Chapter 33
Production and Characterization of Antibodies Against Histamine
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Elisabeth Schneider, Ewald Usleber, and Erwin Märtlbauer Institute for Hygiene and Technology of Food of Animal Orgin, Veterinary Faculty, University of Munich, Schellingstrasse 10, 80799 Munich, Germany
Rabbits were immunized with histamine conjugated to keyhole limpet hemocyanin (KLH). Antibodies against histamine were detected in a competitive direct enzyme immunoassay (EIA), using a histamine-horseradish peroxidase conjugate as the labelled antigen. The detection limit for histamine was 230 ng/mL buffer solution. The assay was very specific for underivatized histamine. Based on the concentrations required for 50% binding inhibition (histamine: 520 ng/mL), relative cross-reactivity of these antibodies with histidine, 1methylhistamine, 3-methylhistamine, andNω-acetylhistaminewas =0.015%, 0.14%, 0.4%, and 0.75%, respectively. The EIA was used to analyse histamine in artificially contaminated salmon meat. The detection limit was in the range of 10 µg/g, recoveries of histamine at levels of 10-1000 μg/g were between 83% and 97%, respectively. Importance of exogenous (dietary) histamine (Figure 1) as a potential risk for human health has been discussed controversely during the last years (7,2). It still is not clear whether histamine, other biogenic amines, or a completely different agent is involved in certain food intoxications such as, e.g., scombrotoxicosis after consumption of mackerel, tuna and related fish (3). However, maximum tolerance levels of 200 mg histamine per kilogram fish tissue have been set within the European Union. A tolerance level for histamine in alcoholic beaverages of 2 mg/L has been suggested. Other foods in which high histamine levels are quite common include certain cheeses and sauerkraut (4). Several physico-chemical methods for histamine, in particular liquid chromatography and fluorescence detection after derivatization with o-phtaldialdehyde or dansyl chloride have been described and so far they are the most commonly used routine assays for biogenic amines in foods (5,6). These methods are usually costly, require extensive sample clean-up, and have a low sample throughput. In order to enhance food control measurements, and possibly to help 0097-6156/96/0621-0413S15.00/0 © 1996 American Chemical Society In Immunoassays for Residue Analysis; Beier, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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clarifying the role of dietary histamine for human health, rapid and easy analytical methods for this compound would be beneficial. Several immunochemical approaches for the detection of histamine in human serum and other biological fluids have been described (7—11). So far, only one report (12) describes the application of an enzyme immuno assay for the detection of histamine in foods, and two other tests are commercially available (13,14). However, since the antibodies used in these tests are not reactive with the parent compound but with a histamine adduct, derivatization of histamine is required before analysis. In this paper we describe a simple method to produce specific polyclonal antibodies against the parent compound, free histamine, and their use in direct competitive EIA of histamine in salmon homogenate. Materials and Methods Materials. Histamine, histidine (D- and L-form), Mx-acetylhistidine, 1-methylhistamine, 3-methylhistamine, Mo-acetylhistamine, tyramine, imidazole, tryptamine, 5-hydroxytryptamine (serotonin), 1-methylimidazole-acetic acid, imidazole 4-acetic acid, tryptophan (D- and L-form), glutaraldehyde 25% (v/v) solution, sodium periodate, sodium borohydride, ^S'^jS'-tetramethylbenzidine (TMB), casein (sodium salt) and polyoxyethylenesorbitan monolaurate (Tween-20) were purchased from Sigma-Aldrich Vertriebs GmbH, Deisenhofen, Germany. Keyhole limpet hemocyanin (KLH), molecular weight 3 - 7 . 5 million, from Megathura crenulataL. was obtained from Boehringer Mannheim GmbH Biochemicals, Mannheim, Germany. EIA-grade horseradish peroxidase (HRP), molecular weight 40,000 (EC 1.11.1.7) was also from Boehringer. Affinity chromatography purified sheep antirabbit immunoglobulin G (IgG) was used as described earlier (15). Salmon meat homogenate with low histamine level ( and L-tryptophan, tryptamine, and tyramine.
A preliminary study, to check the applicability of the EIA for the detection of histamine in salmon meat homogenate, showed that levels as low as 10 μ% histamine per gram tissue could be detected (Table Π). High recoveries between 83 and 97% were obtained in a concentration range from 10-1000 j*g/g, using a simple and rapid sample preparation procedure. A n extract dilution which corresponds to 50 mg of sample per mL was necessary to avoid nonspecific sample matrix inter ference.
Table Π. Recovery of Histamine in Salmon Meat by EIA (n=5) Histamine Added Oig/g) 0 10 100 1000
Mean 0*g/g) „a 8.3 97 870
Histamine Found SD 0*g/g) C V (%)
Recovery (%)
1.5 16 160
83 97 87
18 16.5 19
Histamine negative extracts gave mean % B / B values of 89% ± 8 . 8 , outside the detection limit of the standard curve ( % B / B Q < 75%; Figure 2). a
0
Considering the sample dilution factor of 1:20, histidine concentrations of at least 70 mg/g would be required to give false-positive reactions, a value which is quite above the normal histidine level ( 6 - 7 mg/g) in salmon tissue (25). For histamine determination near the tolerance level of 200 /xg/g, optimum sample
In Immunoassays for Residue Analysis; Beier, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
33. SCHNEIDER ET AL.
Antibodies Against Histamine
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dilutions for EIA are ten times higher (ca 1:200) and correspond to approximately 5 mg of sample per mL. False positive results due to reaction with free histidine can therefore be excluded. In conclusion, the test system for histamine described here could be useful as a simple and inexpensive screening method for histamine in fish and fish products. Further test evaluation, using naturally contaminated sample material and comparing EIA results with those of other methods (e.g, H P L C ) , are under study.
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Acknowledgment We thank Dr. L . Walther, L U A Sudbayern, OberschleiBheim, Germany, for providing the histamine-negative salmon sample material, and M . Djeffal for excellent technical assistance. Literature Cited 1.
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