Multiplex assay kit for cervical cancer precancerous lesion and cancerous lesion detection, preparation method therefor, and use thereof

Abstract

A multiplex assay kit for cervical cancer precancerous lesion and cancerous lesion detection, a preparation method therefor, and the use thereof. The multiplex assay kit at least comprises multiple test strips for detecting the following indexes: the E5 protein, the E6 protein, the E7 protein, and hTERT. A connecting pad (2) of each test strip is coated with a detection antibody-colloidal gold conjugate, and a detection line (3) of the test strip is coated with a specific capture antibody, the specific capture antibodies on the test strips being different. The multiplex assay of the E5 protein, the E6 protein, the E7 protein, and hTERT can effectively improve the sensitivity and specificity of cervical cancer precancerous lesion and cancerous lesion detection. Compared to detection of only the E7 protein, the multiplex assay kit has significantly improved detection rate, sensitivity, and specificity.

Description

A combined detection kit for cervical cancer precancerous lesions and cancerous changes, its preparation method and application. Technical Field

[0001] This invention relates to the field of detection technology for precancerous lesions and carcinogenesis of cervical cancer, and in particular to a combined detection kit for the detection of precancerous lesions and carcinogenesis of cervical cancer, its preparation method and application. Background Technology

[0002] Cervical cancer is the fourth leading cause of cancer death among women worldwide, after breast cancer, and the leading cause of malignant tumors of the female reproductive tract. Statistics show that nearly 500,000 new cases of cervical cancer are diagnosed globally each year, with 83% occurring in developing countries. In my country, there are 131,500 new cases annually, accounting for 28.8%. Approximately 290,000 people die from cervical cancer worldwide each year, including about 30,000 in my country. In 2018, there were 570,000 new cases and 311,000 deaths from cervical cancer globally. Because early symptoms of cervical cancer are often subtle, early screening, diagnosis, prevention, and intervention are crucial. Current screening methods for cervical cancer mainly include cellular and molecular-level detection methods. For example, the clinical thin-layer liquid-based cytology test (TCT) and colposcopy require specific instruments and specialized technicians, and result interpretation requires extensive experience, making them unsuitable for rapid early screening of cervical cancer. In addition, the ThinPrep Cytology Test (TCT) is limited to analyzing whether the cell morphology is normal. Most abnormal cells are cervical inflammation, which does not mean cervical cancer. A biopsy is still needed to determine the precancerous lesions or cancerous changes of the cervix.

[0003] With the development of medical experimental technology and a deeper understanding of the relationship between human papillomavirus (HPV) and cervical lesions, HPV infection detection methods have evolved from the tissue and cellular level to the molecular level. However, HPV testing is limited to detecting the HPV virus itself. HPV infection is transient, and infection with HPV does not equate to cervical cancer; a biopsy is still needed to determine precancerous lesions or cancerous changes in the cervix. Commonly used HPV detection methods include sequencing, hybridization capture, and gene chip methods. However, these require expensive equipment and demand a high level of skill from the operator. Hybridization capture is complex and time-consuming, taking approximately 5 hours per test, and is prone to cross-reactions. Gene chip products (such as PCR-reverse dot blot hybridization and liquid chip methods) have the disadvantage of not being able to achieve real-time detection, requiring post-amplification product analysis, which takes more than 4 hours, is also complex, may be subject to PCR product contamination, and cannot meet the needs of large-scale clinical cervical cancer screening. Summary of the Invention

[0004] The purpose of this invention is to address the cumbersome and time-consuming detection methods for cervical precancerous lesions and cancerous changes in existing technologies, and to provide a combined detection kit for cervical cancer precancerous lesions and cancerous changes.

[0005] Another object of the present invention is to provide a method for preparing the aforementioned combined detection kit.

[0006] Another object of the present invention is to provide an application of the aforementioned combined detection kit.

[0007] The technical solution adopted to achieve the purpose of this invention is:

[0008] A combined detection kit for detecting precancerous lesions and cancerous changes in cervical cancer, comprising at least multiple test strips capable of detecting the following indicators: E5 protein, E6 protein, E7 protein, and hTERT;

[0009] Each test strip has a connector pad coated with a detection antibody-colloidal gold conjugate, and the detection lines of the test strip are coated with specific capture antibodies. The specific capture antibodies on each test strip are different.

[0010] In the above technical solution, the cervical precancerous lesions include low-grade cervical precancerous lesions and high-grade cervical precancerous lesions. The detection rate of low-grade cervical precancerous lesions is greater than 96%, the sensitivity is greater than 90%, and the specificity is greater than 96%. The detection rate of high-grade cervical precancerous lesions is greater than 94%, the sensitivity is greater than 89%, and the specificity is greater than 95%. The detection rate of the cervical cancer detection kit is greater than 91%, the sensitivity is greater than 87%, and the specificity is greater than 92%.

[0011] In the above technical solution, the detection limit of the E5 protein is 35 ng / mL, the detection limit of the E6 protein is 25 ng / mL, the detection limit of the E7 protein is 20 ng / mL, and the detection limit of hTERT is 30 ng / mL.

[0012] In the above technical solution, the quality control line on the test strip is pre-coated with goat anti-mouse IgG antibody.

[0013] In the above technical solution, the test strip includes a sample pad, a connecting pad, a detection pad, and an absorption pad that are sequentially overlapped.

[0014] The above technical solution also includes sample processing reagents, which include Tris, NaCl, Triton X-100 and BSA. The sample processing method is as follows: place the sample in a test tube containing the sample processing reagents, then seal the test tube and shake to mix.

[0015] In another aspect, the present invention provides a method for preparing the aforementioned combined detection kit, comprising the following steps:

[0016] Step 1, Detection line coating: First, prepare a specific capture antibody coating solution, and then coat the detection lines of the test pad of the test strip with the specific capture antibody coating solution.

[0017] Step 2: Perform antibody-colloidal gold coating detection.

[0018] In the above technical solution, the preparation method further includes: step 3, coating of the quality inspection line, wherein the coating amount of each specific capture antibody on the quality inspection line is 1.5 mg / mL.

[0019] In another aspect, the present invention provides a non-diagnostic detection method based on the aforementioned combined detection kit, comprising the following steps:

[0020] Step S1, Sample collection and post-processing;

[0021] Step S2: Add the post-processed sample from Step 1 to the sample well of the combined detection kit and wait for 15 minutes;

[0022] Step S3: Observe and record the results.

[0023] In another aspect, the present invention provides the application of the aforementioned combined detection kit in a product for the detection of precancerous lesions and cancerous changes in the cervix.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] 1. This invention has found that the combined detection of E5 protein, E6 protein, E7 protein, and hTERT (a component of telomerase) can effectively improve the sensitivity and specificity of detecting precancerous lesions and cancerous changes in the cervix. For example, compared with detecting only E7 protein, the detection rate, sensitivity, and specificity of the combined detection kit of this invention are significantly improved.

[0026] 2. The combined detection kit of the present invention greatly reduces false negatives in cervical cancer screening by combining four tests: E5 protein, E6 protein, E7 protein and hTERT. At the same time, it reduces false positives caused by the transient nature of HPV infection. The combined tests complement each other and can be used for cervical cancer screening at different stages, especially for early screening, which greatly improves the accuracy of detecting precancerous lesions and cancerous changes of cervical cancer.

[0027] 3. The detection time using the combined detection kit of this invention is only 15 minutes, which is shorter than that of existing technologies. Sample collection is more convenient, and the detection rate of cervical precancerous lesions is higher than 91%, while the detection rate of cervical cancer is higher than 89%. This improves the accuracy and efficiency of cervical precancerous lesion and cancer detection and diagnosis. Attached Figure Description

[0028] Figure 1 shows a schematic diagram of the test strip structure of the combined detection kit of the present invention.

[0029] Figure 2 shows the expression levels of biomarkers composed of related proteins in cervical precancerous lesions, cancerous tissues, and normal cervical epithelial cell tissues, as verified by immunohistochemistry in an embodiment of the present invention.

[0030] In the diagram: 1-Sample pad, 2-Connecting pad, 3-Detection line, 4-Quality control line, 5-Detection pad, 6-Absorbent pad, 7-Backing, 8-Processed sample. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0032] Example 1

[0033] A combined detection kit for cervical precancerous lesions and cancerous changes includes four test strips as shown in Figure 1, each capable of detecting the following indicators: E5 protein, E6 protein, E7 protein, and hTERT (a component of telomerase).

[0034] Each test strip has a connection pad 2 coated with a detection antibody-colloidal gold conjugate, and the test line 3 of the test strip is coated with a specific capture antibody. The specific capture antibody on each test strip is different.

[0035] Specifically, the test strip includes a sample pad 1, a connecting pad 2 (made of polyester material in this embodiment), a detection pad 5 (made of nitrocellulose membrane in this embodiment), and an absorption pad 6, which are sequentially overlapped on the backing 7. The detection pad 5 is provided with a detection line 3 (T line) and a quality control line 4 (C line), and the quality control line 4 is pre-coated with goat anti-mouse IgG antibody.

[0036] The combined detection kit also includes sample processing reagents, which include Tris, NaCl, Triton X-100 and BSA. The sample processing method is as follows: place the sample in a test tube containing the sample processing reagents, then seal the test tube and shake to mix.

[0037] The coating material on the test strips of the combined detection kit and its detection limit are shown in Table 1.

[0038] Table 1. Coating amount and detection limit of specific capture antibody

[0039]

[0040] The combined detection kit of this invention is developed using colloidal gold immunochromatography and sandwich immunoassay. The test line 3 on the nitrocellulose membrane of the test strip is coated with a specific capture antibody, the quality control line 4 is coated with goat anti-mouse IgG antibody, and the polyester membrane is pre-coated with colloidal gold-labeled specific detection antibodies. During testing, the sample travels upwards along the test strip, dissolving the detection antibody-colloidal gold conjugate on the polyester membrane. If E5, E6, E7, or hTERT proteins are present in the sample, they bind with the detection antibody-colloidal gold conjugate to form an Ag-Ab-colloidal gold conjugate. This conjugate continues to travel to the nitrocellulose membrane and specifically aggregates with the pre-coated specific capture antibody on test line 3, forming Ab-Ag-Ab-colloidal gold, which accumulates and develops into a distinct, slightly reddish band, the T-line, indicating a positive result. If the sample does not contain E5, E6, E7, or hTERT proteins, no obvious red band will form at test line 3, indicating a negative result. The free detection antibody—colloidal gold—continues to migrate upwards on the test strip to test line 4, where it binds to the pre-coated goat anti-mouse IgG antibody, accumulates, and develops a faint red band, representing test line 4, or line C.

[0041] Example 2

[0042] A detection method based on the aforementioned combined detection kit includes the following steps:

[0043] Step S1, Sample collection and post-processing:

[0044] Step S1.1: Remove the sample release agent from the kit;

[0045] Step S1.2: Before collecting cervical exfoliated cells, follow the usage instructions for the cervical sampling brush.

[0046] Step S1.3: Gently invert the test tube containing the sample release agent three times and place it on the table. Then, hold the test tube with one hand and gently unscrew the cap with the other hand, ensuring that both the test tube opening and the cap opening are facing upwards.

[0047] Step S1.4: Insert the cervical sampling brush into the test tube opening, press down gently, and place it in place;

[0048] Step S1.5: Hold the sampling brush with one hand and gently push it with the other hand until the brush head is immersed in the liquid. After the brush head touches the bottom of the test tube, rotate it 10 times to allow the brush head to fully scrape against the bottom ribs of the reagent bottle.

[0049] Step S1.6: Remove the sampling brush from the test tube with one hand, gently screw on the test tube cap, and let the test tube stand upright at room temperature for 20-30 minutes.

[0050] Step S2: Add the post-processed sample 8 from Step 1 to the sample pad 1 of each of the four test strips in the combined detection kit, and wait 15 minutes. Wait for the results of the four indicators.

[0051] Step S3: Observe and record the results. If at least one of the four indicators is positive, it indicates a precancerous lesion or cancerous change of the cervix.

[0052] Example 3

[0053] The detection method of Example 2 was used to detect human samples. The detection results are shown in Tables 2, 3 and 4. The method for calculating linear correlation is as follows: a scatter plot is made of the measurement results of the two reagents, with the measurement value of the comparison reagent on the X-axis and the measurement value of the evaluation reagent on the Y-axis. The linear correlation analysis of the measurement values ​​of the two reagents is performed using Medcalc statistical software to obtain the regression equation y, the correlation coefficient r, and the 95% confidence interval.

[0054] Table 2. Results of CIN1 (low-grade cervical precancerous lesion) detection kit and E7 protein detection.

[0055]

[0056] Table 3. Results of CIN2-3 (high-grade cervical precancerous lesion) detection kit and E7 protein detection.

[0057]

[0058] Table 4. Statistical calculation results of cervical cancer detection kit and E7 protein detection.

[0059]

[0060] As can be seen from the data in Tables 2, 3, and 4, the comparison between the results of the combined detection reagent of the present invention and the single detection of E7 protein indicates that the combined detection results of the present invention improve the sensitivity and specificity of the detection of cervical precancerous lesions and cancer, thereby improving the accuracy of the detection and diagnosis of cervical precancerous lesions and cancer.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A combined detection kit for cervical cancer precancerous lesions and cancerous changes, characterized in that, It includes at least multiple test strips that detect the following indicators: E5 protein, E6 protein, E7 protein, and hTERT; Each test strip has a connector pad coated with a detection antibody-colloidal gold conjugate, and the detection line of the test strip is coated with a specific capture antibody. The specific capture antibody on each test strip is different.

2. The combined detection kit for cervical cancer precancerous lesions and cancerous changes as described in claim 1, characterized in that, The cervical precancerous lesions include low-grade cervical precancerous lesions and high-grade cervical precancerous lesions. The detection rate of low-grade cervical precancerous lesions is greater than 96%, the sensitivity is greater than 90%, and the specificity is greater than 96%. The detection rate of high-grade cervical precancerous lesions is greater than 94%, the sensitivity is greater than 89%, and the specificity is greater than 95%. The detection rate of the cervical cancer detection kit is greater than 91%, the sensitivity is greater than 87%, and the specificity is greater than 92%.

3. The combined detection kit for cervical precancerous lesions and cancerous changes as described in claim 1, characterized in that, The detection limit for E5 protein is 35 ng / mL, the detection limit for E6 protein is 25 ng / mL, the detection limit for E7 protein is 20 ng / mL, and the detection limit for hTERT is 30 ng / mL.

4. The combined detection kit for cervical precancerous lesions and cancerous changes as described in claim 1, characterized in that, The quality control line on the test strip is pre-coated with goat anti-mouse IgG antibody.

5. The combined detection kit for cervical precancerous lesions and cancerous changes as described in claim 1, characterized in that, The test strip comprises a sample pad, a connecting pad, a detection pad, and an absorption pad that are sequentially overlapped.

6. The combined detection kit for cervical precancerous lesions and cancerous changes as described in claim 1, characterized in that, It also includes sample processing reagents, which include Tris, NaCl, Triton X-100 and BSA. The sample processing method is as follows: place the sample in a test tube containing the sample processing reagents, then seal the test tube and shake to mix.

7. A method for preparing a combined detection kit as described in any one of claims 1-6, characterized in that, Includes the following steps: Step 1, Detection line coating: First, prepare a specific capture antibody coating solution, and then coat the detection lines of the test pad of the test strip with the specific capture antibody coating solution. Step 2: Perform antibody-colloidal gold coating detection.

8. The preparation method according to claim 7, characterized in that, It also includes: Step 3, coating of the quality control line, wherein the coating amount of each specific capture antibody on the quality control line is 1.5 mg / mL.

9. A non-diagnostic detection method based on the combined detection kit according to any one of claims 1-6, characterized in that, Includes the following steps: Step S1, Sample collection and post-processing; Step S2: Add the post-processed sample from Step 1 to the sample well of the combined detection kit and wait for 15 minutes; Step S3: Observe and record the results.

10. The use of a combined detection kit as described in any one of claims 1-6 in a product for detecting precancerous lesions and cancerous changes in the cervix.

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