Monoclonal antibody of mfge8 and preparation method and application thereof

Abstract

The application provides a kind of MFGE8 monoclonal antibody and its preparation method and application, the heavy chain and light chain variable region nucleotide sequence of the MFGE8 monoclonal antibody is obtained by constructing rabbit phage library, panning, monoclonal screening and identification, then the heavy chain and light chain variable region nucleotide sequence obtained is respectively subcloned into the expression vector containing IgG constant region nucleotide sequence, is constructed into complete IgG antibody expression vector and is introduced into eukaryotic cell to express and purify, and five strains of monoclonal antibodies with strong affinity are screened out by ELISA experiment.The application also pairs five strains of monoclonal antibodies two by two to screen the antibody pair with the strongest antigen binding capacity, and uses the antibody pair to prepare MFGE8 ELISA detection kit for quantitatively detecting the content of MFGE8 in human biological samples, to assist early diagnosis and screening of breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an MFGE8 monoclonal antibody and a preparation method and application thereof. Background Art

[0002] MFGE8 (milk fat globule-EGF factor 8 protein), also known as milk fat globule protein 8 or milk fat globule membrane protein 8, is a secreted glycoprotein widely expressed in mammals. It has multiple biological functions, primarily related to cell adhesion, clearance of apoptotic cells (phagocytosis), wound healing, and immune regulation. MFGE8 binds to phosphatidylserine (PS) on the surface of apoptotic cells and integrin receptors (such as αvβ3 and αvβ5) on phagocytes (such as macrophages and dendritic cells), acting as a "bridging molecule" to facilitate recognition and phagocytosis of apoptotic cells by phagocytes, thereby maintaining tissue homeostasis. Furthermore, MFGE8 activates integrin signaling pathways, promoting the migration of fibroblasts and endothelial cells, accelerating wound healing and angiogenesis, and participating in the reorganization of the extracellular matrix, thereby aiding in the repair of damaged tissues. Furthermore, by promoting the clearance of apoptotic cells and reducing the release of proinflammatory cytokines, MFGE8 inhibits excessive inflammatory responses and influences macrophage polarization (such as the transition to the anti-inflammatory M2 phenotype), contributing to the formation of immune tolerance. In addition, MFGE8 is abnormally expressed in certain tumors (such as breast cancer) and may participate in tumor progression by promoting angiogenesis or tumor cell adhesion.

[0003] MFGE8 protein is a multifunctional molecule whose core functions are to mediate the clearance of apoptotic cells and regulate immune inflammatory responses. Its abnormal expression is associated with various diseases. MFGE8 protein is abnormally expressed in breast cancer patients and therefore serves as a potential marker for breast cancer. The preparation of MFGE8 antibodies can be used for early diagnosis and screening of breast cancer.

[0004] The main methods for producing MFGE8 monoclonal antibodies include traditional hybridoma technology, phage display technology, and transgenic animal technology. While traditional hybridoma technology is mature and can produce hybridoma cells producing MFGE8 antibodies through screening and cloning, hybridoma cells may experience a decrease in antibody affinity over time. Furthermore, the hybridoma cell production process is lengthy, labor-intensive, and difficult to humanize. MFGE8 antibodies produced through transgenic animal technology are humanized or fully human antibodies with low immunogenicity, making them less susceptible to immune system recognition and clearance in vivo and more stably binding to the MFGE8 antigen. However, the production of transgenic animals is difficult, costly, and time-consuming. Phage display technology, on the other hand, allows the construction of large display libraries, such as mouse, rabbit, and human phage display libraries, increasing the chances of identifying antibodies with high affinity and specificity for the MFGE8 antigen. Furthermore, antibodies can be screened and modified in vitro for diagnostic and detection applications.

[0005] Therefore, there is an urgent need to screen MFGE8 monoclonal antibodies through phage display technology and apply them to the early diagnosis and screening of breast cancer. Summary of the Invention

[0006] The present invention provides an MFGE8 monoclonal antibody, preparation method, and application thereof. The heavy and light chain variable region nucleotide sequences of the MFGE8 monoclonal antibody are obtained by constructing a rabbit phage library, panning, monoclonal screening, and identification. The obtained heavy and light chain variable region nucleotide sequences are then subcloned into expression vectors containing IgG constant region nucleotide sequences to construct a complete IgG antibody expression vector, which is then introduced into eukaryotic cells for expression and purification. Five monoclonal antibodies with strong affinity are screened using an ELISA assay. The present invention also pairs the five monoclonal antibodies to screen for the antibody pair with the strongest antigen binding ability. The antibody pairs are then used to prepare an MFGE8 ELISA detection kit for quantitatively detecting MFGE8 content in human biological samples, assisting in the early diagnosis and screening of breast cancer.

[0007] In one aspect, the present invention provides an MFGE8 monoclonal antibody, comprising a first antibody and a second antibody; the amino acid sequence of the heavy chain variable region of the first antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4.

[0008] The two MFGE8 monoclonal antibodies provided by the present invention were prepared by constructing a rabbit phage display library. Twenty-two phage clones were screened from the rabbit phage display library and sequenced, their antibody gene sequences analyzed, and the nucleotide sequences of the antibodies determined. The specific antibody genes screened were subcloned into a suitable expression vector and transformed into a mammalian cell expression system for expression. After purification, 22 highly pure MFGE8 monoclonal antibodies (up to 98% purity) were obtained. Subsequently, indirect ELISA and antibody pairwise testing were used to screen for a pair of antibodies with strong specificity, high affinity, and high stability, namely the first and second antibodies. This antibody pair was formulated into a test kit for detecting the content of MFGE8, a potential breast cancer marker, in samples. This antibody pair does not cross-react with other substances in the sample and is therefore important for the early screening and diagnosis of breast cancer.

[0009] Furthermore, the nucleotide sequence of the heavy chain variable region of the first antibody is shown as SEQ ID NO.5, and the nucleotide sequence of the light chain variable region is shown as SEQ ID NO.6; the nucleotide sequence of the heavy chain variable region of the second antibody is shown as SEQ ID NO.7, and the nucleotide sequence of the light chain variable region is shown as SEQ ID NO.8.

[0010] Furthermore, the heavy and light chain signal peptides of the first antibody and the second antibody are the same, the amino acid sequence of the heavy chain signal peptide is shown in SEQ ID NO.9, and the amino acid sequence of the light chain signal peptide is shown in SEQ ID NO.10; the heavy and light chain constant regions of the first antibody and the second antibody are the same, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.13, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO.14.

[0011] The length of the signal peptide is generally about 15-30 amino acids. If it is too short, it may not be able to effectively guide protein secretion, while if it is too long, it may affect protein folding or subsequent processing. The amino acid sequence of the signal peptide of the heavy chain and light chain provided by the present invention is 19 amino acids.

[0012] The amino acid sequences of the heavy chain and light chain constant regions are the amino acid sequence of the heavy chain constant region of mouse IgG1 and the amino acid sequence of the light chain constant region of mouse IgG1, respectively.

[0013] As you can understand, IgG has four subtypes: IgG1, IgG2, IgG3, and IgG4. The heavy and light chain constant region sequences of any of these subtypes can serve as the heavy and light chain constant regions of the first, second, and third antibodies, but their abilities to activate complement and bind to Fc receptors differ. For detection and diagnosis, the MFGE8 monoclonal antibodies prepared in this invention utilize IgG1 heavy and light chain constant regions. This is because the IgG1 constant region exhibits excellent adaptability in a variety of detection environments and helps maintain the conformational stability of the entire antibody molecule. Whether detecting low-concentration antigens or in complex biological samples (such as blood and tissue homogenates), this allows the variable region to better perform its function of recognizing and binding the antigen, reducing cross-reactivity with unrelated substances in the sample, thereby improving detection specificity, accurately capturing the target antigen, and reducing false-negative results. For example, in cell-based detection systems, the IgG1 constant region can bind to Fc receptors, amplifying the detection signal through Fc receptor-mediated signaling and enhancing detection sensitivity. For example, in ELISA testing, the structure of IgG1 is relatively stable. Compared to other antibody subclasses, IgG1 is less susceptible to denaturation or degradation under varying environmental conditions, such as pH and temperature. This allows it to maintain its activity and antigen-binding ability throughout the various steps of the ELISA test, ensuring the reliability of the test results. Furthermore, IgG1 binds well to enzyme markers and fluorescent markers commonly used in ELISA testing, and this binding does not significantly affect its own antigen-binding activity or the activity of the marker, facilitating subsequent signal detection and amplification. Furthermore, current biotechnology approaches can efficiently produce IgG1 antibodies in a variety of expression systems, such as Chinese hamster ovary cells (CHO cells) and human embryonic kidney cells (HEK293 cells, HEK293T cells). These mature and stable expression systems enable large-scale cultivation and high-yield expression of IgG1 antibodies, meeting the demand for large quantities of antibodies in scientific research and clinical testing.

[0014] The heavy chain signal peptide nucleotide sequence is shown in SEQ ID NO.11, and the light chain signal peptide nucleotide sequence is shown in SEQ ID NO.12; the heavy chain constant region nucleotide sequence is shown in SEQ ID NO.15, and the light chain constant region nucleotide sequence is shown in SEQ ID NO.16.

[0015] Furthermore, the heavy chain amino acid sequence of the first antibody is shown in SEQ ID NO.17, and the light chain amino acid sequence is shown in SEQ ID NO.18; the heavy chain amino acid sequence of the second antibody is shown in SEQ ID NO.19, and the light chain amino acid sequence is shown in SEQ ID NO.20.

[0016] Furthermore, the heavy chain nucleotide sequence of the first antibody is shown as SEQ ID NO.21, and the light chain nucleotide sequence is shown as SEQ ID NO.22; the heavy chain nucleotide sequence of the second antibody is shown as SEQ ID NO.23, and the light chain nucleotide sequence is shown as SEQ ID NO.24.

[0017] In another aspect, the present invention provides a method for preparing the above-mentioned MFGE8 monoclonal antibody, comprising the following steps:

[0018] (1) Immunize animals with MFGE8 antigen;

[0019] (2) Collecting spleen or bone marrow from immunized animals to construct a phage library;

[0020] (3) obtaining SCFV from a phage library by screening and enrichment, and sequencing to obtain the nucleotide sequence of the SCFV;

[0021] (4) cloning the heavy chain variable region and light chain variable region nucleotide sequences of the SCFV into expression vectors containing a signal peptide and a constant region respectively by subcloning;

[0022] (5) The expression vector was introduced into eukaryotic cells for expression and purification to obtain MFGE8 monoclonal antibody.

[0023] On the other hand, the present invention provides a use of an MFGE8 antibody composition in preparing a reagent for improving the ability to bind to MFGE8 protein, the antibody composition comprising a first antibody and a second antibody, the amino acid sequence of the heavy chain variable region of the first antibody being shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region being shown as SEQ ID NO.2; the amino acid sequence of the heavy chain variable region of the second antibody being shown as SEQ ID NO.3, and the amino acid sequence of the light chain variable region being shown as SEQ ID NO.4.

[0024] In some embodiments, the present invention ultimately prepared 22 MFGE8 monoclonal antibodies by constructing a phage library. The binding ability of the purified 22 monoclonal antibodies to the MFGE8 antigen was detected by indirect ELISA, and it was found that only 5 antibodies bound to the antigen. The 5 antibodies were then paired in pairs and their binding ability to the MFGE8 antigen was detected by the double antibody sandwich method. Finally, two pairs of antibodies with strong antigen binding ability were screened out (one pair was the first antibody and the second antibody).

[0025] In another aspect, the present invention provides a MFGE8 detection kit, comprising the above-mentioned MFGE8 monoclonal antibody.

[0026] The MFGE8 detection kit has various forms, including but not limited to enzyme-linked immunosorbent assay (ELISA) kits, chemiluminescence kits, immunofluorescence kits, etc.

[0027] Furthermore, the kit is an ELISA detection kit.

[0028] Furthermore, the ELISA detection kit includes a capture antibody reagent and a detection antibody reagent, the capture antibody in the capture antibody reagent is the first antibody, and the detection antibody in the detection antibody reagent is the second antibody.

[0029] Furthermore, the ELISA detection kit also includes avidin-biotin-peroxidase complex (ABC) solution, sample dilution buffer, antibody dilution buffer, ABC dilution buffer, TMB color development solution, TMB stop solution, and washing buffer.

[0030] The capture antibody reagent is prepared by diluting the MFGE8 monoclonal antibody (the first antibody) with an antibody dilution buffer to a working concentration; the detection antibody (the second antibody) in the detection antibody reagent needs to be prepared as a biotinylated antibody.

[0031] In some embodiments, the cross-reactivity of the ELISA test kit with factors such as M-CSF, TNF-α, TNF RI / II, VCAM-1, and VEGF is tested, and the cross-reactivity rate is less than 1%.

[0032] Furthermore, the present invention provides a use of the above-mentioned MFGE8 detection kit in a product for improving the efficacy of diagnosing breast cancer.

[0033] In some methods, the MFGE8 detection kit is used to detect serum samples from healthy people and breast cancer patients, and the test results are used to generate a receiver operating characteristic (ROC) curve through software and the AUC value is calculated. An AUC value greater than 0.85 indicates that the detection kit can better distinguish patients from healthy people and can be used for early screening and diagnosis of breast cancer.

[0034] The present invention has the following beneficial effects:

[0035] 1. Two MFGE8 monoclonal antibodies were prepared and screened by immunizing rabbits with MFGE8 and constructing a rabbit phage library. They are the first antibody and the second antibody, which have strong specificity, high affinity and high stability.

[0036] 2. The first and second antibodies not only have strong specific binding ability with MFGE8 and good affinity, but also do not cross-react with other cytokines (<1%);

[0037] 3. The MFGE8 ELISA kit was prepared using the first antibody and the second antibody for quantitative detection of MFGE8 content in human biological samples, which can better distinguish healthy people from breast cancer patients and assist in the early diagnosis of breast cancer, with an AUC value of >0.85. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the RNA electrophoresis diagram of spleen and bone marrow of rabbits after immunization in Example 1;

[0039] Figure 2 This is the electrophoresis of the light chain variable region (VL) sequence and the heavy chain variable region (VH) sequence of the spleen cDNA in Example 1;

[0040] Figure 3 This is the electrophoresis diagram of the scFv in Example 1;

[0041] Figure 4 This is the electrophoresis diagram of the phage vector bComb3x into which the scFv fragment has been inserted in Example 1;

[0042] Figure 5 This is the electrophoresis diagram of the antibody GR-8 in Example 2 after treatment with 4× loading buffer (reducing / non-reducing);

[0043] Figure 6 This is the binding curve of the antibody GR-8 in Example 2. DETAILED DESCRIPTION

[0044] The present invention will be further described in detail below with reference to the accompanying drawings and examples. It should be noted that the following examples are intended to facilitate understanding of the present invention and do not have any limiting effect on the present invention.

[0045] The reagents, consumables and experimental instruments used in the following examples are shown in Tables 1 and 2 below.

[0046] Table 1. Reagents and consumables

[0047]

[0048] Table 2. Experimental instruments

[0049]

[0050] Example 1. Preparation of MFGE8 monoclonal antibody

[0051] 1. Animal immunization

[0052] Two Japanese large-eared white rabbits, numbered 1 and 2, were immunized with an antigen dose of 500 μg per rabbit. For the first immunization, the mouse MFGE8 antigen was emulsified with an equal amount of complete Freund's adjuvant and injected subcutaneously at multiple points on the back. Two weeks later, the same dose of the immunogen was emulsified with an equal amount of incomplete Freund's adjuvant. After four immunizations, the serum titer was determined by indirect ELISA. The serum titer detection method is as follows:

[0053] (1) Collect rabbit serum: Before the first immunization and one week after the fourth immunization, collect 3-4 mL of blood from the rabbit's ear vein, let it stand overnight at 4°C, and then centrifuge at 1500 g for 15 minutes to separate the upper serum for testing;

[0054] (2) Coating: Take an appropriate amount of mouse MFGE8 antigen used in the test and dilute it to 0.1 μg / mL, 1 μg / mL, and 5 μg / mL with coating buffer. Then use a pipette to add 100 μL to each well of a 96-well plate. Tap the plate gently to mix the sample, seal it with plastic wrap, and coat it overnight at 4°C.

[0055] (3) Washing: Wash the plate once with 200 μL / well of washing solution and dry the plate;

[0056] (4) Blocking: Block the ELISA plate with 300 μL / well of blocking solution at room temperature for 1 h;

[0057] (5) Washing: Wash the plate twice with 300 μL / well of washing solution and dry the plate;

[0058] (6) Sample loading: The serum sample from step (1) was serially diluted with sample diluent and loaded at 100 μL / well;

[0059] (7) Adding secondary antibody: Add detection antibody (horseradish enzyme-labeled goat anti-rabbit IgG (H+L)) at 0.08 μg / mL to a 96-well plate at 100 μL / well and incubate at room temperature for 2 h;

[0060] (8) Washing the plate: Wash the plate 5 times with 200 μL / well of washing solution and dry the plate;

[0061] (9) Color development: Add 200 μL / well of color development solution and incubate at room temperature for 12 min;

[0062] (10) Termination and detection: Add 50 μL / well of stop solution to terminate the reaction, and then use a microplate reader to detect the reaction at a wavelength of 450 nm. Calculate the relative OD450 value, which is the relative OD450 = detection OD450 - blank control.

[0063] The serum titer test results are shown in Table 3 below.

[0064] Table 3. Serum test results after four immunizations

[0065]

[0066] According to the data analysis in Table 3, the positive standard was (serum absorbance value after the fourth immunization - blank absorbance value) / (negative control serum absorbance value before immunization - blank absorbance value)>2.1. The results showed that the serum titers of both rabbits reached 1:128000 after four immunizations. In comparison, the serum titer of rabbit No. 2 was higher, so the spleen and bone marrow of rabbit No. 2 were taken for subsequent construction of the phage library.

[0067] 2. Construction of phage library and screening of positive scFv

[0068] The spleen and bone marrow of rabbit No. 2 were taken and RNA was extracted from the spleen and bone marrow using TriPure Isolation Reagent. The RNA electrophoresis pattern is shown in the figure below. Figure 1 As shown, the extracted RNA was subjected to UV quantification, and the experimental results are shown in Table 4 below, indicating that the purity and concentration of the extracted RNA were good.

[0069] Table 4. RNA UV quantification results of spleen and bone marrow

[0070]

[0071] The purpose of constructing the phage library in the present invention is to quickly obtain high-affinity specific antibodies after immunization, so the spleen is preferably used for the next step of the experiment.

[0072] Furthermore, reverse transcription was performed using a homemade reverse transcription kit to obtain spleen cDNA, and PCR was performed to amplify the light chain variable region sequence and heavy chain variable region sequence of the cDNA. The light chain PCR primers are shown in Table 5 below, and the heavy chain PCR primers are shown in Table 6 below.

[0073] Table 5. Light chain PCR primers

[0074]

[0075] Table 6. Heavy chain PCR primers

[0076]

[0077] The light chain variable region (VL) and heavy chain variable region (VH) sequences of cDNA were amplified by PCR, and the electrophoresis results were as follows: Figure 2 As shown, the overlap extension PCR method was used to splice the amplified VL sequence and VH sequence through the linker to form a nucleotide sequence encoding scFv. The electrophoresis results of the spliced ​​scFv are shown in FIG. Figure 3Then, the scFv was digested with the homemade restriction endonuclease SfiⅠ and connected to the phage vector bComb3x. The electrophoresis results of the phage vector bComb3x with the scFv fragment inserted are shown as follows. Figure 4 Then, the phage vector bComb3x with the inserted scFv fragment was electroporated into X-Blue competent medium to construct phage display scFv primary antibody libraries A and B for immunizing rabbits. The primary antibody library capacity was calculated and both reached 2.00×10 8 cfu.

[0078] Furthermore, the primary antibody library was infected with helper phage to prepare the original antibody library. The specific method is as follows: the mouse MFGE8 antigen was coated on an ELISA plate, the original antibody library was added to the ELISA plate, non-specifically bound phage was first washed away with a wash solution, and then the phage bound to the antigen was eluted with an eluent. After amplification and precipitation in Escherichia coli, the next round of enrichment screening was carried out. After 2-4 rounds of "adsorption-elution-amplification", the positive library binding to MFGE8 was screened. The binding of library A and library B, which had undergone multiple rounds of solid-phase screening, to the mouse MFGE8 antigen was tested by ELISA. The test results are shown in Tables 7 and 8 below.

[0079] Table 7. Binding of phage positive library A to mouse MFGE8 antigen

[0080]

[0081] Table 8. Binding of phage positive library B to mouse MFGE8 antigen

[0082]

[0083] From the analysis of the results in Tables 7 and 8 above, the ELISA test results showed that the library was positive for binding to mouse MFGE8 protein, and single clones were selected from the two libraries for expression identification.

[0084] Furthermore, monoclonal phages were selected from the positive phage libraries A and B for expression, and the supernatants were collected and tested by ELISA. The phages were then coated with mouse tumor necrosis factor receptor superfamily member 19 (mTNFRSF19) to detect cross-reactivity. The test data are shown in Table 9 below.

[0085] Table 9. Positive monoclonal phage expression ELISA test data

[0086]

[0087] According to the data analysis in Table 9, the scFv antibodies expressed by the 22 positive monoclonal phages all specifically bound to MFGE8 and did not cross-react with mTNFRSF19. The nucleotide sequences of 22 scFvs were obtained by sequencing companies.

[0088] 3. Preparation of monoclonal antibodies

[0089] Furthermore, primers were designed based on the heavy chain variable region sequences and light chain variable region sequences of the 22 groups of scFvs. The forward primers for the heavy and light chain variable regions are shown in Table 10 below, and the reverse primers for the heavy and light chain variable regions are shown in Table 11 below.

[0090] Table 10. Forward primers for the heavy and light chain variable regions of scFv antibodies

[0091]

[0092] Table 11. Reverse primers for the heavy and light chain variable regions of scFv antibodies

[0093]

[0094] PCR was performed using the primers in Tables 10 and 11 above to obtain the light and heavy chain variable region fragments of the scFv antibody. After electrophoresis, the fragments were cut and recovered from the gel. The heavy and light chain variable region fragments recovered from the gel were added to the membrane binding buffer, mixed thoroughly, and added to the purification column. The column was centrifuged at 13,000 rpm for 1 min, and the liquid was discarded. 700 μL of rinse solution was added, and the column was centrifuged at 13,000 rpm for 1 min, and the liquid was discarded. After spinning for 5 minutes, the column was allowed to stand at room temperature for 5 minutes, and Nuclease-Free Water was added for elution to obtain purified heavy and light chain variable region sequence fragments.

[0095] Furthermore, a signal peptide sequence and a constant region sequence were added to the heavy and light chain variable region fragments by PCR to construct complete heavy chain and light chain nucleotide sequences. The heavy chain signal peptide amino acid sequence is shown in SEQ ID NO.9, and the nucleotide sequence is shown in SEQ ID NO.11. The light chain signal peptide amino acid sequence is shown in SEQ ID NO.10, and the nucleotide sequence is shown in SEQ ID NO.12. The constant region sequence is the constant region sequence of mouse IgG1. The heavy chain constant region amino acid sequence is shown in SEQ ID NO.13, and the nucleotide sequence is shown in SEQ ID NO.15. The light chain constant region amino acid sequence is shown in SEQ ID NO.14, and the nucleotide sequence is shown in SEQ ID NO.16. The complete heavy chain nucleotide sequence and light chain nucleotide sequence were ligated to the pUC expression vector plasmid, respectively. The specific method was as follows: 4 μL of the purified complete heavy chain nucleotide sequence / complete light chain nucleotide sequence and 1 μL of the vector were added with 5 μL of ligase and mixed thoroughly. The mixture was reacted at 50°C for 20 minutes and then added to 100 μL of competent medium. After ice bathing for 30 minutes, the mixture was heat-shocked for 90 seconds. The cells were quickly placed on ice for 3 minutes and then plated. The cells were cultured at 37°C overnight. The obtained single clones were amplified by PCR and sequenced the next day. Software comparison showed that the expression vector plasmid had been successfully linked to the specific gene.

[0096] Furthermore, 22 heavy chain-linked vector plasmids and 22 light chain-linked vector plasmids were paired and delivered to HEK 293 cells for transient expression. The specific method is as follows: HEK 293 cells were subcultured in 293 serum-free CD medium. The plasmid DNA to be expressed was mixed with the transfection reagent TF2 and added to the cells. 293 serum-free feed solution was added on days 1, 3, and 5 after transfection. Shake flask culture conditions were: 5% CO2, temperature, 37°C, and shaker speed of 175 rpm. On the fifth day of cell culture, the supernatant was collected and analyzed by ELISA as follows:

[0097] (1) Coating: Coat with mouse MFGE8 antigen protein at 0.1 μg / mL and 1 μg / mL, 100 μL / well, overnight at 4°C;

[0098] (2) Blocking: Shake off the liquid in the plate and pat dry, add 2% BSA, 300 μL / well, seal and incubate at room temperature for 1 h;

[0099] (3) Washing: 300 μL / well washing solution, wash the plate twice, and pat dry the last time;

[0100] (4) Antibody dilution: Take the supernatant of 22 positive clone cells and dilute them 2-fold. Add 100 μL of each to the corresponding well plate, mix well, and react at room temperature for 2 h.

[0101] (5) Wash the plate: 300 μL / well washing solution, wash the plate 3 times, and pat dry the last time;

[0102] (6) Add secondary antibody: dilute horseradish enzyme-labeled goat anti-mouse IgG Fc secondary antibody to a working concentration of 0.08 μg / mL, 100 μL / well, mix well, and incubate at room temperature for 1 h;

[0103] (7) Washing: 300 μL / well washing solution, wash the plate 3 times, and pat dry the last time;

[0104] (8) Color development: Mix solution A and solution B at a ratio of 1:1, add 200 μL to each well, and incubate at room temperature in the dark for 20 min;

[0105] (9) Add 50 μL of stop solution to each well and immediately measure the OD value at a wavelength of 450 nm. Set up three parallel wells, take the average value, and calculate the relative OD450 value. Relative OD450 = detection OD450 - blank control.

[0106] The ELISA test results are shown in Table 12 below.

[0107] Table 12. ELISA test results of the supernatant of 22 positive clone cells after five days of culture

[0108]

[0109] According to the data in Table 12, the cell supernatants of the 22 positive clone cells after five days of culture were all positive for mouse MFGE8 protein antigen ELISA detection, and all had good binding ability.

[0110] Furthermore, after 7 days of cell culture, the cell supernatant was collected and the antibodies were purified as follows:

[0111] (1) Sample preparation: Centrifuge at 4000 g for 30 min using a tabletop centrifuge and collect the supernatant of 22 monoclonal cell lines;

[0112] (2) Filtration: Select different filtration methods according to the volume of the feed liquid, and use a 0.45 μm filter membrane for filtration;

[0113] (3) Connect to the purification system: Select an affinity filler Protein A column of appropriate specifications based on the expression level and connect it to the purification system;

[0114] (4) Water balance: Wash with ultrapure water for 2 CV to replace the 25% ethanol storage solution;

[0115] (5) Equilibrate the column: Equilibrate with AC Binding buffer for 3 CV until the UV baseline is stable;

[0116] (6) Sample loading: Adjust the flow rate to load the sample;

[0117] (7) Eluent: Elute for 5-10 CV until the UV baseline is stable. The flow rate is the same as the sample flow rate.

[0118] (8) Elution: elution with eluent and collection based on UV peaks;

[0119] (9) Neutralization: Add Tris, pH 8.0 to neutralize the eluted antibody;

[0120] (10) Equilibration: Equilibrate with AC Binding Buffer for 3 CV to neutrality;

[0121] (11) CIP cleaning: CIP regeneration liquid cleaning for more than 5 CV;

[0122] (12) Alkali flushing: flush with AC Binding buffer until the pH at the outlet is neutral, then rinse with ultrapure water for 3CV;

[0123] (13) Storage: Equilibrate the column with 25% ethanol for 2 CV and store it.

[0124] Through purification, 22 purified MFGE8 monoclonal antibodies were obtained.

[0125] Example 2: Monoclonal antibody screening

[0126] In this example, the concentration and purity of the 22 purified monoclonal antibodies in Example 1 were first tested, and then their binding ability to antigens at different concentrations was tested to screen out monoclonal antibodies with strong antigen binding ability. The monoclonal antibodies with strong antigen binding ability were then paired to screen out antibody pairs with strong antigen binding ability.

[0127] 1. Detect antibody concentration

[0128] Start the micro-spectrophotometer and apply the test sample buffer solution. At a wavelength of 280 nm, an absorbance value between ±0.015 indicates a stable instrument baseline. Apply the 22 purified antibody samples sequentially and record the absorbance values. Divide the data by the IgG extinction coefficient (1.414) to obtain the concentration of the test sample, as shown in Table 13 below.

[0129] Table 13, 22 Purified Antibody Concentrations

[0130]

[0131] 2. Detect antibody purity

[0132] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to test the purity of 22 purified antibodies. This method separates the proteins in the test sample according to their molecular weight in the electrophoresis gel, thereby verifying the purity of the target antibody. The specific method is as follows:

[0133] (1) Sample processing: Take 5 μg of the 22 purified antibodies in Table 13 and add them to 5 μL of 4× loading buffer (reducing) and 4× loading buffer (non-reducing), heat in a 100°C water bath, and then centrifuge at 10,000 rpm;

[0134] (2) Prepare SDS-PAGE gels with concentrations of 13% and 7.5%;

[0135] (3) Fix the SDS-PAGE gel prepared in (2) in an electrophoresis tank, add sufficient 1× glycine system electrode buffer to the electrophoresis tank, use a micropipette to add the sample solution and protein molecular weight standard treated with 4× loading buffer (non-reducing) in (1) to the sample wells of the 7.5% SDS-PAGE gel; use a micropipette to add the sample solution and protein molecular weight standard treated with 4× loading buffer (reducing) in (1) to the sample wells of the 13% SDS-PAGE gel;

[0136] (4) Electrophoresis: Connect the power supply and run the electrophoresis at a constant voltage of 100 V until the bromophenol blue dye enters the separation gel from the stacking gel. Then adjust the current to 140 V and continue electrophoresis to the bottom of the gel plate. Turn off the power supply.

[0137] (5) Staining: Soak the gel in Coomassie Brilliant Blue R-250 staining solution, heat it in a microwave oven for 60 seconds, and stain it at room temperature on a gently shaking platform for 2-4 hours. Then, replace the destaining solution and cover the gel, heat it in a microwave oven for 60 seconds, and destain it at room temperature on a gently shaking platform. Repeat the destaining operation until blue bands and a clean background are obtained.

[0138] Observe the protein staining bands after decolorization. The purity of the 22 purified antibody proteins is above 95%. Taking the antibody GR-8 as an example, the electrophoresis diagrams after treatment with 4× loading buffer (reducing) and 4× loading buffer (non-reducing) are shown as follows: Figure 5 As shown, the results showed that the purity of the antibody GR-8 was very high, with almost no other impurities, and the purity was as high as 98.0%.

[0139] 3. Binding ability of purified antibodies to MFGE8

[0140] Furthermore, the binding of 22 purified antibodies to MFGE8 was detected by ELISA. The specific steps are as follows:

[0141] (1) Coating: Coat with mouse MFGE8 antigen protein at 0.1 μg / mL, 1 μg / mL, and 5 μg / mL, 100 μL / well, and coat overnight at 4°C;

[0142] (2) Blocking: Shake off the liquid in the plate and pat dry, add 2% BSA, 300 μL / well, seal and incubate at room temperature for 1 h;

[0143] (3) Washing: 300 μL / well washing solution, wash the plate twice, and pat dry the last time;

[0144] (4) Antibody dilution: dilute the antibody to 0.1 μg / mL, add 100 μL / well to the corresponding well plate, mix well, and react at room temperature for 2 h;

[0145] (5) Washing: 300 μL / well washing solution, wash the plate 3 times, and pat dry the last time;

[0146] (6) Add secondary antibody: dilute horseradish enzyme-labeled rabbit anti-mouse IgG F(ab)2 secondary antibody to a working concentration of 0.09 μg / mL, 100 μL / well, mix well, and incubate at room temperature for 1 h;

[0147] (7) Washing: 300 μL / well washing solution, wash the plate 3 times, and pat dry the last time;

[0148] (8) Color development: Mix solution A and solution B at a ratio of 1:1, add 200 μL to each well, and incubate at room temperature in the dark for 20 min;

[0149] (9) Add 50 μL of stop solution to each well and immediately measure the OD value at a wavelength of 450 nm. Set up three parallel wells, take the average value, and calculate the relative OD450 value. Relative OD450 = detection OD450 - blank control.

[0150] The results showed that only 5 of the 22 purified antibodies bound to mouse MFGE8 antigens coated at different concentrations, namely GR-3, GR-5, GR-8, GR-14, and GR-17. The results of their binding to mouse MFGE8 antigens coated at different concentrations are shown in Table 14 below.

[0151] Table 14. ELISA test results of 5 purified antibodies

[0152]

[0153] Furthermore, the affinity of the 5 monoclonal antibodies screened above was tested by ELISA. The detection method is as follows:

[0154] (1) Coating: Coat with MFGE8 antigen protein 0.1 μg / mL, 100 μL / well, overnight at 4°C;

[0155] (2) Blocking: Shake off the liquid in the plate and pat dry, add 2% BSA, 300 μL / well, seal and incubate at room temperature for 1 h;

[0156] (3) Washing: 300 μL / well washing solution, wash the plate twice, and pat dry the last time;

[0157] (4) Antibody dilution: The antibody was diluted to 300 ng / mL, and then diluted threefold to 10 points, with concentrations of 300 ng / mL, 100 ng / mL, 33.33 ng / mL, 11.11 ng / mL, 3.70 ng / mL, 1.23 ng / mL, 0.41 ng / mL, 0.14 ng / mL, 0.05 ng / mL, 0.02 ng / mL, and 0.01 ng / mL, respectively. The blank control was the sample diluent, 100 μL / well was added to the corresponding well plate, mixed evenly, and reacted at room temperature for 2 h;

[0158] (5) Washing: 300 μL / well washing solution, wash the plate 3 times, and pat dry the last time;

[0159] (6) Add secondary antibody: dilute horseradish enzyme-labeled rabbit anti-mouse IgG F(ab)2 secondary antibody to a working concentration of 0.2 μg / mL, 100 μL / well, mix well, and incubate at room temperature for 1 h;

[0160] (7) Washing: 300 μL / well washing solution, wash the plate 3 times, and pat dry the last time;

[0161] (8) Color development: Mix solution A and solution B at a ratio of 1:1, add 200 μL to each well, and incubate at room temperature in the dark for 20 min;

[0162] (9) Add 50 μL of stop solution to each well and immediately measure the OD value at a wavelength of 450 nm. Set up two parallel wells, take the average value, and calculate the relative OD450 value. Relative OD450 = detection OD450 - blank control.

[0163] The test values ​​were input into GraphPad Prism software to calculate the affinity. The results showed that the affinity of the five monoclonal antibodies to the antigen was very high. They can exert their biological activity at lower concentrations. The five antibodies can be used in immune detection to assist in the diagnosis of diseases. The affinity EC50 values ​​of the five monoclonal antibodies GR-3, GR-5, GR-8, GR-14, and GR-17 are 18.57 ng / mL, 20.61 ng / mL, 15.48 ng / mL, 26.34 ng / mL, and 13.05 ng / mL, respectively. The smaller the EC50 value, the higher the affinity. The affinity of the five monoclonal antibodies from high to low are GR-17>GR-8>GR-3>GR-5>GR-14. Taking the monoclonal antibody GR-8 as an example, its affinity test results are shown in Tables 15 and 16 below. Figure 6 shown.

[0164] Table 15. Affinity test results of monoclonal antibody GR-8

[0165]

[0166] 4. Screening of Antibody Pairs

[0167] Five high-affinity monoclonal antibodies were paired in pairs as capture antibodies and detection antibodies, respectively. The ability of the antibody pairs to bind to the MFGE8 antigen was tested by the double antibody sandwich method to screen the best antibody pair. The detection method is as follows:

[0168] (1) Coating with capture antibodies: Coat with monoclonal antibodies GR-3, GR-5, GR-8, GR-14, and GR-17, 1 μg / mL, 100 μL / well, overnight at 4°C;

[0169] (2) Blocking: Shake off the liquid in the plate and pat dry, add 2% BSA, 300 μL / well, seal and incubate at room temperature for 1 h;

[0170] (3) Washing: 300 μL / well washing solution, wash the plate twice, and pat dry the last time;

[0171] (4) Add antigen: dilute mouse MFGE8 to 100 ng / mL, add 100 μL / well to the corresponding well plate, mix well, and react at room temperature for 2 h;

[0172] (5) Washing: 300 μL / well washing solution, wash the plate 3 times, and pat dry the last time;

[0173] (6) Add detection antibodies: add monoclonal antibodies GR-3, GR-5, GR-8, GR-14, and GR-17, 1 μg / mL, 100 μL / well;

[0174] (7) Washing: 300 μL / well washing solution, wash the plate 3 times, and pat dry the last time;

[0175] (8) Add enzyme-labeled antibody: dilute horseradish enzyme-labeled goat anti-mouse IgG Fc to a working concentration of 0.08 μg / mL, 100 μL / well, mix well, and incubate at room temperature for 1 h;

[0176] (9) Washing: 300 μL / well washing solution, wash the plate 3 times, and pat dry the last time;

[0177] (10) Color development: Mix solution A and solution B at a ratio of 1:1, add 200 μL to each well, and incubate at room temperature in the dark for 20 min;

[0178] (11) Add 50 μL of stop solution to each well and immediately measure the OD value at a wavelength of 450 nm. Set up two parallel wells, take the average value, and calculate the relative OD450 value. Relative OD450 = detection OD450 - blank control.

[0179] The results of the monoclonal antibody pairing test are shown in Table 16 below.

[0180] Table 16. Monoclonal antibody pairing test results

[0181]

[0182] According to the data in Table 16, two pairs of antibodies were screened out with the best ability to bind to antigens, one pair was GR-8 (as capture antibody) and GR-3 (as detection antibody), and the other pair was GR-17 (as capture antibody) and GR-5 (as detection antibody). Among them, the heavy chain variable region amino acid sequence of GR-8 is shown in SEQ ID NO.1, the light chain variable region amino acid sequence is shown in SEQ ID NO.2, the heavy chain variable region nucleotide sequence is shown in SEQ ID NO.5, the light chain variable region nucleotide sequence is shown in SEQ ID NO.6, the heavy chain nucleotide sequence is shown in SEQ ID NO.21, and the light chain nucleotide sequence is shown in SEQ ID NO.22; the heavy chain variable region amino acid sequence of GR-3 is shown in SEQ ID NO.3, the light chain variable region amino acid sequence is shown in SEQ ID NO.4, the heavy chain variable region nucleotide sequence is shown in SEQ ID NO.7, the light chain variable region nucleotide sequence is shown in SEQ ID NO.8, the heavy chain nucleotide sequence is shown in SEQ ID NO.23, and the light chain nucleotide sequence is shown in SEQ ID NO.24; the heavy chain variable region amino acid sequence of GR-17 is shown in SEQ ID NO.25, the light chain variable region amino acid sequence is shown in SEQ ID NO.26, the heavy chain nucleotide sequence is shown in SEQ ID NO.33, and the light chain nucleotide sequence is shown in SEQ ID NO. NO.34; the amino acid sequence of the heavy chain variable region of GR-5 is shown in SEQ ID NO.27, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.28, the heavy chain nucleotide sequence is shown in SEQ ID NO.35, and the light chain nucleotide sequence is shown in SEQ ID NO.36.

[0183] Furthermore, circular dichroism (CD) spectroscopy was used to determine the thermal stability Tm values ​​of the screened monoclonal antibodies to evaluate their stability and compare them with existing monoclonal antibodies. The existing MFGE8 monoclonal antibody was purchased from Thermo Fisher Scientific, Catalog No. MA5-23913. The test results are shown in Table 17 below. Tm represents the midpoint temperature of thermal denaturation of a protein, i.e., the temperature at which the protein unfolds by 50%, reflecting the tendency of the protein to change conformation during temperature changes. A higher Tm indicates better stability.

[0184] Table 17. Tm values ​​of different MFGE8 monoclonal antibodies

[0185]

[0186] According to the results in Table 17, it was found that the Tm values ​​of the GR-8, GR-3, GR-17 and GR-5 monoclonal antibodies screened out in this example were all higher than those of the existing MFGE8 monoclonal antibodies, indicating that the screened MFGE8 monoclonal antibodies had better stability.

[0187] Example 3: Preparation of MFGE8 ELISA Detection Kit

[0188] In this example, the two antibody pairs screened in Example 2 (one pair consisting of GR-8 (as a capture antibody) and GR-3 (as a detection antibody), and the other pair consisting of GR-17 (as a capture antibody) and GR-5 (as a detection antibody)) were separately formulated into MFGE8 ELISA detection kits. MFGE8 ELISA detection kits were also prepared using GR-8 and GR-17 as capture antibodies, respectively, and an existing MFGE8 monoclonal antibody (purchased from Thermo Fisher Scientific, Catalog No. MA5-23913) as a detection antibody. The MFGE8 ELISA detection kit and its corresponding capture and detection antibodies are shown in Table 18 below.

[0189] Table 18. MFGE8 ELISA kit and its corresponding capture and detection antibodies

[0190]

[0191] 1. Preparation of MFGE8 ELISA kit

[0192] The kit includes capture antibody reagent, detection antibody reagent, MFGE8 standard, avidin-biotin-peroxidase complex (ABC) solution, sample dilution buffer, antibody dilution buffer, ABC dilution buffer, TMB color development solution, TMB stop solution, and wash buffer. The above reagents are prepared as follows:

[0193] (1) Antibody dilution buffer: 0.01 mol / L pH 7.2 PBS, weigh 0.39 g NaH2PO4·2H2O, 1.27 g Na2HPO4, 0.85 g NaCl, 200 mg NaN3, and add distilled water to 1000 mL;

[0194] (2) Sample dilution buffer: Weigh 0.27 g of KH2PO4, 1.42 g of Na2HPO4, 8 g of NaCl, and 0.2 g of KCl, add about 800 mL of distilled water and stir thoroughly to dissolve, then add concentrated hydrochloric acid to adjust the pH to 7.2-7.4, add 5 g of BSA, and finally adjust the volume to 1 L;

[0195] (3) ABC dilution buffer: Weigh 0.2 g of KH2PO4, 2.9 g of Na2HPO4·12H2O, 8 g of NaCl, and 0.2 g of KCl, add approximately 800 mL of distilled water, stir thoroughly to dissolve, adjust the pH to 7.4 with hydrochloric acid, add 0.5 mL of Tween-20 and 10 g of BSA, and dilute to 1 L;

[0196] (4) Washing buffer: Weigh 0.2 g of KH2PO4, 2.9 g of Na2HPO4·12H2O, 8.0 g of NaCl, and 0.2 g of KCl, add about 800 mL of distilled water, stir thoroughly to dissolve, adjust the pH to 7.4 with hydrochloric acid, and add 0.5 mL of Tween-20 to make the volume 1 L;

[0197] (5) Prepare capture antibody reagent: dilute GR-8 / GR-17 to a concentration of 1 μg / mL with antibody dilution buffer;

[0198] (6) Preparation of detection antibody reagents: a. MFGE8 monoclonal antibody pretreatment: dilute GR-3 / GR-5 / existing MFGE8 monoclonal antibodies with antibody dilution buffer to a concentration of 1 mg / mL; b. Activated biotin reagent: dissolve N-hydroxysuccinimide biotin (NHS-biotin) with an appropriate amount of DMSO to prepare 5 mg / mL; c. Preparation of biotinylated MFGE8 antibody: slowly add the activated biotin reagent to the antibody solution under stirring or oscillation, the molar ratio of biotin to antibody is 5:1, the reaction is carried out at 4 °C, and the reaction time is 4 hours; after the reaction is completed, add an appropriate amount of 0.1 M glycine solution to terminate the reaction, and glycine reacts with the remaining active biotin reagent to block the unreacted active sites; remove the unreacted biotin reagent, organic solvent and other small molecule impurities by dialysis with antibody dilution buffer to obtain the biotinylated MFGE8 antibody and dilute it to a concentration of 1 μg / mL;

[0199] (7) Preparation of MFGE8 standard solution: Prepare the MFGE8 standard solution to 300 ng / mL using sample dilution buffer, and then dilute to 150 ng / mL, 75 ng / mL, 25 ng / mL, 5 ng / mL, 1 ng / mL, and 0.2 ng / mL;

[0200] (8) Prepare avidin-biotin-peroxidase complex (ABC) solution: dilute the ABC solution to 1 μg / mL with ABC dilution buffer.

[0201] (9) Prepare TMB colorimetric solution: the same as that used for ELISA detection in Example 1;

[0202] (10) Prepare TMB stop solution: the same as the stop solution used for ELISA detection in Example 1;

[0203] The above reagents were combined to prepare the four MFGE8 ELISA detection kits shown in Table 18.

[0204] 2. The specific detection steps of the MFGE8 ELISA kit are as follows:

[0205] (1) Coating: Add 100 μL / well of capture antibody reagent to the microwell ELISA plate and coat at 4°C overnight;

[0206] (2) Blocking: Shake off the liquid in the plate and pat dry, add 2% BSA, 300 μL / well, seal and incubate at room temperature for 1 h;

[0207] (3) Sample pretreatment: 1) Serum samples: Allow serum to coagulate in a serum separator tube at room temperature (approximately 4 hours), centrifuge at approximately 1000 × g for 15 minutes, and measure immediately; 2) Plasma samples: Collect plasma using heparin or EDTA as an anticoagulant, centrifuge at 1000 × g for 15 minutes, and measure immediately;

[0208] (4) Adding standards and samples: Add sample dilution buffer (control well), sample, and seven concentrations of MFGE8 standard to the microwell ELISA plate, 100 μL / well, seal the microwell ELISA plate, and incubate at 37 °C for 90 min;

[0209] (5) Use a paper towel or absorbent material to absorb the liquid in the microwells of the ELISA plate. Be careful not to completely absorb the liquid in the microwells. Add 100 μL / well of the detection antibody solution, seal the microwell ELISA plate, and incubate at 37 °C for 60 minutes.

[0210] (6) Wash the plate three times with 300 μL / well wash buffer and pat dry for the final wash.

[0211] (7) Add 100 μL / well of ABC solution, seal the microplate, and incubate at 37°C for 30 minutes;

[0212] (8) Wash the plate 5 times with 300 μL / well washing buffer and pat dry for the last time;

[0213] (9) Add 90 μL / well of TMB colorimetric solution and incubate at 37°C in the dark for 15-25 minutes;

[0214] (10) Add 100 μL / well of TMB stop solution. The liquid in the well will immediately turn yellow. Within 30 minutes after adding TMB stop solution, measure the OD value at a wavelength of 450 nm. Set up three parallel test wells for the same test sample and take the average value. The relative OD450 = test OD450 - blank control.

[0215] The four kits were used to generate four standard curves by detecting MFGE8 standards. The standard curves were fitted with a 4-parameter logistic curve, and the MFGE8 concentration in the samples was calculated using the standard curves. 2All are ≥0.99, indicating that it is suitable for quantitative analysis. Taking kit 1 (capture antibody is GR-8, detection antibody is GR-3) as an example, the fitted 4-parameter logistic curve is: , R 2 =0.999.

[0216] 2. Kit performance test

[0217] Furthermore, the intra-batch and inter-batch precision of the four kits were evaluated using the spike recovery method. The unspiked sample consisted of healthy human serum, and the spiked sample consisted of healthy human serum spiked with a 5 ng / mL MFGE8 standard. Recovery = (spike concentration - unspiked concentration) / spike concentration. The results are shown in Table 19.

[0218] Table 19. Intra-batch and inter-batch precision of MFGE8 detection kit

[0219]

[0220] According to the data in Table 19, Kit 1 (with GR-8 as the capture antibody and GR-3 as the detection antibody) and Kit 2 (with GR-17 as the capture antibody and GR-5 as the detection antibody) exhibited lower intra- and inter-batch CV values ​​and recoveries closer to 100%, indicating that the MFGE8 antibodies prepared and screened using the present invention are more stable and accurate when used with Kits 1 and 2. In contrast, Kits 3 and 4, prepared using GR-8 and GR-17 as capture antibodies and existing MFGE8 monoclonal antibodies, respectively, exhibited unstable and low accuracy. Therefore, Kits 1 and 2 are preferred MFGE8 ELISA test kits.

[0221] Furthermore, the cross-reactivity of the preferred kit 1 and kit 2 with factors such as M-CSF, TNF-α, TNF RI / II, VCAM-1, and VEGF was detected to be less than 1%.

[0222] Example 4: Application of MFGE8 ELISA Detection Kit in Tumor Diagnosis

[0223] MFGE8 expression is upregulated in breast cancer, so it can be used as a potential diagnostic marker for breast cancer.

[0224] In this example, kits 1-4 prepared in Example 3 were used to test MFGE8 in the serum of healthy subjects and breast cancer patients, respectively, to evaluate their ability to distinguish between diseased populations.

[0225] Venous blood samples from 100 healthy subjects and 100 breast cancer patients at the same hospital were collected as a test group. 5 mL of blood was collected from each sample, and serum was separated by centrifugation (3000 rpm, 15 minutes). The serum was aliquoted and stored at -80°C for future use. Venous blood samples from 80 healthy subjects and 120 breast cancer patients at another hospital were collected as a validation group. 5 mL of blood was collected from each sample, and serum was separated by centrifugation (3000 rpm, 15 minutes). The serum was aliquoted and stored at -80°C for future use. The test and validation samples were independently assayed using kits 1-4. The absorbance values ​​were recorded, and the concentrations were calculated to determine the MFGE8 levels in the serum. The cutoff value for MFGE8 levels used to distinguish breast cancer patients was 80 ng / mL: patients with 80 ng / mL or higher were considered breast cancer patients, while those with less than 80 ng / mL were considered healthy subjects. The ability of the four kits to distinguish between patients with and without breast cancer was evaluated using SPSS or R programming. Receiver operating characteristic (ROC) curves were generated, and area under the curve (AUC) values ​​were calculated. The AUC values, sensitivity (true positive rate), and specificity (true negative rate) of the four kits for the test group and validation group are shown in Table 20 below.

[0226] Table 20. AUC values, sensitivity (true positive rate), and specificity (true negative rate) of the four kits

[0227]

[0228] According to the data in Table 20, only Kit 1 had an AUC value greater than or equal to 0.85 in both the test and validation groups, indicating that it can effectively distinguish patients from healthy subjects. In comparison, Kit 2 had slightly lower diagnostic efficacy than Kit 1, while Kits 3 and 4 had even lower diagnostic efficacy. Therefore, Kit 1 (with GR-8 as the capture antibody (i.e., the primary antibody) and GR-3 as the detection antibody (i.e., the secondary antibody)) is the optimal MFGE8 ELISA kit for the early diagnosis and screening of breast cancer.

[0229] Existing antibodies not only have low detection sensitivity, but also produce significantly different test results depending on whether the antibody is used as a detection antibody or an enzyme-labeled antibody. Furthermore, different antibodies exhibit significant differences in stability (Tm), cross-reactivity with the matrix, and high-dose hook effect, all of which can lead to reduced efficacy in breast cancer diagnosis.

[0230] At the same time, during the diagnosis of breast cancer, MFGE8 antibodies are needed to detect blood samples. There are complex interfering substances in the blood samples, the matrix has a greater impact, and there is a high risk of cross-reactions or interference from some nonlinear fragments. Using different antibodies for breast cancer detection and diagnosis will inevitably produce completely different diagnostic results. Therefore, a better antibody combination must be selected to improve diagnostic efficiency.

[0231] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the scope defined by the claims.

Claims

1. A MFGE8 monoclonal antibody, characterized in that: It includes a first antibody or a second antibody; the amino acid sequence of the heavy chain variable region of the first antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

4.

2. The MFGE8 monoclonal antibody according to claim 1, wherein The heavy and light chain signal peptides of the first and second antibodies are the same, the heavy chain signal peptide amino acid sequence is shown in SEQ ID NO.9, and the light chain signal peptide amino acid sequence is shown in SEQ ID NO.10; the heavy and light chain constant regions of the first and second antibodies are the same, the heavy chain constant region amino acid sequence is shown in SEQ ID NO.13, and the light chain constant region amino acid sequence is shown in SEQ ID NO.

14.

3. The MFGE8 monoclonal antibody according to claim 2, wherein The heavy chain amino acid sequence of the first antibody is shown in SEQ ID NO.17, and the light chain amino acid sequence is shown in SEQ ID NO.18; the heavy chain amino acid sequence of the second antibody is shown in SEQ ID NO.19, and the light chain amino acid sequence is shown in SEQ ID NO.

20.

4. Use of an MFGE8 antibody composition in preparing a reagent for detecting MFGE8 protein, characterized in that: The antibody composition includes a first antibody and a second antibody, wherein the amino acid sequence of the heavy chain variable region of the first antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

4.

5. A MFGE8 detection kit, characterized in that: The invention comprises the MFGE8 monoclonal antibody according to any one of claims 1 to 3.

6. The MFGE8 detection kit according to claim 5, characterized in that The kit is an ELISA detection kit.

7. Use of the MFGE8 detection kit according to claim 5 or 6 in the preparation of a product for diagnosing breast cancer.

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