Application of triiodothyroxine autoantibody as thyroid dysfunction marker in cancer treatment process

Abstract

The invention relates to the technical field of clinical detection, in particular to application of a triiodothyroxine autoantibody as a thyroid dysfunction marker in the cancer treatment process. The method can help a clinician to judge the real thyroid function state of the patient, and improves the diagnosis accuracy.

Description

Technical Field

[0001] The present disclosure relates generally to the field of clinical detection technology, and more particularly to the use of triiodothyronine autoantibodies as markers of thyroid dysfunction during cancer treatment. Background Art

[0002] The thyroid gland is an important organ of the endocrine system. Thyroid dysfunction will affect the normal functioning of the patient's endocrine system, and thyroxine plays an important regulatory role in the body's cardiovascular and metabolic aspects. In recent years, with the changes in people's lifestyles and eating habits, relevant studies have shown that the incidence of patients with thyroid dysfunction has been increasing year by year. When patients have thyroid dysfunction, it will have an adverse effect on the patient's own physical condition and quality of life.

[0003] Clinically, during the diagnosis and treatment of cancer, it is common for thyroid function test results to be inconsistent with clinical symptoms or for TSH, free triiodothyronine (FT3), and free thyroxine (FT4) to change irregularly. This phenomenon has a great negative impact on the doctor's clinical judgment, which in turn will have an adverse effect on the patient's treatment. If these interfering factors can be eliminated, it will help clinicians to more accurately judge the patient's true thyroid function level. Summary of the invention

[0004] This disclosure covers the following technical solutions:

[0005] The purpose of the present disclosure is to provide a detection agent that specifically binds to triiodothyronine autoantibodies (T3-Ab) for use in preparing a kit for diagnosing abnormal thyroid function during cancer treatment.

[0006] Another object of the present disclosure is to provide a kit for diagnosing abnormal thyroid function during cancer treatment, which is the kit defined in the above application.

[0007] The inventors of the present disclosure unexpectedly discovered in the clinic that thyroid dysfunction side effects occurred during tumor treatment and speculated that it was related to T3-Ab. In particular, in some preferred schemes, it is also strongly correlated with thyroid dysfunction during the treatment of tumors with immune checkpoint inhibitors. Therefore, the present disclosure can help clinicians determine the true thyroid function status of patients and improve diagnostic accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0009] Figure 1 is the linear function fitted by the T3-Ab kit;

[0010] Figure 2 is the linear function fitted by the T4-Ab kit;

[0011] Figure 3 This is the distribution diagram of T3-Ab levels in patients receiving ICI tumor treatment;

[0012] Figure 4 This is the curve detected using T3-Ab alone as a marker;

[0013] Figure 5 This is the curve of using T3-Ab and T4-Ab as combined markers for detection;

[0014] Figure 6 This is the curve using T3-Ab, FT3, FT4 and TSH as combined markers;

[0015] Figure 7 This is the curve using T3-Ab, T4-Ab, FT3, FT4 and TSH as the combined index;

[0016] Figure 8 is the curve using FT3, FT4, and TSH as combined markers;

[0017] Fig. 9 To monitor the changing trend of T3-Ab levels during ICI tumor treatment. DETAILED DESCRIPTION

[0018] References to embodiments of the present disclosure will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present disclosure. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present disclosure without departing from the scope or spirit of the present disclosure. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0019] Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present disclosure is the same as that commonly understood by those of ordinary skill in the art to which the present disclosure belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present disclosure. The terms used herein in the specification of the present disclosure are only for the purpose of describing specific embodiments and are not intended to limit the present disclosure.

[0020] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0021] The terms "and / or", "or / and", and "and / or" used herein have a selection range that includes any one of two or more related listed items, and also includes any and all combinations of related listed items, wherein the any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in the present disclosure, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0022] As used in this disclosure, the terms "comprising," "including," and "including" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0023]

[0013] Numerical ranges recited in this disclosure as endpoints include all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0024] As used herein, reference to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, description referring to "about X" includes description of "X".

[0025] The present disclosure relates to concentration values, and the meaning includes fluctuations within a certain range. For example, fluctuations can be allowed within a corresponding precision range. For example, 2%, a fluctuation within the range of ±0.1% can be allowed. For values ​​that are large or do not require too fine control, the meaning is also allowed to include greater fluctuations. For example, 100mM, fluctuations within the range of ±1%, ±2%, ±5%, etc. can be allowed. Involving molecular weight, the meaning is allowed to include fluctuations of ±10%.

[0026] As used herein, the singular articles "a," "an," and "the" include plural referents unless otherwise indicated.

[0027] In the present disclosure, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0028] In the present disclosure, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0029] In the present disclosure, "preferred", "better", "more preferred", and "suitable" are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of the present disclosure. In the present disclosure, "optionally", "optional", and "optional" mean that they are optional, that is, they refer to any one of the two parallel schemes of "yes" or "no". If multiple "optional" items appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "optional" is independent of each other. In the present disclosure, "further", "further", "especially", etc. are used for descriptive purposes to indicate differences in content, but should not be understood as limitations on the scope of protection of the present disclosure.

[0030] As used herein, the term "diagnosis" is used in its broadest sense and includes diagnosis, prognosis, treatment, and monitoring of thyroid dysfunction during cancer treatment. Diagnosis can be accompanied by a determination of the severity of the thyroid dysfunction.

[0031] As used herein, the term "treat" and variations thereof or "ameliorate" refer to therapeutic treatment, wherein the objective is to reverse, alleviate, ameliorate, inhibit, slow or stop the progression or severity of a disease (e.g., cancer), its associated conditions and / or symptoms. The term "treat" includes reducing or alleviating at least one adverse effect or symptom of a disease (e.g., cancer).

[0032] Herein, the "second" and "third" in "second detection agent" and "third detection agent" are only used for descriptive purposes to distinguish between the same (or similar) things or features, and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "second", "third", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0033] As used herein, "autoantibodies" refer to antibodies produced by a subject's immune system that are directed against and specifically bind to its "self-antigens," "self-antigens," or "antigenic epitopes."

[0034] As used herein, the term "specific binding" refers to a higher affinity of a binding molecule for a target molecule than the affinity of the binding molecule for a non-target molecule. A binding molecule that specifically binds to a target molecule does not substantially recognize or bind to a non-target molecule, e.g., an antibody "specifically binds" and / or "specifically recognizes" another molecule, meaning that the interaction is dependent on the presence of a binding specificity in a molecular structure, e.g., an antigenic epitope.

[0035] All documents mentioned in the present disclosure are cited as references in the present disclosure, just as each document is cited as a reference individually. Unless they conflict with the invention objectives and / or technical solutions of the present disclosure, the cited documents involved in the present disclosure are cited with all their contents and all their purposes. When the present disclosure involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present disclosure involves cited documents, the examples and preferred methods of the cited relevant technical features may also be incorporated into the present disclosure as references, but are limited to the implementation of the present disclosure. It should be understood that when the cited content conflicts with the description in the present disclosure, the present disclosure shall prevail or be modified adaptively according to the description of the present disclosure.

[0036] The present disclosure relates to the use of a detection agent that specifically binds to triiodothyronine autoantibodies in the preparation of a kit for diagnosing thyroid dysfunction during cancer treatment.

[0037] The present disclosure discovers for the first time that triiodothyronine autoantibodies can be used as markers for abnormal thyroid function during cancer treatment, and therefore proposes the above application.

[0038] In the present disclosure, unless otherwise stated, the terms "marker", "biochemical marker" or "thyroid dysfunction marker" refer to "triiodothyronine autoantibodies" or fragments thereof that can be specifically detected, according to the general understanding of those skilled in the art, and their meanings can be interchangeable depending on the context.

[0039] This marker can be used to diagnose thyroid dysfunction during cancer treatment. This diagnostic test or method often provides information that is critical to the safe and effective use of the corresponding drug or biological product, and can help healthcare professionals determine whether the benefits of a particular oncology treatment product to the patient will outweigh any potential serious side effects or risks.

[0040] The object detected by the kit provided by the present disclosure is a biological sample. The terms "biological sample", "sample", etc. are used in the broadest sense herein and are usually obtained for subjects. The subject is an animal, preferably at least including mammals, such as primates, including humans. The biological sample is usually obtained from a patient. The sample can be any biological tissue or fluid that can measure the biomarker of the present disclosure, and the sample usually contains triiodothyronine autoantibodies. Such samples include but are not limited to cells that may or may not contain cells, and preferred biological samples are blood, serum or plasma. Such samples also include biopsies (such as thyroid biopsies). The term biological sample also includes any material derived by processing a biological sample. Derived materials include but are not limited to cells (or their descendants) separated from a sample or proteins extracted from a sample. The processing of a biological sample may involve one or more of the following: filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, etc. For example, a biological sample is mixed with at least one component such as a diluent, a preservative, a stabilizer, a buffer, etc. to form a test sample.

[0041] In some embodiments, the subject of the cancer treatment has undergone or is undergoing treatment with an immune checkpoint inhibitor (ICI). ICI activates the body's immune system by intervening in immune checkpoints and enhances the body's anti-tumor ability. However, an overactivated immune system may attack non-tumor tissues and cause immune-related adverse events (irAEs). Although the occurrence of irAEs often indicates that ICI treatment is effective, uncontrollable progression of damage may lead to interruption of ICI treatment. Studies have shown that among the endocrine system AEs related to ICI treatment, such as anti-PD-1 treatment, thyroid immune damage occurs most frequently, including thyrotoxicosis and hypothyroidism. However, since the mechanism of thyroid damage induced by ICI treatment is currently unclear, currently only hormone replacement rather than etiological treatment is given to thyroid irAEs in clinical practice. The inventors accidentally discovered in clinical practice that the side effects of abnormal thyroid function during tumor ICI treatment were related to T3-Ab, so the value of T3-Ab in tumor ICI treatment has far-reaching significance for guiding clinical treatment.

[0042] The term "checkpoint inhibitor" as used herein refers to an agent that inhibits immune checkpoint proteins or pathways to stimulate or promote the body's anti-tumor response. Preferred checkpoint inhibitors of the present disclosure include inhibitors for one or more of the following targets: CTLA-4, PD-1, PD-L1, B7-H3, IDO, KIR, LAG3, TIM-3, TIGIT, VISTA, CD47, GITR, OX40, ADAR1, A2AR, SIRPα, and TLR. Immune checkpoint inhibitors can be commercially available drugs, and are usually antibodies, and therefore further preferred are ipilimumab targeting the CTLA-4 target; at least one of nivolumab, pembrolizumab, cemiplimab, toripalimab, sintilimab, camrelizumab, tislelizumab, zimberelimab, prolgolimab, and dostarlimab targeting the PD-1 target; at least one of atezolizumab, durvalumab, and avelumab targeting the PD-L1 target; and relatlimab targeting the LAG-3 target.

[0043] In some embodiments, the triiodothyronine autoantibody is selected from IgG and / or IgM antibodies. IgG referred to in the present disclosure includes its various subtypes, for example, for samples of human origin, IgG includes IgG1, IgG2, IgG3 and IgG4. IgM referred to in the present disclosure includes its various subtypes, for example, for samples of human origin, IgM includes IgM1 and IgM2.

[0044] In some embodiments, the cancer is caused by any one or more of bone, bone connection, muscle, lung, trachea, heart, spleen, artery, vein, blood, capillary, lymph node, lymphatic vessel, lymph fluid, oral cavity, pharynx, esophagus, cardia, stomach, duodenum, small intestine, colon, rectum, anus, appendix, liver, gallbladder, pancreas, parotid gland, sublingual gland, urinary kidney, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina, vulva, scrotum, testis, vas deferens, penis, eye, ear, nose, tongue, skin, brain, brainstem, medulla oblongata, spinal cord, cerebrospinal fluid, nerve, thyroid gland, parathyroid gland, adrenal gland, pituitary gland, pineal gland, pancreatic islet, thymus gland, gonad, sublingual gland and parotid gland. In some embodiments, the cancer is caused by any one or more of lung cancer, gastric cancer, esophageal cancer, liver cancer, colon cancer, gallbladder cancer and cardia cancer.

[0045] As a method for measuring the marker of the present disclosure, for example, any method can be applied as long as it is a method for specifically measuring triiodothyronine autoantibodies. Preferably, an immunoassay method is used.

[0046] The immunoassay method is not particularly limited, and examples thereof include various enzyme immunoassays, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence (IIF), double monoclonal antibody sandwich immunoassay, polyclonal antibody sandwich immunoassay, immunostaining, immunofluorescence, protein blotting, biotin-avidin method, immunoprecipitation, colloidal gold agglutination, immunochromatography, latex agglutination (LA), immunoturbidimetry (TIA), chemiluminescence immunoassay, immunocolloidal gold method, immunospot method, and the like.

[0047] The general features of immunoassays (e.g., ELISA, radioimmunoassay, etc.) are well known to those skilled in the art (see Immunoassay, E.Diamandis and T.Christopoulus, Academic Press, Inc., SanDiego, CA, 1996, the contents of which are incorporated herein by reference). Immunoassays for detecting autoantibodies with specific immunological specificity generally require the use of reagents (antigens) that exhibit specific immunological reactivity to the relevant autoantibodies. Depending on the form of the assay, the antigen may be fixed on a solid support. The test sample is contacted with the antigen, and if there are autoantibodies with the desired immunological specificity in the sample, the autoantibodies will react immunologically with the antigen to form an antigen / autoantibody complex, which can then be detected or quantitatively measured. Immunoassays for detecting autoantibodies according to the present disclosure may be based on standard techniques known in the art. The detection of autoantibodies may be carried out in any suitable form, preferably in a form that enables contact between a sample suspected of containing autoantibodies and the antigen. Examples of using antigens to detect autoantibodies are given in the examples herein.

[0048] In some preferred embodiments, the immunoassay is based on chemiluminescence.

[0049] In some embodiments, the detection agent comprises triiodothyronine antigen.

[0050] In some embodiments, the triiodothyronine antigen is conjugated to a solid support. Exemplary solid supports include, but are not limited to, filters, membranes, beads (e.g., magnetic or fluorophore-labeled beads), platelets, silicon wafers, glass, metals, plastics, chips, mass spectrometry targets, or substrates. In some embodiments, the solid support is a bead. In some embodiments, the bead is a microsphere. In the present disclosure, the term "microsphere" may be a sphere, a near-sphere, a cube, a polyhedron, or an irregular shape. The diameter of the microsphere is preferably 10 nm to 1 mm, for example 100 nm, 500 nm, 1 μm, 10 μm, 100 μm, 500 μm; preferably 400 nm to 10 μm.

[0051] In some embodiments, the kit further comprises an antibody that can specifically bind to the Fc segment of triiodothyronine autoantibody.

[0052] In some embodiments, the antibody capable of specifically binding to the Fc portion of the triiodothyronine autoantibody is labeled with a signal substance.

[0053] Any detectable signal substance for antibody labeling known in the art can be used in the present disclosure, and exemplary signal substances are fluorescent substances, quantum dots, digoxin-labeled probes, biotin, radioactive isotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense substances, chemiluminescent markers, ultrasound contrast agents, photosensitizers, colloidal gold or enzymes. In some preferred embodiments, the signal substance is a chemiluminescent agent. In some preferred embodiments, the signal substance is at least one of luminol and its derivatives, isoluminol and its derivatives, lucigenin, lophanine, peroxy oxalate esters, and acridinium esters. In some specific embodiments, the signal substance is ABEI.

[0054] In some embodiments, the kit further comprises one or more of triiodothyronine autoantibody standards, reaction buffer, water, and chemiluminescent substrate solution.

[0055] In some preferred embodiments, the detection reagent further comprises a second detection reagent for tetraiodothyronine autoantibodies.

[0056] Compared with T3-Ab detection alone, using T3-Ab and T4-Ab as a combined marker to detect thyroid dysfunction in cancer treatment, such as ICI treatment, can greatly improve the sensitivity and specificity of detection. Figure 5 and its related description, in this embodiment, the sensitivity is 86.9% and the specificity is 71.9%.

[0057] In some embodiments, the tetraiodothyronine autoantibodies are selected from IgG and / or IgM type antibodies.

[0058] In some preferred embodiments, the detection reagent further comprises a third detection agent for quantitatively determining at least one of FT3, FT4 and TSH. That is, for example, the detection object of the detection reagent comprises at least one of triiodothyronine autoantibodies, FT3, FT4 and TSH; or the detection object of the detection reagent comprises at least one of triiodothyronine autoantibodies, tetraiodothyronine autoantibodies, FT3, FT4 and TSH.

[0059] Compared with T3-Ab detection alone, or T3-Ab and T4-Ab detection together, or FT3, FT4 and TSH as combined markers, adding more detection markers can further improve the detection effect. Figure 6 , Figure 7 and related descriptions thereof; in these embodiments, when T3-Ab, FT3, FT4 and TSH are used as combined markers, the sensitivity is 88.9% and the specificity is 72.1%; when T3-Ab, T4-Ab, FT3, FT4 and TSH are used as combined markers, the sensitivity is 94.4% and the specificity is 82.7%.

[0060] According to yet another aspect of the present disclosure, it also relates to a kit for diagnosing abnormal thyroid function during cancer treatment, which is the kit defined in the application described above.

[0061] The term "kit" in the present disclosure may refer to any article (e.g., package or container) comprising at least one device, which includes a detection agent as described in the present disclosure. In some embodiments, the kit may further include instructions for use used in the method described in the present disclosure or its steps, preferably also include supplementary reagents and / or components or components.

[0062] The components in the kit can be packaged separately or in combination as needed. The components in the kit can be packaged in the form of a solution, a solid or a test strip. In some embodiments, at least one component in the kit is a solid, and the solid includes at least one of freeze-dried microspheres, freeze-dried cakes, freeze-dried powders, and spots that depend on the presence of a solid medium. Therefore, the components required for autoantibody detection, particularly various enzymes, antibody components, and reaction buffer components, etc., can be provided in a freeze-dried form.

[0063] According to yet another aspect of the present disclosure, a method for diagnosing thyroid dysfunction during cancer treatment comprises determining the level of triiodothyronine autoantibodies in a patient sample during cancer treatment, and comparing the determined value with a reference value.

[0064] In the context of the present disclosure, the term "reference value" refers to the test result from a control sample. When used to characterize a subject, it refers to a patient who has not seen abnormal thyroid function during cancer treatment, such as ICI treatment. The term "control sample" refers to one or more samples obtained from a patient who has not seen abnormal thyroid function during cancer treatment, such as ICI treatment. In certain embodiments, patients who have not seen abnormal thyroid function during cancer treatment, such as ICI treatment, have similar parameters such as gender, age, and / or body mass index, etc., compared to the subject from whom the biological sample to be tested was obtained. Exemplary reference values ​​can be determined according to the cut-off value in Example 3 of the present disclosure.

[0065] In some embodiments, the level of triiodothyronine autoantibodies in a sample from a patient during cancer treatment is measured using an immunoassay, preferably a chemiluminescent assay.

[0066] In some typical embodiments, the method comprises:

[0067] 1) contacting a patient sample from a cancer treatment process with a detection agent that specifically binds to triiodothyronine autoantibodies under conditions that allow for the formation of a complex;

[0068] 2) adding a substance with a detectable label, which can specifically recognize the complex formed in step 1);

[0069] 3) quantifying the detectable labeled signal to obtain the amount of triiodothyronine autoantibodies.

[0070] As is well known to those skilled in the art, the ideal scenario for diagnosis is a situation in which a single event or process causes various diseases, for example, in infectious diseases. In all other cases, correct diagnosis may be very difficult, especially when the etiology of the disease is not fully understood. As will be understood by those skilled in the art, for a given multifactorial disease, the diagnosis without biochemical markers is 100% specific and 100% sensitive. Determine whether the subject sample is compared with the normal control sample to determine whether the thyroid dysfunction during the cancer treatment process can be performed using statistical methods known in the art, and confirmed using confidence intervals and / or p values. In some embodiments, the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% or 99.99% and the p value is 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001 or 0.0001. In addition, additional biochemical markers can be used to participate in the assessment of the presence or severity of the disease to improve the accuracy of the assessment; in fact, in conventional clinical diagnosis, various clinical symptoms and biological markers are usually considered in combination to diagnose, treat and control potential diseases. As an example, the method further includes determining the level of tetraiodothyronine autoantibodies in patient samples from the cancer treatment process, and comparing the measured value with a reference value. In some embodiments, the method further includes determining the level of at least one of FT3, FT4 and TSH in patient samples from the cancer treatment process, and comparing the measured value with a reference value. In some embodiments, the method further includes determining the level of at least one of tetraiodothyronine autoantibodies, FT3, FT4 and TSH in patient samples from the cancer treatment process, and comparing the measured value with a reference value. The above-mentioned additional biochemical markers can improve the accuracy of the present method.

[0071] The embodiments of the present disclosure will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance given in the present disclosure, and can also be based on the experimental manuals or conventional conditions in the art, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0072] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0073] Example 1 Preparation of T3-Ab Detection Agent

[0074] 1. Preparation of chemiluminescence indirect detection kit for T3-Ab

[0075] 1. Preparation of T3-Ab Calibrator

[0076] (1) Low-point calibration (0.4 AU / mL): Use PBS buffer to dilute the T3-Ab stock solution (0.4 mg / mL) to the required concentration at a dilution ratio of 1:68608.

[0077] (2) High point calibration (25.6 AU / mL): Use PBS buffer to dilute the T3-Ab stock solution (0.4 mg / mL) to the required concentration at a dilution ratio of 1:1072.

[0078] 2. Preparation of T3-Ab quality control

[0079] Quality control (2.2AU / mL): Use PBS buffer to dilute T3-Ab stock solution (0.4mg / mL) to the required concentration at a dilution ratio of 1:12473.

[0080] 3. Reaction Buffer

[0081] Use PBS diluent, formula: phosphate buffer solution 100mmol / L, containing preservatives.

[0082] 4. T3 coated magnetic spheres

[0083] The magnetic spheres were coated with antigens that specifically recognized T3-Ab, the ratio of magnetic spheres to antigen was 1 mg:2 ug, and the concentration of magnetic sphere suspension was 20 mg / mL.

[0084] 5. ABEI labeled secondary antibody

[0085] The anti-human secondary antibody hIgG that labels and recognizes T3-Ab, the feeding ratio of ABEI and the secondary antibody is 1 mg:200 μg.

[0086] 6. Chemiluminescent substrate solution

[0087] Sodium hydroxide NaOH and hydrogen peroxide H2O2.

[0088] 7. Sample addition process

[0089] The detection was performed on the chemiluminescent immunoassay analyzer MAGLUMI X8 of Shenzhen Xinye Biotechnology Co., Ltd. The instrument loading process parameters included: 10 μL sample + 20 μL T3-coated magnetic balls + 150 μL reaction buffer Buffer, incubated for 20 minutes, washed 3 times + 200 μL ABEI-labeled secondary antibody, incubated for 10 minutes, washed 3 times. Finally, the chemiluminescent substrate solution for the fully automatic immunoassay system was added to start the chemiluminescent reaction and generate a light signal. The relative light intensity (RLU) measured by the photomultiplier tube is proportional to the T3-Ab concentration in the sample (or calibrator / quality control product).

[0090] 2. T3-Ab Kit Performance Verification

[0091] 1. Blank limit

[0092] The LOB of the test kit prepared in Example 1 is declared to be 0.08AU / mL. According to YY / T1789.3-2022 "Methods for Performance Evaluation of In Vitro Diagnostic Test Systems Part 3: Limit of Detection and Limit of Quantitation", the test kit prepared in Example 1 and the chemiluminescent immunoassay analyzer MAGLUMI X8 are used to select 3 test days, 2 clinical blank samples, and 4 repeated measurements for each sample. Calculate the percentage of blank sample test results that are less than or equal to the LOB declaration. If the percentage is greater than 87%, the declared LOB is considered to meet the standard.

[0093] 2. Detection limit

[0094] The LOD of the test kit prepared in Example 1 is declared to be 0.12AU / mL. According to YY / T1789.3-2022 "Methods for Performance Evaluation of In Vitro Diagnostic Test Systems Part 3: Limit of Detection and Limit of Quantitation", the test kit prepared in Example 1 and the chemiluminescent immunoassay analyzer MAGLUMI X8 were used to select 3 test days, 2 clinical detection limit concentration samples, and 4 repeated measurements for each sample. Calculate the percentage of the total number of results greater than or equal to the LOB declaration in the detection limit concentration sample test results. If the percentage is greater than 87%, the declared LOD is considered to meet the standard.

[0095] Evaluation results:

[0096] Table 1. Blank limit and detection limit test results

[0097]

[0098] Among them, according to the formula (number of blank samples less than or equal to 0.08 / total number of blank samples)*100%, the LOB evaluation percentage is 95.83%; according to the formula (number of low-concentration samples greater than or equal to 0.08 / total number of low-concentration samples)*100%, the LOD evaluation percentage is 100%, both of which meet the requirements.

[0099] 3. Linear

[0100] It is stated that the linear interval of the T3-Ab detection kit prepared in this embodiment is [0.16, 10.0]. According to YY / T1789.4-2022 "In Vitro Diagnostic Test System Performance Rating Method Part 4: Linear Interval and Reportable Interval", high and low value samples are used to select different proportional coefficients, and the low value sample L is recorded as S1, and the high value sample is recorded as S6. The dilution of each concentration sample is calculated according to the dilution ratio. On the same MAGLUMI X8 instrument within one day, calibration and internal quality control tests are carried out according to the sample addition process in Example 1. After the quality control passes, the S1-S6 samples are tested. Each sample is repeated 4 times, and the data is recorded and the results are statistically analyzed. The "linear regression combined with the offset method" is used to analyze the data. If r>0.99, it can be preliminarily judged that the established linear interval meets the requirements. The specific preparation method is shown in the following table:

[0101] Table 2. Sample preparation method and theoretical concentrations at each point for linearity test

[0102]

[0103] Record the test results of each sample, calculate the test mean of each sample, use the theoretical concentration of each sample as the horizontal axis, and the test mean of each sample as the vertical axis, and fit the linear function y=bx+a and the correlation coefficient r, where b is the slope and a is the intercept. Figure 1 As shown, the linear range of the T3-Ab kit prepared in this scheme is 0.17-9.8AU / mL, and the correlation coefficient r is 0.9963, which meets the linear requirements.

[0104] 3. Preparation of Chemiluminescent Indirect T4-Ab Detection Kit

[0105] 1. Configuration of T4-Ab Calibrator

[0106] (1) Low-point calibration (0.4 AU / mL): Use PBS buffer to dilute the T4-Ab stock solution (0.4 mg / mL) to the required concentration at a dilution ratio of 1:10496.

[0107] (2) High point calibration (25.6 AU / mL): Use PBS buffer to dilute the T4-Ab stock solution (0.4 mg / mL) to the required concentration at a dilution ratio of 1:164.

[0108] 2. Configuration of T4-Ab quality control products

[0109] Quality control (2.2AU / mL): Use PBS buffer to dilute T4-Ab stock solution (0.4mg / mL) to the required concentration at a dilution ratio of 1:1908.

[0110] 3. Reaction Buffer

[0111] 100mmol / L phosphate buffer solution, containing preservatives.

[0112] 4. T4 coated magnetic spheres

[0113] The magnetic spheres were coated with antigens that specifically recognized T4-Ab, the ratio of magnetic spheres to antigen was 1 mg:5 μg, and the concentration of magnetic sphere suspension was 20 mg / mL.

[0114] 5. ABEI labeled secondary antibody

[0115] The anti-human secondary antibody hIgG that labels and recognizes T4-Ab, the feeding ratio of ABEI and the secondary antibody is 1 mg:200 μg.

[0116] 6. Chemiluminescent substrate solution

[0117] Sodium hydroxide NaOH and hydrogen peroxide H2O2.

[0118] 7. Sample addition process

[0119] The detection was performed on the chemiluminescent immunoassay analyzer MAGLUMI X8 of Shenzhen Xinye Biotechnology Co., Ltd. The instrument loading process parameters included: 10 μL sample + 20 μL T4-coated magnetic balls + 150 μL reaction buffer Buffer, incubated for 20 minutes, washed 3 times + 200 μL ABEI-labeled secondary antibody, incubated for 10 minutes, washed 3 times. Finally, the chemiluminescent substrate solution for the fully automatic immunoassay system was added to start the chemiluminescent reaction and generate a light signal. The relative light intensity (RLU) measured by the photomultiplier tube is proportional to the T4-Ab concentration in the sample (or calibrator / quality control product).

[0120] 4. T4-Ab Kit Performance Verification

[0121] 1. Blank limit

[0122] The LOB of the test kit prepared in Example 1 is declared to be 0.08AU / mL. According to YY / T1789.3-2022 "Methods for Performance Evaluation of In Vitro Diagnostic Test Systems Part 3: Limit of Detection and Limit of Quantitation", the test kit prepared in Example 1 and the chemiluminescent immunoassay analyzer MAGLUMI X8 are used to select 3 test days, 2 clinical blank samples, and 4 repeated measurements for each sample. Calculate the percentage of blank sample test results that are less than or equal to the LOB declaration. If the percentage is greater than 87%, the declared LOB is considered to meet the standard.

[0123] 2. Detection limit

[0124] The LOD of the test kit prepared in Example 1 is declared to be 0.12AU / mL. According to YY / T1789.3-2022 "Methods for Performance Evaluation of In Vitro Diagnostic Test Systems Part 3: Limit of Detection and Limit of Quantitation", the test kit prepared in Example 1 and the chemiluminescent immunoassay analyzer MAGLUMI X8 were used to select 3 test days, 2 clinical detection limit concentration samples, and 4 repeated measurements for each sample. Calculate the percentage of the total number of results greater than or equal to the LOB declaration in the detection limit concentration sample test results. If the percentage is greater than 87%, the declared LOD is considered to meet the standard.

[0125] Evaluation results:

[0126] Table 3. Blank limit and detection limit test results

[0127]

[0128] Among them, according to the formula (number of blank samples less than or equal to 0.08 / total number of blank samples)*100%, the LOB evaluation percentage is 91.67%; according to the formula (number of low-concentration samples greater than or equal to 0.08 / total number of low-concentration samples)*100%, the LOD evaluation percentage is 100%, both of which meet the requirements.

[0129] It is stated that the linear range of the T4-Ab detection kit prepared in this embodiment is [0.16, 10.00]. According to YY / T1789.4-2022 "In Vitro Diagnostic Test System Performance Rating Method Part 4: Linear Interval and Reportable Interval", the low-value sample L is recorded as S1, the high-value sample is recorded as S6, and 6 samples are obtained by dilution according to the dilution ratio, and the concentration covers the entire linear interval. The specific preparation method is shown in Table 4. On the same MAGLUMI X8 instrument within one day, calibration and internal quality control tests are carried out according to the sample addition process in Example 1 of the instruction manual. After the quality control passes, the S1-S6 samples are tested, and each sample is repeated 4 times, and the data is recorded and the results are statistically analyzed. The "linear regression combined with the offset method" is used to analyze the data. If r>0.99, it can be preliminarily judged that the established linear interval meets the requirements.

[0130] Table 4. Sample preparation method and theoretical concentration at each point for linear test

[0131]

[0132] Record the test results of each sample, calculate the test mean of each sample, use the theoretical concentration of each sample as the horizontal axis, and the test mean of each sample as the vertical axis, and fit the linear function y=bx+a and the correlation coefficient r, where b is the slope and a is the intercept. The fitted linear function is as follows: Figure 2 As shown, r is 0.9959, r>0.99, that is, the established linear interval meets the requirements.

[0133] Example 2 Discovery of inducing the production of T3-Ab in the body during tumor treatment

[0134] 1. Study population, sample source, and experimental methods

[0135] Study population and source:

[0136] 62 cancer patients receiving chemotherapy and 62 cancer patients receiving ICI treatment; all from the Affiliated Hospital of Jiangsu University.

[0137] Sample Type: Serum

[0138] Experimental method: 5.0 mL of venous blood was collected into a blood collection tube and allowed to stand at room temperature. The blood was centrifuged and the serum was separated and stored at 2°C to 8°C. The kit of Example 1 was used and the signal was detected by MAGLUMI X8 instrument.

[0139] Data analysis: Graphpad 7.0 analysis software was used to perform unpaired T-test statistical analysis of the data, and p < 0.05 indicated that the difference was statistically significant.

[0140] 2. Results Analysis

[0141] Tumor patients who only received chemotherapy were used as the control group, and tumor patients who received ICI treatment were used as the treatment group. The serum T3-Ab levels of patients in the control group (n=62) and the ICI treatment group (n=62) were detected by chemiluminescence to evaluate whether PD-1 blocking immunotherapy would affect the body's T3-Ab expression. Figure 3 As shown in the data, compared with the control group, the serum T3-Ab levels of tumor patients were significantly higher after PD-1 blockade therapy than those of the control group (p<0.0001). The results showed that ICI treatment can induce the body to produce T3-Ab, which may be related to irAE thyroid side effects.

[0142] Example 3 T3-Ab has excellent diagnostic performance in diagnosing adverse thyroid reactions associated with ICI treatment

[0143] 1. Study population and source:

[0144] A total of 320 cancer patients with abnormal thyroid function after ICI treatment and 100 cancer patients without abnormal thyroid function after ICI treatment were recruited from the Affiliated Hospital of Jiangsu University.

[0145] Sample Type: Serum

[0146] Experimental method: 5.0 mL of venous blood was collected into a blood collection tube and allowed to stand at room temperature. The blood was centrifuged and the serum was separated and stored at 2°C to 8°C. The kit of Example 1 was used and the signal was detected by MAGLUMI X8 instrument.

[0147] Data analysis: SPSS software was used to draw ROC curves and perform data analysis.

[0148] 2. Results Analysis

[0149] The cut-off values ​​(critical values ​​for positive and negative results) were statistically analyzed to distinguish whether thyroid dysfunction occurred after ICI treatment. The cut-off values ​​of T3-Ab in the experimental group were 1.315AU / mL, and the cut-off value of T4-Ab was 1.06AU / mL; the cut-off values ​​of FT3, FT4, and TSH in the control group were 6.395pmol / L, 21.85pmol / L, and 4.21uIU / mL, respectively.

[0150] At the same time, the ROC curve is drawn according to the test results, as follows:

[0151] 1) ROC curve of the experimental group:

[0152] ① The curve of using T3-Ab as a marker alone ( Figure 4 shown).

[0153] ② The curve of using T3-Ab and T4-Ab as combined markers ( Figure 5 shown).

[0154] ③ Curves using T3-Ab, FT3, FT4 and TSH as combined markers ( Figure 6 shown).

[0155] ④ The curve using T3-Ab, T4-Ab, FT3, FT4 and TSH as the combined index ( Figure 7 shown).

[0156] 2) ROC curve of control group (conventional laboratory indicators: FT3, FT4 and TSH):

[0157] Curves using FT3, FT4 and TSH as combined markers ( Figure 8 shown).

[0158] Based on the above graph, we can see that:

[0159] In the control group, the area under the curve of FT3, FT4, and TSH as a combined marker was 0.7204, with a sensitivity of 86.9% and a specificity of 71.9%;

[0160] In the experimental group:

[0161] ① The area under the curve of T3-Ab alone was 0.6261, with a sensitivity of 85.6% and a specificity of 62.1%;

[0162] ② The area under the curve of T3-Ab and T4-Ab as a combined marker was 0.8588, with a sensitivity of 87.1% and a specificity of 74.4%;

[0163] ③The area under the curve of using T3-Ab, FT3, FT4 and TSH as a combined marker was 0.8507, with a sensitivity of 88.9% and a specificity of 72.1%;

[0164] ④The area under the curve of the combined test of T3-Ab, T4-Ab, FT3, FT4 and TSH was 0.9306, with a sensitivity of 94.4% and a specificity of 82.7%;

[0165] The area under the curve of T3-Ab alone was 0.6261>0.5, proving that T3-Ab can effectively diagnose abnormal thyroid function during ICI treatment.

[0166] Compared with conventional laboratory indicators (FT3, FT4 and TSH), the sensitivity / specificity / area under the curve of combined detection of T3-Ab and T4-Ab, combined detection of T3-Ab and conventional laboratory indicators, and combined detection of T3-Ab, T4-Ab and conventional laboratory indicators were significantly better than those of conventional laboratory indicators.

[0167] Example 4 Application of T3-Ab in a kit for assisting thyroid function detection during monitoring of tumor treatment

[0168] 1. Research population and source:

[0169] The stages before ICI treatment, after the first course of treatment, after the second course of treatment, and after the third course of treatment for 7 typical patients; the specific information of the 7 typical patients whose T3 Ab levels were tested is shown in Table 3.

[0170] Sample Type: Serum

[0171] Experimental method: 5.0 mL of venous blood was collected into a blood collection tube and allowed to stand at room temperature. The blood was centrifuged and the serum was separated and stored at 2°C to 8°C. The kit of Example 1 was used and the signal was detected by MAGLUMI X8 instrument.

[0172] Data analysis: Graphpad 7.0 analysis software was used for statistical analysis of the data.

[0173] 2. Results Analysis

[0174] In order to more comprehensively examine the dynamic changes of serum T3-Ab in patients during ICI treatment, we used a line graph to analyze the changes in T3-Ab in 7 typical patients before ICI treatment, after the first course of treatment, after the second course of treatment, and after the third course of treatment. The results show (see Table 5 and Figure 8 ), during ICI treatment, patients will experience elevated T3-Ab levels after the first course of treatment, and as the treatment progresses, T3-Ab levels also show a gradual upward trend. The results of this study are consistent with the patient's thyroid function and clinical phenomena. After ICI treatment, the patient first developed hyperthyroidism and then gradually turned to hypothyroidism. At this time, the clinician used levothyroxine tablets for intervention treatment. However, during the progression of thyroid dysfunction, other thyroid autoantibodies (TG-Ab, TPO-Ab, TR-Ab) did not show significant changes, and the pathogenesis of irAE thyroid side effects is not clear. Therefore, we speculate that ICI-related thyroid dysfunction is closely related to T3-Ab. Clinicians are advised to test T3-Ab as a supplement while screening for thyroid dysfunction to improve the accuracy of diagnostic results.

[0175] Table 5. Typical case patient information and T3-Ab levels during ICI tumor treatment

[0176]

[0177] Example 5 T3-Ab has excellent performance in diagnosing abnormal thyroid function in different tumor patients treated with immunotherapy Study population, sample source and experimental method

[0178] 1. Study population and source: Sera from 420 tumor patients who underwent immunotherapy, including 85 patients with gastric malignant tumors, 93 patients with esophageal malignant tumors, 78 patients with lung malignant tumors, 55 patients with liver malignant tumors, 36 patients with colon malignant tumors, 38 patients with gallbladder malignant tumors, and 35 patients with cardia malignant tumors, all from the Affiliated Hospital of Jiangsu University.

[0179] Sample Type: Serum

[0180] Experimental method: 5.0 mL of venous blood was collected into a blood collection tube and allowed to stand at room temperature. The blood was centrifuged and the serum was separated and stored at 2°C to 8°C. The kit of Example 1 and T3, FT4 and TSH kits were used to perform signal detection on a MAGLUMI X8 instrument.

[0181] Data analysis: SPSS statistics 17.0 software was used to analyze the data.

[0182] 2. Results Analysis

[0183] In order to evaluate and prove that T3-Ab has excellent performance in diagnosing abnormal thyroid function in different tumor patients who have undergone immunotherapy, a total of 420 serum specimens of patients with different tumor diseases who have undergone immunotherapy were collected, and the T3-Ab level in the specimens was detected by the kit prepared in Example 1. T3-Ab>1.315AU / mL was defined as positive for abnormal thyroid function. Statistics show that T3-Ab can effectively detect abnormal thyroid function in different tumor patients who have undergone immunotherapy. The AUC of abnormal thyroid function in patients with gastric malignant tumors, esophageal malignant tumors, lung malignant tumors, colon malignant tumors, gallbladder malignant tumors, and cardia malignant tumors during immunotherapy is better than conventional laboratory indicators (FT3, FT4, TSH). Among them, gastric malignant tumors have better diagnostic performance, followed by esophageal malignant tumors and colon malignant tumors. The diagnostic performance of T3-Ab for abnormal thyroid function in different tumor patients who have undergone immunotherapy is as follows:

[0184] Table 6: Diagnostic value of T3-Ab for abnormal thyroid function in patients with different tumors treated with immunotherapy

[0185] Medical conditions AUC of T3-Ab diagnosis AUC of routine laboratory indicators Gastric malignancy 0.809 0.736 Esophageal malignancy 0.798 0.765 Lung malignancy 0.736 0.701 Liver malignancy 0.545 0.628 Colon cancer 0.711 0.699 Gallbladder malignancy 0.606 0.657 Cardiac malignancy 0.634 0.517

[0186] The above-described embodiments only express several implementation methods of the present disclosure, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present disclosure, and these all belong to the protection scope of the present disclosure. Therefore, the protection scope of the patent of the present disclosure shall be based on the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. Use of a detection agent that specifically binds to triiodothyronine autoantibodies in the preparation of a kit for diagnosing thyroid dysfunction during cancer treatment.

2. The use according to claim 1, comprising measuring the level of triiodothyronine autoantibodies in a sample from a patient during cancer treatment, and comparing the measured value with a reference value; Optionally, the step of determining the level of triiodothyronine autoantibodies in a patient sample from a cancer treatment process comprises the following steps: 1) contacting a patient sample from a cancer treatment process with a detection agent that specifically binds to triiodothyronine autoantibodies under conditions that allow for the formation of a complex; 2) adding a substance with a detectable label, which can specifically recognize the complex formed in step 1); 3) quantifying the detectable labeled signal to obtain the amount of triiodothyronine autoantibodies.

3. The use according to claim 1, wherein the sample is blood, serum or plasma.

4. The use according to claim 1, wherein the subject undergoing cancer treatment has undergone or is undergoing immune checkpoint inhibitor treatment; Optionally, the immune checkpoint inhibitor includes inhibitors against one or more of the following targets: CTLA-4, PD-1, PD-L1, B7-H3, IDO, KIR, LAG3, TIM-3, TIGIT, VISTA, CD47, GITR, OX40, ADAR1, A2AR, SIRPα and TLR.

5. The use according to claim 1, wherein the triiodothyronine autoantibody is selected from IgG and / or IgM antibodies.

6. The use according to claim 1, wherein the cancer is caused by lesions in any one or more of bones, bone joints, muscles, lungs, trachea, heart, spleen, arteries, veins, blood, capillaries, lymph nodes, lymphatic vessels, lymph fluid, oral cavity, pharynx, esophagus, cardia, stomach, duodenum, small intestine, colon, rectum, anus, appendix, liver, gallbladder, pancreas, parotid gland, sublingual gland, urinary kidney, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina, vulva, scrotum, testicle, vas deferens, penis, eye, ear, nose, tongue, skin, brain, brainstem, medulla oblongata, spinal cord, cerebrospinal fluid, nerves, thyroid gland, parathyroid gland, adrenal gland, pituitary gland, pineal gland, pancreatic islets, thymus gland, gonad, sublingual gland and parotid gland; Optionally, the cancer is any one or more of lung cancer, gastric cancer, esophageal cancer, liver cancer, colon cancer, gallbladder cancer and cardiac cancer.

7. The use according to any one of claims 1 to 6, wherein the kit is suitable for immunoassay; Optionally, the immunoassay is based on chemiluminescence; Optionally, the detection agent comprises triiodothyronine antigen; Optionally, the triiodothyronine antigen is conjugated to a solid support; Optionally, the kit further comprises an antibody capable of specifically binding to the Fc segment of triiodothyronine autoantibodies; Optionally, the antibody capable of specifically binding to the Fc segment of the triiodothyronine autoantibody is labeled with a signal substance; Optionally, the signal substance is a chemiluminescent reagent; Optionally, the signal substance is at least one of luminol and its derivatives, isoluminol and its derivatives, lucigenin, lophanthine, peroxy oxalate esters, and acridinium esters; Optionally, the signal substance is ABEI; Optionally, the kit further comprises one or more of triiodothyronine autoantibody standard, reaction buffer, water, and chemiluminescent substrate solution.

8. The use according to any one of claims 1 to 7, wherein the detection agent further comprises a second detection agent for tetraiodothyronine autoantibodies; Optionally, the tetraiodothyronine autoantibody is selected from IgG and / or IgM type antibodies.

9. The use according to any one of claims 1 to 8, wherein the detection reagent further comprises a third detection agent for quantitatively determining at least one of FT3, FT4 and TSH.

10. A kit for diagnosing abnormal thyroid function during cancer treatment, which is the kit defined in the use according to any one of claims 1 to 9; Optionally, it is also accompanied by an instruction manual of the kit, which indicates that the kit is used to measure the amount of triiodothyronine autoantibodies in a sample of a subject.

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