A kit for predicting prognosis and adverse reaction of diarrhea in breast cancer targeted therapy

Abstract

The application discloses a kit for predicting prognosis of breast cancer targeted therapy and adverse reactions of diarrhea, and the kit is used for evaluating the curative effect of pyrotinib on HER2 positive breast cancer and the risk of diarrhea by detecting the expression levels of IL-6, HER2, EGFR, JAK2, Src and STAT3; the kit comprises specific antibodies, antibody conjugates and immunohistochemical detection reagents, wherein the antibody conjugate comprises carboxyl functionalized magnetic beads, and is obtained by modification through hexadiene succinic acid, (E)-5-methylhex-3-ene acid and other reagents; qualitative and quantitative analysis of the markers in tumor tissues is carried out by using an immunohistochemical technology, the kit provides a reliable tool for individualized prognosis evaluation and monitoring of adverse reactions of diarrhea in breast cancer targeted therapy, and is helpful for formulating precise treatment plans in the clinic and improving the treatment benefits of patients.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a kit for predicting prognosis of breast cancer targeted therapy and adverse reactions of diarrhea. BACKGROUND

[0002] Malignant tumor is a disease that seriously endangers human health and safety at present, and targeted therapy occupies a very important position in the treatment of various malignant tumors. As a conventional drug in the targeted therapy of lung cancer, breast cancer and other malignant tumors, tyrosine kinase inhibitors can significantly prolong the overall survival and progression-free survival of patients. However, during the treatment, about 80% of patients will have adverse reactions such as diarrhea, vomiting, nausea, oral mucositis and other digestive tract adverse reactions, which seriously affect the quality of life of tumor patients. In addition, severe digestive tract adverse reactions can also lead to drug reduction during treatment, which not only reduces the clinical efficacy of patients during treatment, but also increases the pain of patients.

[0003] Pyrrolnitrin is a new generation of oral, irreversible tyrosine kinase inhibitor, which can covalently bind to the ATP binding site of the kinase domain of intracellular epidermal growth factor receptor and HER2, prevent the formation of homodimer and heterodimer of intracellular epidermal growth factor receptor and HER2, and inhibit the phosphorylation of intracellular epidermal growth factor receptor and HER2, thereby blocking the activation of downstream signaling pathways and inhibiting the growth of tumor cells. However, although targeted therapy significantly improves the treatment effect of HER2-positive breast cancer, long-term use of HER2-targeted drugs can lead to the occurrence of primary or secondary drug resistance, affecting the treatment effect, and the mechanisms of drug resistance include changes in HER2 receptor structure, activation of downstream signaling pathways and changes in tumor microenvironment; the treatment of pyrrolnitrin can also cause drug-related diarrhea, cardiotoxicity and other adverse reactions, which seriously affect the quality of life and treatment compliance of patients.

[0004] At present, the selection of markers for predicting the effect of targeted therapy of HER2-positive breast cancer and predicting diarrhea adverse reactions still faces great challenges. All the biomarkers found and applied to the prognosis of HER2 breast cancer treatment have limitations. Some of the existing biomarkers are not accurate and efficient in diagnosing and predicting the prognosis of HER2-positive breast cancer. Therefore, a feasible marker for predicting pyrrolnitrin treatment-related diarrhea and acquired drug resistance is needed to effectively predict the treatment effect and treatment-related adverse reactions. SUMMARY

[0005] The purpose of the present application is to provide a kit for predicting the prognosis of breast cancer targeted therapy and adverse reactions of diarrhea.

[0006] The technical scheme adopted by the present application to achieve the above-mentioned purpose is as follows:

[0007] A kit comprising specific antibodies, antibody conjugates and immunohistochemical detection reagents.

[0008] Preferably, the specific antibodies comprise one or more of IL-6 antibodies, HER2 antibodies, EGFR antibodies, JAK2 antibodies, Src antibodies and STAT3 antibodies.

[0009] Preferably, the antibody conjugates comprise magnetic bead conjugated antibodies and horseradish peroxidase.

[0010] Preferably, the mass ratio of the magnetic bead conjugated antibodies and the horseradish peroxidase is 10-100:5-50.

[0011] Preferably, the magnetic bead conjugated antibodies comprise carboxyl functionalized magnetic beads and goat anti-mouse IgG antibodies.

[0012] Preferably, the mass ratio of the carboxyl functionalized magnetic beads and the goat anti-mouse IgG antibodies is 1-10g:60-600mg.

[0013] Preferably, the carboxyl functionalized magnetic beads comprise polystyrene modified magnetic nanoparticles; or, (E)-5-methylhex-3-enoic acid and hexadienesuccinate modified silicon hydroxyl functionalized magnetic beads, the silicon hydroxyl functionalized magnetic beads comprise tetraethyl orthosilicate modified magnetic nanoparticles.

[0014] Preferably, the mass ratio of the hexadienesuccinate and the silicon hydroxyl functionalized magnetic beads is 1-10:1-10.

[0015] Preferably, the volume mass ratio of the (E)-5-methylhex-3-enoic acid and the silicon hydroxyl functionalized magnetic beads is 3-30mL:1-10g.

[0016] The tetraethyl orthosilicate modified magnetic nanoparticles form silicon-based functionalized magnetic beads, the hexadienesuccinate and the (E)-5-methylhex-3-enoic acid co-modify the silicon-based functionalized magnetic beads, introduce carboxyl groups to the surface of the magnetic beads, and copolymerize to form a carboxyl functionalized layer; the synergistic effect of the two can regulate the carboxyl density and hydrophobicity of the surface of the magnetic beads, optimize the antibody conjugation rate, enhance the activity and stability of the horseradish peroxidase, and the formed antibody conjugates provide efficient carriers for marker detection, and improve the detection performance of the kit.

[0017] Preferably, the immunohistochemical detection reagents comprise an antibody diluent, the antibody diluent comprises PBS buffer and bovine serum albumin.

[0018] Preferably, the mass volume ratio of the bovine serum albumin and the PBS buffer is 2.5-25g:50-500mL.

[0019] Preferably, the immunohistochemical detection reagents comprise an antigen retrieval buffer.

[0020] Preferably, the antigen repair buffer is a citrate buffer with pH=5.8-6.2.

[0021] Preferably, the immunohistochemical detection reagent comprises a color developing solution and a counterstaining solution, the color developing solution is a diaminobenzidine color developing solution, and the counterstaining solution is a hematoxylin staining solution.

[0022] More preferably, 2-acetamidonon-8-enoic acid can also be added in the modification of the silicon-based functionalized magnetic beads, and the volume-mass ratio of 2-acetamidonon-8-enoic acid to the silicon-hydroxyl functionalized magnetic beads is 3-30 mL:1-10 g. The 2-acetamidonon-8-enoic acid is modified in cooperation with hexadiene succinate and (E)-5-methylhex-3-enoic acid, which can enhance the stability of the carboxyl functionalized layer, reduce non-specific adsorption, and improve the coupling rate with the antibody.

[0023] The kit is used for predicting the prognosis of breast cancer targeted therapy and the adverse reaction of diarrhea, and the breast cancer includes HER2 positive breast cancer.

[0024] The application further provides a preparation method of the magnetic bead coupled antibody, which comprises the following steps:

[0025] Preparation of the magnetic bead coupled antibody: goat anti-mouse IgG antibody is dissolved in a PBS buffer solution with pH=7.3-7.5 to obtain an antibody solution; carboxyl functionalized magnetic beads are dispersed in a 2-morpholinoethanesulfonic acid buffer solution with pH=5.9-6.1, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added, and the mixture is incubated on a shaking table at 36.8-37.2℃ for 20-40 min, the supernatant is discarded, the carboxyl functionalized magnetic beads are washed once with the PBS buffer solution with pH=7.3-7.5, the antibody solution is added, and the mixture is incubated on a shaking table at 36.8-37.2℃ for 2-3 h to obtain the magnetic bead coupled antibody.

[0026] Preferably, the carboxyl functionalized magnetic beads are polystyrene modified nanometer ferroferric oxide, which are purchased from Biyun Tian Biotechnology Co., Ltd., and have a particle size of 0.8-1.2 µm.

[0027] Preferably, the mass-volume ratio of the goat anti-mouse IgG antibody to the PBS buffer solution is 60-600 mg:120-1200 mL.

[0028] Preferably, the mass-volume ratio of the carboxyl functionalized magnetic beads to the 2-morpholinoethanesulfonic acid buffer solution is 1-10 g:100-1000 mL.

[0029] Preferably, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to the carboxyl functionalized magnetic beads is 100-1000 mg:1-10 g.

[0030] Preferably, the mass ratio of N-hydroxysuccinimide to carboxyl-functionalized magnetic beads is 50-500 mg: 1-10 g.

[0031] Preferably, the mass of the antibody solution is measured by the mass of goat anti-mouse IgG antibody therein, and the mass ratio of goat anti-mouse IgG antibody to carboxyl-functionalized magnetic beads is 60-600 mg: 1-10 g.

[0032] The application also provides a preparation method of an antibody conjugate, comprising:

[0033] Preparation of the antibody conjugate: horseradish peroxidase and magnetic bead conjugated antibody are dissolved in PBS buffer solution with pH=7.3-7.5, glutaraldehyde is added, and the reaction is carried out at room temperature in the dark for 1.5-2.5 h, sodium borohydride is added under ice bath conditions to react for 25-35 min to terminate the reaction, and the antibody conjugate is obtained.

[0034] Preferably, the mass-volume ratio of horseradish peroxidase to PBS buffer solution is 10-100 mg: 5-50 mL.

[0035] Preferably, the mass-volume ratio of magnetic bead conjugated antibody to PBS buffer solution is 5-50 mg: 5-50 mL.

[0036] Preferably, the mass ratio of glutaraldehyde to magnetic bead conjugated antibody is 50-500 µg: 5-50 mg.

[0037] Preferably, the mass ratio of sodium borohydride to magnetic bead conjugated antibody is 250-2500 µg: 5-50 mg.

[0038] The application also provides a preparation method of an antibody diluent, comprising:

[0039] Preparation of the antibody diluent: bovine serum albumin, Tween-20 and sodium azide are dissolved in PBS buffer solution with pH=7.3-7.5, and after being fully stirred and dissolved, sterilization is performed by filtering through a 0.20-0.25 μm filter membrane to obtain the antibody diluent.

[0040] Preferably, the mass-volume ratio of bovine serum albumin to PBS buffer solution is 2.5-25 g: 50-500 mL.

[0041] Preferably, the volume ratio of Tween-20 to PBS buffer solution is 5-50 µL: 50-500 mL.

[0042] Preferably, the mass-volume ratio of sodium azide to PBS buffer solution is 10-100 mg: 50-500 mL.

[0043] The application also provides a preparation method of a kit, comprising:

[0044] Preparation of the kit: the kit comprises specific antibodies, antibody conjugates and immunohistochemical detection reagents; the specific antibodies and the antibody conjugates are separately frozen and stored after being divided; the immunohistochemical detection reagents are separately stored in the dark after being divided.

[0045] Preferably, the specific antibodies comprise one or more of IL-6 antibodies, HER2 antibodies, EGFR antibodies, JAK2 antibodies, Src antibodies and STAT3 antibodies.

[0046] Preferably, the immunohistochemical detection reagents comprise antibody diluent, antigen repair buffer, washing solution, blocking solution, color developing solution, counterstaining solution, blueing solution, xylene solution and gradient ethanol solution.

[0047] Preferably, the antigen repair buffer is a citrate buffer with pH = 5.9-6.1.

[0048] Preferably, the washing solution comprises PBS buffer with pH = 7.3-7.5.

[0049] Preferably, the blocking solution is a 2.5-3.5% hydrogen peroxide solution.

[0050] Preferably, the color developing solution is a diaminobenzidine color developing solution.

[0051] Preferably, the counterstaining solution is a hematoxylin staining solution.

[0052] Preferably, the blueing solution is a lithium carbonate blueing solution.

[0053] Preferably, the gradient ethanol solution comprises 80-100% ethanol solution.

[0054] The application also provides a preparation method of a primary antibody working solution, comprising:

[0055] Preparation of the primary antibody working solution: the specific antibodies are diluted with antibody diluent respectively to obtain the primary antibody working solution.

[0056] Preferably, the volume ratio of the specific antibodies to the antibody diluent is 0.5-5: 50-500.

[0057] The application also provides a preparation method of a secondary antibody working solution, comprising:

[0058] Preparation of the secondary antibody working solution: the antibody conjugates are diluted with antibody diluent to obtain the secondary antibody working solution.

[0059] Preferably, the volume ratio of the antibody conjugates to the antibody diluent is 0.5-5: 100-1000.

[0060] The application also provides a preparation method of an immunohistochemical staining pathological section, comprising:

[0061] Preparation of immunohistochemical staining pathological section: the pathological section is baked at 65-75 DEG C for 80-100 min, deparaffinized by immersing in xylene I for 5-15 min and then immersing in xylene II for 5-15 min, hydrated by immersing in gradient ethanol for 10 min each time, the gradient is 99-100%, 94-96% and 84-86%, washed by tap water for 2-4 times and then washed by deionized water for 2-4 times, 2.5-3.5% hydrogen peroxide is added for blocking treatment at room temperature, antigen retrieval buffer is added, and the antigen is retrieved by baking at 75-85 DEG C for 10-20 min, cooled to room temperature, washed by PBS buffer for 2-4 times, the first antibody working solution is added in the range of immunohistochemical oil pen circle and fully covers the tissue, and the slide is incubated in a refrigerator at 3-5 DEG C overnight, washed by PBS buffer for 2-4 times after being balanced at room temperature, the second antibody working solution is added, incubated at room temperature for 50-70 min, washed by PBS buffer for 2-4 times to form antigen-antibody complex, diaminobenzidine color developing solution is added, washed under running water after termination, the slide is re-stained by immersing in hematoxylin, differentiated by immersing in 0.9-1.1% hydrochloric acid alcohol, and returned to blue in lithium carbonate return blue solution, dehydrated by immersing in gradient ethanol, the gradient is 84-86%, 94-96%, 99-100% and 99-100%, and the immunohistochemical staining pathological section is obtained by immersing in xylene I and xylene II for transparency treatment.

[0062] The application further provides a preparation method of the carboxyl functionalized magnetic beads.

[0063] Preparation of the magnetic nanoparticles: FeCl3 6H2O is dispersed in ethylene glycol, ultrasonic dispersed for 20-40 min, polyacrylic acid and urea are added, ultrasonic dissolved for 5-15 min, reacted at 175-185 DEG C under stirring for 20-25 h, magnetically separated after being cooled to room temperature, washed by deionized water until the pH of the washing liquid is 6.9-7.1, and vacuum dried at 55-65 DEG C for 10-15 h to obtain the magnetic nanoparticles.

[0064] Preferably, the mass-volume ratio of FeCl3 6H2O to ethylene glycol is 2-20 g: 75-750 mL.

[0065] Preferably, the mass ratio of polyacrylic acid to FeCl3 6H2O is 0.75-7.5: 2-20.

[0066] Preferably, the mass ratio of urea to FeCl3 6H2O is 4.5-45: 2-20.

[0067] Preferably, the stirring speed is 200-400 rpm.

[0068] Preparation of the silicon hydroxyl functionalized magnetic beads: the magnetic nanoparticles are dispersed in ethanol, deionized water is added, ultrasonic dispersion is performed for 20-40 min, ammonia water is added, ultrasonic dispersion is performed for 5-7 min, tetraethyl orthosilicate is added, ultrasonic dispersion is performed for 5-15 min, reaction is performed at 35-45 DEG C under stirring for 5-7 h, magnetic separation is performed after cooling to room temperature, washing is performed with deionized water until the pH of the washing liquid is 6.9-7.1, vacuum drying is performed at 55-65 DEG C for 10-15 h, and the silicon hydroxyl functionalized magnetic beads are obtained.

[0069] Preferably, the mass-volume ratio of the magnetic nanoparticles to ethanol is 1-10 g: 150-1500 mL.

[0070] Preferably, the mass-volume ratio of the magnetic nanoparticles to deionized water is 1-10 g: 50-500 mL.

[0071] Preferably, the mass-volume ratio of the magnetic nanoparticles to ammonia water is 1-10 g: 60-600 mL.

[0072] Preferably, the mass-volume ratio of the magnetic nanoparticles to tetraethyl orthosilicate is 1-10 g: 1-10 mL, and the stirring speed is 500 rpm.

[0073] Preparation of the carboxyl functionalized magnetic beads: the silicon hydroxyl functionalized magnetic beads are dispersed in acetonitrile, ultrasonic dispersion is performed for 20-40 min, hexadienesuccinate, (E)-5-methylhex-3-enoic acid and azobisisobutyronitrile are added, ultrasonic dispersion is performed for 5-15 min, nitrogen is introduced, reaction is performed at 105-115 DEG C under stirring for 5-15 min, reaction is performed at 85-95 DEG C under stirring for 2-4 h, magnetic separation is performed after cooling to room temperature, washing is performed with ethanol until the supernatant is colorless, vacuum drying is performed at 55-65 DEG C for 10-15 h, and the carboxyl functionalized magnetic beads are obtained.

[0074] Preferably, the mass-volume ratio of the silicon hydroxyl functionalized magnetic beads to acetonitrile is 1-10 g: 50-500 mL.

[0075] Preferably, the mass ratio of hexadienesuccinate to the silicon hydroxyl functionalized magnetic beads is 1-10: 1-10.

[0076] Preferably, the volume-mass ratio of (E)-5-methylhex-3-enoic acid to the silicon hydroxyl functionalized magnetic beads is 3-30 mL: 1-10 g.

[0077] Preferably, the mass ratio of azobisisobutyronitrile to the silicon hydroxyl functionalized magnetic beads is 0.1-1: 1-10.

[0078] The application also provides a preparation method of the carboxyl functionalized magnetic beads, comprising:

[0079] Preparation of carboxyl functionalized magnetic beads: the silicon hydroxyl functionalized magnetic beads were dispersed in acetonitrile, ultrasonic dispersion for 20-40 min, hexadiene succinate, (E)-5-methylhex-3-ene acid, 2-acetylamino non-8-ene acid and azobisisobutyronitrile were added, ultrasonic for 5-15 min, nitrogen was introduced, reaction was carried out at 105-115℃ and stirring for 5-15 min, reaction was carried out at 85-95℃ and stirring for 2-4 h, after cooling to room temperature, magnetic separation was carried out, washed with ethanol until the supernatant was colorless, vacuum drying at 55-65℃ for 10-15 h, to obtain carboxyl functionalized magnetic beads.

[0080] Preferably, the mass-volume ratio of the silicon hydroxyl functionalized magnetic beads to acetonitrile is 1-10 g: 50-500 mL.

[0081] Preferably, the mass ratio of hexadiene succinate to silicon hydroxyl functionalized magnetic beads is 1-10: 1-10.

[0082] Preferably, the volume-mass ratio of (E)-5-methylhex-3-ene acid to silicon hydroxyl functionalized magnetic beads is 3-30 mL: 1-10 g.

[0083] Preferably, the volume-mass ratio of 2-acetylamino non-8-ene acid to silicon hydroxyl functionalized magnetic beads is 3-30 mL: 1-10 g.

[0084] Preferably, the mass ratio of azobisisobutyronitrile to silicon hydroxyl functionalized magnetic beads is 0.1-1: 1-10.

[0085] The present application has the following beneficial effects: high sensitivity and specificity, convenient operation and reliable results, providing quantitative basis for efficacy and risk assessment. Therefore, the present application is a high-efficiency and rapid kit for predicting prognosis and diarrhea adverse reactions of breast cancer targeted therapy. BRIEF DESCRIPTION OF DRAWINGS

[0086] Figure 1 It is a transmission electron microscope schematic diagram of carboxyl functionalized magnetic beads.

[0087] Figure 2 It is a schematic diagram of the effect of pyrotinib on the tumor size of SKBR3 tumor-bearing nude mice.

[0088] Figure 3 It is a schematic diagram of the effect of pyrotinib on the tumor proliferation volume of SKBR3 tumor-bearing nude mice.

[0089] Figure 4The effect of pyrotinib on the tumor weight level of SKBR3 tumor-bearing nude mice.

[0090] Figure 5 The effect of pyrotinib on the tumor structure morphology of SKBR3 tumor-bearing nude mice.

[0091] Figure 6 The effect of pyrotinib on the tumor apoptosis level of SKBR3 tumor-bearing nude mice.

[0092] Figure 7 The effect of pyrotinib on the number of diarrhea within 1h of SKBR3 tumor-bearing nude mice.

[0093] Figure 8 The effect of pyrotinib on the colon length of SKBR3 tumor-bearing nude mice.

[0094] Figure 9 The effect of pyrotinib on the small intestinal structure morphology expression level of SKBR3 tumor-bearing nude mice.

[0095] Figure 10 The effect of pyrotinib on the large intestinal structure morphology expression level of SKBR3 tumor-bearing nude mice.

[0096] Figure 11 The effect of pyrotinib on the colon histological score of SKBR3 tumor-bearing nude mice.

[0097] Figure 12 The effect of pyrotinib on the peripheral blood IL-6, endotoxin, intestinal tissue MPO levels of SKBR3 tumor-bearing nude mice.

[0098] Figure 13 The expression level of EGFR in tumor tissues of nude mice in each group detected by IHC method.

[0099] Figure 14 The expression level of HER2 in tumor tissues of nude mice in each group detected by IHC method.

[0100] Figure 15 The expression level of Src in tumor tissues of nude mice in each group detected by IHC method.

[0101] Figure 16 The expression level of IL-6 in tumor tissues of nude mice in each group detected by IHC method.

[0102] Figure 17 The expression level of JAK2 in tumor tissues of nude mice in each group detected by IHC method.

[0103] Figure 18 The expression level of STAT3 in tumor tissues of nude mice in each group detected by IHC method.

[0104] Figure 19 Schematic diagram of IL-6 expression in IHC staining before treatment of patients.

[0105] Figure 20 Schematic diagram of STAT3 expression in IHC staining before treatment of patients.

[0106] Figure 21 Schematic diagram of IL-6 and STAT3 expression in IHC staining before treatment of patients. DETAILED DESCRIPTION

[0107] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0108] The concepts involved in the present application will be described below in combination with the drawings. It should be pointed out here that the following descriptions of the concepts are only to make the content of the present application easier to understand, and do not represent the limitation of the scope of protection of the present application; meanwhile, the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the drawings and in combination with the embodiments.

[0109] Example 1:

[0110] Preparation of magnetic bead conjugated antibody: dissolve goat anti-mouse IgG antibody in PBS buffer solution with pH=7.4 to obtain antibody solution; disperse carboxyl functionalized magnetic beads in 2-morpholinoethanesulfonic acid buffer solution with pH=6, add 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide, incubate at 37°C for 30 min, discard supernatant, wash once with PBS buffer solution with pH=7.4, add antibody solution, incubate at 37°C for 2.5 h to obtain magnetic bead conjugated antibody. The carboxyl functionalized magnetic beads are polystyrene modified nanometer ferroferric oxide, purchased from Biyun Tian Biotechnology Co., Ltd., with a particle size of 1 µm; the mass-volume ratio of goat anti-mouse IgG antibody to PBS buffer solution is 120 mg: 240 mL, the mass-volume ratio of carboxyl functionalized magnetic beads to 2-morpholinoethanesulfonic acid buffer solution is 2 g: 200 mL, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride to carboxyl functionalized magnetic beads is 200 mg: 2 g, and the mass ratio of N-hydroxysuccinimide to carboxyl functionalized magnetic beads is 100 mg: 2 g; the mass of the antibody solution is measured by the mass of the goat anti-mouse IgG antibody therein, and the mass ratio of goat anti-mouse IgG antibody to carboxyl functionalized magnetic beads is 120 mg: 2 g.

[0111] Preparation of antibody conjugate: dissolve horseradish peroxidase and magnetic bead conjugated antibody in PBS buffer solution with pH=7.4, add glutaraldehyde, react at room temperature in the dark for 2 h, add sodium borohydride under ice bath conditions to react for 30 min to terminate the reaction, and obtain the antibody conjugate. The mass-volume ratio of horseradish peroxidase to PBS buffer solution is 20 mg: 10 mL, the mass-volume ratio of magnetic bead conjugated antibody to PBS buffer solution is 10 mg: 10 mL, the mass ratio of glutaraldehyde to magnetic bead conjugated antibody is 100 µg: 10 mg, and the mass ratio of sodium borohydride to magnetic bead conjugated antibody is 500 µg: 10 mg.

[0112] Preparation of antibody diluent: dissolve bovine serum albumin, Tween-20 and sodium azide in PBS buffer solution with pH=7.4, thoroughly stir and dissolve, filter sterilize through a 0.22 µm filter membrane to obtain the antibody diluent. The mass-volume ratio of bovine serum albumin to PBS buffer solution is 5 g: 100 mL, the volume ratio of Tween-20 to PBS buffer solution is 10 µL: 100 mL, and the mass-volume ratio of sodium azide to PBS buffer solution is 20 mg: 100 mL.

[0113] Preparation of the kit: the kit comprises specific antibodies, antibody conjugates and immunohistochemical detection reagents; the specific antibodies and the antibody conjugates are separately frozen and stored after being dispensed; the immunohistochemical detection reagents are stored in the dark after being dispensed. The specific antibodies comprise IL-6 antibodies, HER2 antibodies, EGFR antibodies, JAK2 antibodies, Src antibodies and STAT3 antibodies; the immunohistochemical detection reagents comprise antibody diluents, antigen retrieval buffer, washing solution, blocking solution, color developing solution, counterstaining solution, bluing solution, xylene solution and gradient ethanol solution; the antigen retrieval buffer is a citrate buffer with pH = 6; the washing solution comprises PBS buffer with pH = 7.4; the blocking solution is 3% hydrogen peroxide solution; the color developing solution is diaminobenzidine color developing solution; the counterstaining solution is hematoxylin staining solution; the bluing solution is lithium carbonate bluing solution; the gradient ethanol solution comprises 85%, 95% and 100% ethanol solutions.

[0114] Preparation of the primary antibody working solution: the specific antibodies are separately diluted with the antibody diluents to obtain the primary antibody working solution. The volume ratio of the specific antibodies to the antibody diluents is 1:100.

[0115] Preparation of the secondary antibody working solution: the antibody conjugates are diluted with the antibody diluents to obtain the secondary antibody working solution. The volume ratio of the antibody conjugates to the antibody diluents is 1:200.

[0116] Preparation of the immunohistochemical staining pathological section: the pathological section is baked at 70℃ for 90 min, soaked in xylene I for 10 min and then soaked in xylene II for 10 min for dewaxing treatment; the section is soaked in gradient ethanol for hydration treatment, with the gradient being 100%, 95% and 85% for 10 min each time; the section is washed with tap water for 3 times and then washed with deionized water for 3 times, 3% hydrogen peroxide is added for blocking treatment at room temperature; the antigen retrieval buffer is added and baked at 80℃ for 15 min for antigen retrieval treatment; the section is cooled to room temperature, washed with PBS buffer for 3 times, and then the primary antibody working solution is added dropwise in the range of the immunohistochemical oil pen circle and covers the tissue completely, the section is placed in a 4℃ refrigerator for incubation overnight; the section is washed with PBS buffer for 3 times after being balanced at room temperature, the secondary antibody working solution is added dropwise, washed with PBS buffer for 3 times after being incubated at room temperature for 60 min, to form an antigen-antibody complex; diaminobenzidine color developing solution is added dropwise, washed under running water after termination; the section is placed in hematoxylin for counterstaining and then rinsed; the section is placed in 1% hydrochloric acid alcohol for differentiation and then blued in lithium carbonate bluing solution; the section is dehydrated in gradient ethanol, with the gradient being 85%, 95%, 100% and 100%; the section is transparentized in xylene I and xylene II to obtain the immunohistochemical staining pathological section.

[0117] Example 2: Compared with Example 1, the only difference is the preparation of the carboxyl functionalized magnetic beads.

[0118] Preparation of magnetic nanoparticles: FeCl3-6H2O was dispersed in ethylene glycol, ultrasonic dispersion for 30 min, polyacrylic acid and urea were added, ultrasonic dissolution for 10 min, reaction at 180℃ under stirring for 24 h, magnetic separation after cooling to room temperature, washing with deionized water until the washing liquid pH=7, vacuum drying at 60℃ for 12 h to obtain magnetic nanoparticles. The mass-volume ratio of FeCl3-6H2O to ethylene glycol was 4g:150mL, the mass ratio of polyacrylic acid to FeCl3-6H2O was 1.5:4, the mass ratio of urea to FeCl3-6H2O was 9:4, and the stirring speed was 300rpm.

[0119] Preparation of silicon hydroxyl functionalized magnetic beads: the magnetic nanoparticles were dispersed in ethanol, deionized water was added, ultrasonic dispersion for 30 min, ammonia was added, ultrasonic for 6 min, tetraethyl orthosilicate was added, ultrasonic for 10 min, reaction at 40℃ under stirring for 6 h, magnetic separation after cooling to room temperature, washing with deionized water until the washing liquid pH=7, vacuum drying at 60℃ for 12 h to obtain silicon hydroxyl functionalized magnetic beads. The mass-volume ratio of magnetic nanoparticles to ethanol was 2g:300mL, the mass-volume ratio of magnetic nanoparticles to deionized water was 2g:100mL, the mass-volume ratio of magnetic nanoparticles to ammonia was 2g:120mL, the mass-volume ratio of magnetic nanoparticles to tetraethyl orthosilicate was 2g:2mL, and the stirring speed was 500rpm.

[0120] Preparation of carboxyl functionalized magnetic beads: the silicon hydroxyl functionalized magnetic beads were dispersed in acetonitrile, ultrasonic dispersion for 30 min, hexadienesuccinate, (E)-5-methylhex-3-ene acid and azobisisobutyronitrile were added, ultrasonic for 10 min, nitrogen was introduced, reaction at 110℃ under stirring for 10 min, reaction at 90℃ under stirring for 3 h, magnetic separation after cooling to room temperature, washing with ethanol until the supernatant was colorless, vacuum drying at 60℃ for 12 h to obtain carboxyl functionalized magnetic beads. The mass-volume ratio of silicon hydroxyl functionalized magnetic beads to acetonitrile was 2g:100mL, the mass ratio of hexadienesuccinate to silicon hydroxyl functionalized magnetic beads was 2:2, the volume-mass ratio of (E)-5-methylhex-3-ene acid to silicon hydroxyl functionalized magnetic beads was 6mL:2g, and the mass ratio of azobisisobutyronitrile to silicon hydroxyl functionalized magnetic beads was 0.2:2.

[0121] Example 3: The difference between this example and Example 2 is only in the preparation of carboxyl functionalized magnetic beads.

[0122] Preparation of carboxyl functionalized magnetic beads: the silicon hydroxyl functionalized magnetic beads were dispersed in acetonitrile, ultrasonic dispersion for 30 min, then hexadiene succinate, (E)-5-methylhex-3-enoic acid and azobisisobutyronitrile were added, ultrasonic for 10 min, nitrogen was introduced, reaction was carried out at 110°C under stirring for 10 min, reaction was carried out at 90°C under stirring for 3 h, magnetic separation was carried out after cooling to room temperature, ethanol was used for cleaning until the supernatant was colorless, vacuum drying was carried out at 60°C for 12 h, and carboxyl functionalized magnetic beads were obtained. The mass-volume ratio of silicon hydroxyl functionalized magnetic beads to acetonitrile was 2 g: 100 mL, the mass ratio of hexadiene succinate to silicon hydroxyl functionalized magnetic beads was 2:2, the volume-mass ratio of (E)-5-methylhex-3-enoic acid to silicon hydroxyl functionalized magnetic beads was 9 mL:2 g, and the mass ratio of azobisisobutyronitrile to silicon hydroxyl functionalized magnetic beads was 0.2:2.

[0123] Example 4: Compared with Example 2, the only difference is the preparation of carboxyl functionalized magnetic beads.

[0124] Preparation of carboxyl functionalized magnetic beads: the silicon hydroxyl functionalized magnetic beads were dispersed in acetonitrile, ultrasonic dispersion for 30 min, then hexadiene succinate, (E)-5-methylhex-3-enoic acid and azobisisobutyronitrile were added, ultrasonic for 10 min, nitrogen was introduced, reaction was carried out at 110°C under stirring for 10 min, reaction was carried out at 90°C under stirring for 3 h, magnetic separation was carried out after cooling to room temperature, ethanol was used for cleaning until the supernatant was colorless, vacuum drying was carried out at 60°C for 12 h, and carboxyl functionalized magnetic beads were obtained. The mass-volume ratio of silicon hydroxyl functionalized magnetic beads to acetonitrile was 2 g: 100 mL, the mass ratio of hexadiene succinate to silicon hydroxyl functionalized magnetic beads was 2:2, the volume-mass ratio of (E)-5-methylhex-3-enoic acid to silicon hydroxyl functionalized magnetic beads was 9 mL:2 g, and the mass ratio of azobisisobutyronitrile to silicon hydroxyl functionalized magnetic beads was 0.2:2.

[0125] Example 5: Compared with Example 2, the only difference is the preparation of carboxyl functionalized magnetic beads.

[0126] Preparation of carboxyl functionalized magnetic beads: the silicon hydroxyl functionalized magnetic beads were dispersed in acetonitrile, ultrasonic dispersion for 30 min, then hexadiene succinate, (E)-5-methylhex-3-ene acid, 2-acetylamino non-8-ene acid and azobisisobutyronitrile were added, ultrasonic dispersion for 10 min, nitrogen was introduced, and the reaction was carried out at 110℃ under stirring for 10 min and at 90℃ under stirring for 3 h. After cooling to room temperature, magnetic separation was carried out, and the magnetic beads were washed with ethanol until the supernatant was colorless. The magnetic beads were dried at 60℃ under vacuum for 12 h to obtain the carboxyl functionalized magnetic beads. The mass-volume ratio of the silicon hydroxyl functionalized magnetic beads to acetonitrile was 2g:100mL, the mass ratio of hexadiene succinate to the silicon hydroxyl functionalized magnetic beads was 2:2, the volume-mass ratio of (E)-5-methylhex-3-ene acid to the silicon hydroxyl functionalized magnetic beads was 6mL:2g, the volume-mass ratio of 2-acetylamino non-8-ene acid to the silicon hydroxyl functionalized magnetic beads was 9mL:2g, and the mass ratio of azobisisobutyronitrile to the silicon hydroxyl functionalized magnetic beads was 0.2:2.

[0127] Comparative Example 1: Compared with Example 1, the only difference is that hexadiene succinate is not used in the preparation of the carboxyl functionalized magnetic beads.

[0128] Comparative Example 2: Compared with Example 1, the only difference is that (E)-5-methylhex-3-ene acid is not used in the preparation of the carboxyl functionalized magnetic beads.

[0129] Test Example 1: Microstructure characterization of the carboxyl functionalized magnetic beads.

[0130] Test sample: the carboxyl functionalized magnetic beads prepared in Example 2.

[0131] Test method: the carboxyl functionalized magnetic beads were dispersed in ethanol, ultrasonic dispersion was carried out until the magnetic beads were uniformly dispersed, a magnetic bead solution was obtained, the magnetic bead solution was dropped on double copper mesh using a pipette, the copper mesh was folded after natural air drying, and transmission electron microscopy was used for observation.

[0132] The transmission electron microscopy image of the carboxyl functionalized magnetic beads prepared in the present application is shown in Figure 1 , which shows that the carboxyl functionalized magnetic beads as shown in the figure are successfully obtained.

[0133] Test Example 2: Coupling rate test of magnetic bead coupled antibody.

[0134] Test sample: the carboxyl functionalized magnetic beads prepared in each example and comparative example.

[0135] Test method: dissolve goat anti-mouse IgG antibody with mass m0 in PBS buffer solution with pH=7.4 to obtain an antibody solution; disperse carboxyl functionalized magnetic beads in 2-morpholinoethanesulfonic acid buffer solution with pH=6, add 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide, incubate at 37℃ for 30 min on a shaking table, discard the supernatant, wash once with PBS buffer solution with pH=7.4, add the antibody solution, incubate at 37℃ for 2.5 h on a shaking table, transfer the reaction solution to a centrifuge tube, place it on a magnetic stand for 5 min, and then adsorb the magnetic beads on the wall of the tube, and then take the supernatant; add PBS buffer solution with pH=7.4 to the magnetic beads, resuspend by gently shaking, separate again on the magnetic stand, and repeat the washing for 3 times, collect the washing liquid each time, combine to the supernatant, and record the total volume V; use an ultraviolet-visible spectrophotometer to measure the absorbance of each standard at a wavelength of 280 nm, obtain the antibody concentration C in the supernatant, calculate the mass m1 of the antibody in the supernatant according to m1=C×V, and calculate the coupling rate of the antibody coupled to the magnetic beads according to the coupling rate (%)=(m0-m1) / m0×100%.

[0136] The coupling rate test results of the magnetic bead coupled antibody prepared in the application are shown in Table 1.

[0137] Table 1 Coupling rate test results of magnetic bead coupled antibody

[0138]

[0139] Example 1 has a simple surface modification, and the carboxyl active site is easily damaged in the activation process, so the number of effective active sites for coupling antibodies is small, and the antibody coupling rate is low; Example 2 forms carboxyl functionalized magnetic beads by silicon hydroxyl functionalization and further modification of (E)-5-methylhex-3-ene acid and hexadiene succinate, the functional layer formed in the modification process is more stable, reducing the damage to the surface structure in the activation process, thereby the antibody coupling rate is significantly improved compared with Example 1; Example 3 increases the amount of (E)-5-methylhex-3-ene acid, further enhancing the stability of the coupling bond, so that the coupling rate is further improved compared with Example 2; Examples 4-5 introduce 2-acetylamino non-8-ene acid, which optimizes the dispersibility of the magnetic beads in the reaction system, reduces non-specific adsorption, and the higher amount of 2-acetylamino non-8-ene acid in Example 5 further improves the coupling rate; Comparative Example 1 does not use hexadiene succinate, the surface functionalization of the magnetic beads is not complete, the number of active sites is small and unevenly distributed, resulting in a significantly lower antibody coupling rate than Example 2; Comparative Example 2 does not use (E)-5-methylhex-3-ene acid, the magnetic bead surface lacks this key modification group, the carboxyl functional layer formed is not stable enough, and the active site is easily inactivated, thereby the antibody coupling rate is low.

[0140] Test Example 3: IL-6 sensitivity test of the kit.

[0141] Test sample: the kit prepared by each example and comparative example.

[0142] Test method: standard solution containing different concentrations of marker IL-6 was prepared, and the concentration gradient was set as 100 ng / mL, 50 ng / mL, 25 ng / mL, 10 ng / mL, 5 ng / mL, 1 ng / mL and 0.1 ng / mL, diluted with PBS buffer, and the negative control was a blank sample containing only PBS buffer; 100 μL of the marker standard solution with different concentrations was taken, 10 μL of magnetic bead coupled antibody was added, 37℃ oscillation incubation was carried out for 30 min, magnetic separation was carried out to discard the supernatant, and PBS washing was carried out for 3 times; 100 μL of secondary antibody working solution was added, room temperature incubation was carried out for 20 min, magnetic separation and washing were carried out for 3 times, diaminobenzidine color developing liquid was added for color development for 5 min, and the reaction was terminated; the positive staining area was analyzed by Image J, and the average optical density was calculated.

[0143] The IL-6 sensitivity test results of the kit prepared by the application are shown in Table 2.

[0144] Table 2 IL-6 sensitivity test results of the kit

[0145]

[0146] Example 1 has poor magnetic bead dispersity, low diaminobenzidine color development intensity, and low average optical density of the finally obtained dyed sample; examples 2-3 are modified by silicon hydroxyl functionalization and (E)-5-methylhex-3-enoic acid and 2-acetylamino non-8-enoic acid to form a more stable carboxyl functional layer, thereby enhancing the diaminobenzidine color development intensity and improving the optical density; examples 4-5 introduce 2-acetylamino non-8-enoic acid to reduce the aggregation of magnetic beads and antibody conjugates, so that the binding of antibody and IL-6 is more sufficient, and the optical density is improved; comparative examples 1-2 lack (E)-5-methylhex-3-enoic acid or 2-acetylamino non-8-enoic acid modification, and the surface active site is insufficient, and the optical density is significantly lower than that of example 2.

[0147] Test Example 4: Detection of prognosis and diarrhea adverse reaction of pyrotinib treatment for human HER2 positive breast cancer SKBR3 tumor-bearing nude mice.

[0148] Test method:

[0149] Preparation of pyrotinib related diarrhea human breast cancer SKBR3 cell tumor-bearing nude mice model: BALB / c-Nude nude mice were purchased and adaptively fed for 1 week, and the feeding conditions were as follows: SPF level, 50 ± 10% constant humidity, 25 ± 3°C constant temperature, free feeding and drinking, and regular replacement of bedding. After adaptive feeding, the mice were continuously gavaged with 40 mg / kg of pyrotinib for 10 days to promote diarrhea, and gavaged for 10 days and then washed out for 3 days; human HER2-positive breast cancer SKBR3 cells in the logarithmic growth phase and in good growth condition were digested, centrifuged, and the cell density was adjusted to 5 × 10 7 million / mL with serum-free medium, and 50% volume of high growth factor Matrigel was mixed; after isoflurane anesthesia of the nude mice, 200 μL of the prepared cell suspension was inoculated under the right mammary pad of each nude mouse using a 1 mL syringe, and after inoculation, the mice were returned to the cage for normal feeding; after 10-14 days of growth, when the tumor volume of each nude mouse reached 100 mm 3 , the nude mice were randomly divided into: blank group and pyrotinib group; the blank group was gavaged with 100 μL of normal saline, 1 time / day, for 21 consecutive days; the pyrotinib group: each nude mouse was gavaged with 100 μL of 5 mg / mL pyrotinib, 1 time / day, for 21 consecutive days.

[0150] Detection of tumors in pyrotinib-treated human HER2-positive breast cancer SKBR3 tumor-bearing nude mice: On days 1, 3, 6, 9, 12, 15, 18, and 21 of the experiment, the tumor volume was recorded and the tumor proliferation curve was plotted, and 24 h after the last administration, the nude mice were sacrificed, the tumors were aseptically removed, and the tumor mass was recorded; the SKBR3 tumor-bearing nude mouse tumors were embedded using paraffin embedding method, and tumor tissue paraffin sections were obtained for HE staining; after staining, whole slide scanning was performed to observe the morphological characteristics of the tumor tissues of the mice in each group.

[0151] The effect of pyrotinib on the tumor size of SKBR3 tumor-bearing nude mice is shown in Figure 2 , the effect of pyrotinib on the tumor proliferation volume of SKBR3 tumor-bearing nude mice is shown in Figure 3 , and the effect of pyrotinib on the tumor weight level of SKBR3 tumor-bearing nude mice is shown in Figure 4 . Except for the blank group, the tumor volume and tumor growth rate of the tumor-bearing nude mice in each group decreased to varying degrees after starting gavage administration; compared with the blank group, the tumor volume and tumor mass of the pyrotinib group decreased to varying degrees; indicating that the use of pyrotinib alone can achieve good anti-tumor effect.

[0152] The effect of pyrotinib on the tumor structure and morphology of SKBR3 tumor-bearing nude mice is shown in Figure 5As shown, the tumor cells in the tumor tissue of the blank group were densely arranged, and most of the cells were multinucleated cells, and the nuclei were round or oval; the effect of pyrotinib on the apoptosis level of the tumor of the SKBR3 tumor-bearing nude mice was as shown in the following table: Figure 6 As shown, the tumor cells in the tumor tissue of the blank group were densely arranged, and most of the cells were multinucleated cells, and the nuclei were round or oval; the effect of pyrotinib on the apoptosis level of the tumor of the SKBR3 tumor-bearing nude mice was as shown in the following table:

[0153] On the 1st, 3rd, 6th, 9th, 12th, 15th, 18th and 21st day of the experiment, the diarrhea of the nude mice in each group within 1 hour was recorded using a multifunctional small animal autonomous activity recorder, and 24 hours after the last administration, the colon tissue of the nude mice was aseptically stripped after the nude mice were sacrificed, and the colon length of the nude mice in each group was measured; the small intestine tissue and colon tissue of the nude mice in each group were embedded using a paraffin embedding method, and the paraffin sections of the small intestine tissue and colon tissue were obtained for HE staining, and after the staining was completed, the morphology of the small intestine tissue and colon tissue in each group was observed using a whole slide scanner, and a Histological score was performed to evaluate the morphology of the mouse tissue in each group.

[0154] The effect of pyrotinib on the number of diarrhea within 1 hour of the SKBR3 tumor-bearing nude mice was as shown in the following table: Figure 7 As shown, the effect of pyrotinib on the colon length of the SKBR3 tumor-bearing nude mice was as shown in the following table: Figure 8 As shown, compared with the blank group, the number of diarrhea within 1 hour of the nude mice in the pyrotinib group was significantly increased, and the colon length was significantly shortened; pyrotinib in the treatment of human HER2-positive breast cancer SKBR3 tumor-bearing nude mice would cause the nude mice to have the adverse reactions of increased number of diarrhea and shortened colon length.

[0155] The effect of pyrotinib on the expression level of the small intestine structure morphology of the SKBR3 tumor-bearing nude mice was as shown in the following table: Figure 9 As shown, the effect of pyrotinib on the expression level of the large intestine structure morphology of the SKBR3 tumor-bearing nude mice was as shown in the following table: Figure 10 As shown, the cells in the small intestine tissue and colon tissue of the blank group were regularly arranged, and the nuclei were large and deeply stained; the effect of pyrotinib on the histological score of the colon of the SKBR3 tumor-bearing nude mice was as shown in the following table: Figure 11As shown, in the mucosa layer, many well-arranged crypt structures can be seen, and the histological score of the colon tissue is low; in the pyrotinib group, inflammatory cells and immune cells infiltrate the colon tissue, the colon mucosa appears mild edema, and the goblet cells and crypts are destroyed or lost, the colon epithelial cells are arranged in a missing manner, only a small amount of inflammatory cells infiltrate the small intestinal tissue, the tissue structure is relatively complete, and the histological score of the colon tissue is increased; it is proved that the increase in the number of diarrhea in the treatment of human HER2-positive breast cancer SKBR3 tumor-bearing nude mice with pyrotinib is mainly due to the inflammatory reaction of "ulcerative colitis" caused by inflammatory infiltration and structural damage of the colon tissue, and the damage to the structure of the small intestine is not obvious during pyrotinib treatment, only a small amount of inflammatory cells infiltrate, confirming that the pathological site of the diarrhea adverse reaction caused by pyrotinib is mainly the colon.

[0156] Before the nude mice were killed, 4kda FITC-dextran was administered by gavage 12h before the nude mice were killed, and after the nude mice were killed, the peripheral blood of the nude mice in each group was collected by retro-orbital venous blood collection method, and after the serum was separated, the serum limulus reagent kit was also used to detect the endotoxin level and IL-6 expression level in the peripheral blood serum of the nude mice in each group; after the colon tissues of the nude mice in each group were obtained, the anti-myeloperoxidase kit was used to detect the inflammation level in the colon tissue of the nude mice.

[0157] The effects of pyrotinib on the IL-6, endotoxin and intestinal tissue MPO levels of the peripheral blood of the SKBR3 tumor-bearing nude mice are shown in Figure 12 As shown in (a), the effect of pyrotinib on the IL-6 level of the peripheral blood of the SKBR3 tumor-bearing nude mice is shown in Figure 12 As shown in (a), the effect of pyrotinib on the IL-6 level of the peripheral blood of the SKBR3 tumor-bearing nude mice is shown in Figure 12 As shown in (b), the effect of pyrotinib on the intestinal tissue MPO level of the SKBR3 tumor-bearing nude mice is shown in Figure 12 As shown in (c), compared with the blank group, the IL-6 level, endotoxin level and intestinal tissue MPO level in the peripheral blood of the pyrotinib group were significantly increased, proving that the increase in the number of diarrhea in the treatment of human HER2-positive breast cancer SKBR3 tumor-bearing nude mice with pyrotinib is mainly due to the increase in the inflammation level of the colon tissue, the increase in the level of myeloperoxidase, the damage to the colon epithelial tissue and the increase in the colon permeability, which in turn causes the increase in the endotoxin level in the peripheral blood, an inflammatory reaction, and also proves that the treatment of pyrotinib can cause the increase in the expression level of IL-6 in the peripheral blood, causing a systemic inflammatory reaction.

[0158] At 24 h after the last administration, the colon of the nude mice was aseptically stripped after sacrifice, and the colon tissue of the SKBR3 tumor-bearing nude mice was embedded by paraffin embedding, and the colon tissue paraffin sections were obtained for IHC staining to detect the protein expression levels of IL-6 / STAT3 signaling pathway and HER2 / STAT3 signaling pathway related proteins, EGFR, HER2, Src, IL-6, JAK2 and STAT3 in the colon tissue and the subcellular localization level of STAT3.

[0159] The IHC method was used to detect the expression levels of EGFR in the tumor tissues of the nude mice in each group, as shown in Figure 13 The IHC method was used to detect the expression levels of HER2 in the tumor tissues of the nude mice in each group, as shown in Figure 14 The IHC method was used to detect the expression levels of Src in the tumor tissues of the nude mice in each group, as shown in Figure 15 The IHC method was used to detect the expression levels of IL-6 in the tumor tissues of the nude mice in each group, as shown in Figure 16 The IHC method was used to detect the expression levels of JAK2 in the tumor tissues of the nude mice in each group, as shown in Figure 17 The IHC method was used to detect the expression levels of STAT3 in the tumor tissues of the nude mice in each group, as shown in Figure 18 Compared with the blank group, the expression levels of EGFR, HER2 and Src proteins in the pyrotinib group were reduced, the expression levels of IL-6 and JAK2 were increased, and the expression level of STAT3 was not obviously changed, but the proportion of STAT3 protein entering the nucleus in the subcellular localization level was lower, indicating that the treatment of pyrotinib can inhibit the HER2 / Src / STAT3 signaling pathway related proteins in the colon tissue, leading to damage of the colon tissue after pyrotinib treatment, and due to the blockage of the HER2 / Src / STAT3 signaling pathway, the self-repairing ability of the colon tissue is reduced; the treatment of pyrotinib activates the expression level of STAT3 through the IL-6 / JAK2 pathway without affecting the HER2 (EGFR) / Src signal transduction; due to the inhibition of pyrotinib on the HER2 (EGFR) / Src signal transduction, STAT3 protein cannot enter the nucleus to play the transcription factor function to transcribe the colon proliferation and invasion related proteins, and a large amount of STAT3 enters the mitochondria to stimulate the occurrence of colon related inflammation and activate the IL-6 / JAK2 signal transduction.

[0160] Test Example 5: Detection of prognosis and diarrhea adverse reactions of pyrotinib treatment in clinical patients.

[0161] Test method:

[0162] The correlation between the degree of diarrhea adverse reaction and the prognosis of patients with advanced HER2-positive BRCA treated with pyrotinib was analyzed using the sample data analysis of PANDORA study. A total of 79 patients were enrolled in the PANDORA study, and the treatment method of pyrotinib 400 mg / d d1-d28 + docetaxel 75 mg / m2 d1 (once every 3 weeks) was used for treatment. The median PFS of the patients was 16.0 months 95% CI (11.2, 20.8). Among all the patients, a total of 49 patients had diarrhea reaction, and 17 patients had diarrhea adverse reaction ≥ grade 3. After all the patients were stratified according to long PFS and short PFS, it was found that in the long PFS patients, there were 21 patients with grade 0 diarrhea, 5 patients with grade I diarrhea, 7 patients with grade II diarrhea, 5 patients with grade III diarrhea, and 1 patient with grade IV diarrhea; in the short PFS patients, there were 8 patients with grade 0 diarrhea, 9 patients with grade I diarrhea, 11 patients with grade II diarrhea, 9 patients with grade III diarrhea, and 2 patients with grade IV diarrhea; 9 patients in the long PFS group and the short PFS group were randomly selected for IL-6 and STAT3 IHC staining.

[0163] The IL-6 expression in IHC staining of the patients before treatment is shown in Figure 19 The STAT3 expression in IHC staining of the patients before treatment is shown in Figure 20 The IL-6 and STAT3 expression in IHC staining of the patients before treatment is shown in Figure 21 In the long PFS group, IL-6 and STAT3 appeared low-level expression; while in the short PFS group, IL-6 and STAT3 appeared high-level expression; which indicated that the degree of diarrhea adverse reaction of patients treated with pyrotinib was related to the prognosis, and appropriate level of adverse reaction could improve the treatment effect of patients and make patients obtain longer PFS; while more serious adverse reaction might mean that patients had lower PFS and worse treatment benefit.

[0164] The above embodiments and / or implementations are only used to illustrate the preferred embodiments and / or implementations of the present technology, and do not limit the embodiments of the present technology in any form. Any person skilled in the art can make some changes or modifications to other equivalent embodiments without departing from the scope of the technology disclosed in the present disclosure, but should be considered as substantially the same technology or embodiments as the present disclosure.

[0165] The principles and implementation manners of the present application are described herein by using specific examples, and the above example descriptions are only used to help understand the method of the present application and its core idea. The above descriptions are only preferred embodiments of the present application, and it should be pointed out that, due to the limited nature of the language expression, there are objectively infinite specific structures, and for ordinary skilled persons in the technical field, some improvements, refinements or changes can be made without departing from the principles of the present application, and the above technical features can also be combined in an appropriate manner; these improvements, refinements, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, shall be regarded as the protection scope of the present application.

Claims

1. A kit comprising a specific antibody, an antibody-drug conjugate, and an immunohistochemical detection reagent, wherein the specific antibody comprises one or more of IL-6 antibody, HER2 antibody, EGFR antibody, JAK2 antibody, Src antibody, and STAT3 antibody; the antibody-drug conjugate comprises a magnetic bead-conjugated antibody and horseradish peroxidase, wherein the magnetic bead-conjugated antibody comprises carboxyl-functionalized magnetic beads and goat anti-mouse IgG antibody, wherein the carboxyl-functionalized magnetic beads comprise silanol-functionalized magnetic beads modified with (E)-5-methylhexanoic acid and hexadiene succinate, and wherein the silanol-functionalized magnetic beads comprise tetraethyl orthosilicate-modified magnetic nanoparticles.

2. The kit according to claim 1, wherein the mass ratio of the magnetic bead-conjugated antibody to horseradish peroxidase is 10-100:5-50.

3. The kit according to claim 1, wherein the mass ratio of the carboxyl-functionalized magnetic beads to goat anti-mouse IgG antibody is 1-10g:60-600mg.

4. The kit according to claim 1, wherein the carboxyl-functionalized magnetic beads comprise polystyrene-modified magnetic nanoparticles.

5. The kit according to claim 1, wherein the mass ratio of hexadiene succinate to silanol-functionalized magnetic beads is 1-10:1-10.

6. The kit according to claim 1, wherein the volume-to-mass ratio of (E)-5-methylhex-3-enoic acid to silanol-functionalized magnetic beads is 3-30 mL: 1-10 g.

7. The kit according to claim 1, wherein the immunohistochemical detection reagent comprises an antibody diluent, the antibody diluent comprising PBS buffer and bovine serum albumin, wherein the mass-to-volume ratio of bovine serum albumin to PBS buffer is 2.5-25 g: 50-500 mL.

8. The kit according to claim 1, wherein the immunohistochemical detection reagent includes an antigen retrieval buffer, wherein the antigen retrieval buffer is a citrate buffer with a pH of 5.8-6.

2.

9. The kit according to claim 1, wherein the immunohistochemical detection reagent comprises a chromogenic solution and a counterstaining solution, wherein the chromogenic solution is a diaminobenzidine chromogenic solution and the counterstaining solution is a hematoxylin staining solution.

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