A kit for predicting pCR in neoadjuvant therapy of HER2-positive breast cancer

Abstract

The invention discloses a HER2-positive breast cancer neoadjuvant therapy pCR prediction kit, belonging to the field of biotechnology. By preparing functional nanomagnetic beads, activated functional nanomagnetic beads and protein antibodies, and establishing a protein detection method, a protein detection kit for detecting TUBB protein, CUTA protein and ATP5MF protein is obtained. The activated functional nanomagnetic beads are fully combined with the antibodies, and the expression levels of TUBB, CUTA and ATP5MF proteins can be well detected and reflected, so as to be applied to the prediction of pCR for the neoadjuvant therapy of HER2-positive breast cancer. And the HER2-positive breast cancer neoadjuvant therapy pCR prediction kit has a low minimum detection limit and coefficient of variation, indicating high sensitivity and good repeatability, high precision, and can quickly and accurately perform detection and prediction.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a HER2-positive breast cancer neoadjuvant therapy pCR prediction kit. Background Art

[0002] Human epidermal growth factor receptor (HER2)-positive breast cancer accounts for 15-30% of invasive breast cancer. HER2 plays a vital role in cell growth, differentiation, motility and invasion, and is one of the receptors (HER1-4) of four transmembrane tyrosine kinases. In HER2-positive breast cancer, an increased pathological complete response (pCR) rate is significantly associated with long-term clinical benefit, regardless of hormone receptor status. For node-positive or high-risk node-negative disease, patients with HER2-positive disease should receive neoadjuvant therapy, including anthracycline and taxane regimens combined with trastuzumab, and pertuzumab can be used in combination with trastuzumab for neoadjuvant therapy. For example, patent CN109908341B uses pertuzumab, trastuzumab and aromatase inhibitors to treat HER2-positive breast cancer, but although HER2-targeted therapy significantly improves the neoadjuvant pCR rate of patients with HER2-positive breast cancer, some patients still show clinical resistance to anti-HER2 treatment, and 40-50% of patients still have residual disease after treatment. Currently, there is no predictive method for neoadjuvant therapy of HER2-positive breast cancer. Clinically, every two neoadjuvant therapy cycles, tumor patients will be evaluated for tumor size and invasiveness using a variety of methods including ultrasound, mammography, CT, and magnetic resonance imaging. The detection of some serum markers, such as circulating tumor DNA and circulating tumor cell counts, may also provide information about the patient's response to treatment. It often takes at least two treatment cycles to evaluate the efficacy of neoadjuvant therapy for patients with HER2-positive breast cancer through clinical auxiliary examinations, and it is impossible to adjust the medication in time according to the tumor's sensitivity to the drug, which consumes a lot of time and money. The detection of serum markers is still not considered to be an accurate and reliable predictive method in clinical practice.

[0003] Therefore, a simple and efficient method to predict the efficacy of neoadjuvant therapy in patients with HER2-positive breast cancer is urgently needed. Summary of the invention

[0004] The purpose of the present invention is to provide a HER2-positive breast cancer neoadjuvant therapy pCR prediction kit to solve the time-consuming and accuracy problems of evaluating the efficacy of neoadjuvant therapy for HER2-positive breast cancer patients.

[0005] In order to solve the above technical problems, the present invention specifically provides the following technical solutions:

[0006] The invention discloses a HER2-positive breast cancer neoadjuvant therapy pCR prediction kit, comprising: activated functional nanomagnetic beads, protein antibodies, HRP-labeled goat anti-rabbit IgG antibodies, TMB enzyme chemiluminescent substrate solution and PBS solution; the activated functional nanomagnetic beads are obtained by reacting functional nanomagnetic beads with glutaraldehyde; the protein antibody is any one of TUBB protein antibody, CUTA protein antibody and ATP5MF protein antibody. The activated functional nanomagnetic beads are combined with the protein antibody as a solid phase carrier, have a large specific surface area, can combine more protein molecules, and improve the detection range. Compared with physical adsorption, the chemical groups on the surface of the magnetic beads can be covalently coupled with the protein, which is more stable, thereby improving the precision and sensitivity of the detection.

[0007] Preferably, the method for preparing functional nanomagnetic beads comprises:

[0008] Fe3O4 magnetic nanoparticles are dispersed in an ethanol aqueous solution, 3-aminopropyltriethoxysilane is added, the pH is adjusted to 10-12 with ammonia water and then reacted, and then 3-(2,3-epoxypropyloxy)propylmethyldiethoxysilane is added to react, and after the reaction is completed, functional nanomagnetic beads are obtained by washing.

[0009] Preferably, Fe3O4 magnetic nanoparticles are prepared from FeCl3·6H2O and FeSO4·7H2O in water under the action of NaOH, wherein the amount of FeCl3·6H2O added to water is 0.017-0.03 g / ml, the amount of FeSO4·7H2O added to water is 0.01-0.02 g / ml, the amount of NaOH used is 0.01-0.02 g / L, the reaction temperature of FeCl3·6H2O, FeSO4·7H2O and NaOH is 70-90°C, and the reaction time is 0.5-1.5 h.

[0010] Preferably, the amount of Fe3O4 magnetic nanoparticles in the ethanol aqueous solution is 0.0004-0.0007 g / ml, the mass ratio of the amount of 3-aminopropyltriethoxysilane added to the ethanol aqueous solution to the amount of Fe3O4 magnetic nanoparticles used is 1:0.4-0.8, and the reaction temperature after adding 3-aminopropyltriethoxysilane is 50-70°C, and the reaction time is 4-8 h.

[0011] Preferably, the mass ratio of 3-(2,3-epoxypropyloxy)propylmethyldiethoxysilane to Fe3O4 magnetic nanoparticles is 1:0.4-0.8, and after adding 3-(2,3-epoxypropyloxy)propylmethyldiethoxysilane, the reaction temperature is 50-70°C and the reaction time is 4-8 h.

[0012] Preferably, the method for detecting protein content in the kit includes:

[0013] A PBS glycerol solution containing protein antibodies is added to an activated functional nanomagnetic bead solution for reaction and separation to obtain a magnetic bead-antibody complex, and the magnetic bead-antibody complex is placed in a PBS solution for mixing; then the supernatant antigen is added for incubation to obtain a magnetic bead-antibody-antigen complex, the magnetic bead-antibody-antigen complex is washed, and then an enzyme-labeled secondary antibody is added for incubation, and then washed after the incubation; finally, TMB enzyme chemiluminescent substrate solution is added for reaction, and the luminescence value is detected after the reaction is completed.

[0014] Preferably, the amount of the protein antibody in the PBS glycerol solution is 2-3 mg / ml, the volume ratio of glycerol to PBS solution in the PBS glycerol solution is 1:1.8-2.2, the volume ratio of the PBS glycerol solution containing the protein antibody to the activated functional nanomagnetic bead solution is 1:4.5-5.5, the reaction temperature is 23-27°C, the reaction time is 1.5-3 h, the amount of the magnetic bead-antibody complex in the PBS solution is 0.008-0.015 g / ml, and the pH of the PBS solution is 7.5-8.5.

[0015] Preferably, the volume ratio of the supernatant antigen to the PBS glycerol solution containing the protein antibody is 1:0.7-0.9, the incubation time after adding the supernatant antigen is 8-15 min, the enzyme-labeled secondary antibody is HRP-labeled goat anti-rabbit IgG antibody, the volume ratio of the enzyme-labeled secondary antibody to the PBS glycerol solution containing the protein antibody is 1:0.9-1.1, and the incubation time is 8-15 min.

[0016] Preferably, the volume ratio of TMB enzyme chemiluminescent substrate solution to PBS glycerol solution containing protein antibody is 1:0.4-0.9, the reaction conditions are room temperature in the dark for 8-15 min, the protein antibody is one of TUBB protein antibody, CUTA protein antibody and ATP5MF protein antibody, and TUBB standard protein, CUTA standard protein and ATP5MF standard protein are used for quantitative analysis.

[0017] The present invention discloses a method for preparing functional nano magnetic beads, which is specifically:

[0018] Preparation of functional nanomagnetic beads: FeCl3·6H2O and FeSO4·7H2O are dissolved in water, and stirred and dissolved under nitrogen to obtain a reactant. Then, NaOH is added to the reactant, and stirred at 70-90 °C for 0.5-1.5 h. The Fe3O4 magnetic nanoparticles and the solution are separated by a magnet to obtain Fe3O4 magnetic nanoparticles, which are then washed and collected. The collected Fe3O4 magnetic nanoparticles are placed in an ethanol aqueous solution to obtain a magnetic ethanol aqueous solution. 3-aminopropyltriethoxysilane is added to the magnetic ethanol aqueous solution, and the pH is adjusted to 10-12 with ammonia water. The reaction is stirred at 50-70 °C for 4-8 h, and then 3-(2,3-epoxypropyloxy)propylmethyldiethoxysilane is added. The reaction is stirred at 50-70 °C for 4-8 h. After the reaction is completed, the functional nanomagnetic beads are washed.

[0019] Preferably, in the preparation of functional nanomagnetic beads, the amount of FeCl3·6H2O added to water is 0.017-0.03 g / ml, the amount of FeSO4·7H2O added to water is 0.01-0.02 g / ml, the amount of NaOH added to the reactant is 0.01-0.02 g / L, the amount of Fe3O4 magnetic nanoparticles in ethanol water is 0.0004-0.0007 g / ml, the mass ratio of the amount of 3-aminopropyltriethoxysilane used to the amount of Fe3O4 magnetic nanoparticles used is 1:0.4-0.8, and the mass ratio of the amount of 3-(2,3-epoxypropyloxy)propylmethyldiethoxysilane used to the amount of Fe3O4 magnetic nanoparticles used is 1:0.4-0.8.

[0020] The present invention also discloses a method for preparing activated functional nanomagnetic beads, which is specifically:

[0021] Preparation of activated functional nanomagnetic beads: functional nanomagnetic beads are dispersed in a PBS solution to obtain a magnetic bead solution, glutaraldehyde is added to the magnetic bead solution, and the reaction is oscillated at 25-30°C for 2.5-5 hours to stop activation. Then, the activated functional nanomagnetic beads are washed to obtain activated functional nanomagnetic beads, and finally, the activated functional nanomagnetic beads are placed in a PBS solution to obtain an activated functional nanomagnetic bead solution.

[0022] Preferably, in the preparation of activated functional nanomagnetic beads, the amount of functional nanomagnetic beads in the PBS solution is 0.01-0.016 g / ml, the volume ratio of glutaraldehyde used to the amount of PBS solution used is 1:0.05-0.3, the pH of the PBS solution is 6.8-7.3, the amount of activated functional nanomagnetic beads placed in the PBS solution is 0.007-0.013 g / ml, and the pH of this PBS solution is 4.7-5.3.

[0023] The present invention discloses a method for preparing a protein antibody, which is specifically:

[0024] Preparation of protein antibodies: Immunize male rabbits with protein. For the first immunization, mix the protein with Freund's complete adjuvant to obtain a mixture, and inject the mixture into the groin and hind paws of the rabbits. After 12-16 days, the same amount of the mixture is used for the second immunization, and the injection site is the same as before. After 24-32 days, the third immunization is performed, and the protein is mixed with Freund's incomplete adjuvant and injected subcutaneously on the back. After 36-48 days, all the blood of the rabbits is collected, salted out with 30-35% saturated ammonium sulfate, centrifuged and precipitated, and dialyzed with physiological saline for 20-30 hours to obtain the dialysate. The dialysate is separated and eluted with DEAE-32 chromatography column, and the eluate is collected to obtain purified antibodies.

[0025] Preferably, in the preparation of protein antibodies, the protein is mixed with Freund's complete adjuvant at a volume ratio of 1:2-3 during the primary and secondary immunizations, and the injection volume of the mixture is 0.5-0.8 ml / mouse; during the third immunization, TUBB protein solution is mixed with Freund's complete adjuvant at a volume ratio of 1:3-3.7, and the injection volume of the mixture is 0.18-0.25 ml / mouse; the protein is one of TUBB protein, CUTA protein and ATP5MF protein.

[0026] The present invention also discloses a method for detecting protein content using a kit, which is specifically:

[0027] The method for detecting protein content in the kit is as follows: add the PBS glycerol solution containing the protein antibody to the activated functional nanomagnetic bead solution, shake at 23-27 ℃ for 1.5-3 h, separate the activated functional nanomagnetic beads and the solution to obtain the magnetic bead-antibody complex, and vortex the magnetic bead-antibody complex with PBS solution. Add the supernatant antigen obtained by crushing and lysing to the magnetic bead-antibody complex and incubate for 8-15 min to obtain the magnetic bead-antibody-antigen complex, wash the magnetic bead-antibody-antigen complex, add the enzyme-labeled secondary antibody to react for 8-15 min, and then wash three times. Finally, add the TMB enzyme chemiluminescent substrate solution, react at room temperature in the dark for 8-15 min, and then detect the luminescence value.

[0028] Preferably, in the method for detecting protein content in the kit, the amount of the protein antibody in the PBS glycerol solution is 2-3 mg / ml, the volume ratio of glycerol to PBS solution in the PBS glycerol solution is 1:1.8-2.2, the volume ratio of the PBS glycerol solution containing the protein antibody to the activated functional nanomagnetic bead solution is 1:4.5-5.5, and the amount of the magnetic bead-antibody complex in the PBS solution is 0.008-0.015 g / ml, the pH of the PBS solution is 7.5-8.5, the volume ratio of the supernatant antigen to the PBS glycerol solution containing the protein antibody is 1:0.7-0.9, the enzyme-labeled secondary antibody is HRP-labeled goat anti-rabbit IgG antibody, the volume ratio of the enzyme-labeled secondary antibody to the PBS glycerol solution containing the protein antibody is 1:0.9-1.1, the volume ratio of the TMB enzyme chemiluminescent substrate solution to the PBS glycerol solution containing the protein antibody is 1:0.4-0.9, the protein antibody is any one of TUBB protein antibody, CUTA protein antibody and ATP5MF protein antibody, and any one of TUBB standard protein, CUTA standard protein and ATP5MF standard protein is used for quantitative analysis.

[0029] Preferably, when the kit is used to detect protein content, astragalus polysaccharide can also be added to the magnetic bead-antibody complex. After the magnetic bead-antibody coupling reaction is completed, the addition of astragalus polysaccharide can block the excess uncoupled active sites or surface hydrophobic sites on the magnetic beads, reduce the nonspecific adsorption of the magnetic beads, and further improve the specificity and sensitivity of protein content detection.

[0030] Preferably, in the detection of protein content, the mass ratio of astragalus polysaccharide added to the magnetic bead-antibody complex to the activated functional nanomagnetic beads is 1:4-10, and the shaking reaction temperature after astragalus polysaccharide is added to the magnetic bead-antibody complex is 23-27°C, and the shaking time is 0.8-1.5 h.

[0031] Preferably, in a preferred embodiment of the present invention, the method for detecting protein content in the kit is as follows: adding a PBS glycerol solution containing a protein antibody to an activated functional nanomagnetic bead solution, shaking at 23-27 ° C for 1.5-3 h, separating the activated functional nanomagnetic beads from the solution after the end to obtain a magnetic bead-antibody complex, adding astragalus polysaccharide to the magnetic bead-antibody complex, shaking at 23-27 ° C for 0.8-1.5 h, and mixing the activated functional nanomagnetic beads with a PBS solution of pH 7.5-8.5 after the end of the shaking. Then, the supernatant antigen obtained by crushing and lysing is added to the magnetic bead-antibody complex and incubated for 8-15 min to obtain a magnetic bead-antibody-antigen complex, and the magnetic bead-antibody-antigen complex is washed and then an enzyme-labeled secondary antibody is added to react for 8-15 min, and then washed 2-3 times. Finally, the TMB enzyme chemiluminescent substrate solution is added, and the luminescence value is detected after the reaction at room temperature and protected from light for 8-15 min.

[0032] More preferably, in the above preferred embodiment, the amount of the protein antibody in the PBS glycerol solution is 2-3 mg / ml, the volume ratio of the PBS glycerol solution containing the protein antibody to the activated functional nanomagnetic bead solution is 1:4.5-5.5, and the amount of the magnetic bead-antibody complex in the PBS solution is 0.008-0.015 g / ml, the pH of the PBS solution is 7.5-8.5, the mass ratio of astragalus polysaccharide added to the magnetic bead-antibody complex to the activated functional nanomagnetic beads is 1:4-10, the volume ratio of the supernatant antigen used to the PBS glycerol solution containing the protein antibody is 1:0.7-0.9, the enzyme-labeled secondary antibody is HRP-labeled goat anti-rabbit IgG antibody, the volume ratio of the enzyme-labeled secondary antibody used to the PBS glycerol solution containing the protein antibody is 1:0.9-1.1, the volume ratio of the TMB enzyme chemiluminescent substrate solution used to the PBS glycerol solution containing the protein antibody is 1:0.4-0.9, the protein antibody is one of TUBB protein antibody, CUTA protein antibody and ATP5MF protein antibody, and TUBB standard protein, CUTA standard protein and ATP5MF standard protein are used for quantitative analysis.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention prepares functional nanomagnetic beads, activated functional nanomagnetic beads and protein antibodies, and establishes a protein detection method, thereby obtaining a protein detection kit for detecting TUBB protein, CUTA protein and ATP5MF protein; the magnetic beads prepared by the present invention can be fully combined with the antibodies, and the obtained kit can quickly test proteins with high sensitivity, good repeatability and high precision, and can well detect the expression levels of the three proteins, thereby being applied to the prediction of pCR in neoadjuvant therapy of HER2-positive breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0036] Figure 1 This is the ROC diagram of the random forest model;

[0037] Figure 2 is the feature importance graph of the random forest model;

[0038] Figure 3 Random forest score plots for TUBB, CUTA, and ATP5MF proteins;

[0039] Figure 4 This is the infrared spectrum characterization of functional nanomagnetic beads. DETAILED DESCRIPTION

[0040] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] The following first describes the concepts involved in the present application in conjunction with the accompanying drawings. It should be noted that the following description of each concept is only to make the content of the present application easier to understand, and does not limit the scope of protection of the present application; at the same time, the embodiments and features in the embodiments of the present application can be combined with each other in the absence of conflict. The present application will be described in detail with reference to the accompanying drawings and in conjunction with the embodiments.

[0042] Screening of protein markers: Samples were obtained from formalin-fixed paraffin-embedded samples of 75 HER2-positive breast cancer patients before and after neoadjuvant therapy in Zhejiang Cancer Hospital. The samples of 35 groups of HER2-positive breast cancer before and after treatment that did not achieve pCR after neoadjuvant therapy and 40 HER2-positive breast cancer samples before treatment that achieved pCR after neoadjuvant therapy were sliced ​​and dewaxed. 4% SDS and crushing beads were added to each sample, the tissue was crushed, and the supernatant was collected by centrifugation. The supernatant was ultrasonically treated at 80 °C for 60 min, and then heated at 90 °C for 60 min. The ultrasonic treatment and heating were repeated 5 times, and the supernatant was collected by centrifugation for 15 min, and acetone was added for incubation for 15 h. After the end, the acetone was removed by centrifugation, and acetone was added again for washing. The precipitate was obtained by repeated centrifugation to remove the acetone, and acetone was added to dissolve the precipitate again to obtain the sample, i.e., the supernatant antigen. The protein in the sample was quantitatively measured using the Bradford protein quantification kit. The amount of 4% SDS added to the sample was 0.15 ml / sample, and the ratio of the amount of 4% SDS added to the sample to the amount of acetone added was 1:0.33.

[0043] According to the quantitative results, the sample was mixed with ABC buffer, DTT was added, and incubated at 60 °C for 20 minutes. Then IAA was added, and then ABC buffer was added for centrifugal filtration to obtain the pretreated sample. The pretreated sample was then transferred to an ultrafiltration tube and washed and centrifuged 3 times to obtain the purified sample. Then trypsin and 10% SDC were added to the purified sample, incubated at 37 °C for 15 h, centrifuged for 15 min, and then ABC buffer was added for washing and centrifugation for 15 min to obtain the enzymatic sample. Then 10% TFA was added to the enzymatic sample to terminate the digestion, and the supernatant sample was obtained by centrifugation for 15 min. Finally, the supernatant sample was desalted using Thermo Scientific PierceC18 Tips, eluted with ACN, and centrifuged at 55 °C for 40 min to obtain the protein sample. The concentration of protein in the sample in ABC buffer was 0.0002 g / ml, the volume ratio of DTT to ABC buffer was 1:50, the volume ratio of IAA added to ABC buffer was 1:25, the volume ratio of purified sample to trypsin was 1:50, the volume ratio of purified sample to 10% SDC was 1:0.4, and the volume ratio of 10% TFA to ABC buffer for washing was 1:10.

[0044] Liquid chromatography-mass spectrometry was used to perform proteomics detection on protein samples, and the data were divided into training set and validation set at a ratio of 3:1. A prediction model was constructed using random forest machine learning method. Figure 1As shown in Figure 2, in the training set, the AUC of the prediction model reached 98.9%, and in the validation set, the AUC of the prediction model reached 100%. Figure 2 As shown in the figure, TUBB, CUTA and ATP5MF are the three proteins with the largest influence coefficients in the prediction model. Figure 3 The results showed that the expression scores of the three proteins TUBB, CUTA and ATP5MF in the pCR population after neoadjuvant therapy were significantly lower than those in the Non-pCR population, so the effect of neoadjuvant therapy can be distinguished by measuring these three proteins.

[0045] Embodiment 1:

[0046] Preparation of functional nanomagnetic beads: FeCl3·6H2O and FeSO4·7H2O were dissolved in water under nitrogen conditions, and then NaOH was added and stirred at 85°C for 1 h. Fe3O4 magnetic nanoparticles and the solution were separated by a magnet to obtain Fe3O4 magnetic nanoparticles, which were then washed with ethanol and distilled water. After washing, the particles were placed in an ethanol-water solution to obtain a magnetic ethanol-water solution. 3-aminopropyltriethoxysilane was added to the magnetic ethanol-water solution, and the pH was adjusted to 11 with ammonia water. The particles were stirred and reacted at 60°C for 5 h, and then 3-(2,3-epoxypropyloxy)propylmethyldiethoxysilane was added. The particles were stirred and reacted at 50°C for 5 h. After the reaction, the particles were washed 5 times with ethanol to obtain functional nanomagnetic beads. The amount of FeCl3·6H2O added to water is 0.0239 g / ml, the amount of FeSO4·7H2O added to water is 0.0139 g / ml, the amount of NaOH added to the reactant is 0.016 g / L, the amount of Fe3O4 magnetic nanoparticles in the magnetic ethanol aqueous solution is 0.0005 g / ml, the mass ratio of the amount of 3-aminopropyltriethoxysilane added to the magnetic ethanol aqueous solution to the amount of Fe3O4 magnetic nanoparticles used is 1:0.55, and the mass ratio of the amount of 3-(2,3-epoxypropyloxy)propylmethyldiethoxysilane used to the amount of Fe3O4 magnetic nanoparticles used is 1:0.74.

[0047] Preparation of activated functional nanomagnetic beads: functional nanomagnetic beads are placed in PBS solution to obtain a magnetic bead solution, glutaraldehyde is added to the magnetic bead solution, and the activation is stopped by shaking at 29 °C for 3 h. Then the activated functional nanomagnetic beads are washed to obtain activated functional nanomagnetic beads, and finally the activated functional nanomagnetic beads are placed in PBS solution to obtain an activated functional nanomagnetic bead solution. The amount of functional nanomagnetic beads in the PBS solution is 0.0125 g / ml, the volume ratio of the amount of glutaraldehyde added to the magnetic bead solution to the amount of PBS solution is 1:0.25, the pH of the PBS solution is 7, the amount of activated functional nanomagnetic beads added to the PBS solution is 0.01 g / ml, and the pH of this PBS solution is 5.

[0048] Preparation of protein antibodies: Male rabbits were immunized with TUBB protein. For the first immunization, TUBB protein was mixed with Freund's complete adjuvant to obtain a mixture, and the mixture was injected into the groin and hind paws of the rabbits. After 14 days, the same amount of the mixture was used for the second immunization, and the injection site was the same as before. After 28 days, the third immunization was performed, TUBB protein was mixed with Freund's incomplete adjuvant, and then injected subcutaneously at 12 o'clock on the back. After 42 days, all the blood of the rabbits was collected, salted out with 33% saturated ammonium sulfate, centrifuged and precipitated, and dialyzed with physiological saline for 24 hours to obtain the dialysate. The dialysate was separated and eluted with DEAE-32 chromatography column, and the eluate was collected to obtain purified antibodies. For the first and second immunizations, TUBB protein solution was mixed with Freund's complete adjuvant at a volume ratio of 1:2.5, and the injection volume of the mixture was 0.7 ml / mouse. For the third immunization, TUBB protein solution was mixed with Freund's complete adjuvant at a volume ratio of 1:3.3, and the injection volume of the mixture was 0.2 ml / mouse.

[0049] The method for detecting protein content in the kit is as follows: add the PBS glycerol solution containing TUBB protein antibody to the activated functional nanomagnetic bead solution, shake at 25 °C for 2 h, and then separate the activated functional nanomagnetic beads and the solution with a magnet to obtain the magnetic bead-antibody complex, and vortex the magnetic bead-antibody complex with PBS solution. Add the supernatant antigen obtained by crushing and lysing to the magnetic bead-antibody complex and incubate for 10 min to obtain the magnetic bead-antibody-antigen complex. After washing the magnetic bead-antibody-antigen complex, add the enzyme-labeled secondary antibody to react for 10 min, and then wash three times. Finally, add the TMB enzyme chemiluminescent substrate solution, react at room temperature in the dark for 10 min, and then detect the luminescence value. The amount of TUBB protein antibody in PBS glycerol solution is 2.5 mg / ml, the volume ratio of glycerol to PBS solution in PBS glycerol solution is 1:2, the volume ratio of the PBS glycerol solution containing TUBB protein antibody to the activated functional nanomagnetic bead solution is 1:5, the amount of magnetic bead-antibody complex in PBS solution is 0.01 g / ml, the pH of PBS solution is 8, the volume ratio of the supernatant antigen to the PBS glycerol solution containing TUBB protein antibody is 1:0.8, the enzyme-labeled secondary antibody is HRP-labeled goat anti-rabbit IgG antibody, the volume ratio of the enzyme-labeled secondary antibody to the PBS glycerol solution containing TUBB protein antibody is 1:1, the volume ratio of the TMB enzyme chemiluminescent substrate solution to the PBS glycerol solution containing TUBB protein antibody is 1:0.5, and TUBB standard protein is used for quantitative analysis.

[0050] Embodiment 2:

[0051] Preparation of functional nanomagnetic beads: The preparation of functional nanomagnetic beads in this embodiment is compared with that in Example 1, except that the mass ratio of the amount of 3-(2,3-epoxypropyloxy)propylmethyldiethoxysilane used in this embodiment to the amount of Fe3O4 magnetic nanoparticles used is 1:0.58, and other conditions and parameters are the same as in Example 1.

[0052] The preparation of activated functional nanomagnetic beads is the same as in Example 1.

[0053] The preparation of protein antibody is the same as in Example 1.

[0054] The method for detecting protein content in the kit is the same as in Example 1.

[0055] Embodiment 3:

[0056] The preparation of functional nanomagnetic beads is the same as in Example 1.

[0057] The preparation of activated functional nanomagnetic beads is the same as in Example 1.

[0058] The preparation of protein antibody is the same as in Example 1.

[0059] The method for detecting protein content in the kit is as follows: add the PBS glycerol solution containing TUBB protein antibody to the activated functional nanomagnetic bead solution, shake at 25 °C for 2 h, and then use a magnet to separate the activated functional nanomagnetic beads and the solution to obtain the magnetic bead-antibody complex. Add astragalus polysaccharide to the magnetic bead-antibody complex, shake at 25 °C for 1 h, and vortex the magnetic bead-antibody complex with PBS solution after the shaking. Add the supernatant antigen obtained by crushing and lysing to the magnetic bead-antibody complex and incubate for 10 min to obtain the magnetic bead-antibody-antigen complex. After washing the magnetic bead-antibody-antigen complex, add the enzyme-labeled secondary antibody to react for 10 min, and then wash three times. Finally, add the TMB enzyme chemiluminescent substrate solution, react at room temperature in the dark for 10 min, and then detect the luminescence value. The amount of TUBB protein antibody in PBS glycerol solution is 2.5 mg / ml, the volume ratio of glycerol to PBS solution in PBS glycerol solution is 1:2, the volume ratio of PBS glycerol solution containing TUBB protein antibody to activated functional nanomagnetic bead solution is 1:5, the mass ratio of astragalus polysaccharide added to magnetic bead-antibody complex to activated functional nanomagnetic beads is 1:8, the amount of magnetic bead-antibody complex in PBS solution is 0.01 g / ml, the pH of PBS solution is 8, the volume ratio of supernatant antigen to PBS glycerol solution containing TUBB protein antibody is 1:0.8, the enzyme-labeled secondary antibody is HRP-labeled goat anti-rabbit IgG antibody, the volume ratio of enzyme-labeled secondary antibody to PBS glycerol solution containing TUBB protein antibody is 1:1, the volume ratio of TMB enzyme chemiluminescent substrate solution to PBS glycerol solution containing TUBB protein antibody is 1:0.5, and TUBB standard protein is used for quantitative analysis.

[0060] Embodiment 4:

[0061] The preparation of functional nanomagnetic beads is the same as in Example 1.

[0062] The preparation of activated functional nanomagnetic beads is the same as in Example 1.

[0063] The preparation of protein antibody is the same as in Example 1.

[0064] The method for detecting protein content by the kit: The method for detecting protein content by the kit in this embodiment is compared with that in Example 3, except that the mass ratio of the amount of Astragalus polysaccharide added to the magnetic bead-antibody complex to the activated functional nanomagnetic beads is 1:5, and the other conditions and parameters are the same as in Example 3.

[0065] Embodiment 5:

[0066] The preparation of functional nanomagnetic beads is the same as in Example 1.

[0067] The preparation of activated functional nanomagnetic beads is the same as in Example 1.

[0068] Preparation of protein antibodies: The preparation of protein antibodies in this example is different from that in Example 1, except that TUBB protein is replaced by CUTA protein, and other conditions and parameters are the same as in Example 1.

[0069] Method for detecting protein content by kit: The preparation of protein antibody in this example is compared with that in Example 1, except that TUBB protein antibody is replaced by CUTA protein antibody, and other conditions and parameters are the same as in Example 1.

[0070] Embodiment 6:

[0071] The preparation of functional nanomagnetic beads is the same as in Example 1.

[0072] The preparation of activated functional nanomagnetic beads is the same as in Example 1.

[0073] Preparation of protein antibodies: The preparation of protein antibodies in this example is different from that in Example 1, except that TUBB protein is replaced by ATP5MF protein, and other conditions and parameters are the same as in Example 1.

[0074] Method for detecting protein content by kit: The preparation of protein antibody in this example is compared with that in Example 1, except that TUBB protein antibody is replaced by ATP5MF protein antibody, and other conditions and parameters are the same as in Example 1.

[0075] Comparative Example 1:

[0076] Preparation of functional nanomagnetic beads: The preparation of functional nanomagnetic beads in this embodiment is compared with that in Example 1, except that the mass ratio of the amount of 3-(2,3-epoxypropyloxy)propylmethyldiethoxysilane used in this embodiment to the amount of Fe3O4 magnetic nanoparticles used is 1:0.2, and other conditions and parameters are the same as in Example 1.

[0077] The preparation of activated functional nanomagnetic beads is the same as in Example 1.

[0078] The preparation of protein antibody is the same as in Example 1.

[0079] The method for detecting protein content in the kit is the same as in Example 1.

[0080] Comparative Example 2:

[0081] Preparation of activated functional nanomagnetic beads: The preparation of functional nanomagnetic beads in this embodiment is compared with that in Example 1, except that the mass ratio of 3-(2,3-epoxypropyloxy)propylmethyldiethoxysilane used in this embodiment to the amount of Fe3O4 magnetic nanoparticles used is 1:1, and other conditions and parameters are the same as in Example 1.

[0082] The preparation of protein antibody is the same as in Example 1.

[0083] The method for detecting protein content in the kit is the same as in Example 1.

[0084] Comparative Example 3:

[0085] The preparation of functional nanomagnetic beads is the same as in Example 1.

[0086] The preparation of activated functional nanomagnetic beads is the same as in Example 1.

[0087] The preparation of protein antibody is the same as in Example 1.

[0088] The method for detecting protein content by the kit: The method for detecting protein content by the kit in this embodiment is compared with that in Example 3, except that the mass ratio of the amount of Astragalus polysaccharide added to the magnetic bead-antibody complex to the activated functional nanomagnetic beads is 1:12, and the other conditions and parameters are the same as in Example 3.

[0089] Comparative Example 4:

[0090] The preparation of activated functional nanomagnetic beads is the same as in Example 2.

[0091] The preparation of protein antibody is the same as in Example 1.

[0092] Method for detecting protein content by the kit: The method for detecting protein content by the kit in this embodiment is compared with that in Example 3, except that the mass ratio of the amount of Astragalus polysaccharide added to the magnetic bead-antibody complex to the activated functional nanomagnetic beads is 1:2, and other conditions and parameters are the same as in Example 3.

[0093] Experimental Example 1:

[0094] The functional nanomagnetic beads prepared in Example 1 were characterized by infrared spectroscopy in a scanning range of 500-3500 cm -1 The results are as follows Figure 4 As shown, 2800-3000cm -1 is the absorption peak of CH, 1630 cm -1 The absorption peak of NH is at 1060 cm -1 The CN absorption peak is at 589 cm -1 Nearby is the absorption peak of Fe-O, 3400-3500 cm -1 Nearby is the absorption peak of OH, 1100-1200 cm -1 It is the absorption peak of Si-O, which is consistent with the characteristics of functional nanomagnetic beads.

[0095] Experimental Example 2:

[0096] The minimum detection limit analysis was performed on the minimum detection limit of the method for detecting protein content of the kits of Examples 1-6 and Comparative Examples 1-4, that is, the absorbance values ​​of 20 blank solutions were measured according to the protein determination method of each embodiment, and the average and standard deviation of the absorbance values ​​of the blank controls were calculated. The average value of the blank controls + 2 standard deviations were used as the judgment standard, and the minimum detection limit of the system, that is, the sensitivity, was calculated according to the standard curve, and the blank solution was PBS solution.

[0097] Table 1 Minimum detection limit of the method for detecting protein content in the kit

[0098]

[0099] The results of the minimum detection limit determination of the method for detecting protein content by the kits of Examples 1-6 and Comparative Examples 1-4 are shown in Table 1. The minimum detection limit of Example 3 is lower than that of Example 1, indicating that the addition of astragalus polysaccharide during protein detection can further improve the protein content detection effect, that is, improve the sensitivity; Compared with Comparative Example 1, the minimum detection limit of Example 1 is lower than that of Comparative Example 1, that is, the protein content detection sensitivity of Example 1 is higher, indicating that the usage amount of 3-(2,3-epoxypropyloxy)propylmethyldiethoxysilane cannot be too high, and too high will reduce the efficacy of the functional nanomagnetic beads, thereby reducing the sensitivity of protein content detection; Compared with Comparative Example 2, the minimum detection limit of Example 1 is lower than that of Comparative Example 2, that is, the protein content detection sensitivity of Example 1 is higher, indicating that 3- The usage of (2,3-epoxypropyloxy)propylmethyldiethoxysilane cannot be too low. If it is too low, the functional nano-magnetic beads will not be well modified with groups, which will also cause the sensitivity of protein content detection to decrease. Therefore, when 3-(2,3-epoxypropyloxy)propylmethyldiethoxysilane is used to modify the functional nano-magnetic beads, the usage amount needs to be at an appropriate value. Too high or too low a value will reduce the sensitivity of the protein content detection method. Compared with Example 1, the minimum detection limit of Comparative Example 3-4 is not much different. Compared with Example 3, the minimum detection limit of Comparative Example 3-4 is higher than that of Example 3, indicating that the usage amount of Astragalus polysaccharide also needs to be at a moderate value. Too high or too low a usage amount will affect the further binding of the magnetic bead-antibody complex and the antigen, thereby inhibiting the sensitivity of protein content detection.

[0100] Experimental Example 3:

[0101] The intra-batch precision was tested according to the method for detecting protein content using the kits of Examples 1-7 and Comparative Examples 1-4, and the same batch of reagents was used to perform repeated tests 20 times, and the coefficient of variation, i.e., CV value, was calculated.

[0102] Table 2 Coefficient of variation values

[0103]

[0104] The results of the determination and calculation of the intra-batch coefficient of variation of Examples 1-6 and Comparative Examples 1-4 are shown in Table 2. The coefficient of variation of Example 3 is lower than that of Example 1, indicating that the addition of astragalus polysaccharide during protein detection can further reduce the discrete degree of protein content detection and make the detection accuracy higher; Compared with Comparative Example 1, the coefficient of variation of Example 1 is lower than that of Comparative Example 1, that is, the protein content detection accuracy of Example 1 is higher, indicating that the usage amount of 3-(2,3-epoxypropyloxy)propylmethyldiethoxysilane cannot be too high, and too high will reduce the efficacy of functional nanomagnetic beads, thereby reducing the precision of protein content detection; Compared with Comparative Example 2, the minimum detection limit of Example 1 is lower than that of Comparative Example 2, that is, the protein content detection accuracy of Example 1 is higher, indicating that 3-( The usage of 2,3-(2,3-epoxypropyloxy)propylmethyldiethoxysilane cannot be too low. If it is too low, the functional nano-magnetic beads will not be well modified with groups, which will also cause the accuracy of protein content detection to decrease. Therefore, when 3-(2,3-epoxypropyloxy)propylmethyldiethoxysilane is used to modify the functional nano-magnetic beads, the usage amount needs to be at an appropriate value. Too high or too low a usage amount will reduce the accuracy of the protein content detection method. Compared with Example 1, the minimum detection limit of Comparative Example 3-4 is not much different. Compared with Example 3, the minimum detection limit of Comparative Example 3-4 is higher than that of Example 3, indicating that the usage amount of Astragalus polysaccharide also needs to be at a moderate value. Too high or too low a usage amount will affect the further binding of the magnetic bead-antibody complex and the antigen, thereby reducing the accuracy of protein content detection.

[0105] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any technical personnel in this field may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0106] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and its core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A kit for predicting pCR in neoadjuvant therapy of HER2-positive breast cancer, comprising: Activate functional nanomagnetic beads, protein antibody, HRP-labeled goat anti-rabbit IgG antibody, TMB enzyme chemiluminescent substrate solution and PBS solution; The activated functional nanomagnetic beads are obtained by reacting functional nanomagnetic beads with glutaraldehyde; the protein antibody is any one of TUBB protein antibody, CUTA protein antibody and ATP5MF protein antibody; and the protein antibody is a rabbit antibody.

2. A HER2-positive breast cancer neoadjuvant therapy pCR prediction kit according to claim 1, characterized in that: The preparation method of the functional nano magnetic beads comprises: Fe3O4 magnetic nanoparticles are dispersed in an ethanol aqueous solution, 3-aminopropyltriethoxysilane is added, the pH is adjusted to 10-12 with ammonia water and then reacted, and then 3-(2,3-epoxypropyloxy)propylmethyldiethoxysilane is added to react, and after the reaction is completed, functional nanomagnetic beads are obtained by washing.

3. A HER2-positive breast cancer neoadjuvant therapy pCR prediction kit according to claim 2, characterized in that: The Fe3O4 magnetic nanoparticles are prepared from FeCl3·6H2O and FeSO4·7H2O in water under the action of NaOH, wherein the amount of FeCl3·6H2O added to the water is 0.017-0.03 g / ml, the amount of FeSO4·7H2O added to the water is 0.01-0.02 g / ml, the amount of NaOH used is 0.01-0.02 g / L, the reaction temperature of FeCl3·6H2O, FeSO4·7H2O and NaOH is 70-90°C, and the reaction time is 0.5-1.5 h.

4. The kit for predicting pCR in neoadjuvant therapy of HER2-positive breast cancer according to claim 2, characterized in that: The amount of the Fe3O4 magnetic nanoparticles in the ethanol aqueous solution is 0.0004-0.0007 g / ml, the mass ratio of the amount of 3-aminopropyltriethoxysilane added to the ethanol aqueous solution to the amount of Fe3O4 magnetic nanoparticles used is 1:0.4-0.8, and after adding 3-aminopropyltriethoxysilane, the reaction temperature is 50-70°C and the reaction time is 4-8 h.

5. The kit for predicting pCR in neoadjuvant therapy of HER2-positive breast cancer according to claim 2, characterized in that: The mass ratio of the amount of 3-(2,3-epoxypropyloxy)propylmethyldiethoxysilane used to the amount of Fe3O4 magnetic nanoparticles used is 1:0.4-0.

8. After adding 3-(2,3-epoxypropyloxy)propylmethyldiethoxysilane, the reaction temperature is 50-70°C and the reaction time is 4-8 h.

6. A kit for predicting pCR in neoadjuvant therapy of HER2-positive breast cancer according to claim 1, characterized in that: The method for detecting protein content in the kit includes: A PBS glycerol solution containing protein antibodies is added to an activated functional nanomagnetic bead solution for reaction and separation to obtain a magnetic bead-antibody complex, and the magnetic bead-antibody complex is placed in a PBS solution for mixing; then the supernatant antigen is added for incubation to obtain a magnetic bead-antibody-antigen complex, the magnetic bead-antibody-antigen complex is washed, and then an enzyme-labeled secondary antibody is added for incubation, and then washed after the incubation; finally, TMB enzyme chemiluminescent substrate solution is added for reaction, and the luminescence value is detected after the reaction is completed.

7. A kit for predicting pCR in neoadjuvant therapy of HER2-positive breast cancer according to claim 6, characterized in that: The amount of the protein antibody in the PBS glycerol solution is 2-3 mg / ml, the volume ratio of glycerol to PBS solution in the PBS glycerol solution is 1:1.8-2.2, the usage amount of the PBS glycerol solution containing the protein antibody and the volume ratio of the activated functional nanomagnetic bead solution are 1:4.5-5.5, the reaction temperature is 23-27°C, the reaction time is 1.5-3 h, the amount of the magnetic bead-antibody complex in the PBS solution is 0.008-0.015 g / ml, and the pH of the PBS solution is 7.5-8.

5.

8. The kit for predicting pCR in neoadjuvant therapy of HER2-positive breast cancer according to claim 6, characterized in that: The volume ratio of the supernatant antigen to the PBS glycerol solution containing the protein antibody is 1:0.7-0.9, the incubation time after adding the supernatant antigen is 8-15 min, the enzyme-labeled secondary antibody is an HRP-labeled goat anti-rabbit IgG antibody, the volume ratio of the enzyme-labeled secondary antibody to the PBS glycerol solution containing the protein antibody is 1:0.9-1.1, and the incubation time is 8-15 min.

9. A kit for predicting pCR in neoadjuvant therapy of HER2-positive breast cancer according to claim 6, characterized in that: The volume ratio of the TMB enzyme chemiluminescent substrate solution to the PBS glycerol solution containing the protein antibody is 1:0.4-0.9, the reaction conditions are room temperature and light-proof reaction for 8-15 min, the protein antibody is one of TUBB protein antibody, CUTA protein antibody and ATP5MF protein antibody, and TUBB standard protein, CUTA standard protein and ATP5MF standard protein are used for quantitative analysis.

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