REAGENTS AND IMMUNOCHEMICAL DOSAGE PROCEDURE OF THE DEGREE OF DENATURATION OF PROTEIN SUBSTANCES.

Abstract

Le réactif immunochimique d'identification et de quantification d'une substance protéique-test contenue dans un produit alimentaire est constitué par au moins un anticorps monoclonal dirigé contre une protéine-test contenue dans ledit produit alimentaire et dénaturée par un traitement thermique effectué à une température de traitement donnée, pendant une durée déterminée. Application dudit réactif à l'identification et à la quantification d'une protéine-test présente dans un produit alimentaire à tester.

Description

This Addition relates to improvements to the reagents and methods for immunochemical assay of the degree of denaturation of protein substances, in accordance with the main patent. The main patent aims to provide immunochemical reagents suitable for measuring the degree of denaturation of protein substances such as those contained in food products subjected to thermal, physical, chemical or enzymatic denaturation treatments. According to the main patent, antibodies are used to identify and quantify a component (mainly protein and polysaccharide components) present in any food product and to determine the temperature at which the food product was heated - in particular to check whether the preservation treatment to which the food product was subjected is sufficient from a technical and hygienic point of view. The main patent proposed to use polyclonal or monoclonal antibodies directed against a test protein present in the food product to be tested, such as ovalbumin, to differentiate by the ELISA technique, the presence of native ovalbumin initially present in the product to be tested, prior to heat treatment, and the presence of ovalbumin denatured by heating, and to determine the temperature at which the product had been heated (50°C, 65°C, 85°C, 100°C, 130°C, in particular). The purpose of this Addition is to provide immunochemical reagents consisting of antibodies capable not only of determining the temperature at which the test protein was heated, but also of determining the duration for which the test protein was heated at that temperature. Furthermore, this Addition aims to provide antibodies directed against a test protein denatured by the heating temperatures to which food products are subjected, which allow the heating temperatures to be determined with an accuracy of at least 5°C, and potentially as low as 1°C. The present Addition also aims to provide a test, in particular a sandwich test which involves the ELISA method, to identify the duration of the heat treatment undergone by the food products to be tested and the temperature of the heat treatment with an accuracy of at least 5°C. The purpose of this Addition is an immunochemical reagent for the identification and quantification of a test protein substance contained in a food product to be controlled, and for the recognition of the temperature and duration of the heat treatment to which said food product to be controlled has been subjected, characterized in that it consists of at least one monoclonal antibody directed against a test protein contained in said food product and denatured by a heat treatment carried out at a given treatment temperature, for a determined duration. According to an advantageous embodiment of the immunochemical reagent according to the present invention, it consists of a battery of monoclonal antibodies, one antibody of which is directed against said test protein in its native state, and at least one other antibody of which is directed against said test protein denatured by a heat treatment carried out at a given treatment temperature, for a determined duration. This Addition further relates to a kit for the identification and quantification of a test protein present in a food product to be tested, by the ELISA method, using antibodies directed against the native protein and against at least one denaturation state of said protein by heat, characterized in that it comprises a battery of monoclonal antibodies directed against various states of said test protein, respectively directed against said test protein in its native state and directed against denaturation states of said test protein caused by heat treatments carried out at different temperatures, possibly close to each other, and against denaturation states of said test protein caused by said heat treatments applied for different durations, which monoclonal antibodies are capable of carrying out the immunocapture of the corresponding antigen,namely of said test protein in the state corresponding to the antibodies directed against it, said kit being further characterized in that it comprises polyclonal antibodies suitable for enabling the identification of the antigen bound to the corresponding specific monoclonal antibody. In addition to the foregoing provisions, the invention includes further provisions, which will become apparent from the description that follows. The invention will be better understood with the aid of the following supplementary description, which refers to examples of implementing a test to determine the temperature and duration of a heat treatment to which a test protein substance present in canned foods is subjected, examples illustrated by the accompanying drawings, in which Figures 1 to 5 represent the recognition of various quantities of a test protein substance treated at a determined temperature for varying durations, using monoclonal antibodies directed against that substance. It must be clearly understood, however, that these examples and drawings and the corresponding descriptive parts are given solely as an illustration of the object of the invention, and do not in any way constitute a limitation of it. Example 1 Temperature Recognition which is a protein substance The test was heated. Several monoclonal antibodies directed against a test protein likely to be contained as an additive in canned foods were produced by the method of KOHLER and MILSTEIN: native ltovalbumin and ltovalbumin denatured by a heat treatment at 75°C applied for 30 minutes were respectively injected into suitably chosen mice which were then sacrificed and whose spleen cells were fused with mouse plasmacytoma cells. After selecting hybridomas secreting antibodies directed respectively against native ovalbumin and against heat-denatured ovalbumin, a plurality of monoclonal antibodies were isolated and used in a sandwich assay. ELISA to determine the heating temperatures to which ovalbumin had been subjected. It is understood that although the test protein used in the present Addition is ltovalbumin, the invention nevertheless applies to any test protein denaturable by heat treatment in the temperature ranges used in canning, whether this test protein is added to the preserved food product or is naturally present in it. The following monoclonal antibodies were isolated and used in the heat treatment temperature determination test, in accordance with this Addendum: Identification Number, Name, Nature of the antibody of the antibody 3 MB4 D8 IgG1 4 QA6 B5 IgG1 6 KA4 B4 IgG1 7 AD6 C4 IgG1 8 BC4 F2 IgM 9 IB4 E5 IgG1 The hybridomas BC4F2, IB4E5, KA4B4, which secrete these monoclonal antibodies, were the subject of a deposit with the CNCM held by the PASTEUR INSTITUTE, under N I-907, I-908 and I-909 dated October 10, 1989. The test used to determine the temperature of the heat treatment is a sandwich test. ELISA using on the one hand the aforementioned monoclonal antibodies for the immunocapture of the antigen and on the other hand NAC2 rabbit polyclonal antibodies to identify the captured antigen. The test protein constituting the antigen to be identified was ovalbumin heated for 10 minutes at different temperatures. Table I below shows the optical densities obtained using four of the aforementioned monoclonal antibodies for immunocapture. This table shows a clear difference between 70°C and 75°C on the one hand, and between 75°C and 80°C on the other, when ovalbumin is present in the medium at a concentration of 50 ng / ml. TABLE I Detection of the presence of 50 ng / ml of ovalbumin after 10 minutes of heat treatment, using monoclonal antibodies, in a sandwich ELISA test Optical densities for Treatment Untreated to heat treated 50"C 65"C 70"C 75"C 80"C 85"C antibody 4 1700 1700 1600 1500 1400 200 0 6 1000 950 950 850 850 200 0 8 0 0 0 0 300 650 650 900000400800800 Example 2 Determining the duration of the athermic treatment. The role of ovalbumin heating time was examined by performing a sandwich test ELISA in which immunocapture was performed using, respectively, five of the monoclonal antibodies mentioned above. An ovalbumin solution (10 Ag / ml) was heated for different periods at 75°C and different dilutions were tested. Figures 1 to 4 attached show the quantities of antigen tested as a function of the monoclonal antibody used for immunocapture, and the durations for which the ovalbumin was heated. Significant differences were observed under such conditions - for the same antigen concentration (3 ng / ml) with a similar heat treatment at 75°C: 1) with antibody 4, 80% of the DO (density) optical) are lost between 3 and 30 minutes 2) with antibody 7, 60% of the activity is lost between 3 and 30 minutes; 3) with antibody 3, 40% of the activity only between 3 and 30 minutes are lost, and 4) with antibody 9, 90% of the activity is lost between 1 and 9 minutes. The lower the antigen concentration, the more sensitive the test, but the time at which the activity disappears is essentially the same. These results are radically different when comparing the results obtained respectively for a heat treatment at 75°C and for a heat treatment at 80°C: the attached figure 5 shows that for 75°C, the epitope expression is not significantly modified, whether the concentration of ovalbumin is 50 or 10 ng / ml, whereas at a temperature of 80°C, 70 to 90% of the epitopes disappear in one minute. It is clear from the preceding examples that if a specific monoclonal antibody is used for immunocapture, a specific rabbit or mouse polyclonal antibody can be used for the detection of the captured antigen, whether it is ovalbumin, as in the aforementioned examples, or another test protein that is denatured at the processing temperatures used in canning, whether the test protein is a protein added to the preserved food product or is normally present in it. These examples show that immunochemical reagents conforming to this Addition allow 1) to detect with an accuracy of 5°C and sometimes even 1°C, the temperature to which the antigen has been heated; 2) to identify the duration for which the heat treatment was applied. As can be seen from the foregoing, the invention is by no means limited to those modes of embodiment and application which have just been described more explicitly; on the contrary, it embraces all variants which may come to mind for the technician in the field, without departing from the framework, or the scope, of the present invention.

Claims

DEMANDS 1 Immunochemical reagent for the identification and quantification of a test protein substance contained in a food product to be tested, according to claims 1 to 4 of the main patent, and for the recognition of the temperature and duration of the heat treatment to which said food product to be tested has been subjected, characterized in that it consists of at least one monoclonal antibody directed against a test protein contained in said food product and denatured by a heat treatment carried out at a given treatment temperature, for a determined duration. 2 Immunochemical reagent according to claim 1, characterized in that it consists of a battery of monoclonal antibodies of which one antibody is directed against said test protein in the native state, and of which at least one other antibody is directed against said test protein denatured by a heat treatment carried out at a given treatment temperature, for a determined time.

3. A kit for the identification and quantification of a test protein present in a food product to be tested, by the ELISA method, using antibodies directed against the native protein and against at least one heat-denatured state of said protein, characterized in that it comprises a battery of monoclonal antibodies directed against various states of said test protein, respectively directed against said test protein in its native state and directed against denatured states of said test protein caused by heat treatments carried out at different temperatures, possibly close to each other, and against denatured states of said test protein caused by said heat treatments applied for different durations, which monoclonal antibodies are capable of carrying out the immunocapture of the corresponding antigen, namely of said test protein in the state corresponding to the antibodies directed against it.said kit being further characterized in that it comprises polyclonal antibodies suitable for enabling the identification of the antigen bound to the corresponding specific antibody. 4 Hybridomas secreting monoclonal antibodies directed against a test protein, which constitute the immunochemical reagent according to any one of claims 1 and 2, filed with the CNCM held by the PASTEUR INSTITUTE on October 10, 1989 under Nos. I-907, I-908 and I-909.