A detection kit for detecting cervical cancer exfoliated cells at home, and its preparation method and application

Abstract

The present invention discloses a detection kit for detecting cervical cancer exfoliated cells at home, and its preparation method and application. The detection kit comprises a test paper card, the test paper card comprises a card shell, a test paper strip located in the card shell, the test paper strip is composed of a sample pad, a colloidal gold binding pad, a nitrocellulose membrane with a detection line and a quality control line, and a water-absorbing pad, which are overlapped and fixed on a bottom plate in sequence, the detection line is coated with a modified capture antibody, the quality control line is coated with a sheep anti-mouse IgG antibody, and the detection antibody labeled with colloidal gold is coated on the binding pad, the heavy chain and light chain amino acid sequences of the capture antibody are shown in SEQ ID NO.5-6, and the heavy chain and light chain amino acid sequences of the detection antibody are shown in SEQ ID NO.7-8. The detection kit can quickly, conveniently and accurately detect cervical cancer exfoliated cells, which is convenient for women to detect themselves at home, thereby detecting cervical cancer at an early stage.

Description

Technical Field

[0001] The present invention belongs to the technical field of antigen protein detection, and particularly relates to a detection kit for detecting cervical cancer exfoliated cells at home, and a preparation method and application thereof. Background Art

[0002] Cervical cancer is one of the common malignant tumors in women, and its incidence rate is increasing year by year. Early cervical cancer often has no obvious symptoms, so early detection and timely treatment are crucial. Human papillomavirus (HPV) infection is the main pathogenic factor for cervical cancer. After HPV virus infection, its genes are integrated into the genome of the host cell and express a variety of viral proteins, among which E7 protein is the key oncogenic protein of HPV. Therefore, detecting the presence of E7 protein can be used as an effective indicator for early screening of cervical cancer. At present, cytological examination, HPV detection and histopathological examination are mainly used in clinical practice for cervical cancer screening. However, these methods have some shortcomings, such as the need for professional operation, high cost, long time, and complex result interpretation. Therefore, it is urgent to develop an early screening method for cervical cancer that is simple to operate, low cost and accurate results.

[0003] In recent years, home testing technology has developed rapidly, providing a new way for early screening of cervical cancer. The home detection kit for cervical cancer exfoliated cells can quickly determine the risk of cervical cancer by detecting E7 protein in vaginal secretions, but the current detection products are not accurate enough and are prone to false positives or false negatives. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention has developed a detection kit for home detection of cervical cancer exfoliated cells based on the carcinogenic properties of E7 protein. The kit is simple to operate and easy to interpret the results. It can quickly and accurately detect the E7 protein in cervical cancer exfoliated cells, providing women with a convenient method for early screening of cervical cancer.

[0005] The technical solution of the present invention is as follows:

[0006] A detection kit for detecting cervical cancer exfoliated cells at home, comprising a test paper card, the test paper card comprising a card shell and a test paper strip located in the card shell, the test paper strip consisting of a sample pad, a colloidal gold binding pad, a nitrocellulose membrane provided with a detection line and a quality control line, and a water-absorbing pad which are sequentially overlapped and fixed on a bottom plate, the detection line is coated with an E7 protein-specific capture antibody, the quality control line is coated with a sheep anti-mouse IgG antibody, the colloidal gold-labeled E7 protein-specific detection antibody is coated on a binding pad made of a polyester film, the heavy chain amino acid sequence of the E7 protein-specific capture antibody is shown in SEQ ID NO.5, the light chain amino acid sequence of the E7 protein-specific capture antibody is shown in SEQ ID NO.6, the heavy chain amino acid sequence of the E7 protein-specific detection antibody is shown in SEQ ID NO.7, and the light chain amino acid sequence of the E7 protein-specific detection antibody is shown in SEQ ID NO.8.

[0007] Furthermore, the kit also includes a test paper card, a sample release agent, and a sampler.

[0008] Furthermore, the test sample includes cervical exfoliated cells collected from the human body or suspended cells prepared from human cervical tissue.

[0009] Furthermore, the E7 protein includes HPV16 E7 protein and HPV18 E7 protein.

[0010] An E7 protein-specific capture antibody, whose heavy chain amino acid sequence is shown in SEQ ID NO.5, and whose light chain amino acid sequence is shown in SEQ ID NO.6.

[0011] An E7 protein-specific detection antibody, whose heavy chain amino acid sequence is shown in SEQ ID NO.7, and whose light chain amino acid sequence is shown in SEQ ID NO.8.

[0012] Beneficial effects of the present invention:

[0013] E7 protein is an important marker for the progression of cervical malignancy and one of the main indicators for cervical cancer risk assessment. The kit of the present invention can accurately detect E7 protein and provide an auxiliary tool for assessing the risk of cervical cancer and subsequent cervical cancer treatment.

[0014] This product can also be used in medical institutions, where hospital professionals are responsible for testing.

[0015] The kit is simple to use, does not require professional equipment and professionals, and has the advantages of being rapid (15 minutes), specific, and low-cost. It can be used for early warning and early screening of cervical cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Schematic diagram of the test strip structure.

[0017] Figure 2 : The test paper card made by embodiment 2 of the present invention. DETAILED DESCRIPTION

[0018] In order to better understand the technical content of the present invention, the present invention is further described below through specific embodiments and drawings.

[0019] Example 1 Antibody modification

[0020] The affinity and specificity of antigen-antibody binding are mainly related to the variable region, but changes in the constant region will also affect changes in antibody conformation. Studies have found that modifications to capture antibodies and detection antibodies can improve the detection effect of E7 protein.

[0021] Antibody amino acid sequence before modification:

[0022] Capture antibody heavy chain

[0023] The underline represents the constant region, and the wavy line represents the variable region:

[0024]

[0025] The underline of the capture antibody light chain represents the constant region, and the wavy line represents the variable region:

[0026]

[0027] The underline of the heavy chain of the detection antibody represents the constant region, and the wavy line represents the variable region:

[0028]

[0029] Detection antibody heavy chain

[0030] The underline represents the constant region, and the wavy line represents the variable region:

[0031]

[0032] The amino acid sequence of the modified antibody:

[0033] Capture antibody heavy chain

[0034] The underline represents the constant region, the wavy line represents the variable region, the bold represents the inserted sequence in the framework region of the variable region, and the shading represents the sequence change:

[0035]

[0036] Capture antibody light chain

[0037] The underline represents the constant region, the wavy line represents the variable region, and the shading represents the amino acid deletion in the hypervariable region:

[0038]

[0039] Detection antibody heavy chain

[0040] The underline represents the constant region, the wavy line represents the variable region, and the bold represents the framework region insertion sequence:

[0041]

[0042] Detection of antibody light chain

[0043] The underline represents the constant region, the wavy line represents the variable region, and three amino acids are deleted from the constant region:

[0044]

[0045] Preparation of modified antibodies:

[0046] According to the sequence information of the modified antibody, the gene is synthesized and constructed into a mammalian cell vector, and after sequencing confirmation, the plasmid is extracted and set aside. CHO cells are taken and the plasmid is transfected. After the plasmid is transfected for 24 hours, the CHO cells are continued to be cultured so that the cells express the desired antibody. The cell culture medium supernatant is collected by centrifugation, filtered, and purified.

[0047] Example 2 Preparation of a detection kit for home detection of cervical cancer exfoliated cells

[0048] The kit includes a test paper card, a sample release agent (Tris buffer), and a sampler.

[0049] The test paper card includes a card shell, a test paper strip (test paper strip width range 3.20±0.50mm) located in the card shell, and the test paper strip is composed of a sample pad (made of absorbent filter paper) overlapped and fixed on the bottom plate from left to right, a colloidal gold binding pad, a nitrocellulose membrane with a detection line and a quality control line, and an absorbent pad. The detection line is coated with E7 protein-specific capture antibody, the quality control line is coated with sheep anti-mouse IgG antibody, and the colloidal gold-labeled E7 protein-specific detection antibody is coated on the binding pad made of polyester film. The capture antibody and the detection antibody are the antibodies described in Example 1.

[0050] Preparation of test strips:

[0051] Colloidal gold and detection antibody were mixed at a ratio of 30 μg antibody / 1 ml colloidal gold. After labeling for 1 hour, 5% BSA was added under stirring to make the final BSA concentration 1%. After incubation for 1 hour, centrifuge at 4°C, 10,000 r / min for 25 minutes and remove the supernatant. 2% BSA solution was added to resuspend and centrifuge at 4°C, 10,000 r / min for 25 minutes. 3 Resuspend in 0.02M TrisHCl pH7.2 buffer, spray 4% BSA solution on glass wool, dry at 42°C for 60 min, then spray gold-labeled antibody on glass wool, dry in a drying oven at 42°C for 60 min to form a colloidal gold binding pad.

[0052] Dilute the capture antibody to 1 mg / ml with 0.01M PBS. Dilute the goat anti-mouse IgG antibody to 1 mg / ml with 0.01M PBS. Spray the two on the nitrocellulose membrane to form the test line and quality control line respectively.

[0053] Glue the sample pad, polyester film, nitrocellulose membrane and absorbent paper to the base plate in sequence, cut the glued base plate materials into test strips, and place the test strips in the card housing to form a test paper card.

[0054] Inspection principle:

[0055] After adding the sample to be tested to the sample pad, the sample crawls up along the test strip and dissolves the colloidal gold conjugate on the polyester membrane of the binding pad. If E7 protein exists in the sample, the E7 protein binds to the detection antibody-colloidal gold conjugate and continues to crawl to the nitrocellulose membrane and specifically binds to the E7 protein-specific capture antibody coated on the detection line, and accumulates and colors to form an obvious detection line band, which represents a positive result. If the sample does not contain E7 protein, no obvious band will be formed at the detection line position, which represents a negative result. The sample continues to crawl to the top of the test paper to the quality control line and binds to the sheep anti-mouse IgG antibody, and accumulates and colors to form a quality control line band. When the sample shows a quality control line band, the test is valid. Read the result within 15 minutes after the sample is added to the sample pad.

[0056] Test result explanation:

[0057] Positive: Both the test line and the quality control line are colored;

[0058] Negative: only the quality control line is colored;

[0059] Invalid: The quality control line has no color.

[0060] In order to compare the effects of antibodies before and after modification on the test results.

[0061] The inventors used the kit combined with the modified antibody of the present invention to detect 50 HPV16 DNA positive and 33 negative cervical swab specimens. The results showed that all positive specimens showed positive results, and all negative specimens showed negative results, with a detection accuracy of 100%. 20 HPV18 DNA positive samples were detected, with a detection rate of 100%.

[0062] The kit combined with the pre-improved antibody was used to test 50 HPV16 DNA positive and 33 negative cervical swab specimens, and 2 positive samples were found to have false negatives and 1 negative sample to have false positives. The results show that the modified product has higher accuracy.

[0063] Example 3 Application of the kit - home detection of cervical cancer exfoliated cells

[0064] This product is used for auxiliary diagnosis of female cervical diseases. Use a cervical brush to take a sample of exfoliated cells at the cervical opening, place it in a sample release agent to release it, add the sample to the sample pad of the test paper card, wait for about 10 minutes, and refer to the result determination method described in Example 2 to detect whether the sample contains E7 protein, so as to preliminarily diagnose cervical epithelial cell diseases.

[0065] Example 4 Kit Quality Test

[0066]

[0067] The above descriptions are only some embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall fall within the protection scope of the present invention.

Claims

1. A detection kit for detecting cervical cancer exfoliated cells at home, comprising a test paper card, wherein the test paper card comprises a card shell and a test paper strip located in the card shell, wherein the test paper strip is composed of a sample pad, a colloidal gold binding pad, a nitrocellulose membrane with a detection line and a quality control line, and a water-absorbing pad which are sequentially overlapped and fixed on a bottom plate, wherein the detection line is coated with an E7 protein-specific capture antibody, the quality control line is coated with a sheep anti-mouse IgG antibody, and the colloidal gold-labeled E7 protein-specific detection antibody is coated on a binding pad made of a polyester film, characterized in that: The heavy chain amino acid sequence of the E7 protein-specific capture antibody is shown in SEQ ID NO.5, the light chain amino acid sequence of the E7 protein-specific capture antibody is shown in SEQ ID NO.6, the heavy chain amino acid sequence of the E7 protein-specific detection antibody is shown in SEQ ID NO.7, and the light chain amino acid sequence of the E7 protein-specific detection antibody is shown in SEQ ID NO.

8.

2. The detection kit according to claim 1, characterized in that The kit also includes a test paper card, a sample release agent, and a sampler.

3. The detection kit according to claim 1, characterized in that The samples detected by the kit include cervical exfoliated cells collected from human body or suspended cells prepared from human cervical tissue.

4. The detection kit according to claim 1, characterized in that The E7 protein includes HPV16 E7 protein and HPV18 E7 protein.

5. The detection kit according to claim 1, characterized in that The preparation method of the test paper card is: The colloidal gold and the detection antibody are mixed, incubated, centrifuged, resuspended with a BSA solution, then sprayed on glass wool and dried to form a colloidal gold conjugate pad; The capture antibody and goat anti-mouse IgG antibody were diluted with PBS and sprayed on the nitrocellulose membrane to form the test line and quality control line respectively; The sample pad, polyester film, nitrocellulose membrane and absorbent paper are sequentially adhered to the bottom plate, and the adhered bottom plate materials are cut into test paper strips; the test paper strips are placed in the card shell to form a test paper card.

6. An E7 protein-specific capture antibody, characterized in that: The heavy chain amino acid sequence is shown in SEQ ID NO.5, and the light chain amino acid sequence is shown in SEQ ID NO.

6.

7. The E7 protein-specific capture antibody according to claim 6, characterized in that: The E7 protein includes HPV16 E7 protein and HPV18 E7 protein.

8. An E7 protein-specific detection antibody, characterized in that: The heavy chain amino acid sequence is shown in SEQ ID NO.7, and the light chain amino acid sequence of the E7 protein-specific detection antibody is shown in SEQ ID NO.

8.

9. The E7 protein-specific detection antibody according to claim 8, characterized in that: The E7 protein includes HPV16 E7 protein and HPV18 E7 protein.

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