ARHGDIB lactylation detection kit for diagnosing bladder cancer lymph node metastasis and predicting curative effect of chemotherapy and use method of ARHGDIB lactylation detection kit

Abstract

The invention relates to the technical field of medical biological detection, and provides an ARHGDIB lactylation detection kit applied to diagnosis of bladder cancer lymph node metastasis and prediction of chemotherapy curative effect, and the ARHGDIB lactylation detection kit comprises a first antibody, a second antibody, a third antibody, a fourth antibody and a fifth antibody, the second antibody is goat anti-rabbit IgG marked by TYR-570; an antigen repair liquid; a confining liquid; a cell nucleus counterstaining agent; an anti-fluorescence quenching mounting medium; a positive reference; a negative reference; and 10 * phosphate buffer (10 * PBST). The invention also provides a use method of the ARHGDIB lactylation detection kit for diagnosing bladder cancer lymph node metastasis and predicting the curative effect of chemotherapy, lymph node metastasis and chemotherapy prognosis can be evaluated by using a bladder cancer first operation specimen, imaging lymph node staging is assisted before a whole bladder radical operation, and guidance is provided for subsequent neoadjuvant chemotherapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical biological detection technology, and particularly relates to an ARHGDIB lactic acidification detection kit applied to diagnosis of lymph node metastasis of bladder cancer and prediction of chemotherapeutic effect and a use method. BACKGROUND

[0002] Bladder cancer is one of the common malignant tumors in human beings, and lymphatic metastasis is the most common metastasis pathway of bladder cancer. Accurate lymph node staging is crucial for guiding the treatment of cancer patients and evaluating the prognosis.

[0003] The existing methods for diagnosing lymph node metastasis of bladder cancer include imaging examination, intraoperative detection and navigation technology, emerging and auxiliary diagnostic methods, etc. Traditional imaging examination, including CT and MRI technologies, is fast, has high popularity, and has good specificity for larger metastatic lymph nodes, but has low sensitivity, and it is difficult to detect micro-metastasis or normal size metastasis, and nearly half of the actual metastasis patients are missed. The superparamagnetic iron oxide contrast agent improves the resolution of MRI detection of lymph nodes, but has greater side effects, and further application is limited.

[0004] Intraoperative detection and navigation technology represented by pelvic lymph node dissection is the most accurate method for diagnosing lymph node metastasis at present, which can provide a gold standard for pathological diagnosis, but this method is a invasive operation with high complication risk, and the surgical range depends on the experience of surgeons, and it cannot be evaluated before radical surgery.

[0005] Emerging and auxiliary diagnostic methods include circulating tumor DNA (ctDNA) detection, which can detect high-frequency sequencing of ctDNA in patient blood, can indicate the presence of lymph node metastasis before imaging is found, has high sensitivity, is non-invasive, but the specificity needs to be further improved, and the cost is high.

[0006] Neoadjuvant therapy refers to systemic treatment such as chemotherapy, radiotherapy or targeted therapy before surgery, which can reduce tumor volume, reduce analysis, eliminate potential micro-metastasis, create better conditions for subsequent surgery, and improve patient prognosis as much as possible. Although neoadjuvant chemotherapy (NACC) based on cisplatin has a positive effect on the overall survival of bladder cancer patients who respond to treatment, a considerable number of patients do not respond to treatment.

[0007] Currently, some clinical and pathological factors can be used to predict the effect of NACC, such as tumor regression grade, pathological stage downgrading, lymph node status, inflammatory markers (ratio of neutrophils and lymphocytes), squamous or glandular differentiation, and carcinoma in situ. However, pathological evaluation (including tumor regression grade, pathological stage downgrading, and lymph node status) needs to be obtained after NACC treatment, or even after subsequent surgery, and pathological sections are required, so that treatment prognosis information cannot be obtained before NACC treatment. Summary of the Invention

[0008] 1. The technical problem to be solved: Tissue samples were obtained during the first transurethral resection or cystoscopy of bladder cancer patients to predict lymph node metastasis and the effectiveness of neoadjuvant chemotherapy.

[0009] 2. Technical Solution: To address the above problems, this invention provides an ARHGDIB lactation detection kit for diagnosing lymph node metastasis in bladder cancer and predicting chemotherapy efficacy, comprising: Primary antibody: Targets amino acid residues 44-56 of ARHGDIB-K50lac; Secondary antibody: TYR-570-labeled goat anti-rabbit IgG; Antigen retrieval solution; Sealing liquid; Nuclear counterstain; Anti-fluorescence quenching mounting medium; Positive reference; Negative reference; 10× phosphate buffer.

[0010] This invention provides a method for using the ARHGDIB lactation detection kit for diagnosing lymph node metastasis in bladder cancer and predicting the efficacy of chemotherapy.

[0011] 3. Beneficial effects: This invention utilizes specimens from the first transurethral resection of bladder cancer or cystoscopic biopsy to assess lymph node metastasis and chemotherapy prognosis, assists in imaging lymph node staging before radical cystectomy, and provides guidance for subsequent neoadjuvant chemotherapy. Attached Figure Description

[0012] Figure 1 This is the specific detection result of the ARHGDIB-K50lac polyclonal antibody.

[0013] Figure 2 This is a demonstration image of ARHGDIB-K50lac polyclonal antibody used for microarray immunofluorescence staining of bladder cancer tissue.

[0014] Figure 3 The expression level of ARHGDIB-K50lac in 60 cases of bladder cancer with positive lymph node metastasis and 24 cases with negative lymph node metastasis is shown in the figure. **p<0.01.

[0015] Figure 4 The expression level of ARHGDIB-K50lac was observed in 21 patients with chemotherapy-sensitive bladder cancer and 19 patients with chemotherapy-resistant bladder cancer. **p<0.01. Detailed Implementation

[0016] The present invention will now be described in detail with reference to the accompanying drawings.

[0017] The kit of the present invention uses human ARHGDIB lysine residue lactation at position 50 (ARHGDIB-K50lac) as a marker to detect its content in electrocautery or cystoscopic biopsy samples with immunofluorescence, thereby diagnosing lymph node metastasis of bladder cancer and predicting the efficacy of cisplatin chemotherapy.

[0018] The human ARHGDIB protein, named Rho-GDP-dissociation inhibitor 2, has the gene name ARHGDIB and consists of 201 amino acids. The complete sequence from the N-terminus to the C-terminus is: MTEKAPEPHVEEDDDDELDSKLNYKPPPQKSLKELQEMDKDDESLIKYKKTLLGDGPVVTDPKAPNVVVTRLTLVCESAPGPITMDLTGDLEALKKETIVLKEGSEYRVKIHFKVNRDIVSGLKYVQHTYRTGVKVDKATFMVGSYGPRPEEYEFLTPVEEAPKGMLARGTYHNKSFFTDDDKQDHLSWEWNLSIKKEWTE.

[0019] This invention provides an ARHGDIB lactation detection kit for diagnosing lymph node metastasis in bladder cancer and predicting the efficacy of chemotherapy, comprising: Primary antibody: Targets amino acid residues 44-56 of ARHGDIB-K50lac; Secondary antibody: TYR-570-labeled goat anti-rabbit IgG; Antigen retrieval solution; Sealing liquid; Nuclear counterstain; Anti-fluorescence quenching mounting medium; Positive reference; Negative reference; 10× phosphate buffer.

[0020] In one embodiment, the amino acid residues targeting ARHGDIB-K50lac at positions 44-56 are diluted with PBST to a concentration of 0.26 mg / mL.

[0021] In one embodiment, the TYR-570-labeled goat anti-rabbit IgG is ready-to-use and has a concentration of 1~2.5 ng / mL.

[0022] In one embodiment, the antigen retrieval solution is a citrate buffer solution at pH 6.0.

[0023] In one embodiment, the blocking solution is a PBS solution containing 5% bovine serum albumin.

[0024] In one embodiment, the nuclear counterstain is a DAPI solution with a concentration of 1 μg / mL.

[0025] In one embodiment, the positive reference is a bladder cancer tissue section known to highly express ARHGDIB-K50lac; the negative reference is a bladder cancer tissue section known not to express ARHGDIB-K50lac.

[0026] In one embodiment, the ARHGDIB-K50lac antibody was prepared by Hangzhou Huaan Biotechnology Co., Ltd.

[0027] In one embodiment, the 10× phosphate buffer is diluted to 1× PBS before use.

[0028] This invention first commissioned Hangzhou Huaan Biotechnology Co., Ltd. to prepare a polyclonal antibody that specifically recognizes ARHGDIB-K50lac, and prepared a positive control: lactated modified peptide SLIKYKK(lac)TLLGDGC, and a negative control: non-lactated modified peptide SLIKYKKTLLGDGC for verification, as shown in the appendix. Figure 1 As shown.

[0029] Subsequently, the expression level of ARHGDIB-K50lac in tumor tissue samples from 84 bladder cancer patients was detected by immunofluorescence. Figure 2 As shown in the attached figure. Specifically, paraffin-embedded tumor tissue samples were collected from 84 bladder cancer patients, including 60 patients with positive lymph node metastases and 24 patients with negative lymph node metastases. Tissue microarrays were fabricated, and immunofluorescence staining analysis was performed through the following steps: dewaxing and hydration, antigen retrieval, blocking, primary antibody incubation, washing, secondary antibody incubation, washing, counterstaining with DAPI and mounting, image acquisition, and result analysis. The results showed that the expression level of ARHGDIB-K50lac in the tumor tissue of patients with positive lymph node metastases was significantly lower than that in patients with negative lymph node metastases. Figure 3 As shown, p < 0.01.

[0030] Finally, tumor samples from 40 bladder cancer patients undergoing chemotherapy were analyzed before treatment. Of these, 21 were treatment-sensitive and 19 were treatment-resistant. The immunofluorescence assay revealed that the expression level of ARHGDIB-K50lac in the tumor tissues of treatment-resistant patients was significantly lower than that in treatment-sensitive patients, as shown in the attached figure. Figure 4 As shown, p < 0.01.

[0031] A method for using the ARHGDIB lactation assay kit for diagnosing lymph node metastasis in bladder cancer and predicting chemotherapy efficacy includes the following steps: Step 1: Sample Preparation: Paraffin-embedded tumor tissue samples from 84 bladder cancer patients and 40 bladder cancer patients before chemotherapy were obtained from the Department of Pathology, Nanjing Drum Tower Hospital, and prepared into 5 μm thick paraffin tissue microarrays. None of the bladder cancer patients had a history of other tumors. The sections were baked in a 60-65℃ oven for 2 hours to melt the paraffin and enhance tissue adhesion.

[0032] Step 2: Dewaxing and hydration: The sections were then immersed in xylene I (15 minutes) and xylene II (15 minutes) in sequence for dewaxing. Then they were immersed in a gradient of alcohols in sequence: 100% ethanol I (5 minutes), 100% ethanol II (5 minutes), 95% ethanol (5 minutes), and 80% ethanol (5 minutes), and finally rinsed with distilled water.

[0033] Step 3: Antigen retrieval: Immerse the slides in preheated antigen retrieval solution and place them in a microwave oven or pressure cooker for heat-induced antigen retrieval (specific conditions: microwave on high until boiling, keep warm for 15-20 minutes, then cool naturally to room temperature). Then wash three times with PBS, 5 minutes each time.

[0034] Step 4: Blocking: Draw a circle around the tissue with an immunohistochemical pen, add an appropriate amount of blocking solution to cover the tissue, and block at room temperature for 30 minutes. Pour off the blocking solution; do not wash.

[0035] Step 5: Primary antibody incubation: Directly add ready-to-use primary antibody (i.e., ARHGDIB-K50lac polyclonal antibody) to completely cover the tissue, place in a humidified chamber, and incubate overnight at 4°C. Then wash three times with PBST, 5 minutes each time.

[0036] Step 6: Secondary antibody incubation: Add TYR-570 labeled secondary antibody to cover the tissue and incubate at room temperature in the dark for 1 hour. Then wash three times with PBST in the dark, 5 minutes each time.

[0037] Step 7: Counterstaining and Mounting: Counterstain cell nuclei with DAPI solution and incubate at room temperature in the dark for 5-10 minutes. Wash three times with PBS in the dark. Add anti-fluorescence quenching mounting medium, cover with a coverslip, avoiding air bubbles, and seal the edges with nail polish.

[0038] Step 8: Image Acquisition and Result Analysis: Observe under a fluorescence microscope. Use excitation light of a specific wavelength to observe the cell nuclei labeled with DAPI (blue fluorescence) and the signals of ARHGDIB-K50lac labeled with TYR-570 (red fluorescence).

[0039] Results Interpretation: A semi-quantitative scoring system (e.g., H-score) was used. H-score = Σ (Pi × i), where Pi represents the percentage of cells with staining intensity i (i = 0, 1, 2, 3 represent negative, weak, moderate, and strong positive, respectively). Five non-overlapping fields of view were randomly selected, and the average H-score was calculated as the protein expression level of the sample. Positive and negative controls were also included to verify the validity of the experiment. A t-test analysis of data from patients with positive and negative lymph node metastases in bladder cancer revealed that patients with positive lymph node metastases had significantly lower ARHGDIB-K50lac levels (p < 0.01), as shown in the attached figure. Figure 3 As shown in the attached figure. A t-test analysis of data from chemotherapy-sensitive and chemotherapy-resistant bladder cancer patients revealed that chemotherapy-resistant bladder cancer patients had significantly lower levels of ARHGDIB-K50lac (p<0.01). Figure 4 As shown.

Claims

1. An ARHGDIB lactating detection kit for diagnosing lymph node metastasis of bladder cancer and predicting the efficacy of chemotherapy, characterized by: Comprising ​ Primary antibody: 44~56 amino acid residues of ARHGDIB-K50lac; Secondary antibody: TYR-570 labeled goat anti-rabbit IgG; Antigen repair solution; Blocking solution; Nuclear re-staining agent; Anti-fluorescence quenching mounting agent; Positive reference; Negative reference; 10× phosphate buffer solution.

2. The ARHGDIB lactation detection kit as described in claim 1, used for diagnosing lymph node metastasis in bladder cancer and predicting chemotherapy efficacy, is characterized in that: The 44~56 amino acid residues of ARHGDIB-K50lac are diluted with PBST to a concentration of 0.26 mg / mL.

3. The ARHGDIB lactation detection kit as described in claim 1, used for diagnosing lymph node metastasis in bladder cancer and predicting chemotherapy efficacy, is characterized in that: The TYR-570 labeled goat anti-rabbit IgG is ready-to-use with a concentration of 1~2.5 ng / mL.

4. The ARHGDIB lactation detection kit as described in claim 1, used for diagnosing lymph node metastasis in bladder cancer and predicting chemotherapy efficacy, is characterized in that: The antigen repair solution is a citrate buffer with a pH of 6.

0.

5. The ARHGDIB lactation detection kit as described in claim 1, used for diagnosing lymph node metastasis in bladder cancer and predicting chemotherapy efficacy, is characterized in that: The blocking solution is a PBS solution containing 5% bovine serum albumin.

6. The ARHGDIB lactation detection kit as described in claim 1, used for diagnosing lymph node metastasis in bladder cancer and predicting chemotherapy efficacy, is characterized in that: The nuclear re-staining agent is a DAPI solution with a concentration of 1 μg / mL.

7. The ARHGDIB lactation detection kit as described in claim 1, used for diagnosing lymph node metastasis in bladder cancer and predicting chemotherapy efficacy, is characterized in that: The positive reference is a bladder cancer tissue section known to have high expression of ARHGDIB-K50lac; the negative reference is a bladder cancer tissue section known to have no expression of ARHGDIB-K50lac.

8. The ARHGDIB lactation detection kit as described in claim 7, used for diagnosing lymph node metastasis in bladder cancer and predicting chemotherapy efficacy, characterized in that: The ARHGDIB-K50lac antibody is prepared by Hangzhou Huaan Biotechnology Co., Ltd.

9. The ARHGDIB lactated detection kit for diagnosing lymph node metastasis of bladder cancer and predicting the efficacy of chemotherapy according to claim 1, wherein The 10× phosphate buffer solution is diluted to 1× PBST when used.

10. A method for using the ARHGDIB lactic acidification detection kit for diagnosing lymph node metastasis of bladder cancer and predicting the efficacy of chemotherapy according to any one of claims 1-9, comprising the following steps: First step: sample preparation: 84 cases of bladder cancer patients and 40 cases of tumor tissue paraffin specimens of bladder cancer patients before chemotherapy were retrieved from the pathology department of Nanjing Drum Tower Hospital, and were prepared into 5 μm thick paraffin tissue chips by the pathology department. Among them, the bladder cancer patients had no history of other tumors. The slices were placed in an oven at 60~65℃ for 2 hours to melt the paraffin and enhance the adhesion of the tissue. Second step: dewaxing and hydration: the slices were immersed in xylene I for 15 minutes, xylene II for 15 minutes for dewaxing, and then immersed in gradient alcohol: 100% ethanol I for 5 minutes, 100% ethanol II for 5 minutes, 95% ethanol for 5 minutes, 80% ethanol for 5 minutes, and finally washed with distilled water. Third step: antigen repair: the slices were immersed in preheated antigen repair solution and placed in a microwave oven or pressure cooker for heat-induced antigen repair, then washed with PBS for 3 times, 5 minutes each time. Fourth step: blocking: draw a circle around the tissue with an immunohistochemical pen, add an appropriate amount of blocking solution to cover the tissue, and block at room temperature for 30 minutes, then pour off the blocking solution. Fifth step: primary antibody incubation: directly add ready-to-use primary antibody to completely cover the tissue, place it in a wet box, and incubate at 4℃ overnight, then wash with PBST for 3 times, 5 minutes each time. Sixth step: secondary antibody incubation: add TYR-570 labeled secondary antibody to cover the tissue, incubate at room temperature in the dark for 1 hour, then wash with PBST in the dark for 3 times, 5 minutes each time. Seventh step: re-staining and mounting: add DAPI solution to re-stain the nucleus, incubate at room temperature in the dark for 5~10 minutes, wash with PBS in the dark for 3 times, add anti-fluorescence quenching mounting agent, cover with a cover glass to avoid air bubbles, and seal the edges with nail polish. Step 8: Image acquisition and result analysis: under the fluorescence microscope, the DAPI labeled nuclei and the TYR-570 labeled ARHGDIB-K50lac signal were observed using excitation light of specific wavelength; Step 9: Result interpretation: the H-score scoring system was used, H-score = Σ (Pi × i), where Pi represents the percentage of cells with staining intensity i, i = 0, 1, 2, 3 representing negative, weak, medium, and strong positive respectively, 5 non-overlapping fields were randomly selected, and the average H-score was calculated as the protein expression level of the sample. At the same time, positive and negative controls were set to verify the effectiveness of the experiment.

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