1. A radioisotope labeled polypeptide for imaging a tumor, wherein: the method comprises the following steps:

the method comprises the following steps: selecting a suitable experimental object;

step two: selecting tumor cells from a biological cell database, culturing the tumor cells in vitro, establishing a tumor model, and carrying out protein detection on the tumor cells in the tumor model to obtain a polypeptide arrangement combination sequence in the tumor cells;

step three: taking out part of tumor cells from the tumor model, inoculating the tumor body into an experimental object body, regularly observing the proliferation condition of the tumor cells, and screening out an experimental group which is successfully inoculated;

step four: preparing chloramine-T oxidant water solution, preparing labeled antibody protein, introducing the labeled antibody protein into an experimental group through a protein carrier, and observing tumor development conditions through a nuclear medicine development device after tumor cells proliferate;

step five: and observing the expression condition of the labeled antibody protein through the tumor imaging condition, and recording the data result.

2. A radioisotope labeled polypeptide for use in imaging of tumors as claimed in claim 1 wherein: in the first step, a young, healthy and vigorous experimental mouse group is selected from the white mouse group, and is placed under an environment suitable condition for breeding.

3. A radioisotope labeled polypeptide for use in imaging of tumors as claimed in claim 1 wherein: in the second step, tumor cells such as lipoma and skin tumor, which are typical and easy to observe, are selected from the mouse group in the biological cell database.

4. A radioisotope labeled polypeptide for use in imaging of tumors as claimed in claim 1 wherein: and in the second step, extracting tumor cells of the biological cell database, placing the tumor cells under an activation condition for activity treatment, placing the tumor cells subjected to the activity treatment in a culture dish filled with a culture solution for culture, and obtaining a plurality of experimental tumor cells after the tumor cells are proliferated.

5. A radioisotope labeled polypeptide for use in imaging of tumors as claimed in claim 1 wherein: and in the second step, taking out part of experimental tumor cells, placing the experimental tumor cells in another group of culture dishes, adding a proper amount of proteolytic enzyme into the culture dishes, and measuring the arrangement sequence of the polypeptides in the tumor cells.

6. A radioisotope labeled polypeptide for use in imaging of tumors as claimed in claim 1 wherein: and in the third step, taking out part of the tumor cells, injecting the tumor cells into the experimental mouse group through a needle tube, observing the experimental mouse group of the injected tumor cells after the proliferation of the tumor cells, and screening the experimental mouse group of which the tumor cells are successfully proliferated by comparing the characteristic performance conditions of the normal mouse group and the experimental mouse group.

7. A radioisotope labeled polypeptide for use in imaging of tumors as claimed in claim 1 wherein: in the third step, when the characteristic expression condition of the experimental mouse group is observed, the external skin or subcutaneous tissue of the experimental mouse group generates massive tissues for successfully proliferating the tumor cells, and conversely, the tumor cells are inactivated in the experimental mouse group.

8. A radioisotope labeled polypeptide for use in imaging of tumors as claimed in claim 1 wherein: in the fourth step, chloramine-T oxidant aqueous solution is prepared, 50ug/120ul of purified antibody is added into a centrifugal tube, 5ul of 0.01mol/L phosphate buffer solution is additionally added, 5ul of 131I/125I mixed solution is added into one group, 10ul of chloramine-T is added into the centrifugal tube, 50ul of Na2S2O3 is added after 45S for 2min, 100ul of KI solution is added into the solution after two minutes, a proper protein carrier is selected, 5ul of 32S/35S mixed solution is added into the aqueous solution in the middle group in the same way, 2.5ul of mixed solution of 131I/125I and 2.5ul of mixed solution of 32S/35S is added into the last group, the solution is collected, the solution is placed into different reagent tubes, the solution in each reagent tube is ensured to be the same volume, and the solution in each reagent tube is subjected to the measurement of the ratio and the labeling rate.

9. A radioisotope labeled polypeptide for use in imaging of tumors as claimed in claim 1 wherein: and in the fourth step, introducing the marked solution into carrier protein, injecting the protein carrier into an experimental mouse group in which tumor cells are successfully proliferated, and observing the expression position of the marked isotope by using nuclear medicine imaging equipment after waiting for a certain time.

10. A radioisotope labeled polypeptide for use in imaging of tumors as claimed in claim 1 wherein: and in the fifth step, displaying a result through an image, and recording the expression position of the isotope in the experimental mouse group.

Background

Tumor refers to a new organism formed by local tissue cell proliferation under the action of various tumorigenic factors, because the new organism mostly presents space-occupying block-shaped protrusions, which is also called neoplasm. It has been found that tumor cells exhibit metabolic changes that differ from normal cells, while tumor cells themselves can adapt to changes in the metabolic environment by switching between glycolysis and oxidative phosphorylation. In 2019, a paper was newly published by Cancer Cell, researchers found that metformin can significantly inhibit tumor growth in a fasting state, and suggested that the PP2A-GSK3 β -MCL-1 pathway may be a new target for tumor therapy.

The tumor receptor imaging is an imaging technology which displays the space distribution, density and affinity of tumor receptors by utilizing the principle that receptor ligands marked by radioactive nuclide are specifically combined with high-expression receptors in tumor tissues. It has the characteristics of high affinity and high specificity, rapid arrival of radioactivity at a target point and blood clearance, strong tissue penetration capacity and the like, so that a tumor image with high contrast can be obtained in a short time, and human immune reaction hardly occurs.

In the existing tumor receptor imaging, the polypeptide in the tumor cell is not studied in detail by using radioactive isotopes, so that the expression position of the isotopes in the polypeptide in the tumor cell cannot be known, and a medicament cannot act on a target position in the tumor treatment process, so that the treatment of a tumor patient is influenced.

Disclosure of Invention

The technical problem to be solved by the present invention is to overcome the existing defects, and provide a radioisotope-labeled polypeptide for tumor imaging, so as to solve the problem that the expression position of the isotope in the polypeptide in the tumor cell cannot be known, which is proposed in the background art.

In order to achieve the purpose, the invention provides the following technical scheme: a radioisotope labelled polypeptide for use in tumour imaging comprising the steps of:

the method comprises the following steps: selecting a suitable experimental object;

step two: selecting tumor cells from a biological cell database, culturing the tumor cells in vitro, establishing a tumor model, and carrying out protein detection on the tumor cells in the tumor model to obtain a polypeptide arrangement combination sequence in the tumor cells;

step three: taking out part of tumor cells from the tumor model, inoculating the tumor body into an experimental object body, regularly observing the proliferation condition of the tumor cells, and screening out an experimental group which is successfully inoculated;

step four: preparing chloramine-T oxidant water solution, preparing labeled antibody protein, introducing the labeled antibody protein into an experimental group through a protein carrier, and observing tumor development conditions through a nuclear medicine development device after tumor cells proliferate;

step five: and observing the expression condition of the labeled antibody protein through the tumor imaging condition, and recording the data result.

Preferably, in the first step, a young, healthy and vigorous experimental mouse group is selected from the white mouse group, and the experimental mouse group is kept under the environment-suitable condition.

Preferably, in the second step, tumor cells such as lipoma and skin tumor, which are typical and easy to observe in the mouse population, are selected from the biological cell database.

Preferably, in the second step, tumor cells in the biological cell database are extracted, the tumor cells are placed under an activation condition for activity treatment, the tumor cells after the activity treatment are placed in a culture dish filled with a culture solution for culture, and after the tumor cells proliferate, a plurality of experimental tumor cells are obtained.

Preferably, in the second step, a part of the experimental tumor cells are taken out, the experimental tumor cells are placed in another group of culture dishes, a proper amount of proteolytic enzyme is added into the culture dishes, and the arrangement sequence of the polypeptides in the tumor cells is determined.

Preferably, in the third step, part of the tumor cells are taken out, the tumor cells are injected into the experimental mouse group through a needle tube, after the tumor cells proliferate, the experimental mouse group of the injected tumor cells is observed, and the experimental mouse group of the tumor cells successfully proliferate is screened by comparing the performance conditions of the normal mouse group and the experimental mouse group.

Preferably, in the third step, when the characteristic expression condition of the experimental mouse population is observed, the external skin or subcutaneous tissue of the experimental mouse population generates massive tissues for successfully proliferating the tumor cells, and conversely, the tumor cells are inactivated in the experimental mouse population.

Preferably, in the fourth step, a chloramine-T oxidant aqueous solution is prepared, 50ug/120ul of purified antibody is added into a centrifugal tube, 5ul of 0.01mol/L phosphate buffer solution is additionally added, 5ul of 131I/125I mixed solution is added into one group, 10ul of chloramine-T is added, 50ul of Na2S2O3 is added for 2min after 45S, 100ul of KI solution is added into the solution after two minutes, a proper protein carrier is selected, 5ul of 32S/35S mixed solution is added into the aqueous solution in the middle group, 2.5ul of mixed solution of 131I/125I and 2.5ul of 32S/35S is added into the last group, the solution is collected, the solution is placed into different reagent tubes, the solution in each reagent tube is ensured to be the same volume, and the ratio and the labeling rate of the solution in the reagent tubes are measured.

Preferably, in the fourth step, the labeled solution is introduced into carrier protein, the protein carrier is injected into a laboratory mouse group in which tumor cells are successfully proliferated, and after a certain time, the expression position of the labeled isotope is observed by a nuclear medicine imaging device.

Preferably, in the fifth step, the result is displayed through images, and the expression positions of the isotopes in the experimental mouse group are recorded.

Compared with the prior art, the invention provides a radioisotope-labeled polypeptide for tumor imaging, which has the following beneficial effects:

1. according to the invention, two different radioactive isotopes are adopted to mark an experimental mouse group with tumor cells, and in the process of proliferation of the tumor cells of the experimental mouse group, radioactive substances are added into amino acid required by proliferation of the tumor cells, so that after the tumor cells are proliferated, the marked isotope expression position can be observed through nuclear medicine imaging equipment, and the tracking of the isotope in the amino acid marked in the tumor cells is realized, so that scientific researchers can know the isotope expression position in the amino acid in the tumor cells;

2. according to the invention, lipoma and skin tumor with typical tumor cell expression are selected, and in the experiment process of the mouse group, the proliferation condition of the tumor cells can be visually observed by observing the appearance of the experiment mouse group, so that the steps of detecting the tumor cells of the experiment mouse group can be effectively reduced, the survival rate of the experiment mouse group is ensured, and the tumor detection time is saved.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention without limiting the invention in which:

FIG. 1 is a flow chart of a system for radiolabeling polypeptides for tumor imaging according to the present invention;

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1, the present invention provides the following technical solutions: a radioisotope labelled polypeptide for use in tumour imaging comprising the steps of:

the method comprises the following steps: selecting a suitable experimental object;

step two: selecting tumor cells from a biological cell database, culturing the tumor cells in vitro, establishing a tumor model, and carrying out protein detection on the tumor cells in the tumor model to obtain a polypeptide arrangement combination sequence in the tumor cells;

step three: taking out part of tumor cells from the tumor model, inoculating the tumor body into an experimental object body, regularly observing the proliferation condition of the tumor cells, and screening out an experimental group which is successfully inoculated;

step four: preparing chloramine-T oxidant water solution, preparing labeled antibody protein, introducing the labeled antibody protein into an experimental group through a protein carrier, and observing tumor development conditions through a nuclear medicine development device after tumor cells proliferate;

step five: and observing the expression condition of the labeled antibody protein through the tumor imaging condition, and recording the data result.

In the present invention, preferably, in the first step, a young, healthy and vigorous laboratory mouse group is selected from the white mouse group, and the laboratory mouse group is raised under an environment-suitable condition, in the second step, tumor cells which are typical and easy to observe of the mouse group, such as lipoma and skin tumor, are selected from the biological cell database, in the second step, tumor cells in the biological cell database are extracted, the tumor cells are placed under an activation condition for an activity treatment, the tumor cells after the activity treatment are placed in a culture dish filled with a culture solution for culture, after the tumor cells proliferate, a plurality of experimental tumor cells are obtained, in the second step, a part of experimental tumor cells are taken out, the experimental tumor cells are placed in another group of culture dishes, a proper amount of proteolytic enzyme is added into the culture dish, the polypeptide arrangement sequence in the tumor cells is determined, in the third step, and in step three, when the characteristic expression condition of the experimental mouse group is observed, the external skin or subcutaneous tissue of the experimental mouse group generates massive tissues to successfully proliferate the tumor cells, otherwise, the tumor cells are inactivated in the experimental mouse group.

In the invention, preferably, in the fourth step, chloramine-T oxidant aqueous solution is prepared, 50ug/120ul of purified antibody is added into a centrifugal tube, 5ul of 0.01mol/L of phosphate buffer solution is additionally added, 5ul of 131I/125I mixed solution is added into one group, 10ul of chloramine-T is added, 50ul of Na2S2O3 is added after 45S for 2min, 100ul of KI solution is added into the solution after two minutes, a proper protein carrier is selected, 5ul of 32S/35S mixed solution is added into the aqueous solution in the middle group in the same way, 2.5ul of mixed solution of 131I/125I and 2.5ul of 32S/35S is added into the last group, the solution is collected, the solution is placed into different reagent tubes, the solution in each reagent tube is ensured to be the same volume, the solution in the reagent tubes is subjected to the measurement of the ratio and the marking rate, the marked solution is introduced into the carrier protein in the fourth step, injecting the protein carrier into the experimental mouse group in which the tumor cells are successfully proliferated, observing the expression position of the marked isotope by using nuclear medicine imaging equipment after waiting for a certain time, and recording the expression position of the isotope in the experimental mouse group by using an image display result.

The first embodiment is as follows:

a radioisotope labelled polypeptide for use in tumour imaging comprising the steps of:

the method comprises the following steps: selecting a suitable experimental object;

step two: selecting tumor cells from a biological cell database, culturing the tumor cells in vitro, establishing a tumor model, and carrying out protein detection on the tumor cells in the tumor model to obtain a polypeptide arrangement combination sequence in the tumor cells;

step three: taking out part of tumor cells from the tumor model, inoculating the tumor body into an experimental object body, regularly observing the proliferation condition of the tumor cells, and screening out an experimental group which is successfully inoculated;

step four: preparing chloramine-T oxidant water solution, preparing labeled antibody protein, introducing the labeled antibody protein into an experimental group through a protein carrier, and observing tumor development conditions through a nuclear medicine development device after tumor cells proliferate;

step five: and observing the expression condition of the labeled antibody protein through the tumor imaging condition, and recording the data result.

In the present invention, preferably, in the first step, a young, healthy and vigorous laboratory mouse population is selected from the white mouse population, and the laboratory mouse population is kept under an environmentally suitable condition.

In the present invention, preferably, in step two, tumor cells such as lipoma and skin tumor, which are typical and easy to observe in the mouse population, are selected from the biological cell database.

In the present invention, preferably, in the second step, tumor cells in the biological cell database are extracted, the tumor cells are placed under activation conditions for activation treatment, the tumor cells after activation treatment are placed in a culture dish filled with a culture solution for culture, and after proliferation of the tumor cells, a plurality of experimental tumor cells are obtained.

In the present invention, preferably, in step two, a part of the experimental tumor cells is taken out, the experimental tumor cells are placed in another set of culture dishes, and a proper amount of proteolytic enzyme is added into the culture dishes to determine the arrangement order of the polypeptides in the tumor cells.

In the present invention, preferably, in the third step, a part of the tumor cells are taken out, the tumor cells are injected into the experimental mouse group through the needle tube, after the tumor cells proliferate, the experimental mouse group of the injected tumor cells is observed, and the experimental mouse group in which the tumor cells successfully proliferate is screened by comparing the behavior of the normal mouse group and the experimental mouse group.

In the present invention, preferably, in step three, when the characteristic expression condition of the experimental mouse population is observed, the external skin or subcutaneous tissue of the experimental mouse population generates massive tissues for successfully proliferating the tumor cells, and conversely, the tumor cells are inactivated in the experimental mouse population.

In the invention, preferably, in the fourth step, chloramine-T oxidant aqueous solution is prepared, 50ug/120ul of purified antibody is added into a centrifugal tube, 5ul of 0.01mol/L phosphate buffer solution is additionally added, 5ul of 131I/125I mixed solution is added into one group, 10ul of chloramine-T is added, 50ul of Na2S2O3 is added for 2min after 45S, 100ul of KI solution is added into the solution after two minutes, a proper protein carrier is selected, the solution is collected, the solution is placed into different reagent tubes, the solution in each reagent tube is ensured to be the same volume, and the ratio and the labeling rate of the solution in the reagent tubes are measured.

In the present invention, preferably, in the fourth step, the labeled solution is introduced into a carrier protein, the protein carrier is injected into a population of experimental mice in which tumor cells are successfully proliferated, and after a certain period of time, the expression position of the labeled isotope is observed by a nuclear medicine imaging device.

In the present invention, preferably, in the fifth step, the result is displayed by an image, and the expression position of the isotope in the experimental mouse population is recorded.

Example two:

a radioisotope labelled polypeptide for use in tumour imaging comprising the steps of:

the method comprises the following steps: selecting a suitable experimental object;

step two: selecting tumor cells from a biological cell database, culturing the tumor cells in vitro, establishing a tumor model, and carrying out protein detection on the tumor cells in the tumor model to obtain a polypeptide arrangement combination sequence in the tumor cells;

step three: taking out part of tumor cells from the tumor model, inoculating the tumor body into an experimental object body, regularly observing the proliferation condition of the tumor cells, and screening out an experimental group which is successfully inoculated;

step four: preparing chloramine-T oxidant water solution, preparing labeled antibody protein, introducing the labeled antibody protein into an experimental group through a protein carrier, and observing tumor development conditions through a nuclear medicine development device after tumor cells proliferate;

step five: and observing the expression condition of the labeled antibody protein through the tumor imaging condition, and recording the data result.

In the present invention, preferably, in the first step, a young, healthy and vigorous laboratory mouse population is selected from the white mouse population, and the laboratory mouse population is kept under an environmentally suitable condition.

In the present invention, preferably, in step two, tumor cells such as lipoma and skin tumor, which are typical and easy to observe in the mouse population, are selected from the biological cell database.

In the present invention, preferably, in the second step, tumor cells in the biological cell database are extracted, the tumor cells are placed under activation conditions for activation treatment, the tumor cells after activation treatment are placed in a culture dish filled with a culture solution for culture, and after proliferation of the tumor cells, a plurality of experimental tumor cells are obtained.

In the present invention, preferably, in step two, a part of the experimental tumor cells is taken out, the experimental tumor cells are placed in another set of culture dishes, and a proper amount of proteolytic enzyme is added into the culture dishes to determine the arrangement order of the polypeptides in the tumor cells.

In the present invention, preferably, in the third step, a part of the tumor cells are taken out, the tumor cells are injected into the experimental mouse group through the needle tube, after the tumor cells proliferate, the experimental mouse group of the injected tumor cells is observed, and the experimental mouse group in which the tumor cells successfully proliferate is screened by comparing the behavior of the normal mouse group and the experimental mouse group.

In the present invention, preferably, in step three, when the characteristic expression condition of the experimental mouse population is observed, the external skin or subcutaneous tissue of the experimental mouse population generates massive tissues for successfully proliferating the tumor cells, and conversely, the tumor cells are inactivated in the experimental mouse population.

In the invention, preferably, in the fourth step, chloramine-T oxidant aqueous solution is prepared, 50ug/120ul of purified antibody is added into a centrifugal tube, 5ul of 0.01mol/L phosphate buffer solution is added, 10ul of chloramine-T is added, 50ul of Na2S2O3 is added after 45S for 2min, 100ul of KI solution is added into the solution after two minutes, a proper protein carrier is selected, 5ul of 32S/35S mixed solution is added into the aqueous solution in the middle group in the same way, the solution is collected, the solution is placed into different reagent tubes, the solution in each reagent tube is ensured to be the same volume, and the ratio and the labeling rate of the solution in the reagent tubes are measured.

In the present invention, preferably, in the fourth step, the labeled solution is introduced into a carrier protein, the protein carrier is injected into a population of experimental mice in which tumor cells are successfully proliferated, and after a certain period of time, the expression position of the labeled isotope is observed by a nuclear medicine imaging device.

In the present invention, preferably, in the fifth step, the result is displayed by an image, and the expression position of the isotope in the experimental mouse population is recorded.

Example three:

a radioisotope labelled polypeptide for use in tumour imaging comprising the steps of:

the method comprises the following steps: selecting a suitable experimental object;

step two: selecting tumor cells from a biological cell database, culturing the tumor cells in vitro, establishing a tumor model, and carrying out protein detection on the tumor cells in the tumor model to obtain a polypeptide arrangement combination sequence in the tumor cells;

step three: taking out part of tumor cells from the tumor model, inoculating the tumor body into an experimental object body, regularly observing the proliferation condition of the tumor cells, and screening out an experimental group which is successfully inoculated;

step four: preparing chloramine-T oxidant water solution, preparing labeled antibody protein, introducing the labeled antibody protein into an experimental group through a protein carrier, and observing tumor development conditions through a nuclear medicine development device after tumor cells proliferate;

step five: and observing the expression condition of the labeled antibody protein through the tumor imaging condition, and recording the data result.

In the present invention, preferably, in the first step, a young, healthy and vigorous laboratory mouse population is selected from the white mouse population, and the laboratory mouse population is kept under an environmentally suitable condition.

In the present invention, preferably, in step two, tumor cells such as lipoma and skin tumor, which are typical and easy to observe in the mouse population, are selected from the biological cell database.

In the present invention, preferably, in the second step, tumor cells in the biological cell database are extracted, the tumor cells are placed under activation conditions for activation treatment, the tumor cells after activation treatment are placed in a culture dish filled with a culture solution for culture, and after proliferation of the tumor cells, a plurality of experimental tumor cells are obtained.

In the present invention, preferably, in step two, a part of the experimental tumor cells is taken out, the experimental tumor cells are placed in another set of culture dishes, and a proper amount of proteolytic enzyme is added into the culture dishes to determine the arrangement order of the polypeptides in the tumor cells.

In the present invention, preferably, in the third step, a part of the tumor cells are taken out, the tumor cells are injected into the experimental mouse group through the needle tube, after the tumor cells proliferate, the experimental mouse group of the injected tumor cells is observed, and the experimental mouse group in which the tumor cells successfully proliferate is screened by comparing the behavior of the normal mouse group and the experimental mouse group.

In the present invention, preferably, in step three, when the characteristic expression condition of the experimental mouse population is observed, the external skin or subcutaneous tissue of the experimental mouse population generates massive tissues for successfully proliferating the tumor cells, and conversely, the tumor cells are inactivated in the experimental mouse population.

In the invention, preferably, in the fourth step, chloramine-T oxidant aqueous solution is prepared, 50ug/120ul of purified antibody is added into a centrifugal tube, 5ul of 0.01mol/L phosphate buffer solution is additionally added, 10ul of chloramine-T is added, 50ul of Na2S2O3 is added for 2min after 45S, 100ul of KI solution is added into the solution after two minutes, a proper protein carrier is selected, 2.5ul of mixed solution of 131I/125I and 2.5ul of 32S/35S is added into the last group in the same way, the solution is collected, the solution is placed into different reagent tubes, the solution in each reagent tube is ensured to be the same volume, and the solution in each reagent tube is subjected to the measurement of the ratio and the labeling rate.

In the present invention, preferably, in the fourth step, the labeled solution is introduced into a carrier protein, the protein carrier is injected into a population of experimental mice in which tumor cells are successfully proliferated, and after a certain period of time, the expression position of the labeled isotope is observed by a nuclear medicine imaging device.

In the present invention, preferably, in the fifth step, the result is displayed by an image, and the expression position of the isotope in the experimental mouse population is recorded.

In view of the above examples, it was found from the results of experiments that isotope expression could be detected in both the cell matrix and the cell membrane surface of the tumor cells of the group of laboratory mice injected with the mixed solution of 2.5ul131I/125I and 2.5ul32S/35S, isotope expression could be detected in the cell matrix and in the cell membrane of the tumor cells of the group of laboratory mice injected with the mixed solution of 5ul131I/125I, and isotope expression could be detected in the cell membrane of the tumor cells of the group of laboratory mice injected with the mixed solution of 5ul 131I/125I.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.