Histamine - Cayman Chemical ?· Endocrinology / Metabolism Infl ammation ... Histamine Enzyme Immunoassay…

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  • Histamine

  • Fabriqu en FranceMade in France

    For research laboratory use onlyNot for human diagnostic use

    This assay has been developed & validatedby Bertin Pharma

    European patent # 89 139 552U.S. patent # 50 47 330

    Bertin Pharma also markets pre-analytical products, EIA kits, antibodies, CYP450s & biochemicals for:

    Cardiology / Hypertension

    Diabetes / Obesity

    Endocrinology / Metabolism

    Infl ammation



    Nitric Oxide

    Oncology / Apoptosis

    Oxidative injury

    Cell signaling

    Drug metabolism

    Do not hesitate to contact our after-sales services for further information at bioreagent@bertinpharma.com

    HistamineEnzyme Immunoassay kit

    A05890.96 wells

    Version: 0116Ref. #A11890

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    96 wellsStorage: -20C

    Expiry date: stated on the package

    DesignationColour of

    capItem #

    Quantityper kit


    Histamine precoated 96-well Strip Plate

    blister with zip

    A08890.1 ea 1Ready to use after thawing

    Histamine Tracer green A04890.100 dtn 1 Lyophilised

    Histamine Standard transparent A06890.1 ea 2 Liquid

    Derivatization Reagentwhite with

    septumA15890.1 ea 2 Powder

    Derivatization Buffer grey/pink A16890.1 ea 1 Liquid

    Histamine EIA Buffer grey/blue A07890.1 ea 1 Lyophilised

    Wash Buffer grey/alu A17000.1 ea 1 Liquid

    Tween 20 transparent A12000.1 ea 1 Liquid

    Histamine Quality Control transparent A10890.1 ea 2 Liuqid

    Ellmans reagentblack with septum

    A09000_50. 100 dtn

    2 Lyophilised

    Instruction Booklet - A11890 1 -

    Well cover sheet - - 1 -

    Each kit contains suffi cient reagents for 96 wells. This allows for the construction of one standard curve in duplicate and the assay of 35 samples in duplicate.

    Precaution for use 6

    Principle of the assay 7

    Materials and equipment required 9

    Sample collection and preparation 10

    Reagent preparation 12

    Assay procedure 16

    Typical results 25

    Assay validation and characteristics 28

    Assay troubleshooting 33

    Bibliography 34

    Table of contents

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    Principle of the assay

    This Enzyme Immunoassay (EIA) is based on the competition between unlabelled derivatized Histamine and acetylcholinesterase (AChE) linked to Histamine (Tracer) for limited specifi c mouse anti-Histamine antibody sites.

    As a former step of this assay, Histamine is derivatized to increase the affi nity of Histamine to the antibody and consequently increase the sensitivity of the assay.

    Tracer and Standard (or sample) are incubated in wells which have been precoated with a mouse anti-Histamine antibody attached to the well. The plate is washed to remove any unbound reagent, and Ellmans Reagent (enzymatic substrate for AChE and chromogen) is added to the wells.

    The AChE tracer acts on the Ellmans Reagent to form a yellow compound that strongly absorbs at 414 nm.

    The intensity of the colour, which is determined by spectrophotometry, is proportional to the amount of tracer bound to the well and is inversely proportional to the amount of free Histamine present in the well during the immunological incubation.

    Precaution for use

    Users are recommended to carefully read all instructions for use before starting work.

    Each time a new pipette tip is used, aspirate a sample or reagent and expel it back into the same vessel. Repeat this operation two or three times before distribution in order to equilibrate the pipette tip.

    > For research laboratory use only > Not for human diagnostic use > Do not pipet liquids by mouth > Do not use kit components beyond the expiration date > Do not eat, drink or smoke in area in which kit reagents are handled

    > Avoid splashing

    The total amount of reagents contains less than 100 g of sodium azide. Flush the drains thoroughly to prevent the production of explosive metal azides.

    Wearing gloves, laboratory coat and glasses is recommended when assaying kit materials and samples.


    Unless otherwise specifi ed, all the experiments are done at room temperature (RT), that is around +20C. Working at +25C or more affects the assay and decreases its effi ciency.

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    Materials and equipment required

    In addition to standard laboratory equipment, the following material is required:

    > Anhydrous N,N-Dimethylformamide (DMF)(*) > Precision micropipettes (20 to 1000 L) > Spectrophotometer plate reader (405 or 414 nm fi lter) > Microplate washer (or wash bottles) > Distilled or deionized water > Polypropylene tubes (no glass tubes)

    (*) Make sure that the bottle of DMF has been opened for

    a short period of time. This point is important to get a good

    derivatization rate.

    Water used to prepare all EIA reagents and buffers must be Ultra Pure, deionized & free from organic contaminants traces.Otherwise, organic contamination can signifi cantly affect the enzymatic activity of the tracer Acetylcholinesterase.Do not use distilled water, HPLC-grade water or sterile water.

    > UltraPure water may be purchased from Bertin Pharma (item #A07001.1L)

    The principle of the assay is summarised below:

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    Sample collection and preparation

    This assay may be used to measure Histamine in samples such as plasma, urine, culture supernatants as well as liquid (e.g. broncho-alveolar lavage fl uids) or solid (brain, nervous tissues) biological samples after extraction. Please refer to the appropriate paragraph for your samples preparation protocol.

    > Blood samplingCollect blood samples in tubes containing EDTA. Centrifuge the samples at 1,600 g for 20 minutes. Collect plasma and keep at -20C until assay. Thaw the sample on the day of the assay, vortex and centrifuge it at 1,600 g for 20 minutes to eliminate the fi brin.

    > PlasmaNo prior extraction procedure is necessary to measure Histamine in plasma samples. If necessary, plasma samples may be diluted in Histamine EIA buffer before derivatization (see below).

    > Urine or culture supernatantsCollect samples in polypropylene tubes. Store the samples at -20C until assay. No prior extraction is necessary to measure Histamine in such samples.

    > Liquid or solid biological samplesFor solid samples, we recommend addition of HClO4 which will precipitate the big proteins, while Histamine will remain in solution. Samples must be extracted at room temperature with 0.1M Perchloric Acid fi nal concentration (10L/mg of tissue).

    Homogenize and then centrifuge at 10,000g for 5 minutes. Histamine is measured in the supernatant, following the protocol for solid samples. Neutralization is performed during derivatization with addition of 20L of NaOH 1.5M.

    Liquid samples can be stored just after collection in polypropylene vials at -20C. Before assaying with #A05890, you have to prepare your samples as below. Filter liquid samples through a 0.22 m fi lter in a tube containing HClO4 1M. So if you fi lter 200L of samples, your tube must contain 20 L HClO4 1M for getting a fi nal HClO4 0.1M. Homogenize and then centrifuge at 10,000g for 5 minutes. Histamine is measured in the supernatant, according to the protocol for liquid samples. Neutralization is performed during derivatization with addition of 20L of NaOH 1.5M (do not add twice NaOH).

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    Reagent preparation

    The coated microtiter plate and reagents are provided ready to use.

    All reagents need to be brought to room temperature (around +20C) prior to the assay

    Histamine EIA Buff er

    Reconstitute the vial #A07890 with 25 mL of UltraPure water. Allow it to stand 5 minutes until completely dissolved and then mix thoroughly by gentle inversion.Stability at 4C: 1 week.

    Histamine Standard

    Open one vial of Standard and dilute it 1:10 by adding 900 L of assay medium directly into the vial. Mix carefully three times with the pipet tip. The obtained solution, called S0, has a concentration of 500 nM.

    The standard preparation depends on the sample to be assayed:

    > for plasma or urine samples, prepare the Standard using the Histamine EIA Buffer,

    > for culture supernatant samples, prepare the Standard using the same culture medium as for the sample,

    > for extracted liquid or solid samples, prepare the Standard in 0.1M HClO4.

    Then take eight polypropylene tubes to prepare the standards S1 to S8. Prepare them as follows:

    > Standard 1 (S1): 100L of S0 + 900 L of assay medium. Vortex.

    > Standards 2 to 8 (S2 to S8): dispense 500L of assay medium in each tube. Add 500L of Standard S1 into the fi rst tube and vortex. Continue this procedure for the other tubes.

    Stability at +4C: 24 hours.

    StandardVolume of Standard

    Volume of Assay Buffer

    Standard concentration nM

    S1 100 L of S0 900 L 50 nM

    S2 500 L of S1 500 L 25 nM

    S3 500 L of S2 500 L 12.5 nM

    S4 500 L of S3 500 L 6.25 nM

    S5 500 L of S4 500 L 3.13 nM

    S6 500 L of S5 500 L 1.56 nM

    S7 500 L of S6 500 L 0.78 nM

    S8 500 L of S7 500 L 0.39 nM

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    Histamine Quality Control

    In one vial # A10890, add 900 L of assay medium, as for the standard. Then dilute 100 L of QC in 900 L of assay medium. The fi nal concentration of this QC is labelled on t