicate.
2) Acylation
ATTENTION: Cover the not-used wells with adhesive
tape. Do not reuse wells.
Add in the acylation plate:
- 100 μl of each standard/control/sample into the wells;
- 25 μl of Acylation Reagent into the wells, using a
multichannel pipette;
- 200 μl of Acylation Buffer into the wells, using a
multichannel pipette.
- Shake the plate gently with rotatory motion for a few
seconds.
- Incubate for 15 minutes at room temperature.
ATTENTION: In case of use of a microplate shaker,
incubate 15 minutes at room temperature at approx. 600
rpm (with a shaking amplitude of 3 mm).
3) First incubation
Maintaining the same assay layout and using the
multichannel micropipette, add in the microtiter
plate coated with histamine:
- 25 μl of each acylated standard/control/sample
drawing from the acylation plate;
- 100 μl of anti-histamine antibody
- Shake the plate gently with rotatory motion for a few
seconds.
- Incubate for 40 minutes at room temperature.
ATTENTION: In case of use of a microplate shaker,
incubate 30 minutes at room temperature at approx. 600
rpm (with a shaking amplitude of 3 mm).
4) Washing sequence:
- Pour the liquid out from the wells.
- Fill completely all the wells with working wash
solution using a squeeze bottle. Pour the liquid out
from the wells.
- Repeat the washing sequence three (3) times.
- Remove the remaining droplets by tapping the
microplate upside down vigorously against
absorbent paper.
Do not allow the wells to dry out.
5) Second incubation
- Using a multichannel pipet, add to the wells 100 µl of
the enzyme conjugate solution.
IFUHU0030203 _v01_EN Eurofins Technologies Hungary Kft., Fóti út 56., 1047 Budapest, Hungary
09 NOV 2020 E-mail: technologies.hu@eurofins.com
Website: www.eurofins-technologies.com
- Shake the plate gently with rotatory motion for few
seconds and cover it with the cover.
- Incubate for 20 minutes at room temperature.
ATTENTION: In case of use of a microplate shaker,
incubate 10 minutes at room temperature at approx.
600 rpm (with a shaking amplitude of 3 mm).
6) Repeat step 4.
7) Developing
- Using the multichannel micropipette, add 100 µl
of developing solution to each well.
- Mix thoroughly with rotatory motion for few
seconds
- Incubate for 15 minutes at room temperature;
protect the plate from direct light.
ATTENTION: In case of use of a microplate shaker,
incubate 15 minutes at room temperature at approx. 600
rpm (with a shaking amplitude of 3 mm).
8) Using a multichannel pipet, add 100µl, of stop
solution to each well and mix thoroughly with rotatory
motion for few seconds.
9) Measure the absorbance at 450 nm.
10) Read within 60 minutes.
9. CALCULATION OF RESULTS
- Calculate the mean absorbance of each control,
standard and sample.
- Divide the mean absorbance value of each standard
and sample by the mean absorbance of the standard
0 and multiply by 100; the standard 0 is thus made
equal to 100% and all the other absorbance values
are expressed as percentages:
Standard (or sample) absorbance B
× 100 = (%)
Standard 0 (B0 ) absorbance B0
- Enter the B/ B0 values calculated for each standard in
a semi-logarithmic system of coordinates and draw
the standard curve.
- Interpolate the B/ B0 value of each sample to the
corresponding concentration from the calibration
curve. For dilution applications multiply this
concentration for the dilution factor, as reported in
chapter 6.
Please note: For results calculation, Excel spreadsheets
are available on Eurofins Tecna website tecna.eurofins-
technologies.com and can be downloaded directly from
the bottom of the product page.
3/4
10. CALIBRATION CURVE EXAMPLE 13. LIABILITY
100
Samples evaluated as positive using the kit have to be re-
90 tested with a confirmation method.
80 Eurofins Technologies Hungary shall not be liable for any
damages to the customer caused by the improper use of
70 the kit and for any action undertaken as a consequence
of results.
60
B/B0 %

Eurofins Technologies Hungary shall not be liable for the

50 unsafe use of the kit out of the current European safety
regulations.
40
30
20
10
0
0.1 1 10 100 1000
ng/ml

11. EVALUATION OF RESULTS

After processing the results, it is necessary to verify the

assay performance. The verification is performed by
comparison of obtained data with those given in kit
specifications (see chapter 12).
If the values are outside the specifications given, then the
results of the test are not assured, therefore the histamine
concentration levels in the samples may not be valid.
In these cases it is advised to check the expiry date of the
kit, the wavelength at which the reading was performed,
as well as the procedure followed.
If operation errors are not identified as cause, contact our
technical assistance.
WARNING: Kit replacement will only be possible in case
of return. The kit must be stored in its integral version at
+2/+8°C.

12. KIT SPECIFICATIONS

12.1 Assay specification

Mean B0 absorbance > 0.7 OD450nm

Std/Ctrl duplicates mean C.V. < 10 %

Control 1 3 ng/ml (+40%)

Control 2 10 ng/ml (+40%)

12.2 Assay performance

Matrix Recovery (%)

Fish 95 - 133

Milk 95 - 146

Wine 87 - 107

IFUHU0030203 _v01_EN Eurofins Technologies Hungary Kft., Fóti út 56., 1047 Budapest, Hungary 4/4
09 NOV 2020 E-mail: technologies.hu@eurofins.com
Website: www.eurofins-technologies.com