A cd93 monoclonal antibody and a preparation method and application thereof

Abstract

The application provides a CD93 monoclonal antibody and a preparation method and application thereof, the heavy chain and light chain variable region nucleotide sequences of the CD93 monoclonal antibody are obtained by screening and enrichment through constructing a rabbit phage library, then the obtained heavy chain and light chain variable region nucleotide sequences are respectively subcloned into an expression vector containing an IgG1 constant region nucleotide sequence, an entire IgG1 antibody expression vector is constructed and introduced into a eukaryotic cell for expression and purification, and the CD93 monoclonal antibody is obtained; three strains of CD93 monoclonal antibodies with the strongest affinity are screened out through ELISA detection. The application further pairs the three strains of screened monoclonal antibodies two by two to screen out an antibody pair with strong antigen binding capacity, and prepares a CD93 ELISA kit from the antibody pair and uses the antibody pair for quantitative detection of the CD93 content in a human biological sample, thereby assisting in the diagnosis and prognosis evaluation of hepatocellular carcinoma.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and in particular relates to a CD93 monoclonal antibody and a preparation method and application thereof. BACKGROUND

[0002] CD93 protein is a transmembrane glycoprotein expressed on endothelial cells, monocytes, macrophages, platelets and some stem cells, and has multiple physiological functions. It mainly participates in angiogenesis, cell adhesion and regulation of inflammatory response in vivo. It promotes the proliferation and migration of vascular endothelial cells, helping the formation of new blood vessels, which is particularly crucial in tumor development, and can enhance tumor blood supply and metastasis ability. At the same time, CD93 can regulate immune cell activity and affect the inflammatory microenvironment. In various cancers such as liver cancer, its abnormally high expression is closely related to tumor malignancy and poor prognosis, and it is both a marker of disease progression and a potential therapeutic target.

[0003] A number of clinical studies have shown that the expression of CD93 in hepatocellular carcinoma (HCC) tissue is significantly higher than that in normal liver tissue adjacent to the cancer, and its high expression is directly related to tumor size, differentiation degree, vascular invasion (a key pathway for liver cancer metastasis) and TNM stage. For example, the positive rate of CD93 is significantly increased in large tumors (diameter > 5 cm) and poorly differentiated hepatocellular carcinoma, suggesting that it is closely related to tumor invasiveness. High expression of CD93 in hepatocellular carcinoma tissue is the main source of elevated CD93 in the blood, therefore, the expression level of CD93 can effectively reflect the malignancy, metastasis risk and prognosis of the tumor, and can be used as a marker for the tumor.

[0004] Detecting the content of CD93 in the blood requires an antibody that specifically binds to it. The methods for preparing CD93 monoclonal antibodies mainly include traditional hybridoma technology, phage display technology, transgenic animal technology, etc. Although the traditional hybridoma technology is mature, it can obtain hybridoma cells producing CD93 antibodies after screening and cloning, but the hybridoma cells may have reduced antibody affinity with passage, in addition, the process of preparing hybridoma cells is long, laborious, and difficult to humanize. The CD93 antibodies prepared by transgenic animal technology are humanized or fully human antibodies, which have low immunogenicity and are not easily recognized and removed by the immune system in vivo, and can more stably bind to CD93 antigens, but the construction of transgenic animals is difficult, costly and time-consuming. The phage display technology can construct a large display library, such as a murine phage display library, a rabbit phage display library, and a human phage display library, etc., which has more opportunities to screen antibodies with high affinity to CD93 antigens, and can further improve the affinity of the antibodies by screening and modifying the antibodies in vitro.

[0005] Therefore, it is urgent to find a CD93 monoclonal antibody by phage display technology and use it to detect the content of CD93 in blood. SUMMARY

[0006] According to the defects of the prior art, the present application provides a CD93 monoclonal antibody and a preparation method and application thereof, the heavy chain and light chain variable region nucleotide sequences of the CD93 monoclonal antibody are obtained by screening and enrichment through constructing a rabbit phage library, then the obtained heavy chain and light chain variable region nucleotide sequences are respectively subcloned into an expression vector containing an IgG1 constant region nucleotide sequence, an entire IgG1 antibody expression vector is constructed and introduced into eukaryotic cells for expression and purification to obtain the CD93 monoclonal antibody; three strains of CD93 monoclonal antibodies with the strongest affinity are screened out through ELISA detection. The present application further pairs the three strains of screened monoclonal antibodies two by two to screen out an antibody pair with strong antigen binding capacity, and prepares a CD93 ELISA kit from the antibody pair and uses it to quantitatively detect the content of CD93 in human biological samples, thereby assisting the diagnosis and prognosis evaluation of hepatocellular carcinoma.

[0007] In one aspect, the present application provides a CD93 monoclonal antibody, including a first antibody, a second antibody and a third antibody; the heavy chain variable region amino acid sequence of the first antibody is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2; the heavy chain variable region amino acid sequence of the second antibody is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4; the heavy chain variable region amino acid sequence of the third antibody is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 6.

[0008] The three CD93 monoclonal antibodies provided by the application are prepared by constructing a rabbit phage display library. The total RNA is extracted from the spleen or bone marrow of a rabbit immunized with CD93, and then the cDNA is synthesized by using the mRNA as a template through reverse transcription PCR (RT-PCR) technology. The gene fragments encoding the variable regions (VH and VL) of the antibodies are linked to the coat protein genes of the phages to construct a fusion gene. Then the fusion gene is inserted into the vector of the M13 phage to form a phage display library. The constructed phage display library is transformed into the host bacteria (X-Blue competent) of Escherichia coli, and the phages can replicate and assemble in the host bacteria. In the process of growth and reproduction of the host bacteria, the antibody genes in the phage display library are amplified in large quantities along with the replication of the phages, and the antibody fragments are expressed and displayed on the surface of the phages by being fused with the coat proteins of the phages. The target antigen is fixed on the surface of an enzyme-linked immunosorbent assay plate, so that the antigen can specifically bind to the corresponding antibodies in the phage display library. The phage display library is incubated with the solid phase carrier coated with the antigen, so that the antibody fragments displayed on the surface of the phages specifically bind to the antigen. The unbound phages are removed by the washing step, and the phages specifically bound to the antigen are retained. The phages bound to the antigen are eluted by using a low-pH buffer, and then the eluted phages are used to infect fresh host bacteria for amplification culture. After 2-4 rounds of screening-elution-amplification process, the phages that can specifically bind to the antigen are highly enriched. The 16 phage clones screened are sequenced, and the antibody gene sequences are analyzed to determine the nucleotide sequences of the antibodies. The antibody genes with specificity screened are subcloned into a suitable expression vector and transformed into a mammalian cell expression system for expression. Then the expressed antibodies are purified by affinity chromatography to obtain 16 CD93 monoclonal antibodies with high purity. The 16 CD93 monoclonal antibodies purified are verified by enzyme-linked immunosorbent assay (ELISA) for function, and the binding specificity and affinity of the antibodies to the antigen are detected. Finally, the three antibodies with the strongest specific binding capacity and the strongest affinity are selected as the first antibody, the second antibody and the third antibody, respectively. Compared with the traditional hybridoma technology, the CD93 monoclonal antibodies obtained by constructing the phage library have the characteristics of short preparation process period, small workload, easy gene modification, and higher specificity, affinity and stability of the antibodies.

[0009] Further, the heavy chain variable region nucleotide sequence of the first antibody is shown as SEQ ID NO. 7, the light chain variable region nucleotide sequence is shown as SEQ ID NO. 8; the heavy chain variable region nucleotide sequence of the second antibody is shown as SEQ ID NO. 9, the light chain variable region nucleotide sequence is shown as SEQ ID NO. 10; the heavy chain variable region nucleotide sequence of the third antibody is shown as SEQ ID NO. 11, the light chain variable region nucleotide sequence is shown as SEQ ID NO. 12.

[0010] Further, the heavy chain signal peptide nucleotide sequence of the first antibody, the second antibody and the third antibody is shown as SEQ ID NO. 15, the light chain signal peptide nucleotide sequence is shown as SEQ ID NO. 16.

[0011] The length of the signal peptide is generally about 15-30 amino acids, which is too short to effectively guide protein secretion, and too long to affect protein folding or subsequent processing. The signal peptide amino acid sequence of the heavy chain and the light chain provided by the present application is 19 amino acids.

[0012] Further, the heavy chain constant region nucleotide sequence of the first antibody, the second antibody and the third antibody is shown as SEQ ID NO. 19, the light chain constant region nucleotide sequence is shown as SEQ ID NO. 20.

[0013] The heavy chain and light chain constant region amino acid sequences are the heavy chain constant region amino acid sequence of murine IgG1 and the light chain constant region amino acid sequence of murine IgG1, respectively.

[0014] It is understood that IgG has four subtypes: IgG1, IgG2, IgG3, and IgG4. The heavy and light chain constant region sequences of all of these subtypes can serve as the heavy and light chain constant regions of the first, second, and third antibodies, but their complement activation and Fc receptor binding abilities differ. For the CD93 monoclonal antibodies prepared in the present invention, IgG1 heavy and light chain constant regions were selected for detection. 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), the variable region is better able to recognize and bind to the antigen, reducing cross-reactivity with unrelated substances in the sample, thereby improving detection specificity, accurately capturing the target antigen, and reducing the occurrence of 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 improving detection sensitivity. For example, in ELISA testing, the structure of IgG1 is relatively stable. Under different environmental conditions, such as pH and temperature, IgG1 is less susceptible to denaturation or degradation than other antibody subclasses. It can 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 methods 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.

[0015] Furthermore, the heavy chain amino acid sequence of the first antibody is shown in SEQ ID NO.27, and the light chain amino acid sequence is shown in SEQ ID NO.28; the heavy chain amino acid sequence of the second antibody is shown in SEQ ID NO.29, and the light chain amino acid sequence is shown in SEQ ID NO.30; the heavy chain amino acid sequence of the third antibody is shown in SEQ ID NO.31, and the light chain amino acid sequence is shown in SEQ ID NO.32.

[0016] Further, 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; the heavy chain nucleotide sequence of the third antibody is shown as SEQ ID NO. 25, and the light chain nucleotide sequence is shown as SEQ ID NO. 26.

[0017] In another aspect, the present application provides a preparation method of the CD93 monoclonal antibody described above, comprising the following steps:

[0018] (1) immunizing an animal with CD93 antigen;

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

[0020] (3) obtaining three SCFVs containing the variable regions of the first antibody, the second antibody and the third antibody respectively from the phage library by screening and enrichment, and sequencing to obtain the nucleotide sequences of the three SCFVs;

[0021] (4) cloning the heavy chain variable region and the light chain variable region nucleotide sequences of the three SCFVs into an expression vector containing a signal peptide and a constant region by subcloning;

[0022] (5) introducing the expression vector into eukaryotic cells for expression and purification to obtain the CD93 monoclonal antibody.

[0023] In another aspect, the present application provides a use of a CD93 monoclonal antibody composition in the preparation of a reagent for improving the binding ability to CD93 protein, wherein the antibody composition is a first antibody and a second antibody, the heavy chain variable region amino acid sequence of the first antibody is shown as SEQ ID NO. 1, and the light chain variable region amino acid sequence is shown as SEQ ID NO. 2; the heavy chain variable region amino acid sequence of the second antibody is shown as SEQ ID NO. 3, and the light chain variable region amino acid sequence is shown as SEQ ID NO. 4.

[0024] In some ways, the present application ultimately prepares 16 strains of CD93 monoclonal antibodies by the method of constructing a phage library, detects the binding ability of 22 strains of purified monoclonal antibodies to CD93 antigen by indirect ELISA, finds that only 3 antibodies have the highest affinity to the antigen, and therefore pairs the 3 antibodies by double antibody sandwich method to detect their binding ability to CD93 antigen, and finally screens three pairs of antibodies with strong antigen binding ability, two of which are the first antibody (as a detection antibody or a capture antibody) and the second antibody (as a detection antibody or a capture antibody).

[0025] In another aspect, the present application provides a CD93 detection kit, comprising the CD93 monoclonal antibody described above.

[0026] The CD93 detection kit can be in various forms, including but not limited to enzyme-linked immunoassay (ELISA) kit, chemiluminescence kit, immunofluorescence kit, etc.

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

[0028] Further, the ELISA detection kit comprises a capture antibody reagent and a detection antibody reagent.

[0029] In some modes, CD93 ELISA detection kits 1-3 are prepared. The capture antibody in the capture antibody reagent of the kit 1 is the first antibody, and the detection antibody in the detection antibody reagent is the second antibody; the capture antibody in the capture antibody reagent of the kit 2 is the third antibody, and the detection antibody in the detection antibody reagent is the first antibody; the capture antibody in the capture antibody reagent of the kit 3 is the second antibody, and the detection antibody in the detection antibody reagent is the first antibody.

[0030] In some modes, the linearity, sensitivity, accuracy, and intra- / inter-batch difference of the CD93 ELISA kits 1-3 are evaluated by experiments, and the detection results show that the kit 3 has better performance in all aspects than the other two kits.

[0031] Further, the ELISA detection kit further comprises an avidin-biotin-peroxidase complex (ABC) solution, a sample dilution buffer, an antibody dilution buffer, an ABC dilution buffer, a TMB developing solution, a TMB stopping solution, and a washing buffer.

[0032] The preparation method of the capture antibody reagent is to dilute the CD93 monoclonal antibody (the first antibody or the second antibody or the third antibody) to the use concentration with the antibody dilution buffer; and the detection antibody (the first antibody or the second antibody) in the detection antibody reagent needs to be prepared into a biotinylated antibody.

[0033] In another aspect, the present application provides a use of the CD93 detection kit described above in a product for improving the efficiency of diagnosing hepatocellular carcinoma.

[0034] In some ways, the serum samples of healthy people and hepatocellular carcinoma patients are detected by using CD93 ELISA detection kits 1-3 and two commercial kits, and the detection results are made into receiver operating characteristic curves (ROC) by software and the AUC values are calculated. The AUC values of kits 1-3 are higher than those of the two commercial kits and are greater than 0.80, indicating that kits 1-3 can better distinguish patients from healthy people and can be used for early screening and diagnosis of hepatocellular carcinoma and prognosis evaluation. However, the detection AUC value of kit 3 is higher, ≥0.85, which can reduce the probability of misjudgment (missed diagnosis or misdiagnosis). Therefore, the kit is preferably kit 3, and the detection antibody of kit 3 is the first antibody, and the capture antibody is the second antibody.

[0035] The present application has the following beneficial effects:

[0036] 1. Three CD93 monoclonal antibodies, first antibody, second antibody and third antibody, are prepared by immunizing rabbits with CD93 and constructing a rabbit phage library, which have high specificity, high affinity and high stability.

[0037] 2. The first antibody, the second antibody and the third antibody are paired to detect their binding capacity with CD93 antigen by double antibody sandwich method, and three pairs of antibodies with strong antigen binding capacity are finally screened and three CD93 ELISA kits are prepared, which have good linearity, high sensitivity, high accuracy and small batch difference.

[0038] 3. The three prepared CD93 ELISA kits are used for quantitative detection of CD93 content in human biological samples, which can better distinguish healthy people from hepatocellular carcinoma patients, and the AUC value can be ≥0.85, which can be used for early screening and diagnosis of hepatocellular carcinoma and prognosis evaluation. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 It is an indirect ELISA detection principle schematic diagram of Example 1;

[0040] Figure 2 It is a RNA electrophoresis map of spleen and bone marrow after immunization of rabbits in Example 1;

[0041] Figure 3 It is an electrophoresis of light chain variable region (VL) sequence and heavy chain variable region (VH) sequence of spleen cDNA in Example 1;

[0042] Figure 4 It is an electrophoresis map of scFv in Example 1;

[0043] Figure 5 It is an electrophoresis map of phage vector bComb3x into which scFv fragment is inserted in Example 1;

[0044] Figure 6 Structure of the expression vector plasmid for Example 2;

[0045] Figure 7 Electrophoresis map of antibody 15 of Example 2 after treatment with 4x loading buffer (non-reduced);

[0046] Figure 8 Electrophoresis map of antibody 15 of Example 2 after treatment with 4x loading buffer (reduced);

[0047] Figure 9 Binding curve of antibody 15 of Example 3. DETAILED DESCRIPTION

[0048] The application will be further described in conjunction with the accompanying drawings and examples. It should be noted that the following examples are intended to facilitate the understanding of the application and do not limit the application in any way.

[0049] The reagents, consumables and experimental instruments used in the following examples are shown in Tables 1 and 2 below.

[0050] Table 1, reagents and consumables

[0051]

[0052] Table 2, experimental instruments

[0053]

[0054] Example 1, preparation of CD93 monoclonal antibody

[0055] 1. Animal immunization

[0056] Two Japanese white rabbits were immunized, with animal numbers 1 and 2. Each rabbit was immunized with 500 μg of antigen. The CD93 immunogen was emulsified with an equal amount of complete Freund's adjuvant for the first immunization, and multiple subcutaneous injections were performed on the back. Two weeks later, the same dose of immunogen was emulsified with an equal amount of incomplete Freund's adjuvant, and immunization was performed four times. The serum titer was determined by indirect ELISA after immunization, as shown in Figure 1 The serum titer detection method is as follows:

[0057] (1) Take rabbit serum: Before the first immunization and one week after the fourth immunization, 3-4 mL of blood was taken from the rabbit ear margin vein, and the serum was separated by centrifugation at 1500 g for 15 min after overnight standing at 4 ℃.

[0058] (2) Coating: Take an appropriate amount of CD93 protein used for detection, dilute it with coating buffer to 0.1 μg / mL, 1 μg / mL and 5 μg / mL respectively, then add 100 μL to each well of the 96-well plate with a pipette, mix the sample by tapping the plate, seal it with plastic wrap, and coat it at 4 ℃ overnight;

[0059] (3) Washing plate: wash the plate once with 200 μL / well of washing solution, and remove the dry enzyme-labeled plate;

[0060] (4) Blocking: block the enzyme-labeled plate with 300 μL / well of blocking solution at room temperature for 1 h;

[0061] (5) Washing plate: wash the plate twice with 300 μL / well of washing solution, and remove the dry enzyme-labeled plate;

[0062] (6) Sample addition: dilute the serum sample of step (1) with sample diluent and add 100 μL / well;

[0063] (7) Addition of secondary antibody: add the detection antibody (horseradish enzyme-labeled goat anti-rabbit IgG (H+L)) to the 96-well plate at 100 μL / well, and incubate at room temperature for 2 h;

[0064] (8) Washing plate: then wash the plate 5 times with 200 μL / well of washing solution, and remove the dry enzyme-labeled plate;

[0065] (9) Color development: add 200 μL / well of color developing solution and let it stand at room temperature for 12 min;

[0066] (10) Termination and detection: add 50 μL / well of termination solution to terminate the reaction, and then detect it with an enzyme-labeled instrument at a wavelength of 450 nm, and calculate the relative OD450 value, relative OD450 = detection OD450 - blank OD450.

[0067] The serum titer detection results are shown in Table 3 below.

[0068] Table 3, serum detection results after four immunizations

[0069]

[0070] According to the data analysis in Table 3, the positive standard is (serum absorbance value after the fourth immunization - blank absorbance value) / (immunization pre-negative control serum absorbance value - blank absorbance value) > 2.1, the results show that the serum titers of the two rabbits after four immunizations reach 1:128000, and the serum titer of the rabbit numbered 1 is higher, so the spleen and bone marrow of the rabbit numbered 1 are used for subsequent construction of phage library.

[0071] 2, Construction of phage library and screening of positive scFv

[0072] The spleen and bone marrow of rabbit No. 1 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 2 As shown, the extracted RNA was subjected to UV quantification, and the experimental results are shown in Table 4 below, indicating that the extracted RNA had good purity and concentration.

[0073] Table 4. RNA UV quantification results of spleen and bone marrow

[0074]

[0075] 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.

[0076] 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.

[0077] Table 5. Light chain PCR primers

[0078]

[0079] Table 6. Heavy chain PCR primers

[0080]

[0081] 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 3 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 Figure 4 Then, 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 5 Then, the phage vector bComb3x with the inserted scFv fragment was electroporated into the X-Blue competent medium to construct the phage display scFv primary antibody library for immunizing rabbits. The primary antibody library capacity was calculated to be 1.51×10 9 cfu.

[0082] Further, the original antibody library is prepared after the auxiliary phage infects the primary antibody library, and the specific method is as follows: the CD93 protein is coated on the ELISA plate, the original antibody library is added to the ELISA plate, the non-specifically combined phage is washed away by the washing solution, and the phage combined with the antigen is eluted by the elution solution, and then the next round of enrichment screening is carried out after amplification and precipitation in E. coli. After 2-4 rounds of "adsorption-elution-amplification", the positive library combined with CD93 is screened. The binding of the library after multiple rounds of solid-phase screening with the mouse CD93 antigen is detected by ELISA, and the detection results are shown in Tables 7-8.

[0083] Table 7, phage positive library ELISA detection data-1

[0084]

[0085] Table 8, phage positive library ELISA detection data-2

[0086]

[0087] From the results of Tables 7-8, the ELISA detection results show that the library is positive combined with the mouse CD93 protein, and a single clone is picked from the library for identification.

[0088] Further, a single clone phage is picked from the positive phage for expression, the supernatant is collected, and the positive single clone phage detection data are shown in Table 9.

[0089] Table 9, positive single clone phage expression ELISA detection data

[0090]

[0091] According to the data analysis of Table 9, it is shown that the scFv antibodies expressed by 16 positive single clone phages are specifically combined with CD93 and have good binding capacity, and 16 groups of scFv nucleotide sequences are obtained by sequencing by a sequencing company.

[0092] 3, Preparation of monoclonal antibody

[0093] Further, primers are designed according to the heavy chain variable region sequences and light chain variable region sequences of the 16 groups of scFv, and the heavy chain variable region primers and light chain variable region primers are shown in Tables 10-11.

[0094] Table 10, heavy chain variable region primers of scFv antibody

[0095]

[0096] Table 11, light chain variable region primers of scFv antibody

[0097]

[0098] 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.

[0099] Furthermore, the signal peptide sequence and constant region sequence were added to the heavy and light chain variable region fragments respectively by PCR to construct a complete heavy chain nucleotide sequence and light chain nucleotide sequence. The heavy chain signal peptide nucleotide sequence is shown in SEQ ID NO.15, and the light chain signal peptide nucleotide sequence is shown in SEQ ID NO.16; the constant region sequence is the constant region sequence of mouse IgG1, the heavy chain constant region nucleotide sequence is shown in SEQ ID NO.19, and the heavy chain constant region nucleotide sequence is shown in SEQ ID NO.20. The complete heavy chain nucleotide sequence and light chain nucleotide sequence were respectively connected to the pUC expression vector plasmid (the structure of the expression vector plasmid is shown in Figure 6 The specific method is 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 min and then added to 100 μL of competent medium. After ice bathing for 30 min, the mixture was heat-shocked for 90 s and quickly placed on ice for 3 min before coating the plate. 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 connected to the specific gene.

[0100] Furthermore, 16 heavy chain-linked vector plasmids and 16 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 fourth or fifth day of cell culture, the supernatant was collected and analyzed by ELISA as follows:

[0101] (1) Coating: Coat with CD93 antigen protein 1 μg / mL, 100 μL / well, overnight at 4°C;

[0102] (2) Sealing: plate in liquid shake off and dry, 2% BSA, 300 μL / well, sealed and incubated at room temperature for 1 h;

[0103] (3) Washing plate: 300 μL / well washing solution, washing plate twice, and the last one was dried;

[0104] (4) Antibody dilution: 16 positive clone cell supernatants were diluted 2 times, and 100 μL of each was added to the corresponding well plate, mixed well, and reacted at room temperature for 2 h;

[0105] (5) Washing plate: 300 μL / well washing solution, washing plate 3 times, and the last one was dried;

[0106] (6) Adding secondary antibody: dilute the horseradish enzyme-labeled goat anti-mouse IgG Fc secondary antibody to a use concentration of 0.08 μg / mL, 100 μL / well, mix well, and incubate at room temperature for 1 h;

[0107] (7) Washing plate: 300 μL / well washing solution, washing plate 3 times, and the last one was dried;

[0108] (8) Color development: mix A and B liquids at a ratio of 1:1, add 200 μL per well, and incubate at room temperature for 20 min in the dark;

[0109] (9) Add 50 μL of stop solution to each well, immediately measure the OD value at 450 nm wavelength, set two parallel wells, take the average value, and calculate the relative OD450 value, relative OD450 = detection OD450 - blank OD450.

[0110] The ELISA detection results are shown in Tables 12-13.

[0111] Table 12, ELISA detection results of supernatant of 15 positive clone cells cultured for five days

[0112]

[0113] Table 13, ELISA detection results of supernatant of 1 positive clone cell cultured for four days

[0114]

[0115] According to the data analysis of Tables 12-13, it is shown that the cell supernatants of 16 positive clone cells cultured for four or five days are all positive in ELISA detection of CD93 protein antigen, and all have good binding capacity.

[0116] Further, the cell supernatant was collected after 7 days of cell culture and the antibody was purified, and the specific method was as follows:

[0117] (1) Sample preparation: centrifugation with a desktop centrifuge, 4000 g for 30 min, and take 16 single clone cell supernatants;

[0118] (2) Filtration: different filtration methods are selected according to the volume of the feed liquid, and 0.45 μm filter membrane is used for filtration;

[0119] (3) Connect the purification system: select the appropriate size of affinity filler Protein A column according to the expression amount, and connect it with the purification system;

[0120] (4) Water balance: wash with ultrapure water for 2 CV, and replace the 25 % ethanol storage solution;

[0121] (5) Equilibrium chromatography column: equilibrate the buffer for 3 CV to the UV baseline stable;

[0122] (6) Sample loading: adjust the appropriate flow rate for sample loading;

[0123] (7) Elution: elution liquid elution for 5-10 CV to the UV baseline stable, and the flow rate is the same as the sample loading flow rate;

[0124] (8) Elution: elution, according to the UV peak collection;

[0125] (9) Neutralization: add Tris, pH 8.0 to neutralize the eluted antibody;

[0126] (10) Equilibrium: equilibrate the buffer for 3 CV to neutral;

[0127] (11) CIP cleaning: CIP regeneration liquid cleaning for more than 5 CV;

[0128] (12) Alkali flushing: balance buffer to flush alkali until the outlet pH is neutral, and then flush with ultrapure water for 3 CV;

[0129] (13) Preservation: 25 % ethanol equilibration for 2 CV, and save the column.

[0130] Through purification, 16 purified CD93 monoclonal antibodies were obtained.

[0131] Example 2, detection of the concentration, purity and specificity of the purified antibody

[0132] 1. Detection of antibody concentration

[0133] Start the microspectrophotometer, and point the sample with the corresponding buffer at 280 nm wavelength. When the absorbance value is between ±0.015, it indicates that the instrument baseline is stable. The 16 purified antibody samples are sequentially spotted, and the absorbance value is recorded. The detection data is divided by the IgG extinction coefficient (1.414), and the resulting value is the concentration of the detection sample, as shown in the following Table 14.

[0134] Table 14, 16 strains of purified antibody concentration

[0135]

[0136] 2, detection of antibody purity

[0137] The purity of 16 strains of purified antibodies was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This method separates the proteins in the sample according to their molecular weights in the electrophoresis gel, thereby verifying the purity of the target antibody. The specific method is as follows:

[0138] (1) Sample treatment: 5 μg of 16 strains of purified antibodies in Table 14 were added to 5 μL of 4x loading buffer (reducing) and 4x loading buffer (non-reducing), respectively, and heated in a 100 ℃ water bath, then centrifuged at 10000 rpm;

[0139] (2) Prepare SDS-PAGE gels with concentrations of 13 % and 7.5 %;

[0140] (3) Fix the SDS-PAGE gels prepared in (2) in the electrophoresis tank, add enough 1x glycine system electrode buffer to the electrophoresis tank, and use a micropipette to add the sample solution treated with 4x loading buffer (non-reducing) in (1) and 13 % protein molecular weight marker to the sample well of the 13 % SDS-PAGE gel; use a micropipette to add the sample solution treated with 4x loading buffer (reducing) in (1) and 7.5 % protein molecular weight marker to the sample well of the 7.5 % SDS-PAGE gel;

[0141] (4) Electrophoresis: connect the power supply, first electrophorese at a constant voltage of 100 V until the bromophenol blue dye enters the separation gel from the concentrated gel, then adjust the current to 140 V and continue electrophoresis until the bottom of the gel plate, turn off the power;

[0142] (5) Staining: immerse the gel in Coomassie Brilliant Blue R-250 staining solution, heat in a microwave oven for 60 s, place on a gently shaking platform for room temperature staining for 2-4 h, then replace the decolorizing solution to cover the gel, heat in a microwave oven for 60 s, place on a gently shaking platform for room temperature decolorization, repeat the decolorization operation until a blue band and clean background are obtained.

[0143] Observe the protein staining band after decolorization. The purity of the 16 strains of purified antibody proteins is above 95 %. Take antibody 15 as an example, the electrophoregrams after treatment with 4x loading buffer (non-reducing) and 4x loading buffer (reducing), respectively, are shown in Figure 7-8 The results show that the purity of antibody 15 is very high, with almost no other bands, and the purity is as high as 95.9 %.

[0144] 3. Detecting the specific binding ability of the antibody to CD93

[0145] Further, the specific binding of 16 purified antibodies to CD93 was detected by ELISA, and the specific steps were as follows:

[0146] (1) Coating: coating CD93 antigen protein 1 μg / mL and 100 μL / well, 4°C coating overnight;

[0147] (2) Blocking: Shake off the liquid in the plate and pat dry, 2% BSA, 300 μL / well, seal and incubate at room temperature for 1 h;

[0148] (3) Washing the plate: 300 μL / well washing solution, washing the plate 2 times, and the last time patting dry;

[0149] (4) Antibody dilution: dilute the antibody to 0.1 μg / mL, 100 μL / well, and add it to the corresponding well plate, mix well, and react at room temperature for 2 h;

[0150] (5) Washing the plate: 300 μL / well washing solution, washing the plate 3 times, and the last time patting dry;

[0151] (6) Adding secondary antibody: dilute the horseradish enzyme-labeled goat anti-mouse IgG Fc secondary antibody to a concentration of 0.08 μg / mL, 100 μL / well, mix well, and incubate at room temperature for 1 h;

[0152] (7) Washing the plate: 300 μL / well washing solution, washing the plate 3 times, and the last time patting dry;

[0153] (8) Color development: mix A and B liquids at a ratio of 1:1, add 200 μL per well, and incubate at room temperature for 20 min in the dark;

[0154] (9) Add 50 μL of stop solution to each well, immediately measure the OD value at 450 nm wavelength, set two parallel wells, take the average value, and calculate the relative OD450 value, relative OD450 = detection OD450 - blank OD450.

[0155] The ELISA detection results of 16 purified antibodies are shown in Tables 15-16.

[0156] Table 15, ELISA detection results of 15 purified antibodies

[0157]

[0158] Table 16, ELISA detection results of 1 purified antibody

[0159]

[0160] According to the data analysis of Table 15-Table 16, it is shown that the ELISA detection results of 16 purified antibodies are all positive, and have good specific binding capacity with CD93; comparing the ELISA detection results of 16 purified antibodies, the relative OD450 values of antibody 1, antibody 3, antibody 7, antibody 11, antibody 13 and antibody 15 all reach 4.10 or more, indicating that they have stronger specific binding capacity with CD93.

[0161] Example 3, detection of affinity of antibodies and screening of antibodies

[0162] The affinity of 16 purified antibodies in Example 2 was detected by ELISA, and the detection method was as follows:

[0163] (1) Coating: coating CD93 antigen protein 0.1 μg / mL, 100 μL / well, 4 ℃ coating overnight;

[0164] (2) Blocking: shake off the liquid in the plate and pat dry, 2% BSA, 300 μL / well, seal after room temperature incubation for 1 h;

[0165] (3) Washing plate: 300 μL / well washing solution, washing plate 2 times, and the last time pat dry;

[0166] (4) Antibody dilution: dilute the antibody to 300 ng / mL, then dilute it by three times gradient for 6 points, the concentrations are 100 ng / mL, 33.33 ng / mL, 11.11 ng / mL, 3.70 ng / mL, 1.23 ng / mL, 0.41 ng / mL, and the blank control is sample diluent, 100 μL / well is added to the corresponding well plate, mixed evenly, and incubated at room temperature for 2 h;

[0167] (5) Washing plate: 300 μL / well washing solution, washing plate 3 times, and the last time pat dry;

[0168] (6) Adding secondary antibody: dilute the horseradish enzyme labeled goat anti-mouse IgG F(ab)2 secondary antibody to a concentration of 0.2 μg / mL, 100 μL / well, mix evenly, and incubate at room temperature for 1 h;

[0169] (7) Washing plate: 300 μL / well washing solution, washing plate 3 times, and the last time pat dry;

[0170] (8) Color development: mix A and B liquids at a ratio of 1:1, then add 200 μL per well, and incubate at room temperature for 20 min in the dark;

[0171] (9) Add 50 μL of termination solution to each well, immediately measure the OD value at 450 nm wavelength, set two parallel wells, take the average value, and calculate the relative OD450 value, relative OD450 = detection OD450 - blank OD450.

[0172] The affinity detection results of the 16 antibodies showed that the affinities of antibodies 15, 13, and 11 were the strongest. The ELISA detection results of the three antibodies diluted to seven concentrations are shown in Table 17, and the binding curve of antibody 15 is shown in Figure 9 .

[0173] Table 17, ELISA detection results of antibodies 15, 13, and 11 diluted to seven concentrations

[0174]

[0175] According to the data analysis in Table 17 and Figure 9 , the results showed that antibodies 15, 13, and 11 bound to CD93 in a concentration-dependent manner, the maximum binding relative OD450 value of antibody 15 was 3.8216, and the EC50 was calculated to be 35.86 ng / mL; the maximum binding relative OD450 value of antibody 13 was 3.7011, and the EC50 was calculated to be 41.24 ng / mL; the maximum binding relative OD450 value of antibody 11 was 3.6839, and the EC50 was calculated to be 31.49 ng / mL. This indicated that the affinities of the three antibodies to CD93 were high, and they could exert their biological activity at a lower concentration, indicating that the three antibodies could be applied to immunodetection and assist in the diagnosis of diseases.

[0176] Further, the three high-affinity mAbs were paired as capture antibodies and detection antibodies, respectively, to detect the ability of the antibodies to bind CD93 antigen by double antibody sandwich method, and the best antibody pair was screened out, and the detection method was as follows:

[0177] (1) Coating capture antibody: coat antibodies 15, 13, and 11, respectively, at 1 μg / mL, 100 μL / well, and 4°C for overnight;

[0178] (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;

[0179] (3) Washing the plate: 300 μL / well of washing solution, wash the plate twice, and pat dry at the end;

[0180] (4) Adding antigen: dilute mouse CD93 antigen to 100 ng / mL, 100 μL / well, and add to the corresponding well plate, mix well, and react at room temperature for 2 h;

[0181] (5) Washing plate: 300 μL / well washing solution, washing plate 3 times, the last one pat dry;

[0182] (6) Adding detection antibody: adding antibody 15, antibody 13, antibody 11 respectively, 1 μg / mL, 100 μL / well;

[0183] (7) Washing plate: 300 μL / well washing solution, washing plate 3 times, the last one pat dry;

[0184] (8) Adding enzyme-labeled antibody: diluting horseradish enzyme-labeled goat anti-mouse IgG Fc to the use concentration of 0.08 μg / mL, 100 μL / well, mixing well, incubating at room temperature for 1 h;

[0185] (9) Washing plate: 300 μL / well washing solution, washing plate 3 times, the last one pat dry;

[0186] (10) Color development: mixing A and B liquids according to 1:1, adding 200 μL to each well, incubating at room temperature for 20 min in dark;

[0187] (11) Adding 50 μL stop solution to each well, immediately measuring OD value at 450 nm wavelength, setting two parallel holes, taking average value, and calculating relative OD450 value, relative OD450=detection OD450-blank control.

[0188] The results of the monoclonal antibody pair detection are shown in Table 18.

[0189] Table 18, results of monoclonal antibody pair detection

[0190]

[0191] According to the results in Table 18, the three groups of antibodies have the best binding ability to the antigen when antibody 13 is used as the detection antibody, antibody 15 is used as the capture antibody; or antibody 15 is used as the detection antibody, antibody 13 is used as the capture antibody; or antibody 15 is used as the detection antibody, antibody 11 is used as the capture antibody. The heavy chain variable region amino acid sequence of antibody 15 is shown as SEQ ID NO. 1, the light chain variable region amino acid sequence is shown as SEQ ID NO. 2, the heavy chain variable region nucleotide sequence is shown as SEQ ID NO. 7, the light chain variable region nucleotide sequence is shown as SEQ ID NO. 8, the heavy chain nucleotide sequence is shown as SEQ ID NO. 21, and the light chain nucleotide sequence is shown as SEQ ID NO. 22; the heavy chain variable region amino acid sequence of antibody 13 is shown as SEQ ID NO. 3, the light chain variable region amino acid sequence is shown as SEQ ID NO. 4, the heavy chain variable region nucleotide sequence is shown as SEQ ID NO. 9, the light chain variable region nucleotide sequence is shown as SEQ ID NO. 10, the heavy chain nucleotide sequence is shown as SEQ ID NO. 23, and the light chain nucleotide sequence is shown as SEQ ID NO. 24; the heavy chain variable region amino acid sequence of antibody 11 is shown as SEQ ID NO. 5, the light chain variable region amino acid sequence is shown as SEQ ID NO. 6, the heavy chain variable region nucleotide sequence is shown as SEQ ID NO. 11, the light chain variable region nucleotide sequence is shown as SEQ ID NO. 12, the heavy chain nucleotide sequence is shown as SEQ ID NO. 25, and the light chain nucleotide sequence is shown as SEQ ID NO. 26.

[0192] Further, the thermal stability Tm value of the obtained monoclonal antibodies was determined by circular dichroism (CD) to evaluate the stability of the monoclonal antibodies, and was compared with existing CD93 monoclonal antibodies purchased from Thermo Fisher Scientific, item number 17-5892-82. The test results are shown in Table 19. Tm represents the midpoint temperature of thermal denaturation of the protein, i.e. the temperature at which the protein unfolds by 50%, reflecting the trend of protein conformation change during temperature change, and the higher the Tm, the better the stability.

[0193] Table 19, Tm values of different CD93 monoclonal antibodies

[0194]

[0195] According to the results in Table 19, it is found that the Tm values of the screened antibody 15, antibody 13 and antibody 11 are higher than those of the existing CD93 monoclonal antibodies, indicating that the screened CD93 monoclonal antibodies have better stability.

[0196] Example 4, a CD93 ELISA detection kit

[0197] Three antibodies screened in Example 3 were respectively made into CD93 ELISA detection kits, named kits 1-3, as shown in the following Table 20.

[0198] Table 20, CD93 ELISA detection kits and their corresponding capture antibodies and detection antibodies

[0199]

[0200] 1. An ELISA kit for detecting CD93 was prepared, which included capture antibody reagent, detection antibody reagent, CD93 standard, avidin-biotin-peroxidase complex (ABC) solution, sample dilution buffer, antibody dilution buffer, ABC dilution buffer, TMB developing solution, TMB stopping solution, and washing buffer.

[0201] 1) Antibody dilution buffer: 0.01 mol / L pH 7.2 PBS, weigh NaH2PO4·2H2O 0.39 g, Na2HPO4 1.27 g, NaCl 0.85 g, and NaN3 200 mg, add distilled water to 1000 mL;

[0202] 2) Sample dilution buffer: weigh KH2PO4 0.27 g, Na2HPO4 1.42 g, NaCl 8 g, and KCl 0.2 g, add about 800 mL of distilled water, stir well to dissolve, then add concentrated hydrochloric acid to adjust pH to 7.2-7.4, add BSA 5 g, and finally make up to 1 L;

[0203] 3) ABC dilution buffer: weigh KH2PO4 0.2 g, Na2HPO4·12H2O 2.9 g, NaCl 8 g, and KCl 0.2 g, add about 800 mL of distilled water, stir well to dissolve, adjust pH to 7.4 with hydrochloric acid, add 0.5 mL of Tween-20 and 10 g of BSA, and make up to 1 L;

[0204] 4) Washing buffer: weigh KH2PO4 0.2 g, Na2HPO4·12H2O 2.9 g, NaCl 8.0 g, and KCl 0.2 g, add about 800 mL of distilled water, stir well to dissolve, adjust pH to 7.4 with hydrochloric acid, add 0.5 mL of Tween-20, and make up to 1 L;

[0205] 5) Preparation of capture antibody reagent: dilute antibodies 11 / 13 / 15 with antibody dilution buffer to a concentration of 1 μg / mL;

[0206] 6) Preparation of detection antibody reagent:

[0207] a. CD93 pretreatment: dilute antibody 13 / 15 to a concentration of 1 mg / mL with antibody dilution buffer;

[0208] b. activated biotin reagent: dissolve N-hydroxysuccinimide biotin (NHS-biotin) with an appropriate amount of DMSO to prepare 5 mg / mL;

[0209] c. preparation of biotinylated CD93 antibody: under the condition of stirring or oscillation, slowly add the activated biotin reagent to the antibody solution, the molar ratio of biotin to antibody is 5:1, the reaction is carried out at 4 ℃, 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 the 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 antibody dilution buffer dialysis to obtain biotinylated CD93 antibody and dilute it to a concentration of 1 μg / mL;

[0210] 7) Preparation of CD93 standard solution: prepare CD93 standard solution with eight concentrations of 5200 pg / mL, 2600 pg / mL, 1300 pg / mL, 650 pg / mL, 325 pg / mL, 162.5 pg / mL, 81.25 pg / mL, 40.625 pg / mL by sample dilution buffer.

[0211] 8) Preparation of avidin-biotin-peroxidase complex (ABC) solution: dilute the ABC solution to 1 μg / mL with ABC dilution buffer.

[0212] 9) Preparation of TMB developing solution: the same developing solution as in Example 1 for ELISA detection.

[0213] 10) Preparation of TMB termination solution: the same termination solution as in Example 1 for ELISA detection.

[0214] The above capture antibody reagent and detection antibody reagent are paired according to Table 20 to configure CD93 ELISA kits 1-3.

[0215] 2, the CD93 ELISA kits 1-3 are used to detect CD93 in the detection sample, and the specific steps are as follows:

[0216] (1) coating: add 100 μL of capture antibody reagent per well to the microplate for coating at 4 ℃ overnight;

[0217] (2) blocking: shake off the liquid in the plate, pat dry, add 300 μL of 2% BSA per well, seal and incubate at room temperature for 1 h;

[0218] (3) Sample pretreatment: 1) Serum sample: let the serum coagulate in the serum separation tube at room temperature (about 4 hours), centrifuge at about 1000xg for 15 minutes, and immediately determine; 2) Plasma sample: collect plasma using heparin or EDTA as an anticoagulant, centrifuge at 1000xg for 15 minutes, and immediately determine;

[0219] (4) Add standard and sample: add sample dilution buffer (control well), sample and eight concentrations of CD93 standard in the microplate, 100 μL / well, seal the microplate, and incubate at 37 °C for 90 minutes;

[0220] (5) Use a paper towel or water-absorbing material to absorb the liquid in the microplate, and make sure that the liquid in the microplate is not completely absorbed, 100 μL / well, add the detection antibody solution, seal the microplate, and incubate at 37 °C for 60 minutes;

[0221] (6) 300 μL / well of washing buffer, wash the plate 3 times, and the last time is dried;

[0222] (7) 100 μL / well of ABC solution, seal the microplate, and incubate at 37 °C for 30 minutes;

[0223] (8) 300 μL / well of washing buffer, wash the plate 5 times, and the last time is dried;

[0224] (9) 90 μL / well of TMB developing solution, incubate at 37 °C in the dark for 15-25 minutes;

[0225] (10) 100 μL / well of TMB stop solution, the liquid in the well immediately turns yellow, and the OD value is measured at 450 nm within 30 minutes after the addition of the TMB stop solution, three parallel detection wells are set for the same detection sample, and the average value is taken, and the relative OD450 = detection OD450-blank OD450.

[0226] The relative OD450 value of the CD93 standard is determined, and the CD93 concentration in the sample is calculated by the standard curve.

[0227] It should be noted that the CD93 detection kit has various forms, and in addition to the enzyme-linked immunosorbent kit provided by the present application, antibodies 11, 13 and 15 can also be prepared into a chemiluminescence kit, an immunofluorescence kit, etc.

[0228] 3. Linear evaluation

[0229] The relative OD450 values of the different concentrations of standard samples of the kit 1-3 are shown in Table 21.

[0230] Table 21, relative OD450 values of different concentrations of standard samples detected by kit 1-3

[0231]

[0232] According to the data in Table 21, the three non-linear regression equations were obtained by Logistic-four parameter method fitting analysis, R 2 > 0.99, and the non-linear regression equations were respectively: , R 2 = 0.9987;

[0233] , R 2 = 0.9984; , R 2 = 0.9992.

[0234] 4. Sensitivity evaluation

[0235] Detection method of blank limit: The zero concentration calibrator was used as the sample for detection, and the detection was carried out by the CD93 ELISA detection kit 1-3 according to the detection method described in Example 4, and the detection was repeated for 20 times. According to the curve equation of the calibrator used in the corresponding kit, the concentration value of the 20 measurement results was obtained, and the average value (M) and the standard deviation (SD) were calculated, and the M+2SD was obtained, which was the blank limit value. The blank limit detection results are shown in Table 22.

[0236] Table 22, blank limit detection results of kit 1-3

[0237]

[0238] According to the results in Table 22, the blank detection limits of kits 1-3 are very low, but the detection limit of kit 3 is lower, and the sensitivity is higher, and the sensitivity can be as high as 0.35 pg / mL.

[0239] Example 5, performance test of kit 1-3

[0240] According to the results of linear evaluation and blank detection line evaluation in Example 4, kits 1-3 were further evaluated for accuracy, stability and batch difference.

[0241] 1. Accuracy evaluation

[0242] Prepare low-value plasma quality control and high-value plasma quality control:

[0243] (1) Preparation of low-value plasma quality control: Through pre-detection of the CD93 content of multiple plasma samples, select the plasma with relatively low CD93 content as the basic sample, dilute the basic sample with sample dilution buffer to reduce the concentration of CD93, and use a pipette to accurately measure the liquid during dilution and fully vortex mix to ensure uniform mixing. The final dilution of the low-value plasma quality control CD93 concentration is 50 pg / mL;

[0244] (2) Preparation of high-value plasma quality control: Through pre-detection of the CD93 content of multiple plasma samples, select the plasma or serum with relatively high CD93 content as the basic sample. According to the content of CD93 in the basic sample and the desired high-value concentration, calculate and accurately add an appropriate amount of CD93 standard. According to the formula C1V1 + C2V2 = C3V3 (C is the concentration, and V is the volume), calculate the volume of standard to be added, then slowly add the standard using a pipette, and vortex mix while adding to fully mix the standard with the basic sample. The final CD93 concentration of the high-value plasma quality control is 4500 pg / mL.

[0245] Use Kit 1-3 to detect the CD93 content in low-value plasma quality control and high-value plasma quality control according to the detection method of Example 4, and compare the results with the Abeam human CD93 ELISA kit (C1qR) (article number ab213762) and Shanghai Zymebio human CD93 ELISA kit (article number ml106328). Each quality control is detected in triplicate, and the average value is taken. The results are shown in Table 23.

[0246] Table 23, concentration results of different detection kits for low-value blood quality control and high-value blood quality control

[0247]

[0248] According to the data analysis in Table 23, the accuracy of Kit 1-3 and two commercially available kits is compared, which shows that Kit 1-3 is more accurate. The reason is that the affinities of antibodies 11, 13, and 15 are very high, so they can improve the detection accuracy of the kit whether they are used as detection antibodies or capture antibodies. In addition, the competition of binding sites and the steric hindrance effect of capture antibodies and detection antibodies in Kit 1-3 are smaller than those of commercially available kits when they bind to the antigen, so they can enhance the ability of subsequent signal amplification, and therefore the detection accuracy is higher. However, compared with the detection accuracy of Kit 1-3, Kit 3 has the highest accuracy.

[0249] 4. Reproducibility evaluation

[0250] The content of CD93 in low-value plasma quality control and high-value plasma quality control was detected by using kits 1-3 according to the detection method provided in Example 4, and each quality control was detected 10 times, the average value M and the standard deviation SD of 10 results were calculated, the coefficient of variation CV was calculated, and the detection results are shown in Table 24.

[0251] Table 24, detection results of kit 1-3 repeatability experiment

[0252]

[0253] According to the analysis of the results in Table 24, the coefficient of variation CV values of the repeatability detection of kits 1-3 are all within 5%, although there is a certain dispersion, but from the confidence limit specified by the standard deviation, this dispersion is reliable, which shows that the nine kinds of kits all have good stability. But compared with the other two kinds of kits, the CV value of kit 3 is significantly smaller, and the stability is the highest.

[0254] 5、Batch difference evaluation

[0255] The kits 1-3 were taken three batches respectively, and the same 1 reference was repeatedly detected according to the detection method provided in Example 4, the reference was low-value plasma quality control, each batch was repeatedly detected 10 times, the average value M and the standard deviation SD of 30 measurement results were calculated, the coefficient of variation CV was calculated, and the batch difference detection results are shown in Table 25.

[0256] Table 25, detection results of kit 1-3 batch difference

[0257]

[0258] According to the analysis of the results in Table 25, although the batch difference CV values of 3 batches of each kit 1-3 are all within 5%, but compared with the other two kinds of kits, the batch difference CV value of kit 3 is lower, which shows that the batch difference of kit 3 is smaller, the stability is higher, the repeatability is stronger, and the consistency is higher.

[0259] In summary, when the detection antibody is antibody 15 and the capture antibody is antibody 13, the detection results of kit 3 prepared are the most accurate, the stability is the highest, and the batch difference is the smallest, which is preferred.

[0260] Example 6, application of kits 1-3 in tumor diagnosis

[0261] Hepatocellular carcinoma tissue highly expresses CD93, which is the main source of the increase of CD93 in blood, therefore, the expression level of CD93 can effectively reflect the malignant degree, metastasis risk and prognosis of the tumor, and can be used as a marker of the tumor.

[0262] The kits 1-3 prepared in Example 4 and two commercially available kits, Abeam Human CD93 ELISA Kit (C1qR) (Cat No. ab213762), Shanghai Enzyme-linked Biological Human CD93 ELISA Kit (Cat No. ml106328) were used to test CD93 in blood samples of healthy subjects and hepatocellular carcinoma patients respectively, and their abilities to distinguish between diseased populations were evaluated.

[0263] Blood samples from healthy subjects (200 cases) and hepatocellular carcinoma patients (200 cases) were collected, 5 mL per case, and serum was separated by centrifugation (3000 rpm, 15 min), aliquoted and stored at -80°C for later use; the healthy subjects and hepatocellular carcinoma patients were divided into a test group (95 healthy subjects, 120 hepatocellular carcinoma patients) and a validation group (105 healthy subjects, 80 hepatocellular carcinoma patients). The samples in the test group and the validation group were independently detected using kits 1-3 and two commercially available kits, and the absorbance values were recorded and the CD93 content in the serum was calculated. The CD93 content cut-off value for distinguishing whether it is a hepatocellular carcinoma patient is 100 pg / mL, and greater than or equal to 100 pg / mL is judged to be a hepatocellular carcinoma patient, and less than 100 pg / mL is judged to be a healthy population. The abilities of the five kits to distinguish between diseased populations were evaluated by R language, i.e. the receiver operating characteristic curve (ROC) was made and the AUC value was calculated. The AUC values, sensitivity (true positive rate), and specificity (true negative rate) of the five kits in the test group and the validation group are shown in Table 26.

[0264] Table 26, AUC values, sensitivity (true positive rate), and specificity (true negative rate) of the five kits

[0265]

[0266] According to the data in Table 26, the detection AUC values of kits 1-3 in the test group and the validation group are all greater than those of the two commercially available kits and exceed 0.80, indicating that they can better distinguish between patients and healthy people. However, compared with kits 1 and 2, kit 3 has higher diagnostic performance and can reduce the probability of misjudgment (missed diagnosis or misdiagnosis). Therefore, kit 3 (the detection antibody is antibody 15 (i.e. the first antibody), and the capture antibody is antibody 13 (i.e. the second antibody)) is the best CD93 ELISA detection kit and can be used for early diagnosis and screening of hepatocellular carcinoma and prognosis evaluation.

[0267] The existing antibodies not only have low detection sensitivity, but even different antibodies are used as detection antibodies or enzyme-labeled antibodies, and the detection results are significantly different. In addition, different antibodies have obvious differences in stability (Tm), cross-reaction with the matrix, high-dose hook effect, etc., which can all lead to a decrease in performance for diagnosing hepatocellular carcinoma.

[0268] Meanwhile, in the process of diagnosing hepatocellular carcinoma, CD93 antibodies are needed to detect blood samples, and there are complex interfering substances in the blood samples, the matrix has a greater influence, and cross-reactions or some non-linear fragment interference may exist, and the detection and diagnosis of hepatocellular carcinoma using different antibodies will inevitably produce completely different diagnostic effects, so a better antibody combination must be selected to improve the diagnostic efficiency.

[0269] Although the present application is disclosed as above, the present application is not limited thereto. Any person skilled in the art, without departing from the spirit and scope of the present application, can make various changes and modifications, therefore the protection scope of the present application should be limited by the scope defined by the claims.

Claims

1. A CD93 monoclonal antibody, characterized in that, The second antibody has a heavy chain variable region amino acid sequence as shown in SEQ ID NO. 3 and a light chain variable region amino acid sequence as shown in SEQ ID NO. 4; the third antibody has a heavy chain variable region amino acid sequence as shown in SEQ ID NO. 5 and a light chain variable region amino acid sequence as shown in SEQ ID NO.

6.

2. The CD93 monoclonal antibody of claim 1, wherein, The second antibody has a heavy chain variable region nucleotide sequence as shown in SEQ ID NO. 9 and a light chain variable region nucleotide sequence as shown in SEQ ID NO. 10; the third antibody has a heavy chain variable region nucleotide sequence as shown in SEQ ID NO. 11 and a light chain variable region nucleotide sequence as shown in SEQ ID NO.

12.

3. The CD93 monoclonal antibody of claim 1, wherein, The second antibody and the third antibody have the same heavy chain signal peptide and light chain signal peptide, the heavy chain signal peptide has an amino acid sequence as shown in SEQ ID NO. 13 and the light chain signal peptide has an amino acid sequence as shown in SEQ ID NO. 14; the heavy chain signal peptide has a nucleotide sequence as shown in SEQ ID NO. 15 and the light chain signal peptide has a nucleotide sequence as shown in SEQ ID NO.

16.

4. The CD93 monoclonal antibody of claim 1, wherein, The second antibody and the third antibody have the same heavy chain constant region and light chain constant region, the heavy chain constant region has an amino acid sequence as shown in SEQ ID NO. 17 and the light chain constant region has an amino acid sequence as shown in SEQ ID NO. 18; the heavy chain constant region has a nucleotide sequence as shown in SEQ ID NO. 19 and the light chain constant region has a nucleotide sequence as shown in SEQ ID NO.

20.

5. The CD93 monoclonal antibody of claim 2, wherein the CD93 monoclonal antibody is an antibody selected from the group consisting of: and. The second antibody has a heavy chain nucleotide sequence as shown in SEQ ID NO. 23 and a light chain nucleotide sequence as shown in SEQ ID NO. 24; the third antibody has a heavy chain nucleotide sequence as shown in SEQ ID NO. 25 and a light chain nucleotide sequence as shown in SEQ ID NO.

26.

6. Use of a CD93 monoclonal antibody composition in the manufacture of a reagent for enhancing the ability to bind to a CD93 protein, characterized in that, The antibody composition is a first antibody and a second antibody, the first antibody has a heavy chain variable region amino acid sequence as shown in SEQ ID NO. 1 and a light chain variable region amino acid sequence as shown in SEQ ID NO. 2; the second antibody has a heavy chain variable region amino acid sequence as shown in SEQ ID NO. 3 and a light chain variable region amino acid sequence as shown in SEQ ID NO.

4.

7. A CD93 detection kit, characterized by, The CD93 monoclonal antibody of any one of claims 1-5.

8. The CD93 detection kit of claim 7, wherein, The kit is an ELISA detection kit.

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