rev: May 8, 2007

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Biogenic Amine Assays for Pharmaceutical and Specialty Research

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The following Elisa kits are distributed by RDI Division of Fitzgerald Industries Intl for in vitro research use only-not for use in or on humans or animals, not for use in diagnostics.

Histamine  (in foodstuffs) ELISA  cat#RDI-RE59211  $625.00/1 kit   $594.003+


 Enzyme Immunoassay for the Quantitative Determination of Histamine in foods

 Catalogue No   : RE 59211                           
 Product group  : Allergy                       Determinations : 12 x 8      
 Porduct name   : Histamine in food             ELISA   96
 Method         : ELISA                                                       
 Incubation time: 2h, 15min                                                   
 Standard curve : 0.8µg/l -  300µg/l                                          
 Sample/Prep.   : 50µl                                                        
 Isotope/Substr.: TMB, 450nm                                                  


Histamine, a heterocyclic primary amine, is produced and metabolized by humans, animals, plants and microorganisms during normal metabolism. Throughout the past years, histamine was not only of interest in the chemical and nutritional field, but has also gained increased importance in medicine and pharmacology. Histamine is mainly found in food which was subject to microbiological and biochemical deterioration due to processing, ripening or storage. Higher concentrations of biogenic amines may be found in food containing protein produced in microbiotic processes as well as in microbiotically deteriorated proteins like fish. The presence of histamine is, therefore, a criterion when controlling the quality of food like fish, meat, etc. Histamine in food is normally harmless since humans have several control functions concerning endogenous biogenic amines as well as exogenous amines which are either ingested or of bacterial origin. If high concentrations of histamine are involved, these control functions may not be sufficient. In this case, the amines may lead to health problems. Therefore, critical histamine concentrations in food have been defined by legal authorities which have to be followed by suppliers and manufacturers of food products

Contents of the Kit

1. Assay Buffer 1 bottle 50 ml, concentrate, phosphate buffer containing Tween, dilute 1 : 20 with bidist. water before use. 2. Acylation Reagent 1 vial 1.5 ml, ready for use, contains dimethylformamide. 3. Indicator Buffer 1 vial 6 ml, ready for use, Tris buffer containing phenol red (colour change < pH 7.5). 4. Microtiter Strips 12 strips 8 wells per strip; coated with goat-anti-rabbit antiserum. 5. Histamine Antiserum 1 vial 6 ml, ready for use, antiserum from rabbit in phosphate buffer, blue coloured. 6. Standard A - F 6 vials each 0.5 ml, ready for use, for the determination of Histamine in food extracts. Standard A B C D E F µg/l 0 3.7 11 33 100 300 7. Controls 1 and 2 2 vials 1.0 ml each, 100 mg/kg and 200 mg/kg, the first dilution is 1:10 in trichlor acetic acid, the subsequent dilution has to be carried out in 0.1 M HCl. 8. Enzyme-Conjugate 1 vial 100 µl, concentrate, Histamine conjugated with peroxidase, dilute 1:100 with Assay Buffer. 9. Substrate Solution-Concentrate 1 vial 1 ml, concentrate, Tetramethylbenzidine (TMB) with stabilizers. 10. Substrate Buffer 1 vial 30 ml, ready for use, Hydrogenperoxide in citrate buffer with stabilizers. Store protected from light! 11. Stop Solution 1 vial 15 ml, ready for use, contains 1 M sulfuric acid. Corrosive! 12. Adhesive Foil 3 pieces

Principle of the Test

The HISTAMINE ELISA kit provides materials for the quantitative measurement of acylated histamine in food extracts. The sample preparation, i.e. acylation of histamine to N-acylhistamine, is part of the sample dilution and is achieved by incubation of the respective sample with the "Acylation Reagent". The assay procedure follows the basic principle of the competitive ELISA: the competition between a peroxidase-conjugated and a non-conjugated antigen for a fixed number of antibody-binding sites (rabbit-anti-histamine). The peroxidase-conjugated antigen-antibody complexes bind to the wells of the microtiter strips which are coated with a goat-anti-rabbit antibody. Unbound antigen is then removed by washing. After the substrate reaction, the optical density is measured at 450 nm. The amount of complexes bound to the plate and the optical density is inversely proportional to the analyte concentration of the sample. Quantification of unknowns is achieved by comparing the enzymatic activity of unknowns with a response curve prepared by using known standards.

Test Procedure

SHORT INSTRUCTION 1. Pipet 50 µl of acylated standards, acylated controls and acylated samples into the appropriate wells. 2. Pipet 50 µl of Peroxidase Conjugate and subsequently 50 µl of Antiserum into each well, shake the plate carefully. 3. Seal the plate with the adhesive foil and incubate 2 hours at room temperature (20-25°C) on a shaker (500 U/min). 4. Wash each well three times with Assay Buffer, 250 µl per well (it is recommended to use washer). Remove Assay Buffer carefully. Invert plate and remove any remaining liquid by tapping on clean blotting paper. Note: The correct performance of the washing procedure is mandatory for the sensitivity and precision of this assay. 5. Pipet 200 µl of freshly prepared TMB Substrate Solution into each well. 6. Seal with the adhesive foil and incubate for 15 min. at room temperature on an orbital shaker (500 1/min). 7. Stop the substrate reaction by adding 100 µl of TMB Stop Solution into each well. 8. Mix contents briefly by shaking the plate gently. 9. Read the optical density at 450 nm (reference wave length 600 - 650 nm) with a microtiter plate reader within 60 min. after stopping. Preparation of Reagents The contents of the kit can be divided into three separate runs. The volumes stated below are for one test procedure with 4 strips (32 determinations). If a larger number of strips is to be used, the volumes have to be changed accordingly. 1. Controls Dilute the Controls 1:10 with 10% trichlor acetic acid. The subsequent dilution with 0.1 M HCl is according to the dilution of sample extracts. 2. Assay Buffer 15 ml of the Assay Buffer (concentrated) have to be diluted 1 : 20 with bidist. water to make up 300 ml. The Assay Buffer is now ready for use. Store at 2-8°C. Crystals may precipitate at 2-8°C. These crystals resolve at room temperature. 3. Peroxidase Conjugate Dilute 20 µl of the Peroxidase Conjugate concentrate with 2.0 ml of diluted Assay Buffer. Use only once! 4. TMB Substrate Solution (should be prepared during the test procedure) Dilute 300 µl of TMB with 9 ml of TMB Substrate Buffer and mix. Prepare just before use and use only once! Sample Collection and Preparation The sample has to be prepared according to national or other legal instructions. According to the German legal requirements, the sample should be treated as follows: The sample has to be cut into small pieces in order to receive an average for the analytical determination. 10.0 g ñ 0.1 g of this sliced material are homogenized with 90 ml of trichlor acetic acid for 1-2 min. at 8,0 equivalent. The homogenate should be filtered with filter paper. Sample Preparation A. Qualitative Determination (Screening method using cut-off value) According to the Council Directive of the European Communities of 1991, the quantitative determination of histamine in fish must be carried out. However, it is possible to apply the assay for qualitative examinations with cut-off values of 100 ppm or 200 ppm as well as quantitative examinations of histamine in fish and a large variety of other foodstuffs. For the qualitative examination, a control included in the kit of 100 mg/kg or 200 mg/kg is used (prediluted 1:10 with trichlor acetic acid). The samples and the prediluted control of either 100 mg/kg or 200 mg/kg is diluted 1:250 in 0.1 M HCl. Pipet 50 µl of diluted control and diluted extracts into glass test tubes. Add 50 µl of Indicator Buffer to each tube. Add 10 µl of Acylation Reagent to each tube, vortex mix, and incubate for 30 min. at room temperature. Add 1 ml of ready for use Assay Buffer and vortex. Withdraw 50 µl of aliquots for ELISA! After measurement in the assay, calculation has to be performed by comparing the extinctions of the control and the samples. In case of smaller values of the sample than measured for the control, the histamine content exceeds the cut-off value, demanded. B. Quantitative Determination 1. Sample preparation for fish According to the value expected of the sample, the trichlor acetic acid extract has to be diluted in 0.1 M HCl. Content 0 - 50 mg/kg Dilution 1:50 Content 5 - 100 mg/kg: Dilution 1:100 Content 10 - 300 mg/kg Dilution 1:250 Content 300-2,500 mg/kg Dilution 1:1,000 Content > 2,500 Dilution 1:5,000 For the quantitative calculation, the dilution factor of 1:10 of the extraction procedure has to be taken into account. 2. Sample Preparation of wine and other foods In the quantitative determination, the dilution with 0.1 M HCl has to be selected in that way that the sample concentration can be measured within the standard range. In case of liquid, clear samples with very low histamine contents, a dilution of 1:20 in 0.1 M HCl is recommended. Example: Red wine from Greece, alcohol content of 11 %, histamine value: 0.82 mg/l Dilution 1:5 1:10 1:20 1:50 1:100 1:200 Value in the standard curve µg/l 211 86.5 42.4 16.1 7.5 4.1 Histamine content mg/l 1.06 0.87 0.85 0.81 0.75 0.82 3. Influence of sample matrix When using this protocol for the determination of fish, we could not see any influence of the sample matrix. However, when working with many different foodstuffs, it is sometimes useful to study the influence of the sample matrix for obtaining correct results.In order to measure the influence of the sample matrix, the sample has to be determined twice (recovery experiment). The first sample has to be added before extraction by a definitive amount of histamine from a histamine stock solution. After multiplication with the dilution factor and subtraction of the value obtained without adding of the stock solution, the value obtained of the added sample has to be the expected value. A deviation of (10 % points at an influence of the matrix. Preparation of the stock solution 10 mg/ml Add 5 ml of 0.1 M HCl to 50 mg of histamine free base. This stock solution is stable for 1 year when stored at 4°C. Expected value Sample dilution Adding of Expected the sample after extraction stock solution value in mg/kg 0 - 50 mg/kg 1:50 25 µl 25 5 - 100 mg/kg 1:100 75 µl 75 10 - 300 mg/kg 1:250 200 µl 200 > 300 mg/kg 1:1000 500 µl 500 4. Acylation Pipet 50 µl standards and diluted extracts into glass test tubes. Add 50 µl of Indicator Buffer to each tube. Add 10 µl of Acylation Reagent to each tube, vortex and incubate for 30 min. at room temperature. Add 1 ml of ready for use Assay Buffer and vortex.

Performance Characteristics

1. Cross reactivity The cross-reactivity of the anti-N-acylhistamine antiserum has been measured against various compounds which were processed like samples Compound Cross-reactivity (%) N-acylated histamine 100.0 N-methyl-histamine 2.42 N-acetyl-histamine 0.61 1-methyl-histamine 0.03 L-Histidine 0.005 5-Hydroxy-3-indole acetic acid <0.005 L-á-Imidazole-lactic acid <0.005 1-Methyl-4-imidazole-acetic acid <0.005 Imidazole-4-acetic acid <0.005 5-Hydroxy-tryptamine <0.005 2. Sensitivity The lowest detectable level that can be distinguished from the zero standard is 0.8 µg/l (defined as 3 x standard deviation of the zero calibrator). This number has to be multiplied by the sample dilution factor. 3. Precision Intra-Assay-Variation (in µg/l) Mean value Standard deviation VK (%) n 11.7 0.94 8.1 10 47.3 1.76 3.7 10 Inter-Assay-Variation (in µg/l) Mean value Standard deviation VK (%) n 11.3 0.87 7.75 10 46.6 3.45 7.39 10 4. Dilution Enriched extracts were diluted with 0.1 M HCl before sample preparation and assayed. The calculated results are shown in mg/kg (ppm): Sample 1/25 1/50 1/100 1/200 1/400 1/800 1/1600 Tuna in juice 20.6 21.4 21.5 - - - - Tuna with vegetables - 120 125 121 118 - - fresh herring - - 251 245 278 278 256 Sardine in oil 24.4 27.3 20.8 - - - - 1/10 1/20 1/40 1/80 1/160 1/320 1/640 fresh herring 27.0 29.0 31.9 33.6 32.0 27.8 30.1 5. Recovery Several samples were enriched with histamine and subsequently measured. Sample Basic value Amount added Amount measured Recovery % mg/kg mg/kg mg/kg Sardine in oil 6.52 100 111 105 Tuna in brew 8.16 10 18.05 99 Tuna in oil 0.85 20 21.6 104 Tuna in oil 0.85 100 97 97 Tuna with vegetable 7.58 100 106 98

Expected Values

Expected Values (Border lines) Fish: According to the legal requirements of the EC guideline, fish or fish products should not be used as food for humans when histamine concentrations exceed 200 mg/kg. Sauerkraut: > 5 mg/kg
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