Application of antibodies against ERBB2 S310F mutation in the preparation of diagnostic tests for gallbladder cancer

Abstract

The present invention provides an application of an antibody for the preparation of a diagnosis and detection of gallbladder cancer, the mutation causes an increase in PD-L1 expression in gallbladder cancer, the antibody specifically recognizes the ERBB2S310F mutation in human gallbladder cancer, and immunohistochemical detection is performed simultaneously; the preparation of the antibody includes: the wild-type serine at position 310 in the ERBB2 amino acid sequence is mutated to phenylalanine, and then rabbits are immunized and purified; the detection of the ERBB2S310F mutation was previously mainly performed by exome sequencing, which requires more time and cost, and is not conducive to the clinical detection of the ERBB2S310F mutation. At the same time, pathological diagnosis is more convenient and is also the gold standard for tumor diagnosis. The antibody prepared according to the ERBB2S310F mutant polypeptide of the present invention can be detected by immunohistochemistry and used for clinical pathological diagnosis of gallbladder cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibody detection, and specifically relates to an application of an antibody of ERBB2 S310F mutation in the preparation and diagnosis of gallbladder cancer. Background Art

[0002] The ERBB family (epidermal growth factor receptor, ERBB, epithelial growth factor family) includes four members: ERBB1 / EGFR, ERBB2 / HER2, ERBB3 and ERBB4. Among them, ERBB2 has mutations in many tumors including breast cancer and gallbladder cancer, among which ERBB2 S310F mutation is a significant mutation in gallbladder cancer. In the past, the detection of ERBB2 mutations was mainly based on whole exome sequencing, which is not convenient in clinical pathological diagnosis. Summary of the invention

[0003] In view of the deficiencies in existing detection, the purpose of the present invention is to provide an antibody against ERBB2 S310F mutation for use in the preparation of a diagnostic test for gallbladder cancer.

[0004] In the early stage, our research group cooperated with the research group of Shanghai Renji Hospital to perform whole exome sequencing on gallbladder cancer samples, confirming that ERBB2 S310F mutation is a significant mutation in gallbladder cancer, and ERBB2 mutation plays an important role in causing increased PD-L1 expression in gallbladder cancer and promoting immune escape of gallbladder cancer. However, there is currently no specific antibody to detect ERBB2 S310F mutation. Therefore, according to the principle of specific reaction of immunological antigen-antibody and the process of antibody preparation, antibodies that specifically recognize ERBB2 S310F mutation are pre-prepared for clinical pathological diagnosis.

[0005] To achieve the above object, the solution of the present invention is:

[0006] In a first aspect, the present invention provides a use of an antibody against the ERBB2 S310F mutation in the preparation of a method for diagnosing and detecting gallbladder cancer.

[0007] Furthermore, ERBB2 S310F mutation causes increased PD-L1 expression in gallbladder cancer.

[0008] Furthermore, the antibody specifically recognized the ERBB2 S310F mutation in human gallbladder cancer.

[0009] Furthermore, the antibodies were subjected to immunohistochemical detection.

[0010] In a second aspect, the present invention provides an application of the above-mentioned ERBB2 S310F mutant antibody in the preparation of a kit for detecting gallbladder cancer. The kit also includes immunohistochemical detection-related reagents, including immunohistochemical DAB display solution (50*), DAB display solution diluent and immunohistochemical rabbit secondary antibody.

[0011] In a third aspect, the present invention provides a method for preparing the above-mentioned ERBB2 S310F mutant antibody, comprising:

[0012] The wild-type serine (S) at position 310 in the ERBB2 amino acid sequence (mRNA sequence is NM_004448.3, amino acid sequence is NP_004439.2) is mutated to phenylalanine (F), and then rabbits are immunized, and the rabbit serum is collected and purified to obtain the ERBB2 S310F mutant antibody.

[0013] Due to the adoption of the above scheme, the beneficial effects of the present invention are:

[0014] Previously, the detection of ERBB2 S310F mutation was mainly carried out through exome sequencing, which consumes a lot of time and costs, and is not conducive to the clinical detection of ERBB2 S310F mutation. At the same time, pathological diagnosis is more convenient and is also the gold standard for tumor diagnosis. The antibody prepared by the present invention based on the ERBB2 S310F mutant polypeptide can be used for immunohistochemical detection and clinical pathological detection of gallbladder cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the first generation sequencing of the present invention to detect the ERBB2 gene map in gallbladder cancer samples.

[0016] Figure 2 Figure 2 shows the specificity of antibodies prepared for the immunohistochemical analysis of the present invention for wild-type ERBB2 and S310F mutant ERBB2.

[0017] Figure 3 Schematic diagram of the antibody of the present invention detecting wild-type ERBB2 and S310F mutated ERBB2 gallbladder cancer. DETAILED DESCRIPTION

[0018] The present invention provides an application of an antibody of ERBB2 S310F mutation in the preparation of a method for diagnosing and detecting gallbladder cancer.

[0019] In order to facilitate the detection of ERBB2 S310F mutation in gallbladder cancer in clinical practice, the mutant peptide of ERBB2 S310F (NYLSTDVG FCTLV) prepared a rabbit polyclonal antibody that can specifically recognize the ERBB2 S310F mutation site, which can be conveniently used for gallbladder cancer pathology detection. In most hospitals, ERBB2 S310F mutations in gallbladder cancer or other tumors can be detected by pathology.

[0020] The antigen used in the antibody prepared by the present invention is NYLSTDVG F This CTLV polypeptide is from position 302 to 314 of the ERBB2 amino acid sequence. The 9th underlined F in this polypeptide, i.e. position 310 of the ERBB2 amino acid sequence, mutated from wild-type serine (S) to phenylalanine (F). After using this polypeptide as an antigen to immunize rabbits, the serum was collected and purified to prepare antibodies that specifically recognize the ERBB2 S310F mutation, which was verified in clinical samples of gallbladder cancer.

[0021] Among them, the invention is a rabbit polyclonal antibody, the main component of which is an antibody, which can specifically recognize the ERBB2S310F mutation in human gallbladder cancer samples, but does not recognize wild-type ERBB2, so it is specific.

[0022] Specifically, the ERBB2 wild-type and S310F mutant samples were identified by first-generation sequencing of gallbladder cancer samples (e.g. Figure 1 ). The screened samples were then subjected to immunohistochemical staining to verify the specificity of the prepared antibodies. Figure 2 and Figure 3 The immunohistochemistry results showed that after testing the clinical samples of gallbladder cancer, the prepared antibody that specifically recognizes the S310F mutation did not react with the wild-type ERBB2, but had a clear positive detection result for the S310F mutant ERBB2, indicating that the prepared antibody has good specificity.

[0023] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Embodiment 1:

[0025] (a) First, the mutant peptide of ERBB2 S310F was prepared: NYLSTDVG F CTLV-C, the serine (S) at position 310 was mutated to phenylalanine (F) (the ninth amino acid of this polypeptide fragment is phenylalanine (F)), and the synthesized polypeptide was purified by HPLC with a purity of >90%.

[0026] (b) The second step is pre-blood collection. Two healthy 6-week-old New Zealand white rabbits (about 2 kg) are selected, the rabbits are fixed and depilated, and 10 mL of blood (5 mL of serum) is drawn through the ear vein as a negative reference.

[0027] (c) Then, the rabbits are immunized and blood is collected. The synthesized polypeptide is dissolved in NaCl to prepare antigens and the rabbits are immunized. Each rabbit is first immunized with 400 μg of antigen, about 1 mL of antigen solution, by intramuscular injection in the back and thigh. Subsequent immunizations can be appropriately reduced each time, with an immunization cycle of about 20 days and 4-5 immunizations. About 10 days after the immunization of the rabbits, blood is collected for testing.

[0028] (d) Antibody titer detection, four groups are: A pre-blood serum, B antiserum, C antiserum + wild-type peptide, D antiserum + mutant peptide. The antigen is pre-added to a 96-well plate, blocked, and dried for later use. Antisera from different groups are added for incubation, and the antisera are diluted in multiples on the original basis, incubated with rabbit secondary antibodies, and a colorimetric substrate is added. The antiserum is detected by an enzyme marker to observe the effect of the antiserum. When the antibody titer reaches more than 10,000, the rabbit can be finally bled.

[0029] (e) Purification of antiserum The antiserum was diluted with PBS, passed through an equilibrium column for elution, and stored at -80°C.

[0030] (f) The purified antiserum can be further used and verified in protein immunoblotting, immunohistochemistry, etc.

[0031] Embodiment 2:

[0032] The application of antibodies against the ERBB2 S310F mutation in clinical diagnosis is mainly through immunohistochemical staining of paraffin sections. The specific experimental steps are as follows:

[0033] (a) Paraffin sections of gallbladder cancer were taken and baked in an oven at 60°C for 1 h to melt the paraffin.

[0034] (b) In a fume hood, the sections were sequentially immersed in xylene for 20 min, xylene for 20 min, 100% ethanol for 5 min, 95% ethanol for 5 min, 80% ethanol for 5 min, 75% ethanol for 5 min, and 50% ethanol for 5 min.

[0035] (c) The cells were then rinsed with tap water for 20 min, added with 3% hydrogen peroxide and placed in a 60°C oven for 10 min, and washed with PBS three times, each time for 5 min.

[0036] (d) Antigen modification was performed with citric acid under high pressure conditions for 10 min, and then the plate was taken out and allowed to cool to room temperature.

[0037] (e) Incubate with the prepared rabbit polyclonal antibody (i.e., antibody against ERBB2 S310F mutation) at 4°C overnight.

[0038] (f) Wash with PBS three times, 5 min each time, and incubate with rabbit polyclonal antibody for 20 min at room temperature.

[0039] (g) Wash with PBS three times, 5 min each time, and perform DAB color development. The appearance of brown precipitate under microscope is a positive result, and the color development can be stopped.

[0040] (h) After staining with hematoxylin for 10 seconds, the samples were immersed in tap water and washed for 20 minutes.

[0041] (i) Then, in a fume hood, the sections were sequentially immersed in 50% ethanol for 5 min, 75% ethanol for 5 min, 95% ethanol for 5 min, 100% ethanol for 5 min, xylene for 20 min, and xylene for 20 min.

[0042] (j) Seal the slides with neutral resin and observe the staining results under a microscope.

[0043] The above description of the embodiments is to facilitate the understanding and use of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. Use of an antibody against ERBB2 S310F mutation in the preparation of a kit for diagnosing or detecting gallbladder cancer, characterized in that: The antibody against the ERBB2 S310F mutation is a serum antibody, specifically a rabbit polyclonal antibody prepared using the NYLSTDVGFCTLV polypeptide as an antigen; The preparation method of the ERBB2 S310F mutant antibody is as follows: the wild-type serine (S) at position 310 in the ERBB2 amino acid sequence is mutated to phenylalanine (F) to prepare a mutant polypeptide NYLSTDVGFCTLV; then the synthesized polypeptide is dissolved with NaCl to prepare an antigen, and the antigen is immunized into rabbits by intramuscular injection; finally, the rabbit serum is collected and purified to obtain the ERBB2S310F mutant antibody; The application method is to use the antibody to perform immunohistochemical detection.

2. The use according to claim 1, characterized in that: The ERBB2 S310F mutation causes elevated PD-L1 expression in gallbladder cancer.

3. The use according to claim 1, characterized in that: The antibody specifically recognizes the ERBB2S310F mutation in human gallbladder cancer.

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