In 2012 Russian scientists have carried out a test on the presence of prions in peptides of animal origin. This study was conducted in the Laboratory of Pathology of Viral Infections of the Research Institute of Flu at the Russian Academy of Medical Sciences. Raw materials for the production of food supplements - peptide complexes with a molecular mass of up to 10 kDa, which were isolated from organs and tissues of young animals such as pigs and calves - were examined. Testing for the presence of prions has been carried out according to methods approved in Russia (1) and recommended by the WHO (2):
- 1. "Guidelines for the patho-histological diagnosis of prion infections" Ministry of Agriculture of the Russian Federation, Department of Veterinary Medicine, No 13-7 / 929, approved by the Director of Veterinary Division V.M. Avilov on May 6, 1997.
- 2. Report of a WHO consultation on public health issues related to human and animal transmissible spongiform encephalopathies / Geneva, 17-19 May 1995/ WHO Veterinary Public Health Unit.
In-depth investigation of the samples was carried out using the most sensitive methods for the detection of prion proteins: Western blot method (immunoblot) using test systems of the company "Prionics AG" and electron microscopy.
Justification for the investigation
Prionfection - slowly progressive disease of animals (sheep scrapie, BSE in cattle) and humans (Kuru, Creutzfeldt-Jakob disease), which runs with disorders of the central nervous system and inevitably leads to death.
Infectious prions (PrPSc) are a pathological form of normal cellular proteins (PrPC) present in mammal body cells. Aggregates of the prion protein in infected cells are similar in structure and properties to the amyloid protein, but in contrast to these, they are resistant to proteolytic processing. The molecular mass of all prion proteins known to date is between 20 and 31 kDa.
Method for the detection of prions
Prion aggregates can be detected histochemically in the tissue or raw material sample by means of the dye Congo red. Dyed samples are examined on a dark background with crossed polarisers. Amyloid aggregates are visible in the form of luminous fibers, but also as green and emerald green clots. This method is recommended by the WHO and the Veterinary Department of the Russian Federation as the primary screening method for the detection of prion infections and the testing of products of animal origin.
Smaller aggregates of single prion molecules, which are not detectable by histochemical methods, can be identified by means of electron microscopy, in the form of so-called prion rods and scrapie-associated fibrils (SAF) at a magnification of 30000 - 50000 times. Treatment of the tested samples with proteinase K allows the infectious prion proteins to be distinguished from non-infectious (cellular) and structurally similar proteins. In combination with the prion concentration method, electron microscopy is more sensitive than histochemical staining.
The most sensitive method for the detection of prions is the Western blot method (immunoblot) using monoclonal antibodies against the prion protein (PrPSc and PrPC), which is used for research purposes as well as for commercial testing of meat for prion infection in the European Union.
Materials and methods
Samples of raw materials were used for the production of food supplements. The samples consisted of cream powder, packaged in hermetically sealed plastic bags. For the identification of prion proteins in the test samples histochemical staining, electron microscopy and Western blot method were used.
Alcohol contained test samples were dyed on an object glass with a 1% aqueous solution of Congo red dye, according to the standard Pierce method, within 3 hours. Thereafter, the test samples were treated with a 1% solution of potassium iodide for 60 seconds and then washed with Ethanola exchange in the ethanol 70%, three times for 10 minutes, to free the unbound dye. The evaluation of the samples was carried out in a dark field with crossed polarizers with a magnification of 300 to 600 times. As a positive control, histological preparations of human amyloid kidney were used.
To increase the sensitivity of the electron microscopic examination, the concentration of fractions of the aggregated protein in the test samples was increased by 3-fold freezing-thawing of the saturated sample solution. Thereafter, 5 ml of solution of each test sample was centrifuged at 120000 g for 2 hours in an ultracentrifuge L-8 ("Beckman", USA). The resulting sediment was dissolved in 50 microliters [μl] of the two-fold distilled (bidistilled) water. Thus, about 200 times the concentration of fractions that can contain prions was achieved. The concentrated sediment of the aggregated protein was coated on form carbon carrier sheets and subjected to proteolytic treatment with proteinase K ("Sigma", USA) at a concentration of 0.2 mg / ml for 20 min at 37 °С. The carrier sheets were treated with a 1.5% solution of sodium phosphate-tungstate (pH 6.7) for contrast enhancement and incubated under an electron microscope (model JEM-100S, JEOL, Japan) with a magnification of 30000 to 100000 times, Aim to identify the presence of "prion rods" and SAF. Yeast prions were used as a positive control. They are very similar in their morphology and stability to the prions of mammals.
The Western blot method was performed according to the guidelines of the company "Prionics" for the test system with monoclonal antibodies of the prionprotein (PrPSc and PrPC). The test samples were homogenized in a special buffer to a concentration of 10% denatured with SDS-mercaptoethanol buffer and then with an amount of 10 microliters [μl] on the surface of a polyacrylamide gel in the cassette (MiniProtean II, "Biorad", USA), electrophoresis was carried out at a voltage of 80 V for 2 h. Coomassie was dyed, and the other was carried out by electrotransport of the material onto a large PVDF membrane with a voltage of 40 V for 10 h at 4 °С. (transfer tank "Biorad", USA) Using acetic acid solution "PonceauS" for 2 minutes, then the membrane was blocked with a BLOCK (PRIONICS check kit) buffer for 1 h at room temperature, incubation with the monoclonal antibodies 6N4 (1: 2500) was performed overnight at 4 °С. After incubation with secondary antibody (goat-antimouse), the signal was identified by means of the chemiluminescent substrate NBT / BCIP, according to the manufacturer's guidelines.
In the investigation of the above-mentioned Raw material samples for the production of food supplements, no amyloid aggregates of proteins, and no prions were identified. No bright aggregate was observed in a dark field after the staining of the specimens. In examining the positive control sample of amyloid kidney, characteristic fibrous yellow-green glowing aggregates of amyloid were observed.
In the electron microscopy examination of the sediment concentrate from the presented samples, no prion structures in the form of so-called "prion rods" and no scrapie-associated fibrils were identified. Only traces of sorption of low molecular weight compounds were detected on the form carbon carrier films. On the positive control samples, yeast prions were detected in the form of characteristic fibril structures (SAF).
In carrying out the electrophoresis with the use of monoclonal antibodies against the prion protein (cellular and infected), proteins with a molecular weight above 20 kDa (molecular weight of prion proteins — 27-30 kDa) were not detected in any of the test samples presented. After the incubation of the PVDF membranes with the samples without proteinase K treatment, with antibody 6N4 treatment and staining, no features of binding were identified in the samples. Thus, no prion proteins could be found in the samples by the Western blot method.
In order to identify prion proteins in samples of raw materials for the production of food supplements, a series of investigations have been carried out at the Russian Institute of Medical Sciences in the Department of Pathology of Viral Infections of the Research Institute of Flu at the Russian Academy of Medical Sciences and the Veterinary Department of the Ministry of Agriculture of the Russian Federation Such as histochemical staining, electron microscopy, and Western blot method (immunoblot) with the use of monoclonal antibodies against prion proteins.
The presence of prions was not determined by any of these methods. These study results for the detection of prions in peptides of animal origin clearly show that biopeptides with a molecular weight of up to 10 kDa are safe substances and can be used in food supplements.