MFGE8 monoclonal antibody as well as preparation method and application thereof

Abstract

The invention provides an MFGE8 monoclonal antibody as well as a preparation method and application thereof. A heavy chain variable region nucleotide sequence and a light chain variable region nucleotide sequence of the MFGE8 monoclonal antibody are obtained through rabbit phage library construction, elutriation, monoclonal screening and identification. And subcloning the obtained heavy chain and light chain variable region nucleotide sequences into an expression vector containing an IgG constant region nucleotide sequence respectively to construct a complete IgG antibody expression vector, introducing the complete IgG antibody expression vector into eukaryotic cells for expression and purification, and screening five monoclonal antibodies with strong affinity through an ELISA (Enzyme-Linked Immunosorbent Assay) experiment. According to the invention, five monoclonal antibodies are paired pairwise for screening antibody pairs with the strongest antigen binding capacity, and the antibody pairs are used for preparing an MFGE8 ELISA detection kit for quantitatively detecting the content of MFGE8 in a human biological sample and assisting in early diagnosis and screening of breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and more particularly, relates to an MFGE8 monoclonal antibody, a preparation method thereof, and an application thereof. Background Art

[0002] MFGE8 (milk fat globule-EGF factor 8 protein), also known as milk fat globulin 8 or milk fat globule membrane protein 8, is a secreted glycoprotein that is widely expressed in mammals and has multiple biological functions, mainly related to cell adhesion, apoptotic cell clearance (phagocytosis), wound healing, and immune regulation. On the one hand, MFGE8 can bind to phosphatidylserine (PS) on the surface of apoptotic cells and integrin receptors (such as αvβ3, αvβ5) on the surface of phagocytes (such as macrophages, dendritic cells), and act as a "bridging molecule" to promote the recognition and phagocytosis of apoptotic cells by phagocytes, thereby maintaining tissue homeostasis. On the other hand, MFGE8 promotes the migration of fibroblasts and endothelial cells, accelerates wound healing and angiogenesis, and participates in the reorganization of the extracellular matrix by activating the integrin signaling pathway, helping to repair damaged tissues. On the other hand, MFGE8 participates in the formation of immune tolerance by promoting the clearance of apoptotic cells, reducing the release of pro-inflammatory factors, thereby inhibiting excessive inflammatory responses and affecting macrophage polarization (such as transformation into the anti-inflammatory M2 phenotype). 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] The MFGE8 protein is a multifunctional molecule whose core role is to mediate the clearance of apoptotic cells and regulate immune inflammatory responses, and its abnormal expression is related to various diseases. The MFGE8 protein is abnormally expressed in breast cancer patients, so it can be used as a potential biomarker for breast cancer, and the preparation of MFGE8 antibodies can be used for the early diagnosis and screening of breast cancer.

[0004] The methods for preparing MFGE8 monoclonal antibodies mainly include traditional hybridoma technology, phage display technology, transgenic animal technology, etc. Although traditional hybridoma technology is mature and hybridoma cells producing MFGE8 antibodies can be obtained through screening and cloning, the antibody affinity may decline during the passage of hybridoma cells. In addition, the process of preparing hybridoma cells has a long cycle, heavy workload, and is difficult for humanization modification. The MFGE8 antibodies prepared by transgenic animal technology are humanized or fully human antibodies with low immunogenicity, which are not easily recognized and cleared by the immune system in vivo and can bind to the MFGE8 antigen more stably. However, the construction of transgenic animals is difficult, costly, and time-consuming. Phage display technology can construct a large display library, such as murine phage display library, rabbit phage display library, human phage display library, etc., and there are more opportunities to screen antibodies with high affinity and high specificity for the MFGE8 antigen. Moreover, antibodies can be screened and modified in vitro and applied to diagnosis and detection.

[0005] Therefore, it is urgent 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, its preparation method and application. The nucleotide sequences of the heavy and light chain variable regions of the MFGE8 monoclonal antibody are obtained by constructing a rabbit phage library, panning, monoclonal screening and identification. Then, the obtained nucleotide sequences of the heavy and light chain variable regions are subcloned into an expression vector containing the nucleotide sequence of the IgG constant region respectively to construct a complete IgG antibody expression vector, which is introduced into eukaryotic cells for expression and purification. Five monoclonal antibodies with strong affinity are screened out through ELISA experiments. The present invention also pairs the five monoclonal antibodies in pairs to screen the antibody pair with the strongest antigen-binding ability, and uses the antibody pair to prepare an MFGE8 ELISA detection kit for quantitatively detecting the content of MFGE8 in human biological samples to assist in the early diagnosis and screening of breast cancer.

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

[0008] The two MFGE8 monoclonal antibodies provided by the present invention are prepared by constructing a rabbit phage display library. 22 phage clones screened from the rabbit phage display library are sequenced, their antibody gene sequences are analyzed, and the nucleotide sequences of the antibodies are determined. The specific antibody genes screened are subcloned into a suitable expression vector and transformed into a mammalian cell expression system for expression. After purification, 22 MFGE8 monoclonal antibodies with high purity (purity up to 98%) are obtained. Then, experiments such as indirect ELISA detection and pairwise antibody pairing detection are carried out, and finally a pair of antibodies with strong specificity, high affinity and high stability, namely the first antibody and the second antibody, are screened out. The antibody pair is made into a detection kit for detecting the content of MFGE8, which is a potential biomarker for breast cancer, in a sample. The antibody pair does not cross-react with other substances in the sample and plays an important role in the early screening and diagnosis of breast cancer.

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

[0010] Further, 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 as shown in SEQ ID NO.9, and the amino acid sequence of the light chain signal peptide is as 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 as shown in SEQ ID NO.13, and the amino acid sequence of the light chain constant region is as 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 effectively guide protein secretion, and if it is too long, it may affect protein folding or subsequent processing. The amino acid sequences of the heavy and light chain signal peptides provided by the present invention are both 19 amino acids.

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

[0013] It is understandable that IgG has four subtypes, namely IgG1, IgG2, IgG3, and IgG4. The constant region sequences of their heavy and light chains can all serve as the constant regions of the heavy and light chains of the first antibody, the second antibody, and the third antibody. However, their abilities to activate complement and bind to Fc receptors are different. The MFGE8 monoclonal antibody prepared in the present invention is used for detection and diagnosis. Therefore, the constant regions of the heavy and light chains of IgG1 are selected because in various detection environments, the constant region of IgG1 exhibits good adaptability, which helps to maintain the conformational stability of the entire antibody molecule. Whether detecting low-concentration antigens or in complex biological samples (such as blood, tissue homogenates), it enables the variable region to better perform the function of recognizing and binding antigens, reduces cross-reactions with irrelevant substances in the sample, thereby improving the specificity of detection, accurately capturing the target antigen in the detection, and reducing the occurrence of false-negative results. For example, in a cell-based detection system, the constant region of IgG1 can bind to the Fc receptor, and by means of the signal transduction mediated by the Fc receptor, the detection signal is amplified, improving the detection sensitivity. Another example is in ELISA detection. The structure of IgG1 is relatively stable. Under different environmental conditions, such as different pH values, temperatures, etc., compared with other antibody subclasses, IgG1 is less likely to denature or degrade, and can maintain its activity and antigen-binding ability during various operation steps of ELISA detection, ensuring the reliability of the detection results. In addition, IgG1 can bind well to commonly used enzyme labels, fluorescent labels, etc. in ELISA detection, and after binding, it will not significantly affect its own antigen-binding activity and the activity of the label, which is conducive to subsequent signal detection and amplification. In addition, current biotechnological means can efficiently produce IgG1 antibodies in a variety of expression systems, such as Chinese hamster ovary cells (CHO cells), human embryonic kidney cells (HEK293 cells, HEK293T cells), etc. These expression systems are mature and stable, enabling large-scale culture and high-yield expression of IgG1 antibodies, meeting the needs of scientific research and clinical detection for a large number of antibodies.

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

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

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

[0017] On the other hand, the present invention provides a method for preparing the above-mentioned MFGE8 monoclonal antibody, comprising the following steps:

[0018] (1) Immunizing an animal with the MFGE8 antigen;

[0019] (2) Collecting the spleen or bone marrow of the immunized animal to construct a phage library;

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

[0021] (4) Subcloning the nucleotide sequences of the heavy chain variable region and the light chain variable region of the SCFV into an expression vector containing a signal peptide and a constant region respectively;

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

[0023] In yet another aspect, the present invention provides the use of an MFGE8 antibody composition in the preparation of a reagent for enhancing the ability to bind to the MFGE8 protein. The antibody composition includes a first antibody and a second antibody. The amino acid sequence of the heavy chain variable region of the first antibody is as shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.2; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.4.

[0024] In some embodiments, the present invention finally prepares 22 MFGE8 monoclonal antibodies by constructing a phage library. The binding ability of the purified 22 monoclonal antibodies to the MFGE8 antigen is detected by indirect ELISA. It is found that only 5 antibodies bind to the antigen. Then, the 5 antibodies are paired pairwise, and their binding ability to the MFGE8 antigen is detected by the double antibody sandwich method. Finally, two pairs of antibody pairs with strong antigen-binding ability are screened out (one pair is the first antibody and the second antibody).

[0025] In yet another aspect, the present invention provides an 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] Further, the kit is an ELISA detection kit.

[0028] Further, 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] Further, the ELISA detection kit also includes an avidin-biotin-peroxidase complex (ABC) solution, a sample dilution buffer, an antibody dilution buffer, an ABC dilution buffer, a TMB chromogenic solution, a TMB stop solution, and a washing buffer.

[0030] The preparation method of the capture antibody reagent is to dilute the MFGE8 monoclonal antibody (the first antibody) with an antibody dilution buffer to the working concentration; the detection antibody (the second antibody) in the detection antibody reagent needs to be prepared into a biotinylated antibody.

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

[0032] Further, the present invention provides the use of the above-mentioned MFGE8 detection kit in products for improving the efficacy of breast cancer diagnosis.

[0033] In some cases, the MFGE8 detection kit is used to detect serum samples of healthy people and breast cancer patients, and the detection results are used to make a receiver operating characteristic curve (ROC) through software and calculate the AUC value. An AUC value > 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, namely the first antibody and the second antibody, are prepared and screened by immunizing rabbits with MFGE8 and constructing a rabbit phage library. They have strong specificity, high affinity, and high stability.

[0036] 2. The first antibody and the second antibody not only have strong ability to specifically bind to MFGE8 and good affinity, but also do not cross-react with other cytokines (<1%).

[0037] 3. The first antibody and the second antibody are used to prepare an MFGE8 ELISA kit for quantitatively detecting the 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 > 0.85. Description of the Drawings

[0038] Figure 1 RNA electrophoresis map of spleen and bone marrow after rabbit immunization in Example 1;

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

[0040] Figure 3 Electrophoresis map of scFv in Example 1;

[0041] Figure 4 Electrophoresis map of phage vector bComb3x into which the scFv fragment has been inserted in Example 1;

[0042] Figure 5 Electrophoresis map of antibody GR-8 after treatment with 4× loading buffer (reducing / non-reducing) in Example 2;

[0043] Figure 6 Binding curve of antibody GR-8 in Example 2. Detailed Description of the Invention

[0044] The present invention will be further described in detail below with reference to the drawings and embodiments. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0045] The reagents, consumables and experimental instruments used in the following embodiments 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 Monoclonal Antibody Against MFGE8

[0051] 1. Animal Immunization

[0052] Two Japanese white rabbits were immunized. The animal numbers were 1 and 2. The antigen dose for each rabbit was 500 μg. For the first immunization, the mouse MFGE8 antigen was made into an emulsifier with an equal amount of complete Freund's adjuvant and injected subcutaneously at multiple points on the back. After 2 weeks, an emulsifier was made with the same dose of immunogen and an equal amount of incomplete Freund's adjuvant, and immunized four times. Then, the serum titer was measured 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, 3 - 4 mL of blood was taken from the marginal ear vein of the rabbit. After standing overnight at 4 °C, the upper serum was separated by centrifugation at 1500 g for 15 min for inspection;

[0054] (2)Coating: Appropriate amount of mouse MFGE8 antigen used for detection was diluted to 0.1 μg / mL, 1 μg / mL, and 5 μg / mL with coating buffer respectively. Then, 100 μL was added to each well of a 96 - well plate using a pipette. The plate was tapped gently to mix the samples, sealed with plastic wrap, and coated overnight at 4 °C;

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

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

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

[0058] (6)Adding samples: The serum samples in step (1) were serially diluted with sample diluent and added at 100 μL / well;

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

[0060] (8)Washing the plate: Then wash the plate five times with 200 μL / well of washing solution and drain the enzyme - labeled plate;

[0061] (9)Color development: Add the chromogenic solution at 200 μL / well and place at room temperature for 12 min;

[0062] (10)Termination and detection: Add the termination solution at 50 μL / well to terminate the reaction, then detect with an enzyme - labeled instrument. The measurement wavelength is 450 nm, and the relative OD450 value is calculated. Relative OD450 = detected OD450 - blank control.

[0063] The serum titer detection 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, taking (absorbance value of serum after the fourth immunization - absorbance value of blank) / (absorbance value of negative control serum before immunization - absorbance value of blank) > 2.1 as the positive standard, the results showed that the serum titers of both rabbits reached 1:128000 after four immunizations. By comparison, the serum titer of the rabbit numbered 2 was higher. Therefore, the spleen and bone marrow of the rabbit numbered 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 the rabbit numbered 2 were taken, and TriPure Isolation Reagent was used to extract the RNA of the spleen and bone marrow. The RNA electrophoresis pattern is as Figure 1 shown. The extracted RNA was quantitatively analyzed by UV, 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. UV quantitative results of RNA from spleen and bone marrow

[0070]

[0071] The phage library of the present invention was constructed to rapidly obtain specific antibodies with high affinity after immunization. Therefore, the spleen was preferably used for the next experiment.

[0072] Furthermore, a self-made reverse transcription kit was used for reverse transcription to obtain the cDNA of the spleen. The light chain variable region sequence and heavy chain variable region sequence of the cDNA were amplified by PCR. 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) sequence and heavy chain variable region (VH) sequence of the cDNA were amplified by PCR. The electrophoresis results are as Figure 2 shown. The amplified VL sequence and VH sequence were spliced into a nucleotide sequence encoding scFv by the overlap extension splicing PCR method. The electrophoresis results of the spliced scFv are as Figure 3The scFv was then 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 in Figure 4 Then, the phage vector bComb3x with the scFv fragment inserted was electroporated into the X-Blue competent medium to construct the 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 original antibody library was prepared by infecting the primary antibody library with auxiliary phages. The specific method was as follows: the mouse MFGE8 antigen was coated on an ELISA plate, the original antibody library was added to the ELISA plate, the non-specifically bound phages were first washed away with a washing solution, and the phages bound to the antigen were then eluted with an elution solution, and the next round of enrichment screening was performed after amplification and precipitation in Escherichia coli. After 2-4 rounds of "adsorption-elution-amplification", the positive library bound to MFGE8 was screened. The binding of library A and library B after multiple rounds of solid phase screening to the mouse MFGE8 antigen was detected by ELISA, and 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 detected by ELISA. They were then coated with mouse tumor necrosis factor receptor superfamily member 19 (mTNFRSF19) to detect cross-reaction. The detection 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, it shows that the scFv antibodies expressed by 22 positive monoclonal phages all specifically bind to MFGE8 and do not cross-react with mTNFRSF19. The nucleotide sequences of 22 groups of scFv were obtained by sending them to a sequencing company for sequencing.

[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 scFv. 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 Table 10 - Table 11 above to obtain the heavy and light chain variable region fragments of the scFv antibody. After electrophoresis, the gel was cut and recovered. The recovered heavy and light chain variable region fragments were added to the membrane-binding solution, thoroughly mixed, and then added to the purification column. After centrifugation at 13000 rpm for 1 min, the liquid was discarded. 700 μL of wash buffer was added, and after centrifugation at 13000 rpm for 1 min, the liquid was discarded. After spinning dry for 5 min and standing at room temperature for 5 min, Nuclease-Free Water was added for elution to obtain the purified heavy and light chain variable region sequence fragments.

[0095] Furthermore, signal peptide sequences and constant region sequences were respectively added to the heavy and light chain variable region fragments by PCR method to construct complete heavy chain nucleotide sequences and light chain nucleotide sequences. The amino acid sequence of the heavy chain signal peptide is shown in SEQ ID NO.9, and the nucleotide sequence is shown in SEQ ID NO.11. The amino acid sequence of the light chain signal peptide 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 murine IgG1. The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.13, and the nucleotide sequence is shown in SEQ ID NO.15. The amino acid sequence of the light chain constant region 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 respectively ligated to the pUC expression vector plasmid. The specific method is as follows: 4 μL of the purified complete heavy chain nucleotide sequence / complete light chain nucleotide sequence, 1 μL of the vector, and 5 μL of ligase were added and mixed well. After reacting at 50 °C for 20 min, it was added to 100 μL of competent cells, ice-bathed for 30 min, heat-shocked for 90 s, quickly placed on ice for 3 min, and then plated. After overnight culture at 37 °C, the obtained monoclonal was subjected to PCR amplification and then sequenced the next day. It was known through software comparison that the expression vector plasmid had been successfully ligated with the specific gene.

[0096] Furthermore, 22 kinds of vector plasmids ligated with heavy chains and 22 kinds of vector plasmids ligated with light chains were paired with each other and transferred to HEK 293 cells for transient expression. The specific method is as follows: HEK293 cells were passaged and cultured with 293 serum-free CD medium. The plasmid DNA to be expressed was mixed with the transfection reagent TF2 and then added to the cells. 293 serum-free feeding solution was added on the 1st, 3rd, and 5th days after transfection. The shaking flask culture conditions were: 5% CO2, temperature 37 °C, and shaking speed 175 rpm. The supernatant was taken on the fifth day of cell culture for ELISA detection. The specific method is as follows:

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

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

[0099] (3) Washing the plate: 300 μL / well washing solution, wash the plate 2 times, and pat dry for the last time;

[0100] (4) Antibody dilution: Take the supernatants of 22 positive clone cells for 2-fold dilution, add 100 μL each to the corresponding well plates, mix well, and react at room temperature for 2 h;

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

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

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

[0104] (8) Color development: Mix solution A and solution B at a ratio of 1:1, then 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, immediately measure the OD value at a wavelength of 450 nm, set three parallel wells, take the average value, and calculate the relative OD450 value. Relative OD450 = detected OD450 - blank control.

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

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

[0108]

[0109] According to the data in Table 12, it shows that the cell supernatants of 22 positive clone cells after five days of culture are all positive in the ELISA detection with mouse MFGE8 protein antigen, and all have good binding ability.

[0110] Furthermore, after 7 days of cell culture, collect the cell supernatant and purify the antibody. The specific method is as follows:

[0111] (1) Sample preparation: Centrifuge using a tabletop centrifuge at 4000 g for 30 min, and take the supernatants of 22 monoclonal cells;

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

[0113] (3) Connect the purification system: Select an appropriate size of affinity packing Protein A column according to 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 chromatography column: Equilibrate with AC Binding buffer for 3 CV until the UV baseline is stable;

[0116] (6) Sample loading: Load the sample at an appropriate flow rate.

[0117] (7) Washing: Wash with the washing solution for 5 - 10 CV until the UV baseline is stable, at the same flow rate as the sample loading flow rate.

[0118] (8) Elution: Elute with the elution solution and collect according to the UV peak.

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

[0120] (10) Equilibration: Equilibrate with AC Binding buffer for 3 CV until neutral.

[0121] (11) CIP cleaning: Clean with CIP regeneration solution for more than 5 CV.

[0122] (12) Rinsing the base: Rinse the base with AC Binding buffer until the pH at the outlet is neutral, and then rinse with ultrapure water for 3 CV.

[0123] (13) Preservation: Equilibrate with 25% ethanol for 2 CV and preserve the column.

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

[0125] Example 2. Screening of monoclonal antibodies

[0126] In this example, first, the concentrations and purities of the 22 purified monoclonal antibodies in Example 1 were detected, then their binding abilities to antigens at different concentrations were detected. Monoclonal antibodies with strong antigen-binding abilities were screened out from them, and then pairs of monoclonal antibodies with strong antigen-binding abilities were paired and screened to obtain antibody pairs with strong antigen-binding abilities.

[0127] 1. Detection of antibody concentration

[0128] Start the micro-spectrophotometer, spot with the corresponding buffer of the test sample. When the absorbance value is between ±0.015 at a wavelength of 280 nm, it indicates that the instrument baseline is stable. Spot the 22 purified antibody samples in sequence, record the absorbance values, divide the detection data by the IgG extinction coefficient (1.414), and the obtained value is the concentration of the test sample, as shown in Table 13 below.

[0129] Table 13. Concentrations of 22 purified antibodies

[0130]

[0131] 2. Detection of antibody purity

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

[0133] (1)Treat the samples: Take 5 μg of each 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) respectively. Heat in a water bath at 100 °C and then centrifuge at 10000 rpm;

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

[0135] (3)Fix the SDS-PAGE gels prepared in (2) in the 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, first electrophorese at a constant voltage of 100 V until the bromophenol blue dye enters the separating gel from the stacking gel, then adjust the current to 140 V and continue electrophoresis until the bottom of the gel plate, and then turn off the power supply;

[0137] (5)Staining: Immerse the gel in Coomassie Brilliant Blue R-250 staining solution, heat in a microwave oven for 60 s, stain at room temperature on a gently shaking platform for 2 - 4 h, then replace with decolorizing solution to cover the gel, heat in a microwave oven for 60 s, and decolorize at room temperature on a gently shaking platform. Repeat the decolorizing 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 all above 95%. Taking antibody GR-8 as an example, its electrophoresis patterns after being treated with 4× loading buffer (reducing) and 4× loading buffer (non-reducing) respectively are as Figure 5 shown. The results show that the purity of antibody GR-8 is very high, with almost no other impurity bands, and the purity is as high as 98.0%.

[0139] 3. Binding ability of the purified antibody 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 respectively, 100 μL / well, and coat overnight at 4 °C;

[0142] (2)Blocking: Discard the liquid in the plate and pat it dry. Add 2% BSA, 300 μL / well, incubate for 1 h at room temperature after sealing;

[0143] (3)Washing the plate: Add 300 μL / well of washing solution, wash the plate 2 times, and pat it dry for 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 the plate: Add 300 μL / well of washing solution, wash the plate 3 times, and pat it dry for the last time;

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

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

[0148] (8)Color development: Mix solution A and solution B at 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, immediately measure the OD value at a wavelength of 450 nm, set three parallel wells, take the average value, and calculate the relative OD450 value. Relative OD450 = detected OD450 - blank control.

[0150] The results showed that only 5 out of 22 purified antibodies could bind to mouse MFGE8 antigen coated at different concentrations, namely GR-3, GR-5, GR-8, GR-14, and GR-17. The binding detection results of them to mouse MFGE8 antigen coated at different concentrations are shown in Table 14 below.

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

[0152]

[0153] Furthermore, the affinity of the above 5 selected monoclonal antibodies was detected by ELISA, and the detection method was as follows:

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

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

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

[0157] (4)Antibody dilution: Dilute the antibody to 300 ng / mL, and then serially dilute it 10-fold in triplicate to 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, 0.01 ng / mL. Use the sample diluent as the blank control. Add 100 μL / well to the corresponding wells of the plate, mix well, and react at room temperature for 2 h;

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

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

[0160] (7)Washing the plate: Add 300 μL / well of washing solution, wash the plate three times, and pat it dry for 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, immediately measure the OD value at a wavelength of 450 nm, set two parallel wells, take the average value, and calculate the relative OD450 value. Relative OD450 = measured OD450 - blank control.

[0163] The detected values were input into GraphPad Prism software to calculate the affinity. The results showed that the affinities of the 5 monoclonal antibodies with the antigen were all very high. They could exert their biological activities at low concentrations, and the 5 antibodies could be applied to immunoassays to assist in the diagnosis of diseases. The EC50 values of the affinities of the 5 monoclonal antibodies GR-3, GR-5, GR-8, GR-14, and GR-17 were 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 affinities of the 5 monoclonal antibodies from high to low were GR-17 > GR-8 > GR-3 > GR-5 > GR-14. Taking the monoclonal antibody GR-8 as an example, the results of its affinity detection are shown in Table 15 and Figure 6 as follows.

[0164] Table 15. Detection results of the affinity of monoclonal antibody GR-8

[0165]

[0166] 4. Screening of antibody pairs

[0167] The 5 high-affinity monoclonal antibodies were paired in pairs and used as capture antibodies and detection antibodies respectively. The ability of the antibody pairs to bind to the MFGE8 antigen was detected by the double antibody sandwich method to screen out the best antibody pairs. The detection method is as follows:

[0168] (1) Coating of capture antibody: Monoclonal antibodies GR-3, GR-5, GR-8, GR-14, and GR-17 were coated respectively at 1 μg / mL, 100 μL / well, and coated overnight at 4 °C;

[0169] (2) Blocking: The liquid in the plate was drained and patted dry, 2% BSA, 300 μL / well, incubated at room temperature for 1 h after sealing;

[0170] (3) Washing the plate: 300 μL / well washing solution, wash the plate 2 times, and pat dry for the last time;

[0171] (4) Adding antigen: Mouse MFGE8 was diluted to 100 ng / mL, and 100 μL / well was added to the corresponding well plate, mixed evenly, and reacted at room temperature for 2 h;

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

[0173] (6) Adding detection antibody: Monoclonal antibodies GR-3, GR-5, GR-8, GR-14, and GR-17 were added respectively at 1 μg / mL, 100 μL / well;

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

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

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

[0177] (10) Color development: After mixing 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, immediately measure the OD value at a wavelength of 450 nm, set two parallel wells, take the average value, and calculate the relative OD450 value. Relative OD450 = detected OD450 - blank control.

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

[0180] Table 16. Detection Results of Monoclonal Antibody Pairing

[0181]

[0182] According to the data in Table 16, two pairs of antibodies were screened with the best ability to bind antigens. One pair is GR-8 (as the capture antibody) and GR-3 (as the detection antibody), and the other pair is GR-17 (as the capture antibody) and GR-5 (as the detection antibody). Among them, the amino acid sequence of the heavy chain variable region of GR-8 is shown in SEQ ID NO.1, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2, the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.5, the nucleotide sequence of the light chain variable region 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 amino acid sequence of the heavy chain variable region of GR-3 is shown in SEQ ID NO.3, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4, the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.7, the nucleotide sequence of the light chain variable region 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 amino acid sequence of the heavy chain variable region of GR-17 is shown in SEQ ID NO.25, the amino acid sequence of the light chain variable region 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.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, the thermal stability Tm value of the monoclonal antibodies obtained by screening was measured by circular dichroism (CD) to evaluate the stability of the monoclonal antibodies and compare them with the existing monoclonal antibodies. The existing MFGE8 monoclonal antibody was purchased from Thermo Fisher Scientific, catalog number MA5-23913. The test results are shown in Table 17 below. Tm therein represents the midpoint temperature of protein thermal denaturation, that is, the temperature at which 50% of the protein unfolds, reflecting the trend of protein conformational change during the temperature change process. The higher the Tm, the better the 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 in this example were all higher than those of the existing MFGE8 monoclonal antibody, indicating that the screened MFGE8 monoclonal antibodies have better stability.

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

[0188] In this example, two pairs of antibodies screened in Example 2 (one pair is GR-8 (as the capture antibody) and GR-3 (as the detection antibody), and the other pair is GR-17 (as the capture antibody) and GR-5 (as the detection antibody)) were respectively made into MFGE8 ELISA detection kits. And MFGE8 ELISA detection kits were made using GR-8 and GR-17 as capture antibodies respectively and an existing MFGE8 monoclonal antibody (purchased from Thermo Fisher Scientific, catalog number MA5-23913) as the detection antibody. The MFGE8 ELISA detection kits and their corresponding capture antibodies and detection antibodies are shown in Table 18 below.

[0189] Table 18. MFGE8 ELISA Detection Kits and Their Corresponding Capture Antibodies and Detection Antibodies

[0190]

[0191] 1. Preparation of MFGE8 ELISA Detection Kit

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

[0193] (1) Antibody dilution buffer: 0.01 mol / L PBS with pH 7.2. Weigh 0.39 g of NaH2PO4·2H2O, 1.27 g of Na2HPO4, 0.85 g of NaCl, and 200 mg of 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 well to dissolve. Then add concentrated hydrochloric acid to adjust the pH to 7.2 - 7.4, add 5 g of BSA, and finally make up 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 about 800 mL of distilled water and stir well to dissolve. Adjust the pH to 7.4 with hydrochloric acid, add 0.5 mL of Tween-20 and 10 g of BSA, and make up the volume 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 approximately 800 mL of distilled water and stir well to dissolve. Adjust the pH to 7.4 with hydrochloric acid, add 0.5 mL of Tween-20, and make up the volume to 1 L.

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

[0198] (6)Preparation of detection antibody reagent: a. Pretreatment of MFGE8 monoclonal antibody: Dilute GR-3 / GR-5 / existing MFGE8 monoclonal antibody with antibody dilution buffer to a concentration of 1 mg / mL; b. Activation of biotin reagent: Dissolve N-hydroxysuccinimide biotin (NHS-biotin) in an appropriate amount of DMSO to prepare a 5 mg / mL solution; c. Preparation of biotinylated MFGE8 antibody: Under stirring or shaking conditions, slowly add the activated biotin reagent dropwise to the antibody solution. The molar ratio of biotin to antibody is 5:1. The reaction is carried out at 4 °C for 4 hours. After the reaction is completed, add an appropriate amount of 0.1 M glycine solution to terminate the reaction. Glycine reacts with the remaining active biotin reagent to block the unreacted active sites. Dialyze through antibody dilution buffer to remove unreacted biotin reagent, organic solvents, and other small molecule impurities to obtain biotinylated MFGE8 antibody and dilute it to a concentration of 1 μg / mL.

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

[0200] (8)Preparation of avidin-biotin-peroxidase complex (ABC) solution: Dilute ABC solution with ABC dilution buffer to 1 μg / mL.

[0201] (9)Preparation of TMB chromogenic solution: The same as the chromogenic solution in the ELISA detection of Example 1.

[0202] (10)Preparation of TMB termination solution: The same as the termination solution in the ELISA detection of Example 1.

[0203] Combine the above reagents to prepare the four MFGE8 ELISA detection kits 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 the capture antibody reagent to the microplate and incubate at 4 °C overnight for coating.

[0206] (2) Blocking: Drain the liquid in the plate and pat dry. Add 300 μL / well of 2% BSA, seal and incubate at room temperature for 1 h.

[0207] (3) Sample pretreatment: 1) Serum samples: Let the serum clot in the serum separator tube at room temperature (about 4 h), centrifuge at about 1000×g for 15 min and measure immediately. 2) Plasma samples: Collect plasma using heparin or EDTA as anticoagulant, centrifuge at 1000×g for 15 min and measure immediately.

[0208] (4) Add standards and samples: Add sample dilution buffer (control wells), samples and seven concentrations of MFGE8 standards to the microplate, 100 μL / well, seal the microplate and incubate at 37 °C for 90 min.

[0209] (5) Use a tissue or absorbent material to blot the liquid in the microplate wells, being careful not to completely dry the wells. Add 100 μL / well of the detection antibody solution, seal the microplate and incubate at 37 °C for 60 min.

[0210] (6) Wash the plate 3 times with 300 μL / well of wash buffer and pat dry for the last time.

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

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

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

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

[0215] The above four kits detect four standard curves by using MFGE8 standards. The standard curves adopt the fitting 4-parameter Logistic curve, and calculate the MFGE8 concentration in the samples through the standard curves. The R of the standard curves of the four kits 2All are ≥ 0.99, indicating that it is suitable for quantitative analysis. Taking Kit 1 (capture antibody is GR-8 and detection antibody is GR-3) as an example, its fitted four-parameter Logistic curve is , R 2 = 0.999.

[0216] 2. Performance detection of the kit

[0217] Furthermore, the within-batch / between-batch precision of the above four kits was detected and investigated by the standard addition recovery method. The unspiked sample was the serum of healthy people, and the spiked sample was the serum of healthy people added with 5 ng / mL of MFGE8 standard. Recovery rate = (detected concentration of spiked sample - detected concentration of unspiked sample) / spiked concentration. The detection results are shown in Table 19 below.

[0218] Table 19. Within-batch / between-batch precision of MFGE8 detection kits

[0219]

[0220] According to the data in Table 19, the within-batch / between-batch CV values of Kit 1 (capture antibody is GR-8 and detection antibody is GR-3) and Kit 2 (capture antibody is GR-17 and detection antibody is GR-5)) are smaller, and the recovery rates are closer to 100%, indicating that the MFGE8 antibodies prepared and screened in the present invention are more stable and accurate for the detection of the prepared Kits 1 and 2. In contrast, the detections of Kits 3 and 4 prepared with the existing MFGE8 monoclonal antibodies using GR-8 and GR-17 as capture antibodies are unstable and inaccurate. Therefore, Kit 1 and Kit 2 are preferably selected for the MFGE8 ELISA detection kit.

[0221] Furthermore, the cross-reactivities of the above-preferred Kits 1 and 2 with factors such as M-CSF, TNF-α, TNF RI / II, VCAM-1, and VEGF were detected, and all were less than 1%.

[0222] Example 4. Application of MFGE8 ELISA detection kit in tumor diagnosis

[0223] The expression of MFGE8 is up-regulated in breast cancer, so it can be used as a potential diagnostic marker for breast cancer diagnosis.

[0224] In this example, Kits 1-4 prepared in Example 3 were used to test MFGE8 in the sera of healthy subjects and breast cancer patients respectively, and their abilities to distinguish diseased populations were evaluated respectively.

[0225] Collect venous blood from healthy subjects (100 cases) and breast cancer patients (100 cases) from the same hospital as the test group, 5 mL per case. Centrifuge to separate serum (3000 rpm, 15 min), aliquot and store at -80 °C for later use. Then collect venous blood from healthy subjects (80 cases) and breast cancer patients (120 cases) from another hospital as the validation group, 5 mL per case. Centrifuge to separate serum (3000 rpm, 15 min), aliquot and store at -80 °C for later use. Independently detect the samples of the test group and the validation group using kits 1-4, record the absorbance values and calculate the concentration to detect the content of MFGE8 in the serum. The cut-off value of the MFGE8 content for distinguishing breast cancer patients is 80 ng / mL. If it is greater than or equal to 80 ng / mL, it is judged as a breast cancer patient; if it is less than 80 ng / mL, it is judged as a healthy person. At the same time, evaluate the ability of the four kits to distinguish diseased populations using SPSS or R language respectively, that is, make the receiver operating characteristic curve (ROC) and calculate the AUC value. The AUC values, sensitivity (true positive rate), and specificity (true negative rate) of the four kits for detecting the test group and the 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 the detected AUC values of Kit 1 for both the test group and the validation group are greater than or equal to 0.85, indicating that it can better distinguish patients from healthy people. In contrast, the diagnostic efficacy of Kit 2 is slightly worse than that of Kit 1, while the diagnostic efficacies of Kit 3 and Kit 4 are poor. Therefore, Kit 1 (the capture antibody is GR-8 (i.e., the first antibody), and the detection antibody is GR-3 (i.e., the second antibody)) as the best MFGE8 ELISA detection kit can be used for the early diagnosis and screening of breast cancer.

[0229] Existing antibodies not only have low detection sensitivity, but even whether different antibodies are used as detection antibodies or enzyme-labeled antibodies, and the detection results will also be significantly different. In addition, there are obvious differences in different antibodies in terms of stability (Tm), cross-reaction with the matrix, high-dose hook effect, etc. All these reasons will lead to a decrease in the efficacy for diagnosing breast cancer.

[0230] At the same time, during the diagnosis of breast cancer, it is necessary to detect its blood samples through MFGE8 antibodies. There are complex interfering substances in the blood samples, and the matrix has a great influence. There are easily cross-reactions or interferences from some non-linear fragments. Using different antibodies for the detection and diagnosis of breast cancer will inevitably produce completely different diagnostic effects. Therefore, a better antibody combination must be selected to improve the diagnostic efficacy.

[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 protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

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

4.

2. The MFGE8 monoclonal antibody according to claim 1, characterized in that, The nucleotide sequence of the heavy chain variable region of the first antibody is as shown in SEQ ID NO.5, and the nucleotide sequence of the light chain variable region is as shown in SEQ ID NO.6; the nucleotide sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.7, and the nucleotide sequence of the light chain variable region is as shown in SEQ ID NO.

8.

3. The MFGE8 monoclonal antibody according to claim 1, characterized in that, 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 as shown in SEQ ID NO.9, and the amino acid sequence of the light chain signal peptide is as 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 as shown in SEQ ID NO.13, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO.

14.

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

20.

5. The MFGE8 monoclonal antibody according to claim 2, characterized in that, The nucleotide sequence of the heavy chain of the first antibody is as shown in SEQ ID NO.21, and the nucleotide sequence of the light chain is as shown in SEQ ID NO.22; the nucleotide sequence of the heavy chain of the second antibody is as shown in SEQ ID NO.23, and the nucleotide sequence of the light chain is as shown in SEQ ID NO.

24.

6. The preparation method of the MFGE8 monoclonal antibody according to any one of claims 1-5, characterized in that, It includes the following steps: (1) Immunize an animal with the MFGE8 antigen; (2) Collect the spleen or bone marrow of the immunized animal to construct a phage library; (3) Obtain the SCFV from the phage library through screening and enrichment, and sequence to obtain the nucleotide sequence of the SCFV; (4) Respectively clone the nucleotide sequences of the heavy chain variable region and the light chain variable region of the SCFV into an expression vector containing a signal peptide and a constant region through subcloning; (5) Introduce the expression vector into eukaryotic cells for expression and purification to obtain the MFGE8 monoclonal antibody.

7. Use of an MFGE8 antibody composition in the preparation of a reagent for enhancing the ability to bind to MFGE8 protein, characterized in that, The antibody composition includes a first antibody and a second antibody. The amino acid sequence of the heavy chain variable region of the first antibody is as shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.2; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.

4.

8. An MFGE8 detection kit, characterized in that, It includes the MFGE8 monoclonal antibody according to any one of claims 1-5.

9. The MFGE8 detection kit according to claim 8, wherein The kit is an ELISA detection kit.

10. Use of the MFGE8 detection kit according to claim 8 or 9 in a product for improving the efficacy of breast cancer diagnosis.

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