CN113030235B - Product and method for differential diagnosis and evaluation of thyroid cancer - Google Patents

Abstract

The invention discloses a product and a method for the differential diagnosis and evaluation of thyroid cancer. In the present invention, in the specific examples, by isolating IgG in body fluid and then releasing N-sugar chains on the IgG by glycosidase, and comparing the difference in the expression level of N-sugar chains on IgG in body fluid of thyroid cancer and a non-cancer control (healthy + benign nodules) using an analysis method based on mass spectrometry, it was found that the expression levels of 3N-sugar chains (H3N5F1, H4N5F1, H5N5F1) in plasma of thyroid cancer patients were significantly increased, respectively, while one of the N-sugar chains H4N4F1 was significantly decreased, as compared to the non-cancer control. The above-described differentially expressed IgG N-sugar chain can effectively distinguish thyroid cancer from non-cancer controls, while the combination of H3N5F1, H4N5F1, and H5N5F1 also has the potential to diagnose thyroid cancer at an early stage. The method has high sensitivity, easy acquisition of required blood samples, high accuracy when being used for the differential diagnosis of the thyroid cancer and important application value.

Description

Product and method for differential diagnosis and evaluation of thyroid cancer

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a product and a method for differential diagnosis and evaluation of thyroid cancer.

Background

Thyroid cancer is the most common malignancy of the endocrine system. In recent years, the incidence of the disease has increased remarkably. More than 19 thousands of thyroid cancer cases are newly added in China, and the health of the population of China is seriously threatened. Thyroid cancer is largely divided into four pathological types. More than 90% of the cancers are Papillary Thyroid Carcinomas (PTC). Epidemiological studies have shown that the palpation rate of thyroid nodules in women is about 5% and in men about 1% in the population. About 5-15% of patients with nodules have malignant lesions and need to be operated or treated in time, and the rest of benign nodules need to be followed up in a standardized way. In addition, although the overall prognosis of differentiated thyroid cancer mainly based on PTC is good, the proportion of foci obviously invading peripheral important structures is 6% -13% when the diagnosis is confirmed, the proportion of distant metastasis is 5.9% -23%, the 10-year survival rate is obviously reduced (26% -70%), and when the distant metastasis foci do not take up iodine after the operation (iodine is difficult to treat), the 10-year survival rate of the patient is less than 10%. Generally, the patients mostly belong to stage III and stage IV patients in the TNM stage (eighth edition) of differentiated thyroid cancer when the diagnosis is confirmed, and related literature reports show that early diagnosis and timely treatment can obviously improve the survival rate of malignant tumor patients. In summary, the judgment of thyroid nodules and the early diagnosis of thyroid cancer are one of the key clinical problems. Currently, there is no diagnostic marker for thyroid cancer in the clinic. Ultrasound and ultrasound-guided puncture are widely used for screening and diagnosing thyroid nodules, are important means for assessing the benign and malignant nodules before surgery, and have obvious defects or limitations, such as strong operation expertise, dependence on the experience of clinicians, invasiveness, reduced accuracy of judging tiny nodules, uncertain cytological results of 20-30% of nodules, and the like. Therefore, a more accurate and noninvasive diagnosis (auxiliary diagnosis) index is sought for judging the benign and malignant thyroid nodules and diagnosing the benign and malignant thyroid nodules at an early stage, so that the method has important significance for avoiding over-treatment of benign patients and enabling malignant patients to obtain definite diagnosis and treatment in time at an early stage of cancer without obvious symptoms. Furthermore, it is worth mentioning that advanced local, recurrent metastasis/iodine refractory PTC remains a difficult point in current clinical diagnosis. Clinical practice shows that the biomarker has important application value in the aspects of differential diagnosis of tumors, discussion of pathogenesis and the like, provides a new way for improving the sensitivity, specificity and accuracy of preoperative diagnosis, and has important potential in patient stratification, prognosis and individualized treatment. These will bring new eosins to the treatment and prognosis of refractory patients and will provide new keys to solve the above clinical problems.

Glycosylation is one of the most common and important post-translational modifications of proteins. Glycoprotein glycosylation is involved in many key physiological and pathological processes such as carcinogenesis, cancer progression, and cancer metastasis. Since sugar chains are involved in various processes associated with cancer (cell differentiation, adhesion, invasion, metastasis, cell signaling, etc.), abnormal glycosylation is considered as one of the hallmarks of cancer. A plurality of tumor markers clinically applied, such as CA125 (applied to ovarian cancer, endometrial cancer and the like), CA19-9 (applied to pancreatic cancer, esophageal cancer and the like), and the like are modified by sugar chains. The AFP kit for glycosylation (fucosylation) was approved by FDA in the united states in 2005 for clinical diagnosis of liver cancer. Interestingly, in addition to the possible changes in glycosylation of glycoproteins of cancer cell origin, the glycosylation of immunoglobulins (Igs) produced by B lymphocytes and of acute phase proteins synthesized by liver cells is also changed, and importantly, this change is detectable in the blood. Thus, sugar chains are potential biomarkers associated with systemic disorders in the humoral circulation of cancer patients. Therefore, a new way is provided for the discovery of the noninvasive serum tumor marker by analyzing the glycosylation spectrum of related glycoprotein in body fluid. The analysis of protein glycosylation is significant for finding thyroid cancer diagnosis (auxiliary diagnosis) markers and realizing accurate patient stratification to further realize individualized treatment. However, there is currently a lack of ways to assess whether thyroid cancer protein glycosylation can be used as a differential diagnosis study for thyroid cancer.

Disclosure of Invention

The invention provides a product and a method for the differential diagnosis and evaluation of thyroid cancer, and provides a new direction for the differential diagnosis and evaluation of thyroid cancer.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides application of a reagent and/or an instrument for detecting N-sugar chains on protein in a sample in preparation of products for diagnosing thyroid cancer and/or identifying early-stage thyroid cancer, wherein the N-sugar chains comprise one or more of H3N5F1, H4N5F1, H5N5F1 and H4N4F 1.

Further, the N-sugar chain is H3N5F 1.

Further, the N-sugar chain is H4N4F 1.

Further, the N-sugar chains are H3N5F1 and H4N5F 1.

Further, the N-sugar chains are H3N5F1 and H5N5F 1.

Further, the N-sugar chain is a combination of H3N5F1, H4N5F1 and H5N5F 1.

The term "sugar chain" refers to a compound formed of one or more unit sugars (monosaccharides and/or derivatives thereof) linked together. When two or more unit sugars are linked, each unit sugar is linked based on a glycosidic bond by dehydration condensation. Examples of the sugar chain include polysaccharides (hexose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, and complexes and derivatives thereof) present in organisms, and a large number of sugar chains degraded from or derived from complex biomolecules such as degraded polysaccharides, glycoproteins, proteoglycans, glycosaminoglycans, and glycolipids, but examples are not limited to those described. In a specific embodiment of the present invention, the sugar chain is a hexose, N-acetylglucosamine, or a compound formed by linking fucose.

The term "monosaccharide" refers to polyhydroxy aldehydes or polyhydroxy ketones and derivatives thereof which cannot be hydrolyzed to simpler molecules than those described and which contain at least one hydroxyl group and at least one aldehyde or ketone group. Monosaccharides are generally represented by the formula: c_nH_2nO_nBut is not limited thereto, and monosaccharides also include fucose (deoxyhexose), N-acetylglucosamine, and the like. The compounds of the above formula in which n is 2, 3, 4, 5, 6, 7, 8, 9 and 10 are referred to as a two-carbon sugar and a three-carbon sugar, respectivelySugars, four carbon sugars, pentoses, hexoses, heptoses, octoses, nonanoses, and decanoses. Generally, the compounds correspond to aldehydes or ketones of chain polyols, the former being referred to as aldoses and the latter as ketoses.

The term "derivative of a monosaccharide" refers to a substance resulting from the substitution of one or more hydroxyl groups on an unsubstituted monosaccharide with another substituent. Derivatives of the monosaccharides include, but are not limited to, saccharides having a carboxyl group (e.g., aldonic acids in which the C-1 position has been oxidized to carboxylic acids (e.g., D-gluconic acid derived from oxidized D-glucose), uronic acids in which the terminal C atom has been converted to carboxylic acids (D-glucuronic acid derived from oxidized D-glucose), saccharides having an amino group or a derivative of an amino group (e.g., acetylated amino group) (e.g., N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, etc.), saccharides having an amino group and a carboxyl group (e.g., N-acetylneuraminic acid (sialic acid), N-acetylmuramic acid (muramidacid), etc.), deoxysaccharides (e.g., 2-deoxy-D-ribose), sulfated saccharides containing a sulfate group, phosphorylated saccharides containing a phosphate group, etc., as used herein, when the compound is referred to as a monosaccharide, the above-mentioned derivatives are also included. Alternatively, for sugars that form hemiacetal structures, glycosides that form acetal structures by reaction with alcohols also fall within the scope of monosaccharides.

The N-sugar chain in the present invention means free NH bonded to aspartic acid of a protein₂Covalently linked glycans.

N-sugar chains have similar core structures, and are classified into three different types according to the difference in branching structure from the core structure: high mannose (High mannose), Complex (Complex), and Hybrid (Hybrid). High mannose means that, outside the core structure, only mannose is comprised; complex sugars mean that the branch structures outside the core structure all begin to extend with N-acetylglucosamine; heterozygote sugars are those in which the branches outside the core structure begin with N-acetylglucosamine and mannose, respectively. The hybrid and complex N-sugar chains may have two or more N-acetylglucosamine-containing branch structures, which are often referred to as antennas. In forming a multi-antenna N-sugar chain, N-acetylglucosamine residues are linked to trimannosyl groups by 6 different N-acetylglucosamine transferases (I-IV). In the N-sugar chain of certain vertebrate glycoproteins, up to 5 branches have been found. Among them, N-acetylglucosamine transferase III links N-acetylglucosamine to β -Man of the core pentasaccharide region, linked by β -1 → 4 bond, and is called bisecting N-acetylglucosamine, and N-sugar chains having such a structure are called bisecting N-sugar chains.

The sample of the present invention includes tissue, body fluid or excrement, and the body fluid includes blood, extracellular fluid, interstitial fluid, lymph fluid, cerebrospinal fluid or aqueous humor. In a preferred embodiment of the present invention, the body fluid is blood, and in a more preferred embodiment, the blood is plasma.

Further, the protein includes immunoglobulin.

The term "immunoglobulin" refers to a class of structurally related glycoproteins that are composed of two pairs of polypeptide chains, a pair of light (L) low molecular weight chains and a pair of heavy (H) chains, all four of which are intrinsically linked by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology Ch.7(Paul, W., eds. Raven Press, N.Y. (1989)). Briefly, each heavy chain is typically composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is typically composed of three domains, CH1, CH2, and CH 3. The heavy chains are linked by disulfide bonds in the so-called "hinge region". Each light chain is typically composed of a light chain variable region (abbreviated as VL in the present invention) and a light chain constant region. The light chain constant region is usually composed of one domain, CL. The VH and VL regions may be further subdivided into hypervariable regions (or hypervariable regions which are highly variable in sequence and/or structurally defined loop form), also known as Complementarity Determining Regions (CDRs), interspersed with more conserved regions, known as Framework Regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and leskj. mol. biol.196,901917 (1987)). Unless otherwise indicated or contradicted by context, the amino acids of the constant region Sequences of the present invention are numbered according to the EU-index (described in Kabat, E.A. et al, Sequences of proteins of immunologicalinterest, fifth edition-US Department of Health and Human Services, NIH publication No.91-3242, pp662,680,689 (1991)).

The immunoglobulins described herein include immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin E (IgE).

In a specific embodiment of the invention, the immunoglobulin is IgG.

Further, the reagent for detecting N-sugar chains on proteins in the sample comprises an antibody and a ligand.

The antibody of the present invention can be isolated from serum and the existing antibody can be screened using an N-sugar chain as an antigen using a conventional method. The antibody of the present invention can also be prepared by a conventional hybridoma method using an N-sugar chain as an antigen.

The antibody described in the present invention may use an antibody or a fragment thereof of any structure, size, immunoglobulin class, origin, etc., as long as it binds to a target protein. The antibodies or fragments thereof included in the assay products of the invention may be monoclonal or polyclonal. An antibody fragment refers to a portion of an antibody (partial fragment) or a peptide containing a portion of an antibody that retains the binding activity of the antibody to an antigen. Antibody fragments may include F (ab ') 2, Fab', Fab, single chain fv (scfv), disulfide-bonded fv (dsfv) or polymers thereof, dimerized V regions (diabodies), or peptides containing CDRs.

Further, the reagent and/or apparatus includes reagents and/or apparatus used in one or more of the following methods: immunoassay, mass spectrometry, liquid chromatography-mass spectrometry, sugar chip technology, or nuclear magnetic resonance.

Further, the immunoassay method includes an enzyme immunoassay method, an immunoturbidimetry assay method, a latex immunoagglutination method, an electrochemiluminescence method, and a fluorescence method.

The term "mass spectrometry" refers to converting particles such as atoms, molecules or clusters into gaseous ions (that is, ionizing) and allowing them to move in a vacuum, and separating and detecting the ions using an electromagnetic force according to a mass-to-charge ratio (m/z).The spectrum obtained based on the ions separated and detected according to m/z (horizontal axis represents m/z and vertical axis represents ion relative intensity) is a mass spectrum. Ions that provide information about molecular weight are often referred to as molecular weight-related ions (there are available: neutral molecules M that lose one electron to give M⁺(ii) a M obtained by adding one electron^-(ii) a Increase of proton derived [ M + H]⁺(ii) a By loss of proton, [ M-H ]]^-(ii) a By loss of negative hydrogen ion, [ M-H ]]⁺(ii) a By addition of alkali metals (e.g. Na) [ M + Na]⁺(ii) a Etc.). Depending on the sample or ionization method (especially in the EI method), molecular weight-related ions may never appear; however, in this case, the ions related to molecular weight can be confirmed by using a mild ionization method. Those ions that occur on the lower mass side compared to the molecular ions are referred to as fragment ions, and these fragment ions are degradation products of the molecular ions and provide structural information of the sample molecules. The ion having the highest ion intensity in the spectrum is referred to as a base peak, and the spectrum is normalized by the peak by taking the relative intensity as 100%.

Ionization methods in mass spectrometry include, but are not limited to:

1) electron ionization method (EI method)

The electron ionization method is an ionization method by bringing thermal electrons into contact with a vaporized sample, and it is the most popular ionization method. Since the ionization is performed after the sample is vaporized, it is necessary to previously vaporize a liquid or solid sample. Because heating is used in the gasification process, it is not possible to test thermally unstable or difficult to volatilize substances. However, tests can be performed if the material is capable of achieving volatility and thermal stability by derivatization, e.g., methylation, silanization, or acylation. Since ionization is generally performed with energy of 70eV, fragment ions are generated by excessive energy with generation of molecular ions (generally, ionization energy of an organic compound is about 12 eV). The information of these fragment ions enables structural analysis of the compound. However, molecular weight information is often not available because of the difficulty in obtaining molecular ions. In this case, the ionization energy must be reduced to about 20eV, or a milder ionization method (CI, DEI, DCI, FAB, or ESI) is selected.

2) Chemical ionization method (CI method)

The chemical ionization method is a method of ionizing a vaporized sample by feeding the sample into a reaction gas (reagent gas) which is ionized in advance. Since this method utilizes an ion-molecule reaction to effect ionization, the ionization energy is close to that of an organic compound, and thus the number of fragment ions is very small, whereas ions ((M + H) having molecular weight information)⁺、(M+NH₄)⁺、(M-H)^-Etc.) appear as reference ions. For the reaction gas, methane, isobutane or ammonia is generally used.

3) Desorption electron ionization method (DEI method)

The DEI method is a method of instantaneously heating in the vicinity of an electron beam to vaporize and ionize a sample before the sample is thermally degraded. Testing of thermally unstable or difficult to volatize substances is made possible. In addition, since molecular ions appear with stronger intensity as compared with the conventional method EI as direct introduction, molecular weight information is more easily obtained. The test method involved placing the sample solution on the tip of a sharp filament (platinum wire with a diameter of 100 μm), inserting the sharp filament into the ion source, and then rapidly heating the filament to vaporize the sample.

4) Desorption chemical ionization method (DCI method)

When the DEI operation is performed with the electron source in the CI state, the operation is DCI.

5) Fast atom bombardment method (FAB method)

The FAB method is a method of ionizing a sample by mixing the sample with matrix molecules thoroughly, placing the mixture on a target, and allowing fast neutral atoms such as Xe to be bombarded thereon. Unlike EI and CI, it does not require vaporization of the sample, and therefore, the method is suitable for testing for thermally unstable or difficult to volatilize substances. However, testing low molecular weight samples can be difficult in some cases because the background peak produced by the strong matrix appears in the lower mass region. The FRIT-FAB test is effective in this case.

6) FRIT-fast atom bombardment method (FRIT-FAB method)

The FRIT-FAB method is also called a continuous flow FAB, and is a method of continuously flowing a sample dissolved in a matrix solution and ionizing the sample by bombarding an outflow port with fast neutral atoms.

7) Electrospray ionization method (ESI method)

The ESI method is an atmospheric pressure ionization method which utilizes a phenomenon that a sample solution spontaneously undergoes spraying and ionization when a high voltage is applied to a capillary. Like FAB, it does not require vaporizing the sample, and therefore, the method is suitable for testing thermally unstable substances or hardly volatile substances. This makes it possible to test peptides and proteins having large molecular weights even for quadrupole rod types having a small mass range. In the conventional ESI test, since fragment ions having structural information cannot be obtained, while when a slightly higher voltage than that generally used is applied to the Capillary/separator-1 (Capillary/Skimmer-1), intracorporeal CID occurs, it makes it possible to test fragment ions having structural information.

8) Matrix-assisted laser desorption ionization method (MALDI method)

MALDI is a technique discovered by Tanaka et al and developed by Hillenkamp et al (Karas M., Hillenkamp, F., anal. chem.1988, 60, 2299-2301). In this method, a sample and a matrix solution are mixed, and then the mixed solution is dried to a solid on a target to be brought into a crystalline state. The ions generated by the sample, e.g., (M + H), can be generated by applying a large amount of energy to the substrate by pulsed laser irradiation⁺And (M + Na) + and desorption of ions produced by the matrix. Even if the sample is contaminated with a trace amount of phosphate buffer solution, Tris buffer solution, guanidine, etc., the analysis can be performed.

Further, the mass spectrometry comprises matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS), fast atom bombardment mass spectrometry or electrospray mass spectrometry.

MALDI-TOF MS is short for matrix-assisted laser desorption ionization time-of-flight mass spectrometry. MALDI-TOF (MS) Mass measurement was performed by MALDI based on the time of flight. When ions are accelerated at a constant acceleration voltage V, and when ion mass is designated as m, ion velocity is designated as V, ion charge number is designated as z, elemental charge is e, and flight time of the ions is t, m/z of the ions can be represented by the following formula:

m/z＝2eVt²/L²

in the MALDI-TOF test, KOMPACT MALDI II/III by Shimadzu/Kratos can be used. At the time of testing, reference may be made to the manufacturer's instructions. In the present specification, the irradiation energy of laser irradiation used in MALDI-TOF test is referred to as "dissociation energy".

Further, the mass spectrometry is matrix assisted laser desorption time-of-flight mass spectrometry.

Further, reagents used in the matrix-assisted laser desorption time-of-flight mass spectrometry comprise standard protein, a matrix and a homogenization reagent.

Further, the Standard protein comprises Calibration Standard II.

Further, the substrate comprises super-2, 5-dihydroxybenzoic acid.

Further, the homogenization reagent comprises ethanol.

In another aspect, the present invention provides a product for diagnosing thyroid cancer and/or identifying early stage thyroid cancer, which comprises a reagent and/or an apparatus for detecting N-sugar chains on a protein in a sample, wherein the N-sugar chains comprise one or more of H3N5F1, H4N5F1, H5N5F1, and H4N4F 1.

Further, the N-sugar chain is H3N5F 1.

Further, the N-sugar chain is H4N4F 1.

Further, the N-sugar chains are H3N5F1 and H4N5F 1.

Further, the N-sugar chains are H3N5F1 and H5N5F 1.

Further, the N-sugar chain is a combination of H3N5F1, H4N5F1 and H5N5F 1.

Further, the protein includes immunoglobulin.

Further, the immunoglobulin is IgG.

Further, the product may further comprise one or more reagents and/or instruments selected from the group consisting of:

(1) reagents and/or instruments for collecting and/or processing a sample;

(2) reagents and/or instruments for isolating and/or purifying proteins in a sample;

(3) reagents and/or instruments for separating, purifying and/or enriching N-sugar chains on proteins in a sample.

Further, the reagent and/or apparatus for separating and/or purifying proteins in a sample includes a reagent and/or apparatus used in separating proteins using any one of the following methods: protein purification column method, salting-out method, organic solvent precipitation method, polyethylene glycol displacement method, liquid chromatography, and affinity chromatography.

Further, the method is a protein purification column method.

Further, the reagents and/or instruments used in the protein purification column method comprise a protein purification column and a purified protein buffer solution.

Further, the Protein purification column comprises Protein A Spin Plate for IgG Screening.

Further, the purified protein buffer solution comprises a binding buffer solution and a leaching buffer solution.

Further, the reagent and/or the apparatus for separating, purifying and/or enriching N-sugar chains on proteins in a sample include reagents and apparatuses used in separating N-sugar chains from proteins using an enzymatic method or a chemical method.

Further, the reagent used in the enzymatic method includes glycosidase.

Further, the glycosidase is glycosidase PNGase F.

Further, the reagent and/or apparatus for separating, purifying and/or enriching N-sugar chains on proteins in a sample further comprises an N-sugar chain enriching material, a reagent for enriching N-sugar chains.

Further, the N-sugar chain-enriched material includes Porous Graphitized Carbon (PGC).

Further, the reagent for enriching N-sugar chain includes acetonitrile, water, trifluoroacetic acid.

Further, the reagent for enriching N-sugar chain is acetonitrile, water and trifluoroacetic acid.

Further, the ratio of the acetonitrile to the water to the trifluoroacetic acid is 80:19.9: 0.1.

Further, the ratio of the acetonitrile to the water to the trifluoroacetic acid is 25:74.95: 0.05.

Further, the reagent for enriching N-sugar chain is water and trifluoroacetic acid.

Further, the ratio of the water to the trifluoroacetic acid is 99.9: 0.1.

In another aspect, the present invention provides a system comprising:

(1) a thyroid cancer evaluation device comprising a control unit and a storage unit for identifying early stage thyroid cancer or evaluating whether a subject has thyroid cancer; and

(2) information communication terminal devices communicatively connected to each other, which provide data on the level of N-sugar chains on proteins in a sample from a subject;

wherein the control unit of the thyroid cancer evaluation device includes:

1) a data receiving unit that receives data regarding the level of N-sugar chains on proteins in the sample transmitted from the information communication terminal device;

2) a discrimination value calculation unit that calculates a discrimination value based on discrimination of the level of N-sugar chains on proteins in the sample received by the data reception unit and the level of N-sugar chains on proteins in the sample with the level of N-sugar chains on proteins stored in the storage unit as an explanatory variable;

3) a discrimination value criterion evaluation unit that evaluates the condition of thyroid cancer in the subject based on the discrimination value calculated by the discrimination value calculation unit; and

4) an evaluation result transmitting unit that transmits the evaluation result of the subject obtained by the discrimination value reference evaluation unit to the information communication terminal device;

further, the N-sugar chain comprises one or more of H3N5F1, H4N5F1, H5N5F1 and H4N4F 1.

Further, the N-sugar chain is H3N5F 1.

Further, the N-sugar chain is H4N4F 1.

Further, the N-sugar chains are H3N5F1 and H4N5F 1.

Further, the N-sugar chains are H3N5F1 and H5N5F 1.

Further, the N-sugar chain is a combination of H3N5F1, H4N5F1 and H5N5F 1.

Further, the protein includes immunoglobulin.

Further, the immunoglobulin is IgG.

Preferred and excellent effects of the present invention:

the invention discovers that the expression level of N-sugar chains is related to thyroid cancer, and the N-sugar chains comprise one or more of H3N5F1, H4N5F1, H5N5F1 and H4N4F 1. Thyroid cancer can be diagnosed and/or early stage thyroid cancer can be identified by detecting the expression level of the N-sugar chain.

The invention also discloses a system for diagnosing the thyroid cancer and/or identifying early thyroid cancer.

Drawings

FIG. 1 is a representative MALDI-TOF MS spectrum of an IgG N-chain: wherein, the graph A is an IgG N-sugar chain MALDI-TOF MS spectrogram of a thyroid benign nodule sample, the graph B is an IgG N-sugar chain MALDI-TOF MS spectrogram of a thyroid cancer sample, H in the graph represents hexose, N represents N-acetylglucosamine, and F represents fucose;

FIG. 2 is a boxplot of IgG N-sugar chains differentially expressed between thyroid cancer and non-cancer controls: wherein, a is a differential expression box plot of H4N4F1, B is a differential expression box plot of H3N5F1, C is a differential expression box plot of H4N5F1, D is a differential expression box plot of H5N5F1, and E is a bisected sugar chain differential expression box plot, in which;

FIG. 3 is a ROC curve for IgG N-sugar chain differentiation between thyroid cancer and non-cancer controls; wherein, the graph A is a ROC curve of a bisecting sugar chain for distinguishing thyroid cancer from a non-cancer control, the graph B is a ROC curve of H3N5F1 sugar chain for distinguishing thyroid cancer from a non-cancer control, the graph C is a ROC curve of H4N5F1 sugar chain for distinguishing thyroid cancer from a non-cancer control, the graph D is a ROC curve of H5N5F1 sugar chain for distinguishing thyroid cancer from a non-cancer control, and the graph E is a ROC curve of H4N4F1 sugar chain for distinguishing thyroid cancer from a non-cancer control;

fig. 4 is a box diagram showing the expression of IgG-bisected N-sugar chains in early thyroid cancer and non-cancer controls, wherein p-value <0.001, TC ═ thyroid cancer (thyroid cancer);

FIG. 5 is a ROC curve for IgG-typing N-sugar chains to distinguish early stage thyroid cancer from non-cancer controls.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. Those of ordinary skill in the art will understand that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.

Examples

First, experimental object and experimental material:

1. test object

400 Healthy Controls (HC), 300 Benign Thyroid Nodules (BTN), 400 Thyroid Carcinomas (TC). In the cases of thyroid cancer, the early stage thyroid cancer accounts for 45.0% and 180 cases.

2. Experimental Material

IgG protein purification column (protein A protein Plate for IgG Screening (96 wells)), purified IgG buffer solution (binding buffer solution, rinsing buffer solution), N-sugar chain-enriched material (porous graphitized carbon (PGC)), reagent for N-sugar chain enrichment (acetonitrile: water: trifluoroacetic acid (80:19.9:0.1), water: trifluoroacetic acid (99.9:0.1), acetonitrile: water: trifluoroacetic acid (25:74.95:0.05)), glycosidase, Standard protein (Calibration Standard II) used for detection, matrix (super-2, 5-dihydroxybenzoic acid (sDHB)), homogenization reagent (ethanol), and sugar chain liberating reagent (PNGase GaF).

Second, Experimental methods

1. Purification of IgG from plasma samples of differentially diagnosed subjects

(1) The purification column and the buffer solutions used (binding buffer solution and rinsing buffer solution) were equilibrated at room temperature for 30 minutes.

(2) Remove the cap at the bottom of the column, place the column on the wash plate, remove the cap at the top of the column, and add 200 μ L of binding buffer to each well for equilibration.

(3) The above assembly was centrifuged in a 96 well plate centrifuge at 1,000 Xg for 1 min. And (5) repeating the steps 2 and 3.

(4) To maintain the protein bound to the column at the best ionic strength and pH, 10. mu.L of each sample serum was diluted with 10. mu.L of binding buffer and the diluted sample serum was added to each well of the column.

(5) The purification column was incubated on a shaker for 30min with slow shaking.

(6) Centrifuging at 1,000 Xg for 1min, collecting centrifuged sample solution, and repeating the sample application for 1 or 2 times.

(7) The purification column was placed on a washing/collection plate, and 500. mu.L of the binding buffer solution was added to each well of the purification column, followed by centrifugation at 1,000 Xg for 1 min. This operation was repeated 3 times.

(8) For repeated use of the wash/collection plates, they were washed once with 70% ethanol and three times with ultrapure water, dried and stored for further use.

(9) Add 20. mu.L of binding buffer to each collection plate, place the purification column on the collection plate, and add 200. mu.L of elution buffer to each well, incubate slowly with shaking for 1min, and centrifuge at 1,000 Xg for 1 min. This step was repeated 2 times and the purified IgG protein was collected by determining in which collection the IgG was in the BCA kit.

(10) The purification column was regenerated by adding 400 μ L of elution buffer to each well of the column, washing 3 times, and then washing three times with 400 μ L of 0.02% sodium azide.

(11) Add 100. mu.L of sodium azide or conjugate buffer to each well of the column, cover it, and place it in a sealable bag for storage at 4 ℃.

2. Glycosidase treatment of IgG to free the sugar chains

(1) The 96-well plate was previously washed once with 70% ethanol and then with ultrapure water 2 times.

(2) Each purified IgG sample solution was mixed with glycosidase, and placed in each well of a 96-well plate in triplicate, and reacted at 37 ℃ for 12 hours.

3. PGC solid phase extraction purification of sugar chain

(1) The filterable 96-well plates were packed with graphitized carbon (1 mg per well).

(2) The graphitized carbon was activated with 0.1% trifluoroacetic acid 80% acetonitrile in water.

(3) 0.1% trifluoroacetic acid in water.

(4) After 150. mu.L of water was added to each well of a 96-well plate for enzymatic reaction, the plate was assembled with graphitized carbon, centrifuged, and loaded. The loading was repeated three times to maximize the binding of the sugar chain to the graphitized carbon.

(5) The impurities and salts were washed with 100. mu.L of water and the washing was repeated 2 times.

(6) The sugar chains were eluted with 0.05% trifluoroacetic acid in 25% acetonitrile in water and the eluted solution was collected for analytical testing.

4. Quantitative analysis by mass spectrometry

And (3) spotting the graphitized carbon enriched N-sugar chain solution and the Calibration solution (Calibration Standard II) in the step (3) on a mass spectrum target plate, airing at room temperature, spotting a matrix (super-2, 5-dihydroxybenzoic acid) on each spot to crystallize the sample, redissolving the sample by ethanol after airing to homogenize the sample, and detecting a sugar chain signal by using MALDI TOF mass spectrum to quantitatively analyze each IgG N-sugar chain.

The mass spectrum is provided with a Smartbeam 3D laser source, signal ions are collected in a positive ion Reflection (RP) mode, FlexControl software is used for controlling, and the m/z range is set as follows during sample detection: 1000 to 3500. The spectrogram acquisition is set as follows: for each sample point on the mass spectrum target plate, the laser completely randomly acquires signals within the range of the sample point, 10K laser shots are accumulated, and a mass spectrum is acquired, wherein the laser frequency is 5000 Hz.

The collected mass spectra were pre-processed using FlexAnalysis and MassyTools software and exported to Microsoft Excel for further analysis. The mass spectrum data is analyzed by sugar chain analysis function auxiliary artificial analysis of GlycoWorkBench, and the identification of the sugar chain structure is mainly based on mass-to-charge ratio, secondary mass spectrum fragment attribution and published documents. The individual sugar chain quantification was obtained from the peak area of the individual sugar chain/the peak area of all sugar chains detected. Differences in N-glycosylation between groups were evaluated by statistical tests, receiver operating characteristic curve (ROC curve). The mass spectrum data quality of the research queue is evaluated by the standard substances randomly distributed on the target plate in the sample detection process, and the average value, the variation coefficient and the standard deviation of each sugar chain of a plurality of obtained standard substances are calculated.

5. Analysis of Experimental data

The difference in the expression level of IgG N-sugar chains in the samples of both groups of thyroid cancer and non-cancer control (healthy + benign nodules) was compared by statistical test, and ROC curve analysis was performed. The diagnostic indicator is considered "highly accurate" when the area-under-the-curve (AUC) ≧ 0.9, is considered "accurate" when 0.8 ≦ AUC <0.9, and is considered "relatively accurate" when 0.7 ≦ AUC < 0.8.

Third, experimental results

1. Thyroid cancer vs. non-cancer control

Compared to benign nodules, H4N4F1 on IgG was significantly reduced in thyroid cancer (as shown in fig. 2A, where P <0.01), 3 sugar chains H3N5F1, H4N5F1, and H5N5F1 (all belonging to bisected sugar chains) were significantly increased in thyroid cancer (as shown in fig. 2B, 2C, and 2D, where P is 0.004 in fig. 2B, P is <0.001 in fig. 2C, and P is 0.001 in fig. 2D), respectively, while bisected sugar chains, i.e., H3N5F1+ H4N5F1+ H5N5F1, were also significantly increased in thyroid cancer (as shown in fig. 2E, where P is < 0.001). Compared to healthy controls, H4N4F1 on IgG was significantly reduced in thyroid cancer, and H3N5F1, H4N5F1, H5N5F1, and H3N5F1+ H4N5F1+ H5N5F1 were significantly increased in thyroid cancer (all p values <0.01, fig. 2). While IgG N-sugar chains did not differ significantly between healthy and benign thyroid nodules (as shown in figure 2). In the sugar chain, H represents hexose, N represents N-acetylglucosamine, and F represents fucose.

The results of ROC curve analysis showed that AUC for H4N4F1, H3N5F1, H4N5F1 and H5N5F1 were 0.883 (95% CI: 0.790-0.976), 0.782 (95% CI: 0.656-0.909), 0.894 (95% CI: 0.802-0.987), 0.766 (95% CI: 0.638-0.895), respectively, when differentiating thyroid adenocarcinoma from non-cancerous controls. In contrast, the bisecting sugar chain combination (H3N5F1+ H4N5F1+ H5N5F1) distinguished the thyroid adenocarcinoma from the non-cancerous control by an AUC of 0.921 (95% CI: 0.848-0.992), a sensitivity of 88% and a specificity of 80%. (see FIG. 3, Table 1)

TABLE 1 diagnostic Performance data

Sugar chain	AUC (accuracy)	Sensitivity of the probe	Degree of specificity
				H4N4F1	0.883(95％CI:0.790–0.976)	88％	80％
H3N5F1,	0.782(95％CI:0.656–0.909)	64％	88％
				H4N5F1	0.894(95％CI:0.802–0.987)	80％	96％
H5N5F1	0.766(95％CI：0.638–0.895)	72％	68％
				H3N5F1+H4N5F1+H5N5F1	0.920(95％CI:0.848–0.992)	88％	80％

In conclusion, the four sugar chains H4N4F1, H3N5F1, H4N5F1 and H5N5F1 and the bisected sugar chain combination (H3N5F1+ H4N5F1+ H5N5F1) have the potential as markers for the differential diagnosis of thyroid cancer.

2. Identification of early stage thyroid cancer

As shown in fig. 4, bisected sugar chains on IgG, i.e., H3N5F1, H4N5F1, H5N5F1, in combination, were significantly elevated in early stage thyroid cancer (P <0.001) compared to non-cancer controls, indicating that IgG glycosylation abnormalities had occurred in early stages of cancer.

As shown in FIG. 5, the results of ROC curve analysis showed that the AUC for bisecting the sugar chain to distinguish early thyroid cancer from non-cancer control was 0.896 (95% CI: 0.824-0.968), the sensitivity was 80%, and the specificity was 87%. The experimental result suggests that bisected sugar chains have the potential to diagnose early thyroid cancer.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims (28)

1. Use of a reagent and/or an apparatus for detecting N-sugar chains on a protein in a sample for the preparation of a product for diagnosing thyroid cancer and/or identifying early stage thyroid cancer, wherein the protein comprises immunoglobulin IgG and the N-sugar chains comprise H3N5F1, H4N4F1, a combination of H3N5F1 and H4N5F1, a combination of H3N5F1 and H5N5F1, or a combination of H3N5F1, H4N5F1 and H5N5F1, wherein H represents hexose, N represents N-acetylglucosamine, and F represents fucose.

2. The use according to claim 1, wherein the reagent for detecting N-sugar chains on proteins in the sample comprises an antibody and a ligand.

3. Use according to claim 1 or 2, wherein the reagents and/or apparatus comprise reagents and/or apparatus for use in one or more of the following methods: immunoassay, mass spectrometry, liquid chromatography-mass spectrometry, sugar chip technology, or nuclear magnetic resonance.

4. The use of claim 3, wherein said immunoassay comprises an enzyme immunoassay, an immunoturbidimetry assay, a latex immunoagglutination assay, an electrochemiluminescence assay or a fluorescence assay.

5. The use of claim 4, wherein said mass spectrometry comprises matrix assisted laser desorption time-of-flight mass spectrometry, fast atom bombardment mass spectrometry or electrospray mass spectrometry.

6. The use of claim 5, wherein said mass spectrometry is matrix-assisted laser desorption time-of-flight mass spectrometry.

7. The use of claim 6, wherein the reagents used in matrix-assisted laser desorption time-of-flight mass spectrometry comprise standard proteins, a matrix, and a homogenization reagent.

8. The use according to claim 7, wherein the Standard protein comprises Calibration Standard II.

9. The use of claim 7, wherein said substrate comprises super-2, 5-dihydroxybenzoic acid.

10. The use of claim 7, wherein the homogenization reagent comprises ethanol.

11. Use according to claim 1, characterized in that the product further comprises one or more reagents and/or instruments selected from the group consisting of:

reagents and/or instruments for collecting and/or processing a sample;

reagents and/or instruments for isolating and/or purifying proteins in a sample;

reagents and/or instruments for separating, purifying and/or enriching N-sugar chains on proteins in a sample.

12. The use according to claim 11, wherein the reagents and/or apparatus for separating and/or purifying proteins in a sample comprise reagents and/or apparatus for use in separating proteins using any one of the following methods: protein purification column method, salting-out method, organic solvent precipitation method, polyethylene glycol displacement method, liquid chromatography, and affinity chromatography.

13. The use according to claim 12, wherein the method is a protein purification column.

14. The use of claim 13, wherein the reagents and/or apparatus used in the protein purification column method comprise a protein purification column, a purified protein buffer solution.

15. The use of claim 14, wherein the Protein purification column comprises Protein a Spin Plate for IgG Screening.

16. The use of claim 14, wherein the purified protein buffer solution comprises a binding buffer solution and a washing buffer solution.

17. The use according to claim 11, wherein the reagent and/or apparatus for separating, purifying and/or enriching N-sugar chains on a protein in a sample comprises a reagent and apparatus used in separating N-sugar chains from a protein by an enzymatic method or a chemical method.

18. The use according to claim 17, wherein the reagent used in the enzymatic process comprises a glycosidase.

19. The use of claim 18, wherein the glycosidase is the glycosidase PNGase F.

20. The use according to claim 17, wherein the reagent and/or apparatus for separating, purifying and/or enriching N-sugar chains on proteins in a sample further comprises an N-sugar chain-enriching material, a reagent for N-sugar chain enrichment.

21. The use according to claim 20, wherein said N-sugar chain-enriched material comprises Porous Graphitized Carbon (PGC).

22. The use according to claim 20, wherein the reagent for enriching N-sugar chain comprises acetonitrile, water, trifluoroacetic acid.

23. The use according to claim 22, wherein the reagent for N-sugar chain enrichment is acetonitrile, water, and trifluoroacetic acid.

24. The use according to claim 23, wherein the ratio of acetonitrile, water and trifluoroacetic acid is 80:19.9: 0.1.

25. The use according to claim 23, wherein the ratio of acetonitrile, water and trifluoroacetic acid is 25:74.95: 0.05.

26. The use according to claim 20, wherein the reagent for N-sugar chain enrichment is water and trifluoroacetic acid.

27. The use according to claim 26, wherein the ratio of water to trifluoroacetic acid is 99.9: 0.1.

28. A system for identifying early stage thyroid cancer or assessing whether a subject has thyroid cancer, comprising:

(1) a thyroid cancer evaluation device comprising a control unit and a storage unit for identifying early stage thyroid cancer or evaluating whether a subject has thyroid cancer; and

(2) an information communication terminal device communicatively connected to the thyroid cancer evaluation device to each other, which provides data on the level of N-sugar chains on proteins in a sample from a subject;

wherein the control unit of the thyroid cancer evaluation device includes:

1) a data receiving unit that receives data regarding a level of N-sugar chains on proteins in the sample transmitted from the information communication terminal device;

2) a discrimination value calculation unit that calculates a discrimination value based on discrimination of the level of N-sugar chains on the protein in the sample received by the data reception unit and the level of N-sugar chains on the protein in the sample stored in the storage unit as an explanatory variable;

3) a discrimination value criterion evaluation unit that evaluates the condition of thyroid cancer in the subject based on the discrimination value calculated by the discrimination value calculation unit; and

4) an evaluation result transmitting unit that transmits the evaluation result of the subject obtained by the discrimination value reference evaluation unit to the information communication terminal device;

the protein comprises immunoglobulin IgG; the N-sugar chain comprises H3N5F1, H4N4F1, a combination of H3N5F1 and H4N5F1, a combination of H3N5F1 and H5N5F1, or a combination of H3N5F1, H4N5F1 and H5N5F1, wherein H represents hexose, N represents N-acetylglucosamine, and F represents fucose.

Images