08/13/2008

Labor Diagnostika Nord GmbH & Co.KG

HistaSure - Histamine Dipstick Test

Interview with Dr. Bonenberger of Cibitest about the joint project between LDN Labor Diagnostika Nord and Cibitest.

“Tuna is an expensive delicacy” says Dr. Bonenberger. “Gourmets all over the world value its raw meat. Vendors will spend tens of thousands of euros for a yellowfin tuna upon the condition that the fish is totally fresh.“

The problem: with earlier tests, freshness could not be determined accurately or fast enough. LDN Nordhorn has developed different technologies to detect Histamine in Food, the Neu-Ulm-based company Cibitest has developed the lateral flow fluorescence technology. “Together we developed a refined procedure that can accurately determine the freshness of this expensive commodity”.

For the fish industry, time is money: the faster a fresh fish goes off, the faster it loses value. Either the product must be prepared immediately by a top chef, or it is only fit to be used as cat food. “With LDN´s HistaSure testing system, the FLORIDA technology of Cibitest inside, we can now precisely determine the histamine concentration in fish meat,” says, Dr. Bonenberger, managing director of Cibitest. The test can also be used for fishmeal, which is used for animal feed.

Simple LED blue light sources may be used for detection.

The HistaSure dipstick test has been developed to test the fish on-site. The histamine test may also be performed with the immunological EIA detection procedure from LDN, but that requires a fully equipped laboratory. The sample arrives at the laboratory, is analysed and the data are sent back to the sampler. „That takes anything from one and several days. However, with fresh fish every minute counts. Analyses that take several days cost money, says Bonenberger“.
“Using HistSure, the sample can be taken at the processing plant or directly on board ship. Both buyers and food inspectors can be qualified to carry out this test. Histamine is an ideal indicator for a protein proteolysis product: the fewer the histamines, the fresher the fish. Ideal, when speed is of the essence”.

Fish factories will continue to perform quality assurance with ELISA, says Bonenberger. “However, when it has to be fast, an on-site rapid test is the ideal solution” says the managing director of Cibitest.

Up until now, on-site rapid testing for histamine has not been sufficient enough to meet needs. The immunoaffinity assay was too insensitive and was unable to recognise if the test sample was histamine-free.

The HistaSure technology was introduced by LDN and its daughter company Rocky Mountain Diagnostics for the first time in November 2006 at a seafood conference coincidentally held in Florida and it attracted a great deal of attention.

The high point: concentrated fluorescence dyes

“FLORIDA stands for Fluorescence Labelled Optical-Read Immuno Dipstick Assay. The immunoassay is not done in a microtitre plate and it replaces the washout of redundant reagents with a chromatography step along the strip“, explains Bonenberger.

”Instead, FLORIDA labels antibodies with a special, patented fluorescence procedure by concentrating as much fluorescence dye as possible on an antibody. Normally, one dye molecule per binding site attaches to the antibody and four, five or even ten dye molecules attach to the antibody; the signal, and thus the sensitivity, is then too weak“, says Bonenberger.

The head of Cibitest outlines the advantages of the new procedure: “This way, 103 to 104 more molecules attach to the antibody so that we achieve a reinforcing effect.” On the test strips, much smaller quantities of antibodies are visible than with conventional labeling procedures. And if fewer antibodies are needed in order to prove the analyte, the sensitivity is increased. The sensitivity can be 50 times higher than the histamine test using ELISA.

On-site mass screening possible

Several test strips are placed next to each other on the blue light screen and photographed through the filter. The photo is scanned for signals using gel analysis software and then processed on an Excel sheet. The advantage of this fluorescence evaluation is the cost saving of several thousand euros because there is no need to buy expensive equipment.

Marketing network already secured

The global marketing by LDN includes the training of distributors who sell directly to fish factories and other prospective customers in the food industry. The United States, Vietnam, Japan, Sri Lanka, Japan, Indonesia and Spain will be the first to use the system. LDN has also made initial contacts in the Gulf Region and Yemen.

Further down the line, LDN and Cibitest intend to develop and market other food-relevant analytes as tests. Histamine is also a quality criterion for wine. Not everyone can tolerate wine with histamine – for example, allergy sufferers get rashes from internal histamine release.

In the case of more expensive wines, Bonenberger anticipates that winegrowers will also be interested in a quality test of this kind. The FLORIDA test could also be applied to other foodstuffs such as cheese or sausage. At the moment, the industrial test for fish has priority but Bonenberger may later decide to develop individual tests for special applications sometime in the future.

Quick, easy handling

All reagents needed to perform 24 tests come with the kit. Fish extracts are derivatized to stabilize histamine. 100 µl are pipetted into the test container, prefilled with the fluorescence-lyophilised labelled antibodies, the extract dissolves the antibodies. The test strip is then placed in the container with the dissolved antibodies. They then either react with the analyte in the sample or not. If they bind, a signal band shows up.

The test requires a 100 µl pipet and blue light, which excites the fluorescence. This is provided by a blue light LED pocket lamp. The use of value orange filter lab glasses is strongly recommended to better recognise the signal.

dbm – May 10, 2008