A HER2 detection kit and a detection method thereof

Abstract

The application relates to a HER2 detection kit and a detection method, and belongs to the technical field of detection. The application relates to a HER2 detection reagent strip, which comprises a sample pad, a combination pad and a nitrocellulose membrane connected in sequence. The combination pad is coated with nanobodies specifically combined with HER2, and the nanobodies comprise CDR sequences shown as SEQ ID NO: 2-4. The kit has the effects of high detection sensitivity, good specificity and short detection time.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical detection, and particularly relates to a HER2 detection kit and a detection method thereof. BACKGROUND

[0002] HER2 (human epidermal growth factor receptor 2) is a member of the epidermal growth factor receptor family with tyrosine kinase activity. Overexpression of HER2 leads to an increase in the formation of HER2 homodimers and heterodimers, thereby causing effects such as cell proliferation, anti-apoptosis, invasion and angiogenesis by activating the PI3K and MAPK pathways. About 15%-30% of breast cancers and 10%-30% of gastric / esophageal cancers have HER2 gene amplification or overexpression.

[0003] Traditional HER2 detection relies on invasive tissue biopsy, mainly using IHC and fluorescence in situ hybridization (FISH) technology. These methods are difficult to use for dynamic and non-invasive HER2 status monitoring of postoperative patients, patients with recurrence or patients with metastatic tumors. Studies have shown that HER2 fragments can be shed from the surface of tumor cells into the blood, and there is a significant correlation between the soluble HER2 extracellular domain fragments in the serum of tumor patients and the HER2 expression level of tumor tissue. As high as more than 50% of metastatic breast cancer patients have elevated HER2 levels in their serum. Therefore, the detection of serum HER2 ECD levels has important clinical application value in the precise typing of tumors, early warning of recurrence and metastasis, efficacy evaluation and prognosis. However, the development in this field is limited by a key bottleneck: there is currently a lack of high-affinity and high-specificity anti-HER2 extracellular region detection antibodies, which seriously hinders the establishment and application of HER2 detection methods based on blood samples with high sensitivity and high specificity, and also hinders related treatment research targeting HER2 ECD targets.

[0004] Therefore, there is an urgent need in the art to develop a HER2 detection kit based on high-affinity antibodies and a detection method thereof to meet the needs of non-invasive and dynamic monitoring of HER2 status, serving the whole process management of tumors. SUMMARY

[0005] In order to overcome the problems existing in the prior art, the application provides a HER2 detection kit and a detection method thereof.

[0006] In a first aspect, the application provides a HER2 detection reagent strip, which comprises a sample pad, a binding pad and a nitrocellulose membrane connected in sequence, the binding pad is coated with a nanobody specifically binding to HER2, and the nanobody comprises CDR sequences as shown in SEQ ID NO: 2-4.

[0007] Optionally, the nanobody comprises a variable region sequence as shown in SEQ ID NO: 1.

[0008] Optionally, the binding pad contains colloidal gold particles labeled with the nanobody.

[0009] Optionally, the nitrocellulose membrane is provided with a detection line coated with a polyclonal antibody against HER2.

[0010] Optionally, the nitrocellulose membrane is further provided with a quality control line, which is farther away from the binding pad than the detection line.

[0011] Optionally, the quality control line is coated with at least one of a mouse anti-goat antibody, avidin, and biotin-BSA.

[0012] In a second aspect, the present application provides a HER2 detection kit, which comprises the HER2 detection reagent strip of the first aspect.

[0013] In a third aspect, the present application provides a method for detecting HER2, which comprises detecting a sample to be tested by the kit of the second aspect; optionally, the sample to be tested is serum, plasma or whole blood.

[0014] Optionally, the method comprises the following steps:

[0015] Step 1: diluting the sample to be tested with a blood sample diluent to obtain a diluted sample;

[0016] Step 2: adding the diluted sample to the sample pad of the HER2 detection reagent strip, and observing the detection line and the quality control line.

[0017] In summary, the present application has at least one of the following beneficial technical effects:

[0018] (1) The HER2 detection kit prepared by the present application is simple to operate, and the detection result can be observed by naked eye without the need for special experimental instruments and professional technical personnel;

[0019] (2) The HER2 detection kit prepared by the present application can show the detection result within 15 minutes, and is suitable for rapid detection of a large number of samples;

[0020] (3) The HER2 detection kit prepared by the present application has a minimum detection limit of 10 ng / mL, and the sensitivity is significantly higher than that of existing methods;

[0021] (4) The high-affinity nanobody obtained by colloidal gold labeling screening is combined with the polyclonal antibody detection line to form a "double antibody sandwich" structure, which greatly improves the signal strength, and clinical sample verification shows that the sensitivity and specificity are both 100%, effectively avoiding cross-reactions. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Figure 1 is a schematic representation of a reagent strip according to the present application. DETAILED DESCRIPTION

[0023] The term "antibody" herein is used in the broadest sense and includes various structures of native antibodies and artificial antibodies, including but not limited to polyclonal antibodies, monoclonal antibodies, single chain antibodies, intact antibodies and antibody fragments, which contain an antigen binding site.

[0024] The term "nanobody" refers to an antibody comprising a single immunoglobulin variable domain (a single variable domain) as a functional antigen binding fragment; like the variable region of full-length antibodies, a single variable domain typically comprises CDR1, CDR2 and CDR3 forming an antigen binding site and a supporting framework region.

[0025] The term "polyclonal antibody" as used herein refers to a mixture of multiple monoclonal antibodies produced by stimulating multiple B cell clones of an animal organism to produce multiple monoclonal antibodies against a plurality of antigenic determinants using an antigen comprising the plurality of antigenic determinants.

[0026] The term "complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is hypervariable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). The CDRs are primarily responsible for binding to an antigenic epitope, and include, in order from N-terminus, CDR1, CDR2, and CDR3. The precise amino acid sequence boundaries of each CDR in a given variable region amino acid sequence can be determined using any of a number of well-known antibody CDR assignment systems, or combinations thereof, including, for example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (http: / / imgt.cines.fr / ), and North CDR definitions based on affinity propagation clustering with a large number of crystal structures. Unless otherwise specified, in the present application, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways. CDRs can also be determined based on having the same AbM numbered positions as a reference CDR sequence, such as any of the CDRs exemplified in the present application. In one embodiment, the CDRs of the single domain antibodies of the present application are determined positions according to the AbM numbering scheme. Unless otherwise specified, in the present application, when referring to residue positions in antibody variable regions and CDRs, including heavy chain variable region residues, the numbered positions are according to the AbM numbering system.

[0027] The following examples are intended to illustrate the present application only and should not be construed as limiting the application in any way.

[0028] Example 1 Screening of anti-HER2 nanobodies

[0029] Recombinant protein HER2-Fc (ECD region sequence of HER2 (UniProt NO: P04626) gene connected with human Fc tag (Uniprot No: P0DOX5, 218-449AA) at C terminal) was used as antigen to immunize alpacas to obtain high-titer antisera. After the end of animal immunization, 50 mL of fresh blood of alpaca was taken, peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Paque density gradient separation solution, RNA was extracted, and after reverse transcription, universal primers were used for amplification, and then cloned into phagemid, transformed into TG1 strain, and phage library was established for screening of monoclonal antibodies. Nanobodies with high specificity and affinity (not cross-reacting with HER1 / 3 / 4, accurately recognizing HER2 ECD) were screened and named Nb-HER2-3.

[0030] The sequence of Nb-HER2-3 was analyzed, and the results showed that the amino acid sequence of VHH of antibody Nb-HER2-3 is shown in SEQ ID NO: 1, and the amino acid sequences of CDR1, CDR2 and CDR3 defined by AbM are shown in SEQ ID NO: 2-4, respectively.

[0031] Example 2: Preparation of detection kit

[0032] The present embodiment provides a HER2 detection kit, which comprises a detection reagent strip and a blood sample diluent sealed in an aluminum foil bag.

[0033] 2.1 Reagent strip

[0034] a) Production of nitrocellulose membrane: mouse anti-goat IgG and rabbit anti-HER2 polyclonal antibody (Bi Yun Tian, AH210) were diluted to 0.5 mg / mL and 1.0 mg / mL respectively with sugar PBS solution, and the solution was uniformly sprayed on the nitrocellulose membrane with a dot membrane instrument, and dried in an oven at 37°C for 12 h. The nitrocellulose membrane is provided with a detection line (T line), and the detection line is anchored with coated rabbit anti-HER2 polyclonal antibody; the nitrocellulose membrane is also provided with a quality control line (C line), and the quality control line is anchored with mouse anti-goat antibody, and the quality control line is farther away from the colloidal gold conjugated pad than the detection line.

[0035] b) Production of colloidal gold conjugated pad:

[0036] Antibody conjugated colloidal gold: Take colloidal gold (boil the solution containing 0.02% gold chloride, add sodium citrate solution, continue boiling for 10 min after the solution turns wine red, cool at room temperature), adjust the pH to 7.8 with potassium carbonate solution, add anti-HER2 antibody Nb-HER2-3 at a dosage of 10 μg / mL, stir for 30 min, then add 10% BSA solution for blocking, centrifuge the solution after 30 min, resuspend the precipitate with a solution containing 0.5% Tris, 2% BSA (pH 8.0), and measure the concentration of the resuspension solution with a spectrophotometer;

[0037] Gold spraying: Dilute the colloidal gold resuspension solution to an OD20 concentration with a solution containing 0.5% Tris, 2% BSA, 15% sucrose, and 3% trehalose, and uniformly spray 2.0 μL / cm on the polyester film with a gold spraying machine, and dry in an oven at 37°C for 12 h.

[0038] 2.2 Assembly and cutting of reagent strips

[0039] First, cut the filter paper into 17*301 mm, cut the colloidal gold binding pad into 10*301 mm, cut the sample pad into 17*301 mm, sequentially paste the sample pad, colloidal gold binding pad, and nitrocellulose membrane, and then combine the PVC bottom sheet and the filter paper to assemble a large card. Figure 1 As shown, cut the assembled large card into 3-4 mm reagent strips, and then place them in a card board together with a desiccant in an aluminum foil bag, and heat seal the opening.

[0040] 2.3 Detection method of the kit

[0041] Preparation of the sample to be tested: Dilute the test serum sample 200 times with serum sample diluent (Aijie, ICT-647). Detection method: Take the detection reagent strip from the sealed bag and place it on a clean and flat surface. Use the provided dropper to drop 3 drops of sample (about 120 μL) onto the sample well (S) of the reagent strip, start timing, and observe the color development of the C line and T line at 15 min. If the C line is colored and the T line is not colored, it is negative. If the C line is not colored, regardless of whether the T line is colored or not, it is an invalid result, indicating that the reagent is invalid or there is an error in the operation process.

[0042] Example 3: Performance evaluation of the detection kit

[0043] 3.1 Limit of detection experiment

[0044] Using the kit obtained in Example 2 to detect serum samples with accurate quantification of HER2 content, dilute the above samples with negative matrix (serum of healthy people) by 5 times to obtain L1-L5, and the detection results are shown in Table 1. The minimum detection limit is 10 ng / mL. In the following table, "+" indicates positive, and "-" indicates negative.

[0045] Table 1

[0046]

[0047] 3.2 Clinical sample experiment

[0048] Serum samples collected from hospitals were tested by the kit of the present application, which were confirmed to be negative (No. N1-N20) or positive (No. P1-P10), and the test results are shown in Table 2. The sensitivity and specificity of the detection were 100% and 100%, respectively, and the kit of the present application had excellent clinical performance.

[0049] Table 2

[0050]

[0051] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details in light of all the teachings of the present disclosure, and such changes fall within the protection scope of the present application. The protection scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. A HER2 detection test strip, characterized in that, The reagent strip comprises a sample pad, a conjugate pad and a nitrocellulose membrane connected in sequence, the conjugate pad is coated with nanobodies specifically binding to HER2, the nanobodies comprise CDR sequences as shown in SEQ ID NO: 2-4.

2. The HER2 detection reagent strip according to claim 1, characterized in that, The nanobodies comprise a variable region sequence as shown in SEQ ID NO:

1.

3. The HER2 detection reagent strip according to claim 1, characterized in that, The conjugate pad contains colloidal gold particles labeled nanobodies.

4. The HER2 detection reagent strip according to claim 1, characterized in that, The nitrocellulose membrane is provided with a detection line coated with polyclonal antibodies against HER2.

5. The HER2 detection reagent strip according to claim 4, characterized in that, The nitrocellulose membrane is further provided with a quality control line, the quality control line is farther away from the conjugate pad than the detection line.

6. The HER2 detection reagent strip according to claim 5, characterized in that, The quality control line is coated with at least one of mouse anti-goat antibody, avidin, biotin-BSA.

7. A HER2 detection kit, characterized by, The kit comprises a HER2 detection reagent strip according to any one of claims 1-6.

8. Use of a HER2 detection kit according to claim 7 in the manufacture of a product for monitoring tumorigenesis and / or disease progression.

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