P.L. Rogers

W.F. Staruszkiewicz

 

ABSTRACT

Six commercially available test kits designed to determine histamine in canned and raw fish were selected to compare the performance of each kit.  The histamine concentration in seven tuna and eight mahimahi test samples as determined by each test kit was compared to the histamine concentrations as determined by the official AOAC method 977.13.  All the kits tested were acceptable for use as screening tests for histamine and were able to distinguish between products that contained less than 50 ppm and those that contained more than 50 ppm.  [Article copies available for a fee from the Haworth Document Delivery Service: 1-800-342-9678.  E-mail address: ,getinfo@haworthpressinc.com> Website: http://www.haworthpressinc.com]

 

Keywords: Histamine, test kits, fish, decomposition, ELISA

 

P. L. Rogers, Ph.D., is Chemist and W. F. Staruszkiewicz is Research Chemist, US Food & Drug Administration, 200 C St. S.W., Washington, D.C. 20204.

 

The results reported in this study do not constitute an endorsement by FDA of any of the products tested.         

 

*Source: Journal of Aquatic Food Product Technology, Vol. 9 (2) 2000 p. 5-17.
Copyright © 2000 by The Haworth Press, Inc. - Posted with permission from The Haworth Press, Inc.

                                                                          

INTRODUCTION

     Histamine testing is a possible control strategy that can be used by seafood processors in their HACCP program to address the hazard of scombrotoxin formation (Fish and Fisheries Products Hazards and Controls Guide, 1998).  Histamine is a product of  decomposition caused by the growth of certain bacteria in seafood.  The amount of the amine that forms is a function of bacterial species, the temperature and time of exposure, and may exceed 1000 ppm.  Fish containing high levels of histamine have been associated with many instances of poisoning commonly referred to as “scombroid poisoning,” a major health problem for consumers (Seafood Safety, 1991).  Scombrotoxic fish usually contains levels of histamine in excess of 200 ppm but such fish may be randomly dispersed within a lot.  For large fish, histamine is found at variable levels even within individual fish (Frank, et al, 1981; Lerke, et al, 1978).  Quality control measures designed to minimize the occurrence of scombrotoxic fish require the determination of histamine levels in the range of approximately 10 to 200 ppm.  Good quality fish contain less than 10 ppm histamine, a level of 30 ppm indicates significant deterioration, and 50 ppm is considered to be evidence of definite decomposition (Compliance Policy Guideline # 540.525, Federal Register, 1995).  The defect action level (DAL), the level at which regulatory actions are taken, for histamine is 50 ppm.

 

     A wide variety of procedures for the determination of histamine have been published.  An official AOAC fluorometric method (977.13) has been used routinely for 20 years as a basis for taking regulatory action on fish containing histamine (Staruszkiewicz, et al, 1977).  A recent modification, changing  the analyte extraction solvent to 75% methanol from 100% methanol, was approved in a second collaborative study so that other biogenic amines such as cadaverine and putrescine could be determined from the same extract (Rogers and Staruszkiewicz, 1997).  Other analytical procedures  have been used primarily to determine histamine in research applications.  Thin-layer chromatography is inexpensive and permits many analyses on one plate, but is only semiquantitative  (Lieber & Taylor, 1978). HPLC with either precolumn or postcolumn derivatization can be used to determine several amines at the same time and can be automated.  Precolumn derivatization involves liquid/liquid extractions which are a limiting factor in quantitation (Meitz & Karmas, 1978; Hui & Taylor, 1983).  In postcolumn methods the pumps are a frequent source of variation particularly if buffers are part of the mobile phase (Veciania-Noques, 1995).  HPLC with oncolumn fluorescence derivatization eliminates much of the analyte isolation, but some background problems remain (Saito, 1992).  Flow injection analysis does not require pre-treatment of the extract.  It does however, require careful selection of reagent concentrations and careful control of four pumps to maintain specificity for the histamine derivative (Hungerford, 1990).  Use of an oxygen-based sensor electrode requires a trichloracetic acid extraction and an enzyme which is not commercially available (Ohashi, 1994).  Another published enzyme-based method is too tedious for routine analysis because it requires two perchloric acid extractions and a 2-hr incubation period (Lerke, 1983; Lopez & Sabater, 1993). A dip stick test developed for histamine is rapid, but is only applicable over a very narrow range (Hall, et al, 1995).  Enzyme-linked immunosorbant assay (ELISA) tests are very specific and sensitive, but also require a trichloroacetic acid analyte extraction, derivatization of the analyte extract, and extended incubation periods (Serrar, et al, 1995).

 

     The limitations of the laboratory-based methods, suggest a need for a simple, rapid test for histamine that can be used in a commercial setting.  Several companies have produced test kits which have been advertised as rapid, easy to use, and capable of providing accurate results at low cost, but little comparative data is available.  Six commercially available test kits marketed by three companies to determine histamine in canned and raw fish were selected to evaluate the performance of  each kit.  The kits are classified as either qualitative or quantitative in the range of one to 500 ppm.  Five of the kits are based on an enzyme-linked  immunosorbent assay (ELISA) that measures the direct competition between the histamine to be assayed and enzyme-labeled histamine conjugate. The sixth kit is based on a chemical colormetric analysis. 

 

MATERIALS AND METHODS

Test Kits

     “Histamarine Enzyme Immunoassay Kit” from Immunotech (Coulter Corp., OPA Locka, FL) is designed to be a quantitative measure of histamine.  In the method, histamine extracted from a fish test portion is determined by use of high affinity specific monoclonal antibodies coated to the wells in a microtiter plate.  There is competitive binding between the acylated histamine in the extract of the test portion and the enzyme conjugate.  The bound enzyme conjugate catalyzes a color change in a reaction with the substrate.  The specific reaction and color product produced are not specified by the manufacturer.  The intensity of the color is inversely proportional to the concentration of histamine in the test solution. 

 

     Immuno-diagnostic Reagents (IDR), Vista, CA, manufactures two different test kits:  one for the quantitative measure of histamine (K1-HTM) and the other for a qualitative measure (K3-HTM).  Both are based on the principle of an enzyme immunoassay.  The histamine that is being assayed and the histamine-alkaline phosphatase conjugate compete  for binding to the antibodies coated onto microtiter wells.  The color change induced by the phosphatase hydrolysis of a chromogenic substrate is inversely proportional to the concentration of histamine in the test solution.

           

     Neogen Corporation, Lansing, MI, manufactures the  ALERT® kit and the Veratox  histamine kit.  Both kits are direct competitive enzyme-linked immunosorbent assays (ELISA).   The histamine in the extract competes with the histamine conjugate for antibody binding.  The color change induced by the action of the bound conjugate on an enzyme substrate is inversely proportional to the concentration of histamine in the test solution.  The ALERT® kit is for qualitative measurement of histamine and the Veratox histamine kit is a modification of the ALERT® test kit for quantitative results.

 

     The Agrimeter II test kit from Neogen is no longer available, but the results are included because the chemistry is unique and may be used at some time.  The Agrimeter II Kit is a screening test for  the presence or absence of histamine in raw, frozen, or canned tuna.  The test utilizes ion exchange chromatography for the purification and isolation of histamine followed by reaction with a diazo dye.    

 

     The test kits were chosen to represent a variety of types of assays available in kit form.  There are at least three other kits available, each based on enzyme immunoassay, which were not tested.  They include RIDASCREEN from R-Biopharm, Inc., Marshall, MI, Transia Tube Histamine from GENE-TRAK Systems, Hopkinton, MA, and Histamine ELISA from Immuno Biological Laboratories, Hamburg, Germany.  The kits were evaluated by comparing the histamine concentration determined by each test kit to the histamine concentration using official AOAC method 977.13. 

 

Test Samples

     The fish used in this comparison and the histamine concentration in each unit, determined by AOAC method 977.13,  are described in Table 1.  Fifteen samples ( seven tuna and eight mahimahi) were chosen to provide a range of histamine concentrations to use in comparing the test kits.  Nine fish contained histamine levels less than 50 ppm, two  contained histamine greater than 50 ppm but less than 100 ppm, and four contained histamine greater than 100 ppm.  The histamine in these samples occurred naturally.  Samples were analyzed by standard FDA sensory procedures for the presence of decomposition odors (U.S. Food and Drug Administration, 1976). Sensory analysis tests for the presence of odors and is not a quantitative procedure.  Raw frozen steaks were prepared for analysis by thawing in a spray of cold water.  Canned tuna was prepared by draining and discarding the liquid packing media.  Odors were evaluated by breaking apart and warming the product in the analyst’s hands.  Samples which contained definite decomposition odors were classified as failed, while samples not containing odors were classified as passable. 

 

AOAC Method

    Histamine was determined by AOAC Official Method 977.13 (Rogers & Staruszkiewicz, 1997).  The method involves an extraction with 75% methanol, removal of interfering compounds by an ion exchange column, derivatization with o-phthaldialdehyde (OPT), and measurement of fluorescence.  The fluorescence intensity of tests and standards was measured using a Turner Fluorimeter Model 112 at an excitation wavelength of 350 nm and an emission wavelength of 444 nm.

 

Methods

 

     The manufacturer’s instructions, included with each test kit, were followed as directed.

 

     Histamarine Kit – Immunotech.  The test portion was extracted with water (one g fish/8g water) and an aliquot of the  supernatant was mixed with an acylation reagent. No incubation time was necessary for this step.  The acylated analyte  and enzyme conjugate were added to the microtiter plate wells  and incubated for 30 minutes.  The microtiter plate was washed and then  incubated for 30 minutes with the substrate.  After the addition of the stop solution, the plate was read at 405-414 nm. The concentration of histamine was calculated by use of a standard curve obtained with standards supplied with the kit.  The conjugate and the wash solution were diluted to the specified volume before use and the substrate was dissolved and diluted to the specified volume.  Adjustable micropipettes or three fixed-size micropipettes were required.

 

     Immuno-diagnostic Reagents - K1-HTM (quantitative) and K3-HTM (qualitative).  The test portion was extracted with 0.03 N HCl solution and  neutralized with Tris-base. Two filtrations followed.  The extract solution and the conjugate were added to the microtiter plate.  After a 60-minute incubation time (30 minutes for the qualitative test), the wells were rinsed to remove nonbound component.  The bound enzymatic activity was then measured by the addition of a chromogenic substrate (p-nitrophenyl phosphate) which turned yellow after a 30-minute incubation period (10 minutes for the qualitative test).  The quantitative test  measured the solution at 405 nm by using an ELISA plate reader; the qualitative test was measured by a visual comparison of test wells  to the negative and positive calibrator wells.  The intensity of the color developed was inversely proportional to the concentration of histamine in the test sample.  The standard solutions, negative control, a histamine-alp conjugate, and p-nitrophenyl phosphate substrate were all supplied ready to use.  The wash buffer had to be diluted before use.  Three sizes of micropipettes were needed.  The HCl solution and the Tris-base were not supplied, and no instructions other than a specified concentration were given to make these solutions. 

 

     The qualitative measure of histamine was estimated by visual comparison with a single standard histamine solution equivalent to 50 ppm histamine in the fish.  For kit K1-HTM, the quantitative measure of  histamine was calculated on the basis of a standard curve.  The average absorbance of two readings for each of five histamine standards was calculated.  The values of percent inhibition for each standard were then plotted versus their corresponding concentration on log paper.  The histamine concentration of the test sample was determined by interpolation from the graph.  The resulting concentration was multiplied by a variable dilution factor which was determined by the amount of Tris-base necessary to neutralize the HCl. 

 

     ALERT® Test Kit – Neogen.  The test portion was extracted with water (10 g fish/90 ml water), filtered, and diluted with extract buffer.  A standard 100 µl volume was used for the addition and transfers of all solutions.   All needed solutions were supplied and only one dilution was required. The extract and the histamine conjugate were added to a mixing well,  then 0.1 ml of the mixture was added to the antibody well and incubated for 10 minutes. Unbound substances were removed by washing.  An enzyme substrate was added and incubated for 10 minutes.  The substrate turned blue as a result of the action of the bound conjugate.  The color was inversely proportional to the amount of histamine in the test sample.  The color was observed visually or by using a colorimeter and compared to a control equivalent to 50 ppm histamine in the fish.

 

     Veratox Histamine Kit – Neogen.  The analyte isolation and test procedure were the same as the ALERT® test kit.  Five standards were included for a standard curve.  The concentration of histamine in each test sample was interpolated from the standard curve. All reagents were supplied, incubation times were short, and only one volume was needed for all the reagent additions using the Veratox kit.

 

     Agrimeter II Test Kit –Neogen.  The test portion was extracted with 100% methanol and the extract filtered.  The extract  was pretreated by mixing with an ion-exchange resin and filtered.  First the pretreated extract was added dropwise to the “detector” provided with the kit.  This was followed by addition of the color reagent and wash buffer.  The reflectance was measured using the AgriMeter^TM within five minutes.  Four sizes of pipettes were needed.  All solutions, reagents, and most of the containers and filters were provided in the kit.

 

RESULTS AND DISCUSSION

     Sensory classifications and histamine levels of the samples used in this study are given in Table 1.  As many as 5 sensory classes have been used in research studies of some seafood samples (Duggan, 1946), while a 3 class system was developed for regulatory applications on unprocessed products (Barry, 1956).  It is now recognized that a sensory test is not quantitative and determines only the presence of retained decomposition odors which has led to a 2 class system as used for the samples in Table 1.

 

     The relationship between sensory class and histamine level depends upon the type of decomposition present in a seafood and the level of expertise of the sensory analyst.  Training of sensory analysts to recognize odors and standardize their decisions is a complex process which requires formal training by experts and experience on samples of unknown composition.  Training samples are prepared from fresh starting materials as well as commercial products of various qualities (Subhapholsiri, 1995).

 

     Samples taken from shipments of commercial fish do not always exhibit definite odors in all portions especially in canned tuna.  If high temperature spoilage has occurred there may be high levels of histamine in some portions with relatively small amounts of odors (Baranowski, 1990).  A chemical test for histamine provides an improved level of safety but some exceptions have been found and further investigations are continuing (Fish and Fisheries Products Hazards and Controls Guide, 1998).

 

     The histamine values determined on 15 test samples by using the AOAC method and the six test kits are shown in Table 2.  Table 3 summarizes a general comparison of all six test kits.

 

     Histamine results determined by the  “Histamarine Test” in fish containing histamine less than 100 ppm by the AOAC method (nine total) were less than the AOAC value in four tests, higher than the AOAC value in three tests, and equivalent (+/- 1 ppm) to the AOAC value in two tests.  The higher values for histamine were found using the “Histamarine Test” in the four tests containing more than 100 ppm histamine by AOAC and in the two tests containing between 50 ppm and 100 ppm histamine.

 

     By using the qualitative IDR K3-HTM tests, results were reported as less than,  much less than,  equivalent to, or more than 50 ppm.  Fish containing histamine less than 50 ppm by the AOAC method were reported as much less or less than 50 ppm by the IDR K3-HTM test.  Fish containing histamine more than 100 ppm by the AOAC method were reported as greater than 50 ppm by the IDR K3-HTM test kit.  Two fish containing 67.9 and 58.1 ppm histamine by the AOAC method were reported as equivalent to 50 ppm by the IDR K3-HTM test kit. 

 

     In general the histamine values from IDR K1-HTM test kit were higher than the histamine values determined by the AOAC method (12 of 15 test samples).  The histamine values determined by the AOAC method on the three test samples that had lower histamine values from IDR K1-HTM test kit were 0.2 and 2 ppm on two of the tests and 67.9 ppm on the third test.  The K1-HTM result was 60.7 for the third test.  The fish containing 58.1 ppm by AOAC was 95.7 ppm by K1-HTM.

 

     The ALERT® test kit used a scale of -5 to zero to +5 where  zero was equivalent to 50 ppm. Using the ALERT® test, fish containing less than 50 ppm histamine by the AOAC method gave a reading of -5.  Two fish containing 67.9 and 58.1 ppm, respectively, gave a reading of +1, and fish with more than 100 ppm gave a reading of +5. 

 

     Histamine values reported with the Veratox kit agreed within two ppm with the histamine values on six of nine test samples containing 50 ppm or less determined by the AOAC method.  The histamine values determined on the other three of nine test samples were within five to 12 ppm of the AOAC method results.  The histamine results on four test samples with histamine values of more than 100 ppm by the AOAC method were also determined to be >100 ppm by the Veratox kit.  The two test samples containing 67.9 and 58.1 ppm histamine by the AOAC method were reported to contain 64 and 66 ppm respectively using the Veratox kit.  

 

     All the fish that contained less than 30 ppm histamine by the AOAC method gave readings of 30 ppm or less using the Agrimeter II test.  The fish containing 58.1 ppm was reported as 75 ppm when the Agrimeter II test was used, and the fish containing 67.9 ppm was reported as 45 ppm when the Agrimeter II test was used.  All fish containing more than 100 ppm were greater than 100 ppm when the Agrimeter II test was used.

 

     Three additional tuna test portions were analyzed in triplicate by each of the three quantitative test kits.  The average, standard deviation, and the coefficient of variation were calculated on these three test sample.  The reproducibility of the three quantitative test kits is found in Table 4.  The histamine concentrations on the three test samples determined by the AOAC method were 30.0 ppm for test a, 71.7 ppm for test b, and 62.7 ppm for test c.   The coefficient of variation ranged from 1.8% to 10.8% for the Veratox kit, from 4.7% to 8.4% for the K1-HTM kit, and from 17.3% to 24.1% for the Histamarine kit.

 

CONCLUSIONS

     These test kits are marketed as screening tests for histamine.  As 50 ppm histamine is evidence of definite decomposition, the performance of each test kit in determining  histamine at or near the 50 ppm is particularly important.  When used as a screening test, each kit will indicate when a test sample is >50 ppm or < 50 ppm, the defect action level (see Table 3).  In this set of samples none of the passable samples would be rejected and none of the rejected samples would be called acceptable using any of the test kits, with one exception.  Using the Agrimeter II test kit on this set of samples, one of the rejected samples (histamine >50 ppm) would be acceptable.  Accuracy of the determination is a limitation of the Agrimeter test kit (Neogen), the Histamarine test kit (Immunotech), the K1-HTM test kit (IDR) and the K3-HTM test kit (IDR), particularly around the 50 ppm level.

 

     The coefficient of variation of the Varieties kit and of the IDR K1-HTM kit was less than 11 percent.  The coefficient of variation of the Histamarine kit was 2 to 10 times greater than the coefficient of variation of the other two quantitative test kits.

 

     Both the ALERT® and the Veratox kit can accurately measure very low values of histamine, values near 50 ppm histamine, and very high values of histamine.  In addition, the ALERT® or Veratox tests have several practical advantages over the other test kits.  Extraction of the test sample with water is easier and involves fewer steps than extraction with dilute acid.  This also eliminates the variable dilution factor that is necessary when the extract must be neutralized with variable amounts of Tris-base.  Both kits contain all required reagents and plasticware to do the analysis, all volumes are 100 µl (requiring only one pipette), the difference in intensity in blue color is visually easier to distinguish than the difference in intensity in the yellow color, and the results can easily be read visually or with a plate reader. The incubation times are significantly less than those in the other ELISA-based tests which reduces the total analysis time.

 

REFERENCES

 

Baranowski, J.D., Frank, H.A., Brust, H.A., Premarathe, R.J. 1990. Decomposition and histamine content in mahi-mahi (Coryphaena hippurus). Journal of Food Protection. 53:217-222.

 

Barry, H.C., Weeks, J.F., and Duggan, R.E. 1956. Effect of storage on decomposed canned shrimp. JAOAC. 39(3):801-805.

 

Duggan, R.E., Strasburger, L.W. 1946. Indole in shrimp. JAOAC. 29(2):177-188.

 

Federal Register. Aug 3, 1995. Decomposition and histamine--raw, frozen tuna and mahi-mahi; canned tuna; and related Species; 60 (149):39754-30956, CPG 540.525.

 

Fish and Fisheries Products Hazards and Controls Guide, 2nd edition. 1998. Chapter 7-8.

 

Frank, H.A., Yoshinaga, D.H., and Nip, W.K. 1981. Histamine formation and honeycombing during decomposition of skipjack tuna, Katsuwonus pelamis,  at elevated temperatures. Marine Fisheries Review. 43(10):9-14.

 

Hall, M., Eldridge, D.B., Saunder, R.D., Fairclough, D.L, and Bateman, R.C. 1995. A rapid dipstick test for histamine in tuna.  Food Biotechnol. 9 (1 & 2):39-57.

 

Hui, J.Y. and Taylor, S.L. 1983. High pressure liquid chromatographic determination of putrefactive amines in foods.  J. Assoc. Off. Anal. Chem. 66:853-857.

 

Hungerford, J.M., Walker, K.D., Wekell, M.M., LaRose, J.E., and Throm, H.R. 1990. Selective determination of histamine by flow-injection analysis. Anal. Chem. 62:1971-1976.

 

Lerke, P.A., Werner, S.B., Taylor, S.L., and Guthertz, L.S. 1978. Scombroid poisoning. A report of an outbreak.  West J. Med. 129:381-386.

 

Lieber, E.R. and Taylor, S.L. 1978. Thin-layer chromatographic screening methods for histamine in tuna fish.  J. Chromatogr. 153:143-152.

 

Lopez-Sabater, E.I., Rodriguez-Jerez, J.J., Roig-Sagues, A.X., and Mora-Ventura, M.T. 1993. Determination of histamine using an enzymic method.  Food Addit. Cont. 10:593-602.

 

Meitz, J.L., Karmas, E. 1978.  Polyamine and histamine content of rockfish, salmon, lobster, and shrimp as an indicator of decomposition.  JAOAC 61(1):139-145.

 

Ohashi, M., Numura, F., Suzuki, M., Otsuka, M., Adachi, O., and Arakawa, N. 1994.  Oxygen-sensor-based simple assay of histamine in fish using purified amine oxidase.  J. Food Sci. 59:519-522.

 

Lerke, P.A., Porcuna, M.N., and Chin, H.B. 1983.  Screening test for histamine in fish.  J. Food Sci. 48:155-157.

 

Rogers, P.L., and Staruszkiewicz, W.F. 1997. Gas chromatographic method for putrescine and cadaverine in canned tuna and mahimahi and fluorometric method for histamine (minor modification of AOAC official method 977.13): collaborative study. J. AOAC Intl. 80(3):591-602.

 

Saito, K., Horie, M., Nose, N., Nakagomi, K., and Nakazawa, H. 1992.  Determination of polyamines in foods by liquid chromatography with on-column fluorescence derivatization.  Anal. Sci.8:675-680.

 

Seafood Safety. 1991. Farid Ahmed, Ed. Chapter 4:93-96.

 

Serrar, D., Brebant, R., Bruneau, S., Denoyel, G.A. 1995.  The development of a monoclonal antibody-based ELISA for the determination of histamine in food: application to fishery products and comparison with the HPLC assay.  Food Chem. 54:85-91.

 

Staruszkiewicz, W.F., Waldron, E.M., and Bond, J.F. 1977.  Fluorometric determination of histamine in tuna:development of method. JAOAC 60:1125-1131.

 

Subhapholsiri, S. Jan 1955. A nose for quality and How does a nose know. Business Review. 24:36-40; 40-41.

 

U.S. Food and Drug Administration. 1976. Preparation of authentic packs of decomposed seafood for training purposes. Laboratory Information Bulletin. No. 1912, FDA, Washington, D.C.

 

Veciana-Nogues, M.T., Hernandez-Jover, T., Marine-Font, A., and Vidal-Carou, M.C. 1995.  Liquid chromatographic method for determination of biogenic amines in fish and fish products. J. AOAC Intl. 78: 1045-1050.

 

   Table 1.  Characteristics of Test Samples of Fish Used for Comparisons

Sample #	Composition	Sensory Evaluation (pass/fail)	Histamine (ppm) AOAC method
1	water packed yellowfin tuna	P	3.0
2	water packed yellowfin tuna	P	3.0
3	Water packed skipjack tuna	P	2.5
4	Frozen yellowfin tuna steaks	F	67.9
5	Frozen yellowfin tuna steaks	F	58.1
6	Frozen yellowfin tuna steaks	F	190
7	Frozen yellowfin tuna steaks	F	300
8	Frozen mahimahi steaks from regulatory samples	P	0.2
9	Frozen mahimahi steaks from regulatory samples	P	2.0
10	Frozen mahimahi steaks from regulatory samples	P	8.8
11	Frozen mahimahi steaks from regulatory samples	P	2.2
12	Frozen mahimahi steaks from regulatory samples	P	19.7
13	Frozen composites of several decomposed mahimahi steaks	F	300
14	Portion of a mahimahi loin received fresh from Hawaii and then frozen	P	0.9
15	Frozen composites of several decomposed Mahimahi steaks	F	158

 

   Table 2.  Histamine Results (ppm) from Test Kits

Sample #	AOAC Histamine	Neogen Agrimeter II	Neogen ALERT® -5 - 0 - +5 0 equiv. to 50	Neogen Veratox	Immunotech Histamarine	IDR K1-HTM	IDR K3-HTM
1	3.0	10	-5	2.6	1	22.4	<<50
2	3.0	0	-5	2.5	0.1	24.4	<50
3	2.5	4	-5	8.4	0.8	6.4	<<50
4	67.9	45	+1	64	71.7	60.7	=50
5	58.1	75	+1	66	72.6	95.7	=50
6	190	>100	+5	>100	309	323	>50
7	300	>100	+5	>100	372	435	>50
8	0.2	0	-5	5.5	0.9	0	<<50
9	2.0	0	-5	2.5	0.8	1.7	<<50
10	8.8	20	-5	9.4	17.1	17.2	<<50
11	2.2	10	-5	3.6	2.3	5.0	<<50
12	19.7	30	-5	31	43.6	35.6	<<50
13	300	>100	+5	>100	366	455	>50
14	0.9	20	-5	2.6	1.0	2.1	<<50
15	158	>100	+5	>100	191	323	>50

 

 

  Table 3.  General Comparison of Test Kits

	Neogen Agrimeter II	Neogen Veratox	Neogen ALERT®	Immunotec Histamarine	IDR K3-HTM	IDR K1-HTM
Confirms histamine levels <50 ppm	yes	yes	yes	yes	yes	yes
Confirms histamine levels ³50 ppm	5 out of 6 times	yes	yes	yes	yes	yes
Extraction	water	water	water	water	0.03% HCl	0.03 % HCl
filtration	easy	easy	easy	easy	difficult, colloidal suspension	difficult, colloidal suspension
Supplied Reagents	Everything needed including dilution & extraction bottles, filtration and sample tubes.	Everything needed including dilution & extraction bottles, filtration and sample tubes. 2 dilutions required	Everything needed including dilution & extraction bottles, filtration and sample tubes. 1 dilution required	Acylation reagent, conjugate, substrate buffer, stop solution - most have to be reconstituted	Buffer, negative and positive calibrator solutions.	Standards are made
Addition of reagents	dropper bottles, 100 ul pipette for test solution	all volumes 100 ul	all volumes 100 ul	Each step requires a different volume, must have adjustable pipettes	dropper bottles, 50 ul pipette for test solutions	25, 50 and 100 ul pipettes required.
Incubation times	none	10 min wash 10 min stop	10 min wash 10 min stop	30 min wash 30 min stop	30 min wash 10 min stop	60 min wash 30 min stop
Reading Results	Agri-Meter	visual color comparison or read @ 650 nm	visual color comparison or read @ 650 nm	read @ 405 nm	visual color comparison	read @ 405 nm
Stability of Reagents	stable @ 4°C	stable @ 4°C	stable @ 4°C	stable @  -20°C	stable @ 4°C	stable @ 4°C
Comments	simple	simple	Definite color change easy to see with the eye	Time consuming reagent preparation	Many transfer steps using 4 plastic tubes or vials per sample.  Buffer is supplied	Many transfer steps using 4 plastic tubes or vials per sample.  Dilution factor not specified, no direction for making buffer and extraction solution
Costa	NA	$5.31/test $7.00/testb	$5.11/test $5.95/testb	$6.00/test	$3.28/test	$1.94/test

   ^a - Cost per test is based on using the entire kit at one time with one set of standards.  The cost would be

   more if only part of the test kit is used at a time because a set of standards would have to be run each time

   a set of sample is run.  The cost per test decreases when the test kits are purchased in larger quantities.

 

   ^b - Cost per test with extraction kit included. 

 

  Table 4.  Reproducibility of Quantitative Test Kits

Test Portiona	Statisticsb	Neogen Veratox	Immunotech Histamarine	IDR K1-HTMc
a	avg.	34.3	10.0	33.3
a	stdev.	3.7	2.4	1.6
a	coef var.	10.8	24.1	4.7
b	avg.	70.0	85.7	63.9
b	stdev.	4.6	16.9	3.3
b	coef var.	6.5	19.8	5.2
c	avg.	69.4	69.8	61.5
c	stdev.	1.2	12.1	5.2
c	coef var.	1.8	17.3	8.4

  ^a - Histamine values by AOAC:  a = 30.0 ppm, b = 71.7 ppm, c = 62.7 ppm

  ^b - Average of three extractions of each sample.

  ^c - Extraction procedure modified by manufacturer between the time of the analysis of the samples and the triplicate analysis for reproducibility.