DE102004007085A1 - To detect the presence of contamination by animal brain or spinal cord tissue, in foodstuffs and pharmaceuticals, a test kit uses a specific antibody for the protein gene product 9.5 - Google Patents

Abstract

To detect the presence of central nervous tissue in products and on surfaces, especially from the brain and the spinal cord, a test kit is used with a specific antibody to determine the presence of protein gene product 9.5 (PGP9.5) which indicates any such contamination. The antibody uses two anti-PGP9.5 antibodies with at least one specific monoclonal antibody. The test kit includes an enzyme linked immunoabsorbant assay (ELISA) test.

Description

The The invention relates to a qualitative and quantitative detection system specified risk material, in particular central nervous tissue, especially brain and spinal cord, in products, in particular food and pharmaceutical products Industry, and on surfaces. Furthermore, a test kit using the detection method shown.

In the years 1920 and 1921 have Creutzfeldt [About a peculiar hearth-shaped spongiform Central nervous system disease, Z. Ges. Neurol. Psychat, 57: 1-20 (1920) Creutzfeldt, HG.] And Jakob [About peculiar diseases of the central nervous system with remarkable anatomical findings, Z. Ges. Neurol. Psychat, 64: 147-228 (1921) Jacob, A.] the first Cases one as yet unclassified central nervous system disease described. The following year, the term was used for this disease Creutzfeldt-Jakob Disease (CJD) introduced [The histopathological Research in Psychiatry, Clinical Weekly, 2: 175-177 (1922) Spielmeyer, W.]. In 1968, the proof of transferability the CJK performed on the example of primates [Creutzfeldt-Jakob Disease (spongiform encephalopathy): transmission to the chimpanzee, Science, 161: 388-389 (1968), Gibbs, CJ, DC Gajdusek, DM Asher, MP Alpers, E Beck, PM Daniel, et al.].

In In the 1990s, sporadic cases of CJD in adolescents increased in Great Britain and France on [Sporadic Creutzfeldt-Jakob Disease in a 16-year Old in UK, Lancet, 146: 1155 (1995) Britton, TC, S Al-Sarraj, C Shaw, T Campbell, J Collinge]. These were characterized by so far histopathological changes not found in CJK and are therefore considered a new variant of Creutzfeldt-Jakob Disease (nvCJK) [A new variant of Creutzfeldt-Jakob disease in the UK, Lancet, 347: 921-925 (1996) Will RG, JW Ironside, M Zeidler, SN Cousens, K Estibeiro, A Alperovitch, S Poser, M Pocchiari, A Hofman, PG Smith].

in the 1985 was the first time a novel spongiform brain disease in cattle - the Bovine spongiform encephalitis (BSE) - described [A novel progressive spongiform encephalopathy in cattle, Vet Rec, 121: 419-420 (1987) GAH Wells, AC Scott, CT Johnson, RF Gunning, RD Hancock, M Jeffrey, M Dawson, R Bradley]. In particular, the spatial and temporal clash the described sporadic cases the nvCJK in adolescents and BSE in the UK and the transmissible Character of the two diseases [Transmission of bovine spongiform encephalopathy in mice, Vet Rec, 123: 472 (1988) Fraser, H, I McConnell, GAH Wells, M Dawson] to the realization that the nCJK evoked by the BSE agent becomes.

When most likely cause of the expression of transferable Spongiform diseases of the central nervous system are considered today certain "infectious" proteins called as Are called prion proteins [Novel proteinaceous infectious particles causes scrapie, Science, 216: 136-144 (1982) Prusiner SB; Identification of prion amyloid filaments in scrapie-infected brain, Cell, 41: 221-235 (1985) DeArmond, SJ, MP McKinley, RA Barry, MB Braunfeld, JR McColloch, SB Prusiner]. Due to the high concentration of prion proteins in brain, spinal cord and nerves, these tissues pose a high risk of infection and become common in addition to lymphatic tissue to the "specified risk material" (SRM).

Therefore regulate legislation such as Regulation (EC) 1326/2001 of 29.06.2001 the removal and separate removal of the SRM. such Legislation also regulates technological changes of the slaughter process involving contamination of the meat Prevent SRM.

These legally prescribed consumer protection measures all over Europe lead to additional costs the meat processing in height of several billion euros. In addition, the addition of brain material provides the processing of meat products has technological advantages, such as better emulsifiability in cooked and boiled sausages and increased stability in minced meat products. To protect the consumer it is therefore necessary to have quality assurance systems inside to introduce the production, the specific detection systems for the detection of risk material to control compliance with the bans on use.

For the detection of central nervous Tissues are known for numerous examination methods, their disadvantages however, in their lack of sensitivity, specificity or elaborate sample preparation or investigation methodology.

It this is light microscopic, enzymatic and immunological Method. Especially when using 10 μm cryostat sections or ultrathin sections and their coloring with usually conducted histological staining techniques (Picroindigo carmine / Karmalaun, Hematoxylin / Eosin) can CNS Tissue constituents often due to structural denaturation not be recorded.

A similar Uncertainty is the enzymatic - photometric method for the detection of the cholesterol content, which also the Cholesterol content of other non-prohibited additives such as egg yolk, Liver or herbal 3-β-hydroxysterine [Method for the detection of bovine spongiforme Encephalopathy (BSE) unwanted Ingredients in meat products-1. Enzymatic cholesterol determination as a rapid method of detecting brain tissue. Meat industry 77 (9): 836-840 (1997) Lucker E, M Bülte].

Next The said method exists a gas chromatographic and Mass spectrometric analysis method that analyzes the brain-specific fatty acid profile for the Detection of CNS tissue [Niederer and Bollhaber identification of species specific central nervous system tissue by gaschromatography mass spectometry (GC-MS) - a possible method for supervision of meat products and cosmetics (2001) Mitt. Hyg. 92: 133-144]. This very sensitive method (0.01 % Detection limit) is only feasible in special laboratories and can not be used as a routine method.

The currently safest routine method for the detection of components of the nervous system are immunological methods using antibodies against γ, γ-enolase [patent DE 198 04 216 C2 ] and against the acid glial fiber protein (GFAP) [patent DE 198 14 088 C1 ]. Their detection limit is 0.25% proportion of brain tissue, depending on the material investigated (scalded sausage or boiled sausage). In order to achieve this sensitivity, however, a fats extraction with downstream filtration was previously necessary in order to reduce the high fat content, which proved to be disturbing for the immunological detection of the selected marker proteins. Without this sample preparation preceding the immunological detection, the detection limit in cooked sausages was 1% with the aid of γ, γ-enolase antibodies. In the study of cooked sausages even amounts of up to 32% added brain material with the aid of γ, γ-enolase antibodies were not detected. The procedure using GFAP antibodies yielded no detection of 1% added brain material in boiled sausage and boiled sausage without upstream fat extraction and filtration.

disadvantageous is the cross-reactivity the used γ, γ-enolase antibodies with Tissues of the poultry, in the inspection of poultry meat products led to false positive results has [Horlacher H et al. Comparison of two CNS detection methods in food (2002) Meat industry 82/1: 91-93).

Currently, two commercially available test systems using γ, γ-enolase antibodies (Brainostic ^TM, ScheBo -Biotech ^® AG) and GFAP antibodies (Ridascreen ^® Risk material R-Biopharm) exist. The Brainostic test is a Westernblot test kit and the Ridascreen test is an ELISA procedure. Both kits were validated in an international study. With the ridascreen method, an admixture of 0.5% nerve tissue was certainly detectable in unheated, moderately heated and strongly heated samples. While the Brainostic test was just as sensitive in unheated and moderately heated samples, the safe detection limit in highly heated samples in this kit was 2% of added nerve tissue [Agazzi et al. Pertormance comparison of two analytical methods for the detection of the central nervous system in sausages: results of an interlaboratory study (2002) Eur Food Res Technol 215: 334-339].

Of the Invention is based on the object, improved methods for the immunological Detection of central nervous Tissues in articles and on surfaces, especially of brain and spinal cord, to provide and this in the form of a test kit, which quickly automatable feasible is to offer.

According to the invention Object by the method described in claim 1 and in the Claim 3 described test kit solved. Procedure and test kit will be in further claims designed advantageous.

essential Feature of the inventive solution the use of antibodies against the protein Gene Product 9.5 (PGP9.5) for the detection of central nervous tissue, especially brain and spinal cord.

It was surprising found that central nervous tissue by the content to PGP9.5, its proven content being proportional to the one to be detected Nerve tissue mass is.

protein Gene Product 9.5 is a neuron-specific protein with a Molecular weight of 27 kD and belongs to the family of ubiquitin-carboxyl-terminal Hydrolases (UCHL1) [The immunolocalization of a protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies. Br. J. Exp. Pathol. 69: 91-104 (1988), Wilson P O G et al.]. It is structurally different and immunologically from the neuron-specific enolase (NSE) and of the acid glial fiber protein (GFAP). PGP9.5 is almost in the cytoplasm of is specifically expressed in all neurons and comprises 1-2% of the whole brain protein [isolation of PGP 9.5, a new human neuronespecific protein detected by high-resolution two-dimensional electrophoresis. J. Neurochem. 40: 1542 to 1547 (1983), Doran J F et al.].

According to the invention was now found that is the addition of tissue of the central nervous system determined by the proportion of PGP9.5 in the sample. Surprisingly is the evidence of central nervous Tissues, especially of the brain and spinal cord, in products despite one during the Processing process completed denaturing of proteins possible. Especially PGP9.5 protein was also stored in bulk after an extreme denaturation (Fc value = 3.3) sensitive and proportional to the added Amount of risk material detected. Preferably, the detection becomes performed with monoclonal PGP9.5-specific antibodies for which no Binding to other proteins could be detected. The invention to be used antibody are commercially available. For production, native PGP9.5 proteins are isolated from brain tissue, as in Doran et al. [Isolation of PGP 9.5, a new human neurone-specific protein detected by high-resolution two-dimensional electrophoresis. J. Neurochem. 40: 1542-1547 (1983)] and Thompson et al. [PGP 9.5-a new marker for vertebrate neurons and neuroendocrine cells. Brain Res. 278: 224-228 (1983)]. By standard method as with Köhler and Milstein are BALB / C mice immunized, their spleen cells isolated and fused with myeloma cells. The resulting hybridoma cells form the antibodies under suitable conditions. Such antibodies are for example from Biogenesis and Novocastra (both UK). Surprisingly It was also found that the detection of nerve tissue as well without additional Fat extraction via standardized phase separation, ideally by centrifugation after heating the sample, feasible is. Using PGP9.5 antibodies, it is possible to use 90 ng brain tissue of bovine. With the help of PGP9.5 antibodies is it still possible still shares of 0.1% brain tissue in cooked sausage and 0.2% brain tissue in full preserves (Fc value = 3.3).

Farther It was found that a certain amount of PGP9.5 protein in defined amounts of homogenized and lyophilized brain tissue a specific amount of PGP9.5 protein in samples with a defined Proportion of added Correspond to brain tissue.

A important property of appropriate PGP9.5 antibodies is in addition to the missing Cross-reactivity with tissues of the ruminant and pigs, the lack of cross-reactivity with tissues, in particular Brain tissue, of the poultry, in the context of the BSE problem reinforced used in food (Embodiment 3).

The The invention also includes a kit for the detection of central nervous tissue in articles and on surfaces in conjunction with an immunological method of determination, preferred a process in which at least one specific antibody immobilized against PGP9.5 protein. This becomes the antigen after sample preparation in a suitable buffer system with that on the carrier immobilized antibody brought in contact and bound there specifically. After a washing step a second labeled antibody is used which binds to a another epitope of the antigen binds. According to the invention is thus the amount of label is proportional to that present in the sample Amount of PGP9.5.

The inventive kits exist next to the coated support of different standards, preferably prepared from homogenized and lyophilized brain material pig, the quantitative classification of the examined Serve samples and the control of the analytical system.

Based following embodiments the invention will be closer explained.

Embodiment 1

Procedure using of anti-PGP9.5 antibodies to determine the detection limit for brain tissue

Homogenates of brain tissue in homogenization buffer (0.05M tris- (hydroxymethyl) -aminomethane, 10 mM EDTA, 10 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride) were prepared for the assay. Both bovine and porcine tissues were used. The protein concentration was preferably determined by the method of Lowry using a commercial assay system (eg, DC protein assay; BioRad). The organ homogenates (1-30 μg total protein) were added to sample buffer. After a brief denaturation (95 ° C, 3 min), the organ homogenates were separated by SDS electrophoresis in 12% polyacrylamide gel (PAGE) (Laemmli, Nature 1970). As molecular weight standard prestained served Roti-Mark / ^® (Roth).

To the electrophoretic separation, the organ homogenates in Tankblotverfahren (BioRad) on nitrocellulose membranes (0.45 microns, Schleicher & Schull) or transferred to PVDF membranes (0.45 μm, Schleicher & Schull). The membrane-immobilized PGP9.5 protein was challenged with PGP9.5 directed antibodies (primary Antibody) bound. As primary antibody Preferably clones 13C4 and 31A3 (both Biogenesis) were used. To the primary antibodies were directed against such primary antibodies Bound secondary antibody. The bound to the secondary antibody Enzymes horseradish peroxidase or alkaline phosphatase put in a color reaction the colorless chromogen 3,3 ', - diaminobenzidine tetrahydrochloride hydrate in a brown dye or Tetranitrozoliumtetrablau in one blue dye around.

The 1 shows a representative Western blot to determine the detection limit of PGP9.5 antibody clone 31A3 for bovine neural tissue. Various amounts of bovine brain homogenate were reported (data in μg total protein). M-molecular weight standard
The antibody clearly detected 90 ng total protein bovine brain homogenate.

Embodiment 2

Procedure using of anti-PGP9.5 antibodies for the detection of central nervous system Fabric especially in cooked sausage

From the federal research institute for Meat research in Kulmbach (BAFF) became reference material in form of cooked sausage with defined additions to brain tissue (from the pig) produced.

For the proof became commercial antibodies against Protein Gene Product 9.5 (PGP9.5) clone 13C4 (Biogenesis) and Clone 31A3 (Biogenesis).

From the sausage samples, one gram was weighed into a centrifuge tube (15 ml, Greiner), added with 5 ml of extraction buffer (5% sodium dodecyl sulfate / SDS, 50 mM tris- (hydroxymethyl) -aminomethane, 10 mM EDTA) and mixed well. Thereafter, the sample was boiled for 15 min at 100 ° C in a water bath and mixed well again after cooling. Subsequently, the homogenate was centrifuged at 10 ° C for 10 min. Under the resulting fat layer, the clear homogenate was removed. In the extracts obtained, the protein concentration was determined using a commercially available protein assay from BioRad (DC protein assay) based on the Lowry method. Defined protein levels (10-60 μg) were separated by SDS electrophoresis in 12% polyacrylamide gel (PAGE) according to Laemmli. As molecular weight standard prestained served Roti-Mark / ^® (Roth). In addition to the sausage extracts, similarly processed brain tissue was used as a positive control. After electrophoretic separation, the sample extracts were transferred by tank blot (BioRad) to nitrocellulose membranes (0.45 μm, Schleicher & Schüll) or to PVDF membranes (0.45 μm, Schleicher & Schull). The PGP9.5 protein immobilized on the membrane was bound with antibodies (primary antibody) directed against PGP9.5. Secondary antibodies directed against such primary antibodies were bound to the primary antibodies. The horseradish peroxidase or alkaline phosphatase enzymes bound to the secondary antibody convert the colorless chromogen 3,3 ', - diaminobenzidine tetrahydrochloride hydrate in a brown dye or tetranitro blue tetrazolium into a blue dye in a color reaction.

Representative Western blots for determining the detection limit of brain tissue in cooked sausage using the PGP9.5 antibody clone 13C4 are in the 2 and clone 31A3 in the 3 shown. In the Western Blot in the 2 in each case 60 .mu.g total protein cooked sausage extract with different proportions of brain tissue (percentages) and as a positive control (K) 1.1 ug bovine brain homogenate was applied. In the Western Blot in the 3 In each case, 30 μg total protein sausage extract with different proportions of brain tissue (percentages) and 1.1 μg total protein of a bovine brain homogenate were separated as positive control (K).

With the PGP9.5 antibodies Clone 13C4 and clone 31A3 were still PGP9.5 in cooked sausage with 0.12 % Brain supplement can be clearly demonstrated.

Embodiment 3

Procedure using of anti-PGP9.5 antibodies for the detection of central nervous system Tissues, in particular in solid preserves

From the federal research institute for Meat research in Kulmbach (BAFF) became reference material in form of full canned food (Fc value = 3.3) with defined additives Brain tissue (produced by the pig).

The Samples were as in the embodiment 1 worked up and examined.

4 shows a representative Western blot to determine the detection limit of PGP9.5 in whole cerebral with the PGP antibody clone 13C4. In each case, 30 μg total protein of whole-conserve extract with different proportions of brain tissue were separated (data in percent); Control (K) 1.7 μg total protein of bovine brain homogenate; M - Molecular weight standard With the PGP9.5 antibody clone 13C4, PGP9.5 was clearly detected in 0.5% brain tissue in extracts from canned foods. In the range of the corresponding molecular weight, 0.1% of added brain tissue had to be differentiated from the 0% sample.

Embodiment 4

Differentiation of tissue of the poultry and of domestic mammals with the help of PGP9.5 antibodies

organs of domestic fowl (Gallus demesticus) and bovine were in one homogenized suitable buffer. As described in embodiment 1, defined protein amounts (5-30 μg) were electrophoretically separated and transferred to membranes. Detection of the PGP9.5 protein was subsequently performed in the manner described (see Example 1). There were the monoclonal antibodies Clones 13C4 and 31A3.

Representative Western blots to study the cross-reactivity of PGP9.5 antibodies with domestic chicken tissues are for the clone 13C4 in the 5 and for clone 31A3 in the 6 shown. For the Westernblot in 5 30 μg total protein heart muscle (Hz), liver (Le), spleen (Mi), kidney (Ni) and brain tissue (HiHu) of the domestic chicken and as a positive control 2.5 ug total protein bovine brain (HiRi) were separated (M-molecular weight standard (data in kD)). For the Westernblot in 6 5-30 μg total protein skeletal muscle (Mu), liver (Le), skin (Ht), heart muscle (He), brain tissue (HiHu) of the domestic chicken and 2.5 μg total protein bovine brain (HiRi) were separated (M - molecular weight standard) ,

In none of the examined chicken organs a PGP9.5-specific signal was obtained.

Embodiment 5

Preparation of a solid phase carrier and Used in a test kit for the detection of central nervous tissue, especially brain and spinal cord, in articles and on contaminated surfaces

5.1 Production of a Solid phase support using PGP9.5 antibody

The Coating of a microtiter plate (Maxisorb, Nunc) is carried out by Incubate of 100 μl / well with the PGP9.5 protein antibody Clone 31A3 over 12 hours at 4 ° C.

After incubation unbound PGP9.5 protein antibody is aspirated and the plate is washed 3x at 250 μl / well with SRM buffer (see below). This is followed by blocking of free binding sites with 120 ul Stable Coat ^® (Sur Modics) for 60 minutes at room temperature.

in the The coated and blocked microtiter plates are connected 4 hours at 37 ° C dried.

5.2 Test kit and method of determination for the detection of central nervous Tissue using the described solid phase carrier

The kit consists of the following reagents:

Sample preparation for the proof of central nervous Tissues, especially brain and spinal cord, in articles

• 1. Crush the sample (at least 20 g) and mix with Hand blender or similar homogenize very finely.
• 2. Weigh 0.5 g sample in 2 ml Eppendorf tube, add 1.25 ml SRM buffer and mix well (Vortex, min. 30 s) (or 2 g sample to 5 ml buffer in centrifuge glass). Perhaps crush small lumps with the help of a spatula!
• 3. 15 min at 100 ° C Boil (heating block or water bath).
• 4. Cool samples let and mix again (Vortex).
• 5. Centrifuge for 10 min at 10,000 rpm and 10 ° C.
• 6. Using a disposable syringe with cannula (or pipette) remove clear homogenate (under the upper fat layer) and add cavity 100 μl in Insert test.

Sample preparation at Detection of central nervous Tissue, especially brain and spinal cord, on contaminated surfaces

• 1. thorough crosswise wiping a ca. 10 × 10 cm area with one with SRM buffer wetted swabs,
• 2. thorough Wetting the entire surface of the swab by turning the dry swab when wiping Sample material,
• 3. Sample by repeated expressions of the swab at the edge of the vessel in 2 ml Eppendorf reaction tube with SRM Transfer buffer.
• 3. 15 min at 100 ° C Boil (heating block or water bath).
• 4. Cool samples to let.
• 5. Centrifuge samples at 10,000 rpm and 10 ° C.
• 6. Remove the clear homogenate and use directly in the test.

preparation the standards

From The supplied brain tissue standards are used after dissolution in deionized water 100 μl standard solution in transferred a reaction vessel and as well as the homogenized samples dissolved in SRM buffers treated.

Test implementation

• 1. Wash 3 times the cavities with 300 μl each prepared SRM buffer and then remove the wash solution Knocking out and through vigorous Knock on clean, absorbent Laboratory towels.
• 2. 100 μl each pipette the standards and samples into the appropriate wells and put the test plate covered in a moist chamber or Close tightly with cover foil.
• 3. The test plate for 2 hours with gentle shaking (10 rpm).
• 4. The cavities Drain well by knocking out and the remaining liquid by vigorous knocking on clean, absorbent laboratory towels remove. The cavities 3x washing each with 300 ul SRM buffer and then Wash buffer carefully remove.
• 5. Pipette 100 μl of prepared antibody conjugate into the wells.
• 6. Covered for 60 min at room temperature in a humid chamber or incubate sealed with cover foil and then through vigorous Remove knock on filter paper.
• 7. Wash 3 times each with 300 ul SRM buffer.
• 8. 100 μl each TMB substrate solution into the cavities Pipette and incubate for 30 min at room temperature in the dark.
• 9. 100 μl each stop solution into every cavity pipette and mix.
• 10. Measurement of absorbance at 450 nm and a reference wavelength of 620 nm in a commercial Microtiter plate reader (e.g., Sunrise, Tecan).

evaluation

to Quantitative evaluation is the extinction of the examined material samples compared with the extinction of the supplied standards. When Standards are defined amounts of homogenized, lyophilized Nervous tissue used by the pig. These defined amounts of Nerve tissue corresponds to defined admixtures of risk material in the samples to be examined. An evaluation of the samples takes place over the Comparison of the absorbances of the samples with the test standards. It it is determined whether the measured absorbance value of the sample is above or below the measured extinction values of the standards.

Claims (9)

Method for the detection of central nervous tissue, especially brain and spinal cord, in articles and on surfaces, characterized in that added brain and / or spinal cord or contamination of surfaces with central nervous Tissue by their content of protein Gene Product 9.5 (PGP9.5) using specific antibody certainly. Method according to claim 1, characterized in that that as an antibody used two anti-PGP9.5 antibodies become. Test kit for the detection of central nervous tissue, especially brain and spinal cord, in articles and on surfaces, characterized in that it comprises at least one specific monoclonal antibody against PGP9.5 proteins contains. Test kit according to claim 3, characterized that the antibody is an anti-PGP9.5 antibody. Test kit according to claims 3 and 4, characterized that he has at least one specific bound to a solid phase antibody against PGP9.5 proteins contains. Test kit according to one of Claims 3 to 5, characterized that he contains an ELISA test. Test kit according to one of Claims 3 to 6, characterized that he is for the analytical quality assurance Lyophilized brain tissue in different concentrations as reference material. Test kit according to claims 3 to 7, characterized in that it consists of the following components: - antibody-coated microtiter plate (96 wells) - 90 ml SRM buffer (0.1 M phosphate buffered saline with 0.05% Tween20), 10-fold concentrated, preserved with thiomersal - 12 ml tetramethylbenzidine substrate solution, ready for use - 12 ml Stop Solution - 1M Hydrochloric Acid, ready to use - 450 ml Antibody Conjugate, Peroxidase-conjugated antibody, in buffer with protein stabilizers - Standard 1 (= zero control) - Standards 2-4, lyophilised Use of at least one specific antibody against PGP9.5 proteins for the detection of central nervous tissue, especially brain and spinal cord, in articles and on surfaces, especially the brain tissue of mammals and poultry and Fully preserved added Brain tissue.