CN114217066A - Prostate cancer/breast cancer bone metastasis small molecular marker and clinical detection kit - Google Patents
Prostate cancer/breast cancer bone metastasis small molecular marker and clinical detection kit Download PDFInfo
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Abstract
The invention provides a prostate cancer/breast cancer bone metastasis small molecular marker and a clinical detection kit. Biomarkers for bone metastasis from prostate cancer, breast cancer, including IL-8 or S100a 4. The invention also prepares an IL-8/S100A4 magnetic antibody, and further develops an IL-8/S100A4 medical detection kit, which is suitable for early diagnosis of tumor bone metastasis and monitoring of tumor bone metastasis in the tumor treatment process.
Description
Technical Field
The invention belongs to the technical field of tumor medical detection, and relates to a prostate cancer/breast cancer bone metastasis small molecular marker and a clinical detection kit.
Background
Bone metastasis is one of the common complications of malignant tumors, seriously affects the survival and life quality of patients, and particularly has the characteristics of high incidence rate and high disabling fatality rate in three high malignant tumors of breast Cancer, prostate Cancer and lung Cancer (Siegel RL, Miller KD, Fuchs HE, Jemal A. CA Cancer J Clin. 2021;71(1):7-33., Hernandez R K, Wade S W, Reich A, et al BMC Cancer, 2018;18(1): 1-11.), as shown in FIG. 1.
Tumor bone metastasis leads to bone tissue destruction, which in severe cases can lead to the occurrence of related adverse events (SER) such as pain, spinal cord compression, hypercalcemia, and pathological fractures. Furthermore, tumor cells can cause resistance to chemoradiotherapy after invading bones, increase the malignancy of tumors, and accelerate the process of tumor lethal (Lipton A, et al cancer.2000;88: 1082. 1090., Saad F, et al J Natl Cancer Inst. 2004; 96: 879. 882., Yin JJ, et al Cell Res. 2005;15(1): 57-62.). Hormone receptor inhibitory endocrine therapy is selected for the treatment of breast Cancer and Prostate Cancer, and distant metastasis frequently occurs when drug resistance recurs, especially bone injury is caused by bone metastasis to 50% of the upper and lower (Howard LE, et al. State Cancer pathological Dis 2016;19(4): 380-.
The current diagnostic techniques for tumor bone metastasis mainly include whole body bone imaging (technetium 99 labeled MDP nuclide imaging) and MRI (magnetic resonance imaging) (Krishnmurthy GT, Blahd WH. Nuclear Med (Stuttg): 1975;13(4):330-40., Cook GJ. Cancer imaging 2010;10(1): 1-8.), but lack specific biological detection indexes for prediction and evaluation of tumor bone metastasis. Therefore, clinically only the occurrence of bone metastasis events can be determined, but the trend and severity of the occurrence of bone metastasis cannot be predicted. Endocrine therapy aimed at inhibiting the activity of male and female receptors (AR, ER) is effective in reducing the primary focus of prostate and breast cancer and reducing the associated biochemical markers (e.g., PSA) but at the same time has the potential risk of promoting tumor metastasis (DiNatale A, Fatatis A. Adv Exp Med biol. 2019;1210: 171-.
Therefore, the tumor bone metastasis mechanism is still unclear, the molecular targeting is not clear, the effective diagnosis and treatment scheme is deficient, and the method is a serious challenge in the global medical community. Especially, before and at the early stage of tumor bone metastasis, an experimental detection method with high specificity and good sensitivity is clinically lacked, and a tumor bone metastasis early diagnosis technology needs to be developed.
Disclosure of Invention
Aiming at the technical problems, the invention provides a prostate cancer/breast cancer bone metastasis small molecular marker and a clinical detection kit.
In order to realize the technical purpose of the invention, the invention adopts the technical scheme that:
the invention provides application of a small molecular marker IL-8 or S100A4 in preparation of a prostate cancer and breast cancer bone metastasis detection reagent.
The invention also provides a preparation method of the IL-8/S100A4 magnetic antibody, which comprises the following steps:
1) washing carboxyl magnetic beads with MES buffer solution with pH =6.0, and then re-suspending the magnetic beads in MES buffer solution with pH =6.0 until the concentration of the magnetic beads is 100 mg/ml;
2) adding EDC-MES buffer solution and Sulfo-NHS-MES buffer solution into the magnetic beads obtained in the step 1), uniformly mixing, adjusting the concentration of the magnetic beads to be 50 mg/ml, and activating for 30 minutes at 25 ℃;
3) removing supernate from the activated magnetic beads, adding MES buffer solution with pH =6.0 for washing, adding IL-8 or S100A4 antibody with the concentration of 2mg/ml into the magnetic beads after activation and washing, immediately mixing uniformly, and reacting for 12-18 hours at 25 ℃;
4) after the reaction is finished, removing the supernatant of the magnetic beads, adding PBS buffer solution with pH =7.4 and Tween20, washing, keeping the magnetic beads in the buffer solution until the final concentration is 10mg/ml, and uniformly mixing for 30 minutes at 25 ℃;
5) the supernatant was removed and resuspended in 10mM PBS buffer pH =7.4 containing 0.1% Tween20+0.1% BSA to a final concentration of 10mg/ml magnetic beads and an antibody concentration of 200. mu.g/ml, to give IL-8/S100A4 magnetic antibody.
Preferably, the concentration of MES buffer pH =6.0 is 15 mM.
Preferably, in step 2), the concentration of EDC in EDC-MES buffer is 20mg/ml and the concentration of Sulfo-NHS in Sulfo-NHS-MES buffer is 24 mg/ml.
Preferably, in step 4), the concentration of PBS buffer of pH =7.4 is 10mM, Tween20 is 0.1% Tween 20.
The invention also provides an HRP immunodetection kit for IL-8/S100A4, which comprises the IL-8/S100A4 magnetic antibody prepared by the preparation method, a horseradish peroxidase HRP labeled antibody, a luminescent substrate and a Tris washing buffer solution, wherein the luminescent substrate consists of luminol, hydrogen peroxide and an enhancer p-2 iodophenol, the antibody concentration of the IL-8/S100A4 magnetic antibody is 200 mug/ml, and the antibody concentration of the horseradish peroxidase HRP labeled antibody is 100 mug/ml.
Preferably, the Tris wash buffer comprises 200mM Tris, 200mM NaCl, 0.2% w/v TritonX-100, 0.3% BND (5-bromo-5-nitro-1, 3-dioxane), 0.1% silicone oil type antifoam agent, HCl adjusted to pH 7.6.
Preferably, the HRP immunoassay kit for IL-8/S100a4 further comprises a dilution of a test sample, wherein the dilution of the test sample comprises 10mM PBS buffer with pH =7.4, 0.1% Tween20 and 0.1% BSA.
The invention also provides an ALP immunodetection kit of IL-8/S100A4, which comprises an IL-8/S100A4 magnetic antibody and an alkaline phosphatase ALP labeled antibody, wherein the antibody concentration of the IL-8/S100A4 magnetic antibody is 200 mug/ml, and the antibody concentration of the alkaline phosphatase ALP labeled antibody is 100 mug/ml.
Preferably, chemiluminescent agents AMPPD and Tris wash buffer are also included.
Preferably, the Tris wash buffer comprises 200mM Tris, 200mM NaCl, 0.5% w/v TritonX-100, 0.3% BND (5-bromo-5-nitro-1, 3-dioxane), 0.1% silicone oil type antifoam agent, HCl adjusted to pH 7.6.
Preferably, the kit further comprises a test dilution, wherein the test dilution comprises 10mM PBS buffer with pH =7.4, 0.1% Tween20 and 0.1% BSA.
The invention has the beneficial effects that:
the invention discloses that the cell inflammatory factor Interleukin 8 (IL-8, Interleukin-8) and the calcium ion binding protein S100A4 are abnormally increased before and after the tumor bone metastasis, and the bone metastasis can be effectively inhibited by reducing the expression level of the two proteins in tumor cells. Therefore, IL-8 and S100A4 can be used to monitor and intervene in the biological targets of tumor bone metastasis. Meanwhile, the IL-8/S100A4 magnetic antibody is prepared, and an IL-8/S100A4 detection kit is further developed, so that the kit is suitable for early diagnosis of tumor bone metastasis and monitoring of tumor bone metastasis in the tumor treatment process.
Drawings
FIG. 1. incidence of bone metastasis from malignant tumors, whole body bone imaging and NMR show that bone injury is caused by bone metastasis from tumors.
FIG. 2. malignant tumor bone metastasis results in bone injury frequency (SER) and mortality. A. Frequency of bone damage caused by tumor bone metastasis; B. prostate cancer SER is significantly associated with mortality; C. breast and lung cancer SER cause a significant reduction in patient survival.
FIG. 3 IL-8 promotes prostate tumor proliferation and bone metastasis. A. After the human prostate tumor LNCaP cell is transfected with the IL-8 expression plasmid, carrying out immunoblot analysis and IL-8 quantitative kit detection; B. inoculating tumor cells to the prostate of the male nude mouse for 20 days, and then tracking and detecting the result by using a fluorescence image; C. and (5) detecting results by a fluorescence image and nuclear magnetic resonance technology 30 days after inoculation. p <0.001 indicates that the two group comparisons reached a very significant level.
Figure 4. S100a4 promotes prostate tumor proliferation. A. After the human prostate tumor LNCaP cells transfect S100A4 expression plasmids, the results of immunoblot analysis and the influence of increasing the expression quantity of S100A4 on the invasion rate and the moving distance of the cells are obtained; B. and (4) inoculating the tumor cells to the prostate of the male nude mouse, and then tracking and detecting the result by using a fluorescence image. p <0.001 indicates that the two group comparisons reached a very significant level.
Fig. 5. S100a4 promotes prostate tumor bone metastasis. A. After the transfection of shRNA plasmid by human prostate malignant tumor PC-3 cells silences (reduces) S100A4, carrying out immunoblot analysis; B. after tumor cells are inoculated to the tail artery of a male nude mouse for 20 days, the result of quantitative analysis of bone density is obtained; C. and (3) inoculating the tumor cells to the tail artery of the male nude mouse 20 days later, and quantitatively analyzing the result of the bone injury. p <0.001 indicates that the two groups compared to a very significant level.
FIG. 6 is the PSA concentration detection report in serum of prostate cancer patients. p <0.05, p <0.01, or p <0.001 indicates a statistically significant difference level between the two groups.
FIG. 7 measurement report of serum IL-8 and S100A4 concentration of prostate cancer patients and breast cancer patients. p <0.01 or p <0.001 indicates a statistically significant difference level between the two groups.
FIG. 8 is a flow chart of the preparation of the IL-8, S100A4 magnetic antibody of the present invention.
FIG. 9 is a reaction schematic diagram of the HRP immunoassay kit of the present invention.
FIG. 10 is a flow chart of the operation of HRP immunoassay according to the present invention.
FIG. 11 is a reaction scheme of an ALP immunoassay kit according to the present invention.
FIG. 12 is a flow chart of the operation of ALP immunoassay according to the present invention.
Detailed Description
In order to more clearly illustrate the present invention, the present invention will be described in further detail below with reference to examples and the accompanying drawings. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Examples
The research process of the invention comprises the following aspects:
effects of IL-8 and S100A4 on prostate cancer and breast cancer bone metastasis and serological detection
IL-8 role in prostate cancer bone metastasis
Interleukin 8 (IL-8) is an 8 kDa small molecule chemokine (or a cytokine) secreted by cells, and its binding receptors are mainly CXCR1 and CXCR2 (Hedges JC, et al, Am J Respir Cell Mol biol. 2000;23(1): 86-94.). IL-8, an important inflammatory regulator, acts primarily on neutrophil recruitment and degradation (Harada A, et al J Leukoc biol. 1994;56(5): 559-64.). We find that the expression level of IL-8 in prostate cancer and breast cancer cells is increased, and sex hormone independent cell proliferation and migration are promoted, and further find that IL-8 can be activated through a RelB-mediated NF-kB non-canonical pathway, and the increase of the expression level of IL-8 can promote prostate cancer proliferation and bone metastasis in nude mice. As shown in FIG. 3, the intracellular IL-8 expression level was improved by immunoblotting (beta-actin is an internal reference control); after the IL-8 plasmid is verified and transfected by using an IL-8 quantitative kit, the expression level of IL-8 in a cell culture solution is increased, and the cell proliferation and the moving distance are enhanced; tumor cells are inoculated to the prostate of a male nude mouse, and fluorescent image tracking detection shows that the increase of IL-8 promotes tumor growth and metastasis.
2. S100A4 function in prostate cancer bone metastasis
S100 calcium-binding protein A4 (S100A 4) is another small 12 kDa protein secreted by cells and plays an important regulatory role in cytoskeleton formation (Jaiswal JK, Nylandsted J. Cell cycle. 2015;14(4): 502-9.). We studied systematically the function of S100a4 in bone metastasis of prostate tumors. S100A4 is over-expressed in human androgen-dependent prostate tumor LNCaP cells, and the infiltration and migration capacity of the cells are enhanced. As shown in fig. 4, after LNCaP cells were transfected with S100a4 expression plasmid, intracellular S100a4 expression level enhancement was determined using immunoblotting technique (β -actin is an internal reference control); increasing the expression quantity of S100A4 to enhance the invasion rate and the moving distance of the cells; tumor cells are inoculated to the prostate of a male nude mouse, and the fluorescent image tracking detection shows that the increase of S100A4 promotes the tumor growth. On the contrary, the secretion of S100A4 by human androgen-independent PC-3 malignant cells is reduced, so that the bone transfer capability of the malignant cells is remarkably reduced. As shown in FIG. 5, after transfection of shRNA plasmid into PC-3 cells to silence (reduce) S100A4, the level of S100A4 expressed in cells was determined to be reduced using immunoblotting techniques (β -actin is an internal reference control); after 20 days of inoculating tumor cells to the tail artery of a male nude mouse, a pathological analysis method is used for detecting the bone density and the bone injury degree. Compared with normal saline, the injection of PC-3 cells can significantly reduce the bone density and cause bone injury. However, decreasing the expression level of S100A4 in PC-3 cells improved bone density and reversed bone damage.
3. Serological assay for PSA, IL-8 and S100A4 in prostate cancer patients
IL-8 and S100A4 are small-molecule proteins secreted by cells, particularly high and secreted by tumor cells and tumor-associated stromal cells, change the tumor microenvironment and promote tumor growth and metastasis (Tan C, et al. Cytokine 2018;108: 151-gene 159. Ishikawa M, et al. oncogene 2019;38(24): 4715-gene 4728.). Our earlier studies revealed that prostate tumors develop to a hormone-independent type and tend to lymphatic metastasis following conventional androgen suppression therapy, with abnormal elevation of IL-8 in the patient's peripheral blood; when tumor bone metastasis ensues, S100A4 is abnormally elevated. Therefore, 20 peripheral blood samples of patients with prostate cancer non-metastasis, lymphatic metastasis and bone metastasis and healthy male controls were collected, and after serum was centrifuged, the concentrations of PSA, IL-8 and S100a4 were measured using antigen-antibody specific reaction. The pathological data and the test results of the patients are detailed in table 1.
TABLE 1 clinical pathological data and serum sample test report of prostate cancer patients
| Index (I) | Number of detected people | PSA (ng/ml) | IL-8 (pg/ml) | S100A4 (ng/ml) |
| Gleason grading | ||||
| 3+3 | 4 | 50.38 | 48.75 | 7.44 |
| 3+4 | 6 | 45.03 | 46.82 | 7.67 |
| 4+3 | 10 | 35.83 | 48.79 | 7.29 |
| 4+4 | 10 | 20.23 | 73.17 | 8.82 |
| 4+5 | 10 | 13.99 | 87.55 | 7.99 |
| 5+4 | 10 | 27.37 | 107.67 | 19.02 |
| 5+5 | 10 | 28.99 | 117.98 | 17.73 |
| Pathological grading | ||||
| 1 | 4 | 50.38 | 48.75 | 7.44 |
| 2 | 6 | 45.03 | 46.82 | 7.67 |
| 3 | 12 | 33.32 | 53.97 | 7.70 |
| 4 | 14 | 16.57 | 78.74 | 8.62 |
| 5 | 24 | 26.35 | 108.40 | 16.48 |
| Metastasis of tumor | ||||
| Is not transferred | 20 | 41.50 | 48.19 | 7.434 |
| |
20 | 17.11 | 80.36 | 8.406 |
| |
20 | 28.18 | 112.8 | 18.37 |
| PSA (ng/ml) | ||||
| >4<10 | 8 | 8.38 | 108.12 | 11.61 |
| >10<30 | 22 | 18.28 | 77.50 | 10.96 |
| >30 | 30 | 42.23 | 75.25 | 11.67 |
| Age (age) | ||||
| <60 | 28 | 29.59 | 85.15 | 11.28 |
| ≥60 | 32 | 28.36 | 76.35 | 11.51 |
| Total of | 60 | |||
| |
20 | 0.4430 | 12.15 | 1.497 |
Grading Standard of prostate cancer according to the International urinary society (ISUP) approved by the 2016 World Health Organization (WHO)
Prostate Specific Antigen (PSA) is an important indicator of current prostate cancer diagnosis. Compared with a healthy male control group, the content of PSA in the serum of the prostate cancer patient is obviously increased. However, there was a downward trend in tumor lymphatic metastasis and a slight return in tumor bone metastasis, as shown in FIG. 6. The results indicate that PSA is an important indicator for diagnosing early stage androgen-dependent prostate cancer (primary lesion), but cannot be used as a diagnostic indicator in the progression of cancer to androgen-independent malignant prostate cancer (metastatic lesion). Clinical serum sample detection reports show that compared with a healthy male control group, the serum contents of IL-8 and S100A4 in the prostate cancer patients are generally increased; especially in the course of primary tumor focus-tumor lymphatic metastasis-tumor bone metastasis, IL-8 and S100A4 levels were gradually increased (FIG. 7).
Accordingly, the present invention discloses that IL-8 and S100A4 are biomarkers for metastasis of malignant prostate bone tumors. Therefore, the detection of the concentrations of IL-8 and S100A4 in the serum of a tumor patient can effectively predict the tendency of tumor bone metastasis, and provides a reliable basis for clinical intervention or treatment of bone metastasis.
4. Serological examination of IL-8 and S100A4 in breast cancer patients
Breast and prostate cancers are two major sex hormone-related malignancies, with morbidity first among the same grade malignancies and mortality second, second only to lung Cancer (Siegel RL, Miller KD, Fuchs HE, Jemal A. CA Cancer J Clin. 2021;71(1): 7-33.). The breast cancer onset in China has the characteristics of early onset age and higher difficulty in early screening technology. The two malignant tumors have a certain common point in occurrence and development, namely, early (or primary focus) tumors are mainly sex hormone receptor (AR, ER) dependent, and the tumor proliferation can be effectively controlled by operation, radiotherapy and hormone treatment. However, drug-resistant relapse remains a prominent problem, especially with high rates of bone metastasis and high mortality. The sex hormone-dependent, stress-independent development is a common feature of malignant development of breast and prostate cancers. Therefore, we performed serological examination of breast cancer at the same time, and the pathological data and examination results of the patients are detailed in table 2.
TABLE 2 clinical pathological data and serum sample detection report of breast cancer patients
| Index (I) | Number of detected people | IL-8 (pg/ml) | S100A4 (ng/ml) |
| ER1 | |||
| Negative of | 28 | 109.59 | 26.23 |
| Positive for | 32 | 67.87 | 18.11 |
| PR2 | |||
| Negative of | 27 | 112.35 | 26.48 |
| Positive for | 33 | 66.87 | 18.15 |
| Her-23 | |||
| Negative of | 36 | 80.19 | 20.63 |
| Positive for | 24 | 98.06 | 23.80 |
| TNM staging4 | |||
| Stage I | 6 | 48.54 | 14.01 |
| Stage II | 24 | 70.88 | 17.14 |
| Stage III | 14 | 98.14 | 25.19 |
| |
16 | 117.13 | 29.11 |
| Metastasis of tumor | |||
| Is not transferred | 20 | 59.94 | 13.91 |
| |
20 | 85.35 | 22.33 |
| |
20 | 116.70 | 29.45 |
| Age (age) | |||
| <60 | 28 | 85.15 | 11.28 |
| ≥60 | 32 | 76.35 | 11.51 |
| Total of | 60 | ||
| Healthy female controls | 20 | 24.51 | 4.21 |
1 hormone receptor (ER);
2 the progestogen receptor PR (progasterone receptor, PR);
3 human epidermal growth factor receptor-2 (Her-2) according to the American Society of Clinical Oncology (ASCO) guidelines, and 4 according to the grading standards defined by the International Association for anticancer Association (UIAC). T, primary tumor T0, no primary tumor, T1, <2cm, T2, 2-5cm, T3, >5cm, T4, invasive chest or skin, N, regional lymph node N0, no lymph node metastasis, N1, ipsilateral lymph node metastasis, N2, ipsilateral metastasis fused with the lymph node of fluid, N3, ipsilateral lymph node metastasis of intra-mammary region, M, distant metastasis M0, no distant metastasis, M1, distant metastasis.
The result shows that the content of IL-8 and S100A4 is gradually increased along with the increase of the malignancy degree of the tumor; particularly, the serum content of S100A4 in the breast cancer bone metastasis patients is higher than that in the prostate cancer bone metastasis patients, and the result is basically consistent with the clinical observation phenomenon that the breast cancer is mainly bone-dissolving type bone metastasis (figure 7).
In conclusion, IL-8 and S100A4 can be used as biological indicators of the progression of malignancy and bone metastasis in two sex hormone-related tumors. The serological detection indexes of IL-8 and S100A4 can be used for clinically diagnosing breast cancer, prostatic cancer and monitoring malignant development of tumors.
Second, clinical serological diagnosis kit research and development scheme
At present, only ELISA kits for IL-8 and S100A4 scientific research are sold in domestic and foreign markets for laboratory detection of IL-8 and S100A4 in cell culture solution and human and animal blood. The serological detection of IL-8 and S100A4 is not included in clinical diagnosis indexes, and medical IL-8 and S100A4 kits are lacked in the market. Therefore, the invention patent comprises the development of IL-8 and S100A4 clinical serological detection kits and the development of relevant clinical detection experiments. According to the principle of antigen-antibody specificity reaction and according to the conventional serological clinical detection specification and matched instrument and equipment used in domestic hospitals, a detection kit labeled by a chemiluminescent enzyme HRP (or ALP) antibody is developed. The specific implementation process is as follows:
1. preparation of IL-8, S100A4 magnetic antibody (core Material)
At present, no medical humanized IL-8 and S100A4 magnetic bead antibody is sold in the market, the EDC & NHS two-step method is used for crosslinking carboxyl magnetic beads and the antibody to prepare the medical core material, and the technical operation flow is shown in figure 8.
a. Buffer solution preparation:
15mM MES pH = 6.0: 2.928g MES were weighed out and dissolved in 900ml ddH2And O, adjusting the pH value to 6.0 by adopting NaOH, and fixing the volume to 1L. The mixture was filtered through a 0.22 μm filter and stored at 4 ℃.
EDC-MES buffer (20 mg/mL): 200mg of EDC was weighed, dissolved in 10ml of 15mM MES buffer and stored at 4 ℃.
Sulfo-NHS-MES buffer (24 mg/mL): 240mg of EDC was weighed, dissolved in 10ml of 15mM MES buffer and stored at 4 ℃.
10mM PBS buffer preparation: weighing: 8g NaCl, 0.2g KCl, 1.44g Na2HPO4，0.24gKH2PO4(ii) a Adjusting the pH value to 7.4 by HCl; add ddH2O is constant volume to 1L; filtering with 0.22 μm filter.
0.1% BSA: 0.01g of BSA powder was weighed and dissolved in the above 10mM PBS buffer.
0.1% Casein: 0.01g of Casein powder was weighed and dissolved in the above 10mM PBS buffer.
b. The preparation method comprises the following specific steps:
1) antibody buffer displacement and adjustment of concentration:
replacing an antibody buffer solution with a MES buffer solution with 15mMpH = 6.0;
② adjusting the antibody concentration to 2 mg/ml.
2) Magnetic bead activation
Taking 10mg of magnetic beads (1 μm, MERCK), washing 3 times by using 15mM MES buffer with pH =6.0, and suspending the magnetic beads in 0.1ml of 15mM MES buffer with pH =6.0 to reach the concentration of the magnetic beads to be 100 mg/ml;
(vii) weighing EDC and Sulfo-NHS and dissolving in 15mM MES buffer pH = 6.0: preparing EDC with the concentration of 20mg/ml and Sulfo-NHS with the concentration of 24 mg/ml; adding 50 μ L EDC and 50 μ L Sulfo-NHS into the washed magnetic beads; mixing evenly immediately, wherein the concentration of magnetic beads is about 50 mg/ml;
③ 30 minutes at 25 ℃.
3) Crosslinking of magnetic beads with antibodies
Removing supernatant of activated magnetic beads, and adding 1ml of cold 15mM MES buffer solution with pH =6.0 for washing for 2 times;
adding 200 mul (with the concentration of 2 mg/ml) of the antibody with the adjusted concentration into the activated and cleaned magnetic beads, and immediately mixing the mixture;
③ reacting for 12 to 18 hours under the condition of uniform mixing at 25 ℃.
4) Sealing and preservation after crosslinking
After the reaction is finished, removing supernatant of magnetic beads, adding 10mM PBS buffer solution with pH =7.4 and containing 0.1% Tween20, and washing for 2 times;
keeping the magnetic beads in PBS buffer solution containing 0.1% Tween20, 0.1% BSA and 0.01% Casein with pH =7.4 to a final concentration of 10mg/ml, and uniformly mixing for 30 minutes at room temperature;
③ remove the supernatant, resuspend the magnetic beads in PBS buffer containing 0.1% Tween20+0.1% BSA, pH =7.4, adjust the final concentration of the magnetic beads to 10mg/ml, adjust the antibody concentration to 200. mu.g/ml, and store in the buffer for later use.
2. Development of HRP (horse radish peroxidase) immunoassay kit
a. The supporting equipment adopts Yapei-i 2000SR, and the principle is shown in figure 9.
The material components of the kit comprise:
antibody (primary antibody, 200. mu.g/ml) coated magnetic microparticles, i.e., the aforementioned IL-8/S100A4 magnetic antibody;
horseradish peroxidase HRP-labeled antibody (secondary antibody, 100 μ g/ml);
luminol (chemiluminescent agent) + hydrogen peroxide (H)2O2) (luminol reagent);
enhancer (p-2 iodophenol);
tris wash buffer (200 mM Tris, 200mM NaCl, 0.2% w/v Triton X-100, 0.3% BND (5-bromo-5-nitro-1, 3-dioxane), 0.1% silicone oil type antifoam, HCl to pH 7.6);
standard (diluted concentration gradient).
The technical operation flow of the HRP immunoassay is shown in FIG. 10, and comprises the following steps:
1) adding 20 mu L of primary anti-coated magnetic particles, 20 mu L of sample (or standard) to be detected and 160 mu L of diluent of the sample to be detected, mixing uniformly, and incubating for 30 minutes;
2) separating magnetic beads in a magnetic field, sucking to remove residual liquid, and adding 500 mul of buffer solution to wash for 3 times;
3) adding 150 μ l of HRP-labeled antibody (secondary antibody), mixing, and incubating for 15 min;
4) repeating the step 2);
5) adding a luminescent substrate: luminol + hydrogen peroxide + enhancer (p 2 iodophenol) was added and incubated for 5 min;
6) the machine reads the value.
Wherein, the HRP secondary antibody labeling implementation step (simple sodium periodate method) comprises the following steps:
weighing 5mg of HRP and dissolving in 1ml of distilled water.
② adding 0.2 ml of newly-prepared 0.1M NaIO into the above-mentioned solution4The solution was stirred at room temperature for 20 minutes in the absence of light.
③ filling the above solution into dialysis bag, dialyzing against 1mM sodium acetate buffer pH =4.4, and standing overnight at 4 ℃.
Mu.l of 0.2M carbonate buffer, pH9.5, was added to raise the pH of the above-hydroformylated HRP to 9.0-9.5, and then 10mg of IgG (antibody, or SPA 5 mg) was immediately added to 1ml of 0.01M carbonate buffer, and the mixture was gently stirred at room temperature for 2 hours in the dark.
Fifthly, 0.1ml of newly-prepared 4mg/ml NaBH is added4Mixing the solutions, and standing at 4 deg.C for 2 hr.
Sixthly, the solution is filled into a dialysis bag and dialyzed against PBS buffer solution with 0.15M and pH =7.4, and the temperature is over night at 4 ℃.
Seventhly, dropwise adding equal volume of saturated ammonium sulfate under stirring, and standing at 4 ℃ for 1 hour.
Eighty percent (3000 rpm) centrifugation is carried out for half an hour, and the supernatant is discarded. The precipitate was washed twice with half-saturated ammonium sulfate and finally dissolved in a small amount of 0.15M PBS pH 7.4.
Ninthly, putting the solution into a dialysis bag, dialyzing 0.15M PBS (phosphate buffer solution) with pH =7.4, removing ammonium ions (detecting by using a naphthalene reagent), centrifuging at 10,000rpm for 30 minutes to remove precipitates, wherein the supernatant is an enzyme conjugate, subpackaging, and freezing for storage.
3. Development of ALP (alpha-amyloid peptide) immunodetection kit
a. The complete equipment adopts Beckmann-DXI 800, and the principle is shown in figure 11.
The kit comprises the following components:
antibody (primary antibody, 200. mu.g/ml) coated magnetic microparticles, i.e., the aforementioned IL-8/S100A4 magnetic antibody;
alkaline phosphatase ALP-labeled antibody (secondary antibody, 100. mu.g/ml);
AMPPD (chemiluminescent agents);
tris wash buffer (200 mM Tris, 200mM NaCl, 0.5% w/v Triton X-100, 0.3% BND (5-bromo-5-nitro-1, 3-dioxane), 0.1% silicone oil type antifoam, HCl to pH 7.6);
standard (diluted concentration gradient);
test dilutions (10 mM PBS buffer pH =7.4 +0.1% Tween20+0.1% BSA).
The technical operation flow of ALP immunodetection is shown in FIG. 12, and the detection step comprises:
1) adding 20 mul of primary anti-coated magnetic particles, 20 mul of samples (or standard products) to be detected and 160 mul of dilution of the samples to be detected, mixing uniformly, and incubating for 30 minutes;
2) separating magnetic beads in a magnetic field, sucking to remove residual liquid, and adding 500 mul of buffer solution to wash for 3 times;
3) adding 150. mu.l ALP-labeled antibody (secondary antibody), mixing, and incubating for 15 min;
4) repeating the step 2);
5) adding 100 mu L of luminescent substrate AMPPD, mixing uniformly, and incubating for 5 minutes;
6) the machine reads the value.
Wherein the ALP-labeled antibody performing step (sodium periodate method) comprises:
dissolving 5mg of alkaline phosphatase in a NaAc solution with the concentration of 1ml and the pH value of 4.75 and the concentration of 0.05M to ensure that the concentration of the alkaline phosphatase is 5 mg/ml;
adding 1ml of sodium periodate solution into the enzyme solution in the step I, and oxidizing at the temperature of 4 ℃ for 20 minutes;
③ adding 1ml of glycol-NaCl solution into the oxidized enzyme solution;
fourthly, adding ice-cold absolute ethyl alcohol with the content of 1.5ml into the solution in the third step, uniformly mixing, standing for 1 hour at the room temperature of minus 20 ℃, and centrifuging for 10 minutes at 4000g to obtain the well-oxidized enzyme;
fifthly, dissolving the enzyme obtained in the precipitation step (iv) by using 0.5ml of carbonate buffer solution with the concentration of 0.5M and the pH value of 9.0, adding 0.1ml of glutaraldehyde, and uniformly mixing;
sixthly, enzyme: the ratio of antibodies was 1: 1 proportion, adding an antibody solution to be marked for reaction, wherein the reaction time is 2 hours;
seventhly, according to the mass ratio, NaBH4: the ratio of enzyme was 0.1: 1 ratio, adding NaBH4Standing the solution at room temperature for 2 hours;
adding 0.6 ml of ice-cold absolute ethyl alcohol, standing for 1 hour at the room temperature of minus 20 ℃, and centrifuging for 10 minutes at 4000 g;
ninthly, precipitating to obtain the marked antibody, and dissolving with PBS according to the concentration of the enzyme-labeled antibody or freeze-drying and storing.
It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made in the above embodiments without departing from the spirit and scope of the invention.
Claims (9)
1. Application of small molecular marker IL-8 or S100A4 in preparing a detection reagent for bone metastasis of prostate cancer and breast cancer.
A method for preparing an IL-8/S100A4 magnetic antibody, comprising:
1) washing carboxyl magnetic beads with MES buffer solution with pH =6.0, and then suspending the magnetic beads in MES buffer solution with pH =6.0 until the concentration of the magnetic beads is 100 mg/ml;
2) adding EDC-MES buffer solution and Sulfo-NHS-MES buffer solution into the magnetic beads obtained in the step 1), uniformly mixing, adjusting the concentration of the magnetic beads to be 50 mg/ml, and activating for 30 minutes at 25 ℃;
3) adding the deactivated magnetic bead supernatant into MES buffer solution with pH =6.0 for washing, adding IL-8 or S100A4 antibody with the concentration of 2mg/ml into the magnetic beads after activation and washing, immediately mixing uniformly, and reacting for 12-18 hours at 25 ℃;
4) after the reaction is finished, removing the supernatant of the magnetic beads, adding PBS buffer solution with pH =7.4 and Tween20 for washing, keeping the final concentration of the magnetic beads in the buffer solution to be 10mg/ml, and uniformly mixing for 30 minutes at 25 ℃;
5) the supernatant was removed and resuspended in 10mM PBS buffer pH =7.4 containing 0.1% Tween20+0.1% BSA to a final concentration of 10mg/ml magnetic beads and an antibody concentration of 200. mu.g/ml, to give IL-8/S100A4 magnetic antibody.
An IL-8/S100A4 HRP immunoassay kit, comprising IL-8/S100A4 magnetic antibody prepared by the preparation method of claim 2, horseradish peroxidase HRP-labeled antibody, luminescent substrate and Tris washing buffer, wherein the luminescent substrate comprises luminol, hydrogen peroxide and enhancer p-2 iodophenol, the IL-8/S100A4 magnetic antibody has an antibody concentration of 200 μ g/ml, and the horseradish peroxidase HRP-labeled antibody has an antibody concentration of 100 μ g/ml.
4. The HRP immunoassay kit for IL-8/S100A4 according to claim 3, wherein the Tris wash buffer comprises 200mM Tris, 200mM NaCl, 0.2% w/v Triton X-100, 0.3% BND, 0.1% silicone oil type antifoaming agent, and HCl adjusted to pH 7.6.
5. The IL-8/S100A4 HRP immunodetection kit according to claim 3, comprising a test sample dilution comprising 10mM PBS buffer pH =7.4, 0.1% Tween20, and 0.1% BSA.
The ALP immunoassay kit for IL-8/S100A4, comprising an IL-8/S100A4 magnetic antibody and an alkaline phosphatase ALP-labeled antibody, wherein the IL-8/S100A4 magnetic antibody has an antibody concentration of 200. mu.g/ml, and the alkaline phosphatase ALP-labeled antibody has an antibody concentration of 100. mu.g/ml.
7. The ALP immunoassay kit for IL-8/S100A4 according to claim 6, further comprising the chemiluminescent agents AMPPD and Tris washing buffer.
8. The kit for ALP immunoassay of IL-8/S100A4 according to claim 7, wherein said Tris wash buffer comprises 200mM Tris, 200mM NaCl, 0.5% w/v Triton X-100, 0.3% BND, 0.1% antifoam agent of silicone oil type, HCl adjusted to pH 7.6.
9. The kit for ALP immunoassay of IL-8/S100A4 according to claim 7, further comprising a test substance dilution containing 10mM PBS buffer pH =7.4, 0.1% Tween20 and 0.1% BSA.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100150946A1 (en) * | 2008-08-28 | 2010-06-17 | Cell Genesys, Inc. | Methods and Compositions for Treating Prostate Cancer or Inducing a Humoral Immune Response Against Prostate Cancer |
| US20110114490A1 (en) * | 2006-04-18 | 2011-05-19 | Advanced Liquid Logic, Inc. | Bead Manipulation Techniques |
| CN106226513A (en) * | 2016-08-02 | 2016-12-14 | 北京乐普医疗科技有限责任公司 | A kind of method of immunomagnetic beads detection by quantitative related antigen and application thereof |
| CN110161226A (en) * | 2019-02-27 | 2019-08-23 | 杭州晟康细胞生物科技有限公司 | A kind of antibody, kit and detection method detecting senile cell |
-
2022
- 2022-02-22 CN CN202210160200.8A patent/CN114217066A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110114490A1 (en) * | 2006-04-18 | 2011-05-19 | Advanced Liquid Logic, Inc. | Bead Manipulation Techniques |
| US20100150946A1 (en) * | 2008-08-28 | 2010-06-17 | Cell Genesys, Inc. | Methods and Compositions for Treating Prostate Cancer or Inducing a Humoral Immune Response Against Prostate Cancer |
| CN106226513A (en) * | 2016-08-02 | 2016-12-14 | 北京乐普医疗科技有限责任公司 | A kind of method of immunomagnetic beads detection by quantitative related antigen and application thereof |
| CN110161226A (en) * | 2019-02-27 | 2019-08-23 | 杭州晟康细胞生物科技有限公司 | A kind of antibody, kit and detection method detecting senile cell |
Non-Patent Citations (7)
| Title |
|---|
| HAEMIN KIM ET AL.: "S100A4 released from highly bone-metastatic breast cancer cells plays a critical role in osteolysis", 《BONE RESEARCH》 * |
| KIM ET AL.: "The role of S100A4 for bone metastasis in prostate cancer cells", 《BMC CANCER》 * |
| STEVEN LEHRER, M.D.: "Serum Interleukin-8 is Elevated in Men with Prostate Cancer and Bone Metastases", 《TECHNOLOGY IN CANCER RESEARCH & TREATMENT》 * |
| 李博等: "乳腺癌肝转移机制研究进展", 《实用肿瘤学杂志》 * |
| 杨士军等: "CA153、IL-6及IL-8与乳腺癌骨转移相关性探讨", 《重庆医学》 * |
| 王燕等: "基质金属蛋白酶与前列腺癌侵袭及转移的关系", 《实用癌症杂志》 * |
| 许宇媚等: "血清白介素和ALP水平与乳腺癌骨转移相关性探讨", 《中华肿瘤防治杂志》 * |
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