IMMUNE ANALYSIS METHODS IN HALAL CONTROL - RESULTS AND PROSPECTS Y. Lebedin

Today, for the objective halal control there are several methods offered for determination of pork in food products and industries.
The most accessible are the tests based on immune analysis. Using a specific antibody response it is possible to determine "pork antigens", i.e. the unique pork muscle tissue proteins, even at very low concentrations.

Dr. Yun-Hwa Peggy Hsie at Florida State University (USA) and the author of this article- in the research department of XEMA company (Russia), were working on this in the last decade. The result of this research was obtaining of, specific for pork, monoclonal antibodies, on its basis were made highly sensitive pork determination methods using immune analysis.

XEMA company has developed an instrumental version of the immune analysis (IFA) designed for laboratory use. Its detection sensitivity is approximately 1 to 10 nanograms of pork antigen per gram of material (1:100000000), while the determination is about 2,5 hours. This method is recommended by the manufacturer for using in halal control laboratories in production or expert bodies. IFA method is less effective than expert method of PCR (DNA identification) by sensitivity in fresh meat, but significantly greater in speed setting, capacity and stability of the material to the culinary and technological processing. Thus, the determination by the IFA remains satisfactory sensitivity for cooking meat under a temperature of 250 degrees for 4 hours.

Another option of immune analytical definition of pork is "express" test based on immune chromatography method. Such tests are also offered by Perkin-Elmer company (USA), moreover there are two different versions-for raw and cooked materials. The XEMA company (Russia) uses a single test for raw and processed meat analysis (XEMA Test Pork). "Express" test is the strip to immerse in liquid, manufacturers offer to chop the investigated sample in water for having it in a liquid state or place an aqueous extract into a disposable tube attached to the test set.

After that, the analysis is carried out for 1-2 minutes, registration is carried out visually, like a pregnancy test. "Express" test results can be photo documented by taking picture of the test-strip. Manufacturers suggest using such tests as for professional control of halal (when there are no conditions, in the field, for example), and so for private control by interested consumers "at their own kitchen". Simple storage conditions (room temperature) and a long shelf life (1.5-2 years) are other advantages of the "express" tests over laboratory IFA

Antigens of pork fat (bacon) are currently under intensive investigation. There have not been in the world practice of immune analysis an analogue for a species-specific determination of fat (lipid) antigens, this problem used to be solved only by methods of liquid and gas-liquid chromatography using sophisticated and expensive devices. In addition, the currently available studies about the comparative structure of the pork fat and other animals leave little hope for obtaining specific antibodies, which recognize exact fat component of swine bacon.

However, laboratory at Florida State University and XEMA research laboratories in Russia and Ukraine are working now on solving this problem. The target for the first version of the test on swine fat, developed by XEMA (tentative title XEMA Test Bacon) is a swine blood albumin, closely associated with fat (albumin is a "carrier" of fat elements in the bloodstream). The first version of the test in April 2011 has shown a basic possibility of creation of hypodermic and internal fat specific belonging immune analysis methods.

The obvious defect of the current version of the developed test is instability of determined complex antigen during heating, XEMA Test Bacon as opposed to XEMA Test Pork loses its sensitivity even under gentle heat treatment - for example, 15-20 minutes of cooking. Today, this test can be recommended only for the examination of raw fatty meat in the halal products manufacturing. In addition, due to the extremely high level of sensitivity (breeding swine blood 1 to 10 million times) XEMA Test Bacon is useful for controlling the contamination of working surfaces of cutting rooms and technological equipment BEFORE the stage of heat treatment.

Pork products of the internal organs (as liver) are not determined by XEMA Test Pork. The first version of the XEMA Test Bacon determines liver extract with a very high sensitivity, which is normal due to its high concentration of fat, but, as mentioned above, XEMA Test Bacon ineffective for boiled and roasted material. Therefore, XEMA is currently developing a test, determining pig’s blood antigen. The blood is inevitably present in all internal organs, there is a hope that some of the blood components can withstand continuous heat treatment (in sausages and canned products) and will be determined by species-specific antibodies. There is an assumption that these while prospective blood components are in a fatty tissue, and test for both components will turn out to be general.

In addition, it is already possible to combine the definition of pork meat and blood in the ONLY (single) test. Such a test with the working title XEMA Test Pork Combo is currently in internal testing at XEMA company.

Antigens of pig connective tissue and skin is a serious challenge as for the Halal control in general (as it’s widely used in the form of food gelatin and most of the time not declared by food products manufacturers) and for the immune analysis. The interesting thing is that gelatin (collagen) is a structural protein, unique in its structure and amino acid composition, highly evolutionarily conservative – which is similar in structure to all mammals and vertebrates in general. For this reason, it is a difficult task to obtain antibodies, or immune response to porcine gelatin in conventional laboratory animals (as mice, rabbits, goats, sheep).

Nevertheless, the XEMA company works on that and looks forward to its successful completion.

The intended outcomes of the "Immune analysis of pig antigens" project of the XEMA company must also become the laboratory tests (IFA) for the three or four antigen variants, and "express" tests for the same antigens - either three individual, or a combination of three-four at one strip and a control one.

The XEMA laboratory experience in application of the developed "express" tests and EIA kit, revealed important facts related to the control of halal. Even the first experiments carried out on the corporate kitchen in the central office in Moscow, cleared out the sources of pork antigen "contamination". First of all, it's a dishwasher and slaughter, and then common cutting boards and knives for different kinds of meat . According to the results of these experiments XEMA recommends those Muslims eating in corporate canteens, to use their own utensils and refrain from eating minced meat or insist on using a separate kitchen equipment for pork.

Also for a period of one year and half XEMA laboratory conducts random sampling control of meat products purchased in major supermarkets in Moscow. The observations are mostly the same as made at home kitchen: almost all samples of minced meat and sausage products produced without pork, according to a recipe stated by manufacturers, include pork in different, but defined in immune analysis quantities.

It should be noted that this observation applies only to manufacturers (small and big) that are not positioning themselves as the specialized on halal production but use pork as a feedstock for other products. Few cases of detection of pork antigen in the raw pork products (pieces on the tray), showed an interesting observation which also confirms the assumption that not only the industrial meat grinders, but cutting surfaces on large-scale production can also be a source of cross contamination of different types of raw materials.

The only way to avoid this danger, in the opinion of the author, is a complete separation of the technological process streams of halal products from other products, as well as continuous monitoring of production equipment and technological surfaces washings using "express" tests.

Determination of antigen markers of the halal slaughtering rules violation at most is still a theoretical problem. As a "target" for the scientific development can be identified the following substances candidates:

- Residual blood
- Residual stress hormones
- Stress-induced muscle tissue antigens

Antigens of residual blood (hemoglobin, proteins, plasma) are the actual target of heat-treated meat only, because detection of residual blood in the fresh meat is not difficult either for visual or for laboratory control. There are widely represented antiserums and antibodies against cow, sheep, horses, chickens and other farm animals plasma proteins in the biotech products market.

The hormones of stress in the traditional sense belong to a class of steroids. The main hormone of stress in the cattle, unlike man, is not cortisol, but corticosterone. Corticosterone determination in the serum or in extracts of fresh muscle tissue is not difficult. However, the effect of cooking (particularly under high temperatures) for the safety of corticosterone and its derivatives is almost not investigated. In addition, there is no clear experimental data on the threshold of permissible concentration of the hormone, indicating a significant violation of the halal slaughter rules.

Search for stress-induced antigens of muscle tissue, resulting in twitching muscle in non halal slaughter is the most difficult task in theoretical term. Besides searching for specific antigens for each kind of halal cattle, additional factor of complexity is the requirement for the stability of this trait to all kinds of cooking.

ХEMA company is working on all of the abovementioned three areas in their laboratories in Moscow, Minsk and Kiev.

Do you usually scrutinize a label or foodstuff chemical composition every time you go shopping? No matter how closely we scrutinize all label information, it is impossible to identify the product genuine composition. Only experts in laboratory conditions can tell you for sure what the foodstuffs contain. Food producers do not specify all ingredients in the composition of the foodstuffs lest they lose their customers. Therefore foodstuff adulteration is rather widespread.
Muslims face similar problems when an issue of halal foodstuffs arises. The customers in Arabic countries are used to the fact that all produce in the market is completely halal foodstuff products, whereas in the countries where the Muslims constitute a minority, one cannot guarantee and claim that halal foodstuffs is fully controlled. There were cases on more than one occasion when pork was produced under the halal branded foodstuffs. Or, due to some process disturbances halal meat contained traces of the meat prohibited in Islam.
Producers tend to disguise the produce composition under “E” code. You need to have a reference book to learn what this or that code means.
A similar situation happens to the individuals allergic to particular foodstuffs. Besides, if a person is allergic to fish he ultimately faces lethal outcome. The USA performs statistical analysis of fatal casualties amidst the human population as a result of anaphylactic shock due to allergy to various foodstuffs. According to the data, annual death toll related to food allergy amount to 250, whereas antibiotics are the cause of 10 000 fatal casualties per year. The life of allergic individuals becomes more complicated due to their failure to accept invitations of their friends to go out for a meal in a restaurant; it’s quite unsafe to take allergic children out to a café or to children’s festivals. Sure enough, allergic individuals have antihistaminics with them. But isn’t it better sometimes to prevent the allergic fit than to combat harmful consequences?
To feel certain about the quality of the product, to make no doubt that the product is halal and to avoid contacts with allergy-causing agents some xematests were developed to detect the presence of fish, soybean and pork in foodstuffs. In addition, XEMA company offers antibiotics detecting tests. Underway is the development of xematests to detect other allergy-causing agents in foodstuffs.
Given the current situation, when the amount of semi-finished products and the number of producing companies are increasing, people have an option to choose the food products brand with no allergy-causing agent dangerous to health. The xematests are easy to use either at home or in public catering facilities. An allergic individual can easily test his meal in a café prior to eating it. A Muslim can assure himself right on the spot of the foodstuff “purity”.