Kit for predicting breast cancer targeted therapy prognosis and diarrhea adverse reaction

Abstract

The invention discloses a kit for predicting breast cancer targeted therapy prognosis and diarrhea adverse reaction, which evaluates the curative effect of pyrrotinib in treating HER2 positive breast cancer and diarrhea risk by detecting the expression levels of IL-6, HER2, EGFR, JAK2, Src and STAT3. The kit comprises a specific antibody, an antibody conjugate and an immunohistochemical detection reagent, the antibody conjugate comprises carboxyl functionalized magnetic beads, and the carboxyl functionalized magnetic beads are obtained by modifying reagents such as hexadiene succinate and (E)-5-methylhex-3-olefine acid; an immunohistochemical technology is utilized to qualitatively and quantitatively analyze markers in tumor tissues, and the kit provides a reliable tool for individualized prognosis evaluation and diarrhea adverse reaction monitoring of breast cancer targeted therapy, and is beneficial to clinically formulating a precise treatment scheme and improving the treatment income of a patient.

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, not only reducing the clinical efficacy of patients during treatment, but also increasing 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 for 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: A kit comprising specific antibodies, antibody conjugates and immunohistochemical detection reagents. Preferably, the specific antibodies comprise one or more of IL-6 antibodies, HER2 antibodies, EGFR antibodies, JAK2 antibodies, Src antibodies and STAT3 antibodies.

[0007] Preferably, the antibody conjugates comprise magnetic bead conjugated antibodies and horseradish peroxidase.

[0008] Preferably, the mass ratio of the magnetic bead conjugated antibodies and the horseradish peroxidase is 10-100:5-50.

[0009] Preferably, the magnetic bead conjugated antibodies comprise carboxyl functionalized magnetic beads and goat anti-mouse IgG antibodies.

[0010] Preferably, the mass ratio of the carboxyl functionalized magnetic beads and the goat anti-mouse IgG antibodies is 1-10g:60-600mg.

[0011] 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.

[0012] Preferably, the mass ratio of the hexadienesuccinate and the silicon hydroxyl functionalized magnetic beads is 1-10:1-10.

[0013] 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.

[0014] 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.

[0015] Preferably, the immunohistochemical detection reagents comprise an antibody diluent, the antibody diluent comprises PBS buffer and bovine serum albumin.

[0016] Preferably, the mass volume ratio of the bovine serum albumin and the PBS buffer is 2.5-25g:50-500mL.

[0017] Preferably, the immunohistochemical detection reagents comprise an antigen retrieval buffer.

[0018] Preferably, the antigen repair buffer is a citrate buffer with pH = 5.8-6.2.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] The application further provides a preparation method of the magnetic bead-coupled antibody, which comprises the following steps: Preparation of the magnetic bead-coupled antibody: dissolve goat anti-mouse IgG antibody in a PBS buffer solution with pH = 7.3-7.5 to obtain an antibody solution; disperse carboxyl functionalized magnetic beads in a 2-morpholinoethanesulfonic acid buffer solution with pH = 5.9-6.1, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and shake and incubate at 36.8-37.2°C for 20-40 min, discard the supernatant, wash once with the PBS buffer solution with pH = 7.3-7.5, add the antibody solution, and shake and incubate at 36.8-37.2°C for 2-3 h to obtain the magnetic bead-coupled antibody.

[0023] 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.

[0024] Preferably, the mass-volume ratio of the goat anti-mouse IgG antibody to the PBS buffer solution is 60-600 mg:120-1200 mL.

[0025] 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.

[0026] Preferably, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to the carboxyl functionalized magnetic beads is 100-1000 mg:1-10 g.

[0027] Preferably, the mass ratio of N-hydroxysuccinimide to the carboxyl functionalized magnetic beads is 50-500 mg:1-10 g.

[0028] Preferably, the mass of the antibody solution is measured by the mass of the goat anti-mouse IgG antibody therein, and the mass ratio of the goat anti-mouse IgG antibody to the carboxyl-functionalized magnetic beads is 60-600 mg:1-10 g.

[0029] The application also provides a preparation method of an antibody conjugate, comprising: Preparation of the antibody conjugate: dissolve the horseradish peroxidase and the magnetic bead-coupled antibody in a PBS buffer solution with pH=7.3-7.5, add glutaraldehyde, react for 1.5-2.5 h under room temperature and in the dark, add sodium borohydride under ice bath conditions, react for 25-35 min to terminate the reaction, and obtain the antibody conjugate.

[0030] Preferably, the mass-volume ratio of the horseradish peroxidase to the PBS buffer solution is 10-100 mg:5-50 mL.

[0031] Preferably, the mass-volume ratio of the magnetic bead-coupled antibody to the PBS buffer solution is 5-50 mg:5-50 mL.

[0032] Preferably, the mass ratio of the glutaraldehyde to the magnetic bead-coupled antibody is 50-500 µg:5-50 mg.

[0033] Preferably, the mass ratio of the sodium borohydride to the magnetic bead-coupled antibody is 250-2500 µg:5-50 mg.

[0034] The application also provides a preparation method of an antibody diluent, comprising: Preparation of the antibody diluent: dissolve the bovine serum albumin, Tween-20, and sodium azide in a PBS buffer solution with pH=7.3-7.5, filter sterilization through a 0.20-0.25 µm filter membrane after fully stirring and dissolving, and obtain the antibody diluent.

[0035] Preferably, the mass-volume ratio of the bovine serum albumin to the PBS buffer solution is 2.5-25 g:50-500 mL.

[0036] Preferably, the volume ratio of the Tween-20 to the PBS buffer solution is 5-50 µL:50-500 mL.

[0037] Preferably, the mass-volume ratio of the sodium azide to the PBS buffer solution is 10-100 mg:50-500 mL.

[0038] The application also provides a preparation method of a kit, comprising: Preparation of the kit: the kit comprises specific antibodies, antibody conjugates, and immunohistochemical detection reagents; the specific antibodies and the antibody conjugates are divided and stored after freeze-drying; and the immunohistochemical detection reagents are divided and stored after avoiding light.

[0039] Preferably, the specific antibodies include one or more of IL-6 antibodies, HER2 antibodies, EGFR antibodies, JAK2 antibodies, Src antibodies and STAT3 antibodies.

[0040] Preferably, the immunohistochemical detection reagents include antibody diluent, antigen retrieval buffer, washing solution, blocking solution, color developing solution, counterstaining solution, bluing solution, xylene solution and gradient ethanol solution.

[0041] Preferably, the antigen retrieval buffer is a citrate buffer with pH=5.9-6.1.

[0042] Preferably, the washing solution includes PBS buffer with pH=7.3-7.5.

[0043] Preferably, the blocking solution is a 2.5-3.5% hydrogen peroxide solution.

[0044] Preferably, the color developing solution is a diaminobenzidine color developing solution.

[0045] Preferably, the counterstaining solution is a hematoxylin staining solution.

[0046] Preferably, the bluing solution is a lithium carbonate bluing solution.

[0047] Preferably, the gradient ethanol solution includes 80-100% ethanol solution.

[0048] The application further provides a preparation method of the primary antibody working solution, comprising: Preparation of the primary antibody working solution: dilute the specific antibodies with the antibody diluent respectively to obtain the primary antibody working solution.

[0049] Preferably, the volume ratio of the specific antibodies to the antibody diluent is 0.5-5: 50-500.

[0050] The application further provides a preparation method of the secondary antibody working solution, comprising: Preparation of the secondary antibody working solution: dilute the antibody conjugate with the antibody diluent to obtain the secondary antibody working solution.

[0051] Preferably, the volume ratio of the antibody conjugate to the antibody diluent is 0.5-5: 100-1000.

[0052] The application further provides a preparation method of the immunohistochemical staining pathological section, comprising: 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.

[0053] The application further provides a preparation method of the carboxyl functionalized magnetic beads. 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.

[0054] Preferably, the mass-volume ratio of FeCl3 6H2O to ethylene glycol is 2-20 g: 75-750 mL.

[0055] Preferably, the mass ratio of polyacrylic acid to FeCl3 6H2O is 0.75-7.5: 2-20.

[0056] Preferably, the mass ratio of urea to FeCl3 6H2O is 4.5-45: 2-20.

[0057] Preferably, the stirring speed is 200-400 rpm.

[0058] 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.

[0059] Preferably, the mass-volume ratio of the magnetic nanoparticles to ethanol is 1-10 g: 150-1500 mL.

[0060] Preferably, the mass-volume ratio of the magnetic nanoparticles to deionized water is 1-10 g: 50-500 mL.

[0061] Preferably, the mass-volume ratio of the magnetic nanoparticles to ammonia water is 1-10 g: 60-600 mL.

[0062] 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.

[0063] 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, hexadiene succinate, (E)-5-methylhex-3-ene 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.

[0064] Preferably, the mass-volume ratio of the silicon hydroxyl functionalized magnetic beads to acetonitrile is 1-10 g: 50-500 mL.

[0065] Preferably, the mass ratio of hexadiene succinate to the silicon hydroxyl functionalized magnetic beads is 1-10: 1-10.

[0066] Preferably, the volume-mass ratio of (E)-5-methylhex-3-ene acid to the silicon hydroxyl functionalized magnetic beads is 3-30 mL: 1-10 g.

[0067] Preferably, the mass ratio of azobisisobutyronitrile to the silicon hydroxyl functionalized magnetic beads is 0.1-1: 1-10.

[0068] The application also provides a preparation method of the carboxyl functionalized magnetic beads, comprising: 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.

[0069] Preferably, the mass-volume ratio of the silicon hydroxyl functionalized magnetic beads to acetonitrile is 1-10 g: 50-500 mL.

[0070] Preferably, the mass ratio of hexadiene succinate to silicon hydroxyl functionalized magnetic beads is 1-10: 1-10.

[0071] 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.

[0072] 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.

[0073] Preferably, the mass ratio of azobisisobutyronitrile to silicon hydroxyl functionalized magnetic beads is 0.1-1: 1-10.

[0074] 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

[0075] Figure 1 It is a transmission electron microscope schematic diagram of carboxyl functionalized magnetic beads.

[0076] Figure 2 It is a schematic diagram of the effect of pyrotinib on the tumor size of SKBR3 tumor-bearing nude mice.

[0077] Figure 3 It is a schematic diagram of the effect of pyrotinib on the tumor proliferation volume of SKBR3 tumor-bearing nude mice.

[0078] Figure 4The effect of pyrotinib on the tumor weight level of SKBR3 tumor-bearing nude mice.

[0079] Figure 5 The effect of pyrotinib on the tumor structure morphology of SKBR3 tumor-bearing nude mice.

[0080] Figure 6 The effect of pyrotinib on the tumor apoptosis level of SKBR3 tumor-bearing nude mice.

[0081] Figure 7 The effect of pyrotinib on the number of diarrhea within 1h of SKBR3 tumor-bearing nude mice.

[0082] Figure 8 The effect of pyrotinib on the colon length of SKBR3 tumor-bearing nude mice.

[0083] Figure 9 The effect of pyrotinib on the small intestinal structure morphology expression level of SKBR3 tumor-bearing nude mice.

[0084] Figure 10 The effect of pyrotinib on the large intestinal structure morphology expression level of SKBR3 tumor-bearing nude mice.

[0085] Figure 11 The effect of pyrotinib on the colon histological score of SKBR3 tumor-bearing nude mice.

[0086] Figure 12 The effect of pyrotinib on the peripheral blood IL-6, endotoxin, intestinal tissue MPO levels of SKBR3 tumor-bearing nude mice.

[0087] Figure 13 The expression level of EGFR in tumor tissue of nude mice in each group detected by IHC method.

[0088] Figure 14 The expression level of HER2 in tumor tissue of nude mice in each group detected by IHC method.

[0089] Figure 15 The expression level of Src in tumor tissue of nude mice in each group detected by IHC method.

[0090] Figure 16 The expression level of IL-6 in tumor tissue of nude mice in each group detected by IHC method.

[0091] Figure 17 The expression level of JAK2 in tumor tissue of nude mice in each group detected by IHC method.

[0092] Figure 18 The expression level of STAT3 in tumor tissue of nude mice in each group detected by IHC method.

[0093] Figure 19 Schematic diagram of IL-6 expression in IHC staining before treatment of patients.

[0094] Figure 20 Schematic diagram of STAT3 expression in IHC staining before treatment of patients.

[0095] Figure 21 Schematic diagram of IL-6 and STAT3 expression in IHC staining before treatment of patients. DETAILED DESCRIPTION

[0096] The technical solutions in the embodiments of the present application will be apparently and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0097] The concepts involved in the present application will be described below in combination with the drawings. It should be pointed out here that the descriptions of the concepts below are only for the purpose of making the content of the present application easier to understand, and do not represent the limitation on the protection scope 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.

[0098] Embodiment 1 Preparation of magnetic bead coupled antibody: dissolve goat anti-mouse IgG antibody 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-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and incubate on a shaking table at 37°C for 30 min, discard the supernatant, wash once with PBS buffer solution with pH=7.4, add the antibody solution, and incubate on a shaking table at 37°C for 2.5 h to obtain the magnetic bead coupled antibody. The carboxyl functionalized magnetic beads are polystyrene modified nanometer ferroferric oxide, which are purchased from Biyun Tian Biotechnology Co., Ltd., and the particle size is 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-ethylcarbodiimide 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 based on 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 120 mg: 2 g.

[0099] Preparation of antibody conjugate: horseradish peroxidase and magnetic bead conjugated antibody were dissolved in PBS buffer solution with pH=7.4, glutaraldehyde was added, and the reaction was carried out at room temperature in the dark for 2h, sodium borohydride was added under ice bath condition for 30min to terminate the reaction, and the antibody conjugate was obtained. The mass-volume ratio of horseradish peroxidase to PBS buffer solution was 20mg:10mL, the mass-volume ratio of magnetic bead conjugated antibody to PBS buffer solution was 10mg:10mL, the mass ratio of glutaraldehyde to magnetic bead conjugated antibody was 100µg:10mg, and the mass ratio of sodium borohydride to magnetic bead conjugated antibody was 500µg:10mg.

[0100] Preparation of antibody diluent: bovine serum albumin, Tween-20 and sodium azide were dissolved in PBS buffer solution with pH=7.4, and after being fully stirred and dissolved, sterilization was performed by 0.22μm filter membrane to obtain the antibody diluent. The mass-volume ratio of bovine serum albumin to PBS buffer solution was 5g:100mL, the volume ratio of Tween-20 to PBS buffer solution was 10µL:100mL, and the mass-volume ratio of sodium azide to PBS buffer solution was 20mg:100mL.

[0101] Preparation of kit: the kit includes specific antibodies, antibody conjugates and immunohistochemical detection reagents; the specific antibodies and antibody conjugates are divided and freeze-dried for storage; and the immunohistochemical detection reagents are divided and stored in the dark. The specific antibodies include IL-6 antibody, HER2 antibody, EGFR antibody, JAK2 antibody, Src antibody and STAT3 antibody; the immunohistochemical detection reagents include antibody diluent, antigen repair buffer, washing solution, blocking solution, color developing solution, re-staining solution, blue returning solution, xylene solution and gradient ethanol solution; the antigen repair buffer is citrate buffer with pH=6; the washing solution includes PBS buffer solution with pH=7.4; the blocking solution is 3% hydrogen peroxide solution; the color developing solution is diaminobenzidine color developing solution; the re-staining solution is hematoxylin staining solution; the blue returning solution is lithium carbonate blue returning solution; and the gradient ethanol solution includes 85%, 95% and 100% ethanol solutions.

[0102] Preparation of primary antibody working solution: the specific antibodies were respectively diluted with antibody diluent to obtain the primary antibody working solution. The volume ratio of specific antibody to antibody diluent was 1:100.

[0103] Preparation of secondary antibody working solution: the antibody conjugate was diluted with antibody diluent to obtain the secondary antibody working solution. The volume ratio of antibody conjugate to antibody diluent was 1:200.

[0104] Preparation of immunohistochemical staining pathological section: the pathological section was baked at 70°C for 90 min, deparaffinized by soaking in xylene I for 10 min and then in xylene II for 10 min, hydrated by soaking in gradient ethanol for 10 min each at 100%, 95% and 85%, washed with tap water for 3 times and then with deionized water for 3 times, blocked by adding 3% hydrogen peroxide at room temperature, subjected to antigen retrieval by adding antigen retrieval buffer and baking at 80°C for 15 min, cooled to room temperature, washed with PBS buffer for 3 times, and then the primary antibody working solution was added to the immunohistochemical oil pen circle range and fully covered the tissue, and the slide was incubated in a 4°C refrigerator overnight; after equilibration at room temperature, the slide was washed with PBS buffer for 3 times, and then the secondary antibody working solution was added, incubated at room temperature for 60 min, washed with PBS buffer for 3 times to form an antigen-antibody complex; the diaminobenzidine color developing solution was added, washed under running water after termination; the slide was stained with hematoxylin, differentiated in 1% hydrochloric acid alcohol, and subjected to blue returning in lithium carbonate blue returning solution; the slide was dehydrated in gradient ethanol at 85%, 95%, 100% and 100%, and then transparentized in xylene I and xylene II to obtain the immunohistochemical staining pathological section.

[0105] Example 2: Compared with Example 1, the only difference is the preparation of the carboxyl functionalized magnetic beads.

[0106] Preparation of magnetic nanoparticles: FeCl3·6H2O was dispersed in ethylene glycol, ultrasonically dispersed for 30 min, polyacrylic acid and urea were added, ultrasonically dissolved for 10 min, reacted at 180°C under stirring for 24 h, magnetically separated after cooling to room temperature, washed with deionized water until the washing liquid pH=7, vacuum dried at 60°C for 12 h to obtain the magnetic nanoparticles. The mass-volume ratio of FeCl3·6H2O to ethylene glycol was 4 g:150 mL, 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 300 rpm.

[0107] Preparation of the silicon hydroxyl functionalized magnetic beads: the magnetic nanoparticles were dispersed in ethanol, deionized water was added, ultrasonic dispersion was performed for 30 min, ammonia water was added, ultrasonic dispersion was performed for 6 min, tetraethyl orthosilicate was added, ultrasonic dispersion was performed for 10 min, reaction was performed at 40°C under stirring for 6 h, magnetic separation was performed after cooling to room temperature, washing was performed with deionized water until the pH of the washing liquid was 7, vacuum drying was performed at 60°C for 12 h, and the silicon hydroxyl functionalized magnetic beads were obtained. The mass-volume ratio of the magnetic nanoparticles to ethanol was 2 g:300 mL, the mass-volume ratio of the magnetic nanoparticles to deionized water was 2 g:100 mL, the mass-volume ratio of the magnetic nanoparticles to ammonia water was 2 g:120 mL, the mass-volume ratio of the magnetic nanoparticles to tetraethyl orthosilicate was 2 g:2 mL, and the stirring speed was 500 rpm.

[0108] Preparation of the carboxyl functionalized magnetic beads: the silicon hydroxyl functionalized magnetic beads were dispersed in acetonitrile, ultrasonic dispersion was performed for 30 min, hexadienesuccinate, (E)-5-methylhex-3-enoic acid and azobisisobutyronitrile were added, ultrasonic dispersion was performed for 10 min, nitrogen was introduced, reaction was performed at 110°C under stirring for 10 min, reaction was performed at 90°C under stirring for 3 h, magnetic separation was performed after cooling to room temperature, washing was performed with ethanol until the supernatant was colorless, vacuum drying was performed at 60°C for 12 h, and the carboxyl functionalized magnetic beads were obtained. The mass-volume ratio of the silicon hydroxyl functionalized magnetic beads to acetonitrile was 2 g:100 mL, the mass ratio of hexadienesuccinate to the silicon hydroxyl functionalized magnetic beads was 2:2, the volume-mass ratio of (E)-5-methylhex-3-enoic acid to the silicon hydroxyl functionalized magnetic beads was 6 mL:2 g, and the mass ratio of azobisisobutyronitrile to the silicon hydroxyl functionalized magnetic beads was 0.2:2.

[0109] Example 3: Compared with Example 2, the only difference is the preparation of the carboxyl functionalized magnetic beads.

[0110] Preparation of the carboxyl functionalized magnetic beads: the silicon hydroxyl functionalized magnetic beads were dispersed in acetonitrile, ultrasonic dispersion was performed for 30 min, hexadienesuccinate, (E)-5-methylhex-3-enoic acid and azobisisobutyronitrile were added, ultrasonic dispersion was performed for 10 min, nitrogen was introduced, reaction was performed at 110°C under stirring for 10 min, reaction was performed at 90°C under stirring for 3 h, magnetic separation was performed after cooling to room temperature, washing was performed with ethanol until the supernatant was colorless, vacuum drying was performed at 60°C for 12 h, and the carboxyl functionalized magnetic beads were obtained. The mass-volume ratio of the silicon hydroxyl functionalized magnetic beads to acetonitrile was 2 g:100 mL, the mass ratio of hexadienesuccinate to the silicon hydroxyl functionalized magnetic beads was 2:2, the volume-mass ratio of (E)-5-methylhex-3-enoic acid to the silicon hydroxyl functionalized magnetic beads was 6 mL:2 g, and the mass ratio of azobisisobutyronitrile to the silicon hydroxyl functionalized magnetic beads was 0.2:2.

[0111] Example 4: Compared with Example 2, the only difference is the preparation of the carboxyl functionalized magnetic beads.

[0112] Preparation of carboxyl functionalized magnetic beads: the silicon hydroxyl functionalized magnetic beads were dispersed in acetonitrile, ultrasonic dispersion for 30 min, then hexadienesuccinate, (E)-5-methylhex-3-enoic acid, 2-acetamidonon-8-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, after cooling to room temperature, magnetic separation was carried out, washed with ethanol until the supernatant was colorless, vacuum drying at 60°C for 12 h to obtain the 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-enoic acid to silicon hydroxyl functionalized magnetic beads was 6mL:2g, the volume-mass ratio of 2-acetamidonon-8-enoic 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.

[0113] Example 5: the difference between this example and example 2 is only in the preparation of carboxyl functionalized magnetic beads.

[0114] Preparation of carboxyl functionalized magnetic beads: the silicon hydroxyl functionalized magnetic beads were dispersed in acetonitrile, ultrasonic dispersion for 30 min, then hexadienesuccinate, (E)-5-methylhex-3-enoic acid, 2-acetamidonon-8-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, after cooling to room temperature, magnetic separation was carried out, washed with ethanol until the supernatant was colorless, vacuum drying at 60°C for 12 h to obtain the 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-enoic acid to silicon hydroxyl functionalized magnetic beads was 6mL:2g, the volume-mass ratio of 2-acetamidonon-8-enoic acid to silicon hydroxyl functionalized magnetic beads was 9mL:2g, and the mass ratio of azobisisobutyronitrile to silicon hydroxyl functionalized magnetic beads was 0.2:2.

[0115] Comparative Example 1: the difference between this comparative example and example 1 is only in the preparation of carboxyl functionalized magnetic beads, which does not use hexadienesuccinate.

[0116] Comparative Example 2: the difference between this comparative example and example 1 is only in the preparation of carboxyl functionalized magnetic beads, which does not use (E)-5-methylhex-3-enoic acid.

[0117] Test Example 1: microstructure characterization of carboxyl functionalized magnetic beads.

[0118] Test sample: carboxyl functionalized magnetic beads prepared in Example 2.

[0119] Test method: The carboxyl functionalized magnetic beads were dispersed in ethanol, and ultrasonic dispersion was performed until uniform, to obtain a magnetic bead solution; the magnetic bead solution was dropped on double copper mesh using a pipette gun; after natural air drying, the copper mesh was folded; and transmission electron microscopy was used for observation.

[0120] The transmission electron microscopy image of the carboxyl functionalized magnetic beads prepared in the application is shown in Figure 1 The transmission electron microscopy image of the carboxyl functionalized magnetic beads prepared in the application is shown in

[0121] Test example 2: coupling rate test of magnetic bead coupled antibody.

[0122] Test sample: carboxyl functionalized magnetic beads prepared in each example and comparative example.

[0123] Test method: goat anti-mouse IgG antibody with a mass of m0 was dissolved in PBS buffer solution with pH=7.4 to obtain an antibody solution; the carboxyl functionalized magnetic beads were dispersed in 2-morpholinoethanesulfonic acid buffer solution with pH=6, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added, and incubation was performed at 37℃ for 30 min on a shaking table; the supernatant was discarded, and the PBS buffer solution with pH=7.4 was used for cleaning once; the antibody solution was added, and incubation was performed at 37℃ for 2.5 h on a shaking table; the reaction liquid was transferred to a centrifuge tube, and was placed on a magnetic stand for 5 min; when the magnetic beads were completely adsorbed on the wall of the tube, the supernatant was sucked; the PBS buffer solution with pH=7.4 was added to the magnetic beads, and resuspension was performed by gently shaking; the magnetic beads were separated again on the magnetic stand, and the washing was repeated for 3 times; the washing liquid was collected each time, and was combined to the supernatant; the total volume V was recorded; the ultraviolet-visible spectrophotometer was used to determine the absorbance of each standard at a wavelength of 280 nm, to obtain the antibody concentration C in the supernatant; according to m1=C×V, the mass m1 of the antibody in the supernatant was calculated; and according to the coupling rate (%)=(m0-m1) / m0×100%, the coupling rate of the magnetic bead coupled antibody was calculated.

[0124] The coupling rate test results of the magnetic bead coupled antibody prepared in the application are shown in Table 1.

[0125] Table 1: Coupling rate test results of magnetic bead coupled antibody

[0126] Example 1 has a simple surface modification, and the carboxyl active site is easily damaged during activation, so the number of effective active sites for antibody coupling is small, and the antibody coupling rate is low; Example 2 forms a carboxyl functionalized magnetic bead by functionalizing the silicon hydroxyl group and further modifying it with (E)-5-methylhex-3-ene acid and hexadiene succinate. The functional layer formed in this modification process is more stable, reducing the damage to the surface structure during activation, thereby significantly improving the antibody coupling rate compared to Example 1; Example 3 increases the amount of (E)-5-methylhex-3-ene acid, further enhancing the stability of the coupling bond, resulting in a further increase in the coupling rate compared to Example 2; Examples 4-5 introduce 2-acetylamino non-8-ene acid, which optimizes the dispersibility of the magnetic beads in the reaction system and reduces non-specific adsorption. In Example 5, a higher amount of 2-acetylamino non-8-ene acid further improves the coupling rate; Comparative Example 1 does not use hexadiene succinate, and the surface functionalization of the magnetic beads is incomplete, with a small number of active sites and uneven distribution, resulting in a significantly lower antibody coupling rate than Example 2; Comparative Example 2 does not use (E)-5-methylhex-3-ene acid, and the magnetic bead surface lacks this key modification group, resulting in an unstable carboxyl functional layer and easily inactivated active sites, thereby resulting in a low antibody coupling rate.

[0127] Test Example 3: IL-6 sensitivity test of the kit.

[0128] Test sample: kits prepared in each example and comparative example.

[0129] Test method: Prepare standard solution containing different concentrations of marker IL-6, with concentration gradient 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 is a blank sample containing only PBS buffer; take 100 μL of different concentrations of marker standard solution, add 10 μL of magnetic bead coupled antibody, incubate at 37℃ for 30 min, magnetic separation and discard the supernatant, wash with PBS for 3 times; add 100 μL of secondary antibody working solution, incubate at room temperature for 20 min, wash 3 times by magnetic separation, add diaminobenzidine color developing solution for color development for 5 min, terminate the reaction; analyze the pixel of the positive staining area by Image J, and calculate the average optical density.

[0130] The IL-6 sensitivity test results of the kit prepared by the present application are shown in Table 2.

[0131] Table 2 IL-6 sensitivity test results of the kit

[0132] The average optical density of the dyed sample is low due to poor dispersion of the magnetic beads and low color intensity of the diamino benzidine in Example 1; the carboxyl functional layer is more stable in Examples 2-3 through silicon hydroxyl functionalization and (E)-5-methylhex-3-enoic acid and 2-acetamidyl non-8-enoic acid modification, thereby enhancing the color intensity of the diamino benzidine and improving the optical density; the aggregation of the magnetic beads and the antibody conjugate is reduced in Examples 4-5 by introducing 2-acetamidyl non-8-enoic acid, making the binding of the antibody and IL-6 more sufficient, and the optical density is improved; the optical density is significantly lower than that of Example 2 in Comparative Examples 1-2 due to the lack of (E)-5-methylhex-3-enoic acid or 2-acetamidyl non-8-enoic acid modification and insufficient active sites on the surface of the magnetic beads.

[0133] Test Example 4: Detection of prognosis and adverse reactions of diarrhea in human HER2-positive breast cancer SKBR3 tumor-bearing nude mice treated with pyrotinib.

[0134] Test method: Preparation of human breast cancer SKBR3 cell tumor-bearing nude mice model with pyrotinib-related diarrhea: 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 bedding change. After adaptive feeding, the mice were continuously gavaged with 40 mg / kg of pyrotinib for 10 days to promote diarrhea, 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 μL 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 then the nude 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.

[0135] Detection of tumors in human HER2-positive breast cancer SKBR3 tumor-bearing nude mice after pyrotinib treatment: the tumor volume was recorded on the 1st, 3rd, 6th, 9th, 12th, 15th, 18th and 21st days of the experiment, and the tumor proliferation curve was drawn. After 24 h of the last administration, the nude mice were sacrificed, the tumors were aseptically removed, and the tumor weight was recorded; the SKBR3 tumor-bearing nude mouse tumors were embedded using paraffin embedding method, and the tumor tissue paraffin sections were obtained for HE staining. After staining, the morphology of the tumor tissue of each group of mice was observed by whole slide scanning.

[0136] The effect of pyrotinib on the tumor size of SKBR3 tumor-bearing nude mice was as shown in Figure 2 The effect of pyrotinib on the tumor proliferation volume of SKBR3 tumor-bearing nude mice was as shown in Figure 3 The effect of pyrotinib on the tumor weight level of SKBR3 tumor-bearing nude mice was as shown in Figure 4 As shown in the figure, except for the blank group, the tumor volume and tumor growth rate of tumor-bearing nude mice in each group appeared to decrease to varying degrees after starting gavage administration; compared with the blank group, the tumor volume and tumor weight of the pyrotinib group appeared to decrease to varying degrees; it is proved that the use of pyrotinib alone can achieve good anti-tumor effect.

[0137] The effect of pyrotinib on the tumor structure morphology of SKBR3 tumor-bearing nude mice was as shown in Figure 5 The tumor tissue of the blank group of nude mice had dense tumor cells, and most of the cells presented multinucleated cell morphology, and the cell nucleus presented round or oval shape; the tumor apoptosis level of SKBR3 tumor-bearing nude mice treated with pyrotinib was as shown in Figure 6 The tumor tissue of the pyrotinib group of nude mice had dispersed tumor cells, and part of the tumor tissue appeared liquefactive necrosis, and a small number of tumor cells had broken cell membranes, and the tumor cells showed apoptosis performance, proving that pyrotinib can change the dense cell structure of tumor tissue, and the cell morphology shows apoptosis characteristics.

[0138] 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 being 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 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 of each group was observed using whole slide scanning, and the Histological score was scored to evaluate the morphology of the tissue of each group of mice.

[0139] The effect of pyrotinib on the number of diarrhea within 1 hour of SKBR3 tumor-bearing nude mice was as shown in Figure 7 The effect of pyrotinib on the colon length of SKBR3 tumor-bearing nude mice was as shown in Figure 8 Compared with the blank group, the number of diarrhea within 1 hour of the nude mice in the pyrotinib group increased significantly, and the colon length was significantly shortened; pyrotinib in the treatment of human HER2-positive breast cancer SKBR3 tumor-bearing nude mice can cause the nude mice to have the adverse reactions of increased number of diarrhea and shortened colon length.

[0140] The effect of pyrotinib on the small intestine structure morphology expression level of SKBR3 tumor-bearing nude mice was as shown in Figure 9 The effect of pyrotinib on the large intestine structure morphology expression level of SKBR3 tumor-bearing nude mice was as shown in Figure 10As shown, the blank group of nude mice small intestine tissue and colon tissue cell arrangement regular, cell nucleus is larger and nuclear deep dye; the influence of pyrotinib on the histological score of colon tissue of SKBR3 tumor-bearing nude mice is as shown in Figure 11 As shown, in the mucosa layer, many arranged neat crypt structure can be seen, and the colon tissue Histological score is low; the colon tissue of pyrotinib group mice appears inflammatory cell and immune cell infiltration, and the colon mucosa appears mild edema, and the goblet cell and crypt appear destruction or loss, and the colon epithelial cell arrangement is missing, and only a small amount of inflammatory cell infiltration exists in the small intestine tissue, and the tissue structure is relatively complete, and the colon tissue Histological score is increased; it is proved that the increase of diarrhea times of pyrotinib in the treatment of human HER2 positive breast cancer SKBR3 tumor-bearing nude mice is mainly due to the inflammatory infiltration and structural damage of colon tissue, leading to the inflammatory reaction of "ulcerative colitis", and the damage of pyrotinib treatment to the tissue structure of small intestine is not obvious, and only slight inflammatory cell infiltration appears, which confirms that the pathological site of the adverse reaction of diarrhea caused by pyrotinib is mainly the colon.

[0141] FITC-dextran of 4kda was given by gavage 12h before the nude mice were killed, and the peripheral blood of each group of nude mice was collected by using the retro-orbital venous blood collection method after the nude mice were killed. After the serum was separated, the endotoxin level and IL-6 expression level in the peripheral blood serum of each group of nude mice were detected by using the serum limulus reagent kit; after the colon tissue of each group of nude mice was obtained, the inflammation level in the colon tissue of nude mice was detected by using the anti-myeloperoxidase kit.

[0142] The influence of pyrotinib on the peripheral blood IL-6, endotoxin and intestinal tissue MPO levels of SKBR3 tumor-bearing nude mice is as shown in Figure 12 As shown, the influence of pyrotinib on the peripheral blood IL-6 level of SKBR3 tumor-bearing nude mice is as shown in Figure 12 As shown in (a), the influence of pyrotinib on the peripheral blood endotoxin level of SKBR3 tumor-bearing nude mice is as shown in Figure 12 As shown in (b), the influence of pyrotinib on the intestinal tissue MPO level of SKBR3 tumor-bearing nude mice is as 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 are obviously increased, which proves that the increase of diarrhea times of pyrotinib in the treatment of human HER2 positive breast cancer SKBR3 tumor-bearing nude mice is mainly due to the increase of colon tissue inflammation level and the increase of myeloperoxidase level, leading to the damage of colon epithelial tissue and the increase of colon permeability, thereby causing the endotoxin level in the peripheral blood to be an inflammatory reaction, and also proving that the treatment of pyrotinib can cause the increase of IL-6 expression level in the peripheral blood, causing a systemic inflammatory reaction.

[0143] At 24 h after the last administration, the colon of the nude mice was removed aseptically 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.

[0144] 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.

[0145] Test Example 5: Detection of prognosis and diarrhea adverse reactions of pyrotinib treatment in clinical patients.

[0146] Test method: The correlation between the degree of diarrhea adverse reactions and the prognosis of patients with advanced HER2-positive BRCA treated with pyrotinib was analyzed using the sample data analysis of the 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 reactions, and 17 patients had adverse reactions of diarrhea ≥ 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.

[0147] The IL-6 expression in the IHC staining of the patients before treatment is shown in Figure 19 The STAT3 expression in the IHC staining of the patients before treatment is shown in Figure 20 The IL-6 and STAT3 expression in the 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 reactions of patients treated with pyrotinib was related to the prognosis, and appropriate level of adverse reactions could improve the treatment effect of patients and make patients obtain longer PFS; while more serious adverse reactions might mean that patients had lower PFS and worse treatment benefits.

[0148] The above-described embodiments and / or implementations are merely 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, and such changes or modifications should be considered as substantially the same technology or embodiments as the present disclosure.

[0149] 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 specific antibodies, antibody conjugates and immunohistochemical detection reagents, wherein the specific antibodies include one or more of IL-6 antibodies, HER2 antibodies, EGFR antibodies, JAK2 antibodies, Src antibodies and STAT3 antibodies.

2. A kit according to claim 1, wherein the antibody conjugate comprises magnetic bead-coupled antibody and horseradish peroxidase, and the mass ratio of the magnetic bead-coupled antibody to the horseradish peroxidase is 10-100:5-50.

3. A kit according to claim 2, wherein the magnetic bead-coupled antibody comprises carboxyl-functionalized magnetic beads and goat anti-mouse IgG antibody, and the mass ratio of the carboxyl-functionalized magnetic beads to the goat anti-mouse IgG antibody is 1-10g:60-600mg.

4. A kit according to claim 3, wherein the carboxyl-functionalized magnetic beads comprise polystyrene-modified magnetic nanoparticles; or, (E)-5-methylhex-3-enoic acid and hexadiene succinate-modified silanol-functionalized magnetic beads, wherein the silanol-functionalized magnetic beads comprise ethyl orthosilicate-modified magnetic nanoparticles. 5 . The kit according to claim 4 , wherein the mass ratio of the hexadienyl succinate to the silanol-functionalized magnetic beads is 1-10:1-10. 6 . The kit according to claim 4 , wherein the volume-to-mass ratio of the (E)-5-methylhex-3-enoic acid to the silanol-functionalized magnetic beads is 3-30 mL:1-10 g.

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

8. A kit according to claim 1, wherein the immunohistochemical detection reagent comprises an antigen retrieval buffer, and 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 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.

10. Use of the kit according to any one of claims 1 to 9 for predicting the prognosis of targeted therapy for breast cancer and the adverse reaction of diarrhea, wherein the breast cancer includes HER2-positive breast cancer.

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