A camelid single-domain antibody against crp and its preparation method and application

Abstract

This invention discloses a camel-derived CRP single-domain antibody, its preparation method, and its applications, relating to the biomedical field. The method involves enriching CRP antigen in alpaca leukocytes immunized with the antigen, followed by single-cell screening and sequencing. Through sequencing structural analysis, a recombinant plasmid of the camel-derived CRP antibody is constructed. This plasmid is cloned and transformed into eukaryotic cells to express related antibodies. Monoclonal antibodies with human CRP resistance are screened for expression. The VHH domain of the monoclonal antibody is cloned and expressed, and paired screening yields CRP-VHH monoclonal antibodies with antibody activity. This antibody can be used in ELISA kits, chemiluminescence kits, and latex kits, possessing the same functionality as existing kits. Furthermore, this antibody also has applications in the preparation of antitumor drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a CRP camel-derived single-domain antibody, its preparation method, and its application. Background Technology

[0002] Immunoglobulins (Ig) are globulins with antibody activity or chemical structures similar to antibody molecules. Antiserum can bind to relevant antigens for immune responses primarily because it contains immunoglobulins, products of immune cells (except for monoclonal antibodies). These substances are also distributed in body fluids, exocrine fluids, and on the membranes of lymphocytes. Immunoglobulins have various components, primarily IgG, which accounts for about 70% of the total, at approximately 7-16 g / L. In mammals, Ig molecules have a symmetrical structure, composed of four peptide chains linked by disulfide bonds. Figure 1 As shown, the heavy chain (H chain) consists of two long chains, and the light chain (L chain) consists of two short chains. Disulfide bonds between the chains form a classic Y-shape. Regardless of whether it's the heavy or light chain, the amino acid sequence of the first domain is diverse, known as the variable region for antigen recognition in immunoglobulins. Following the variable region are regions with relatively fixed sequences, called constant regions. Except for the hinge region, each domain consists of approximately 110 amino acid residues and has its unique function. The variable regions of immunoglobulins include four framework regions (FRs) that provide the backbone structure of the domains and maintain spatial stability, and three complementarity determining regions (CDRs) that form a complementary relationship with the antigen surface. The amino acid sequences of the CDR regions vary greatly and are also known as hypervariable regions. Compared to the variable regions, the constant regions are more conserved within the species. There are four skeletal regions in both VH and VL, denoted as FR1, FR2, FR3 and FR4, respectively.

[0003] In 1993, Hammers et al. isolated a heavy-chain antibody from camel peripheral blood lymphocytes, differing in structure from traditional antibodies. This antibody naturally lacked a light chain but possessed a variable region CH1 in the heavy chain. By amplifying this variable region via PCR, an antibody fragment, termed a single-domain antibody, could be obtained. This single-domain antibody had a molecular weight of only about 15 kDa, while conventional antibodies typically have a molecular weight of around 155 kDa. Because the molecular weight of the single-domain antibody is only about 1 / 10 of the conventional antibody, it is also called a nanobody (VHH, variable domain of heavy chain of heavy-chain antibody). Even though this antibody contains only a heavy chain, it still possesses complete antigen-binding properties. VHH antibodies not only have complete antigen affinity but also possess many advantages. For example, the CDR3 region of the VHH antibody has an exposed large convex ring (stable by disulfide bonds), which can penetrate deep into the cracks or cavities of the antigen, enhancing the binding ability of the VHH antibody to the antigen. This antibody has advantages such as small molecular weight, ease of acquisition and expression, high water solubility and stability, and simple targeting and humanization. VHH antibodies have great potential applications as research tools, disease diagnostic tools, and disease treatment strategies.

[0004] C-reactive protein (CRP) is an acute-phase reactive protein produced by the body in response to inflammation, infection, or injury. Elevated levels indicate an inflammatory response. Human CRP plays important functions, such as participating in host defense, antigen clearance, and metabolism, all of which require interaction with a range of exogenous and autogenous factors. CRP can interact with DNA and histones, and it can clear nuclear material released from damaged circulating cells. In addition to these functions and effects, CRP also has pathogenic effects. For example, elevated CRP levels have been shown to be closely associated with coronary atherosclerosis. CRP is not only a marker of atherosclerosis but may also directly participate in its formation. CRP binds to oxidized LDL (low-density lipoprotein), exacerbating tissue damage in coronary artery infarction and inhibiting the repair of damaged vascular endothelium. Summary of the Invention

[0005] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a CRP camel-derived single-domain antibody, its preparation method and application.

[0006] The technical solution of the present invention is as follows:

[0007] A CRP camel-derived single-domain antibody includes a framework region and a complementarity-determining region, wherein the complementarity-determining region has a first complementarity-determining region, a second complementarity-determining region, and a third complementarity-determining region; the first complementarity-determining region is the amino acid region of positions 28-34 on the amino acid sequence SEQ ID NO:3, the second complementarity-determining region is the amino acid region of positions 52-58 on the amino acid sequence SEQ ID NO:3, and the third complementarity-determining region is the amino acid region of positions 97-106 on the amino acid sequence SEQ ID NO:3.

[0008] This invention also discloses a method for preparing camel-derived CRP single-domain antibodies. The method involves enriching CRP antigen in alpaca leukocytes immunized with the antigen, followed by single-cell screening and sequencing. Through sequencing structure analysis, a recombinant plasmid for camel-derived CRP antibodies is constructed. This plasmid is cloned and transformed into eukaryotic cells to express related antibodies. Monoclonal antibodies with human CRP resistance are screened for expression. The VHH domain of the monoclonal antibody is then cloned and expressed, and the antibody is obtained through pairing screening.

[0009] The present invention also discloses an ELISA kit comprising the CRP camel-derived single-domain antibody as described above or the CRP camel-derived single-domain antibody prepared by the preparation method described above.

[0010] The present invention also discloses a chemiluminescence reagent kit comprising the CRP camel-derived single-domain antibody described above or the CRP camel-derived single-domain antibody prepared by the preparation method described above.

[0011] The present invention also discloses a latex kit containing the CRP camel-derived single-domain antibody as described above or the CRP camel-derived single-domain antibody prepared by the preparation method described above.

[0012] The present invention also discloses the application of the CRP camel-derived single-domain antibody as described above or the CRP camel-derived single-domain antibody prepared by the preparation method as described above in the preparation of antitumor drugs.

[0013] The beneficial effects of this invention are as follows: This invention enriches alpaca leukocytes immunized with CRP antigen, followed by single-cell screening and sequencing. Through sequencing structure analysis, a recombinant plasmid of camel-derived CRP antibody is constructed, cloned and transformed into eukaryotic cells to express related antibodies, and monoclonal antibodies with human CRP resistance are obtained through screening. The VHH domain of the monoclonal antibody is cloned and expressed, and CRP-VHH monoclonal antibodies with antibody activity are obtained through pairing screening. This antibody can be used in ELISA kits, chemiluminescence kits, and latex kits, and has the same standard function as existing related kits for detection. At the same time, this antibody still has applications in the preparation of anti-tumor drugs. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the structure of the Ig molecule in mammals in the background art;

[0015] Figure 2 This is a standard curve graph for the CRP-VHH antibody kit.

[0016] Figure 3 This is a graph showing the results of CRP-VHH antibody chemiluminescence assay.

[0017] Figure 4 This is a graph showing the detection results of CRP-VHH antibody-conjugated latex microspheres. Detailed Implementation

[0018] This section will describe specific embodiments of the present invention in detail, but it should not be construed as limiting the scope of protection of the present invention.

[0019] Example 1

[0020] Alpaca leukocytes were enriched with CRP antigen through immunization, and then single-cell screening was performed using CytoFLEX LX flow cytometry, followed by sequencing using the 10X Genomics single-cell sequencing platform. Based on the sequencing structure analysis, 100 recombinant plasmids of camel-derived CRP antibodies were constructed, cloned, transformed into eukaryotic cells to express the relevant antibodies, and screened by ELISA to obtain 20 monoclonal antibodies with human CRP resistance (see Table 1 for specific numbering). Finally, the VHH domain of the 20 CRP monoclonal antibodies was cloned and expressed, and 6 CRP-VHH monoclonal antibodies with antibody activity were obtained through ELISA pairing screening (see Table 1 for specific numbering).

[0021] Table 1. Recombinant vectors expressing CRP and CRP-VHH antibody activity

[0022]

[0023] The antibody includes a framework region and a complementarity-determining region, wherein the complementarity-determining region has a first complementarity-determining region, a second complementarity-determining region, and a third complementarity-determining region; wherein having at least 80% homology with the complementarity-determining region and / or having at least 60% homology with the framework region is within the scope of protection of this invention.

[0024] The amino acid sequence of this antibody is as follows: (Bold and underlined parts are complementation-determining regions, the rest are framework regions)

[0025] As shown in SEQ ID NO: 1, the first complementarity-determining region is the amino acid region from position 28 to 35, the second complementarity-determining region is the amino acid region from position 52 to 58, and the third complementarity-determining region is the amino acid region from position 96 to 108; specifically, SEQ ID NO: 1:

[0026] MAEVQLVESGGGLVQAGESLRLSCAAS GSMFSDNV MGWYRQAPGKQRELAF IRTGGST YADSVKGRFTISRDNSVNTVYLQMDSLKPEDTAIYFC RHTIPVPSTPYDY WGQGTQVTVSS

[0027] As shown in SEQ ID NO: 2, the first complementarity-determining region is the amino acid region from position 28 to 34, the second complementarity-determining region is the amino acid region from position 52 to 58, and the third complementarity-determining region is the amino acid region from position 97 to 106; specifically, SEQ ID NO: 2: MAEVQLVESGGGLVQAGESLRLSCAAS TSIFDRL MGWYRQAPGKQRELAAF ITPSGR T TYADSVKGRFTISRDNSVNTVYLQMDSLKPEDTAIYFC YYRLNPDQNY WGQGTQVTVSS

[0028] As shown in SEQ ID NO: 3, the first complementarity-determining region is the amino acid region from position 28 to 34, the second complementarity-determining region is the amino acid region from position 52 to 58, and the third complementarity-determining region is the amino acid region from position 97 to 106; specifically, SEQ ID NO: 3: MAEVQLVESGGGLVQAGESLRLSCAAS QSIFDRL MGWYRQAPGKQRELAAF ITPGGR T TYADSVKGRFTISRDNSVNTVYLQMDSLKPEDTAIYFC YYRLNPDSNY WGQGTQVTVSS

[0029] As shown in SEQ ID NO: 4, the first complementarity-determining region is the amino acid region from position 28 to 34, the second complementarity-determining region is the amino acid region from position 52 to 58, and the third complementarity-determining region is the amino acid region from position 97 to 106; specifically, SEQ ID NO: 4: MAEVQLVESGGGAVQAGESLRLSCAAS TSIFDLL MGWYRQAPGKQRELAAF INPSGR T TYADSVKGRFTISRDNSVNTVYLQMDSLKPEDTAIYFC RNRLNPDQNY WGQGTQVTVSS

[0030] As shown in SEQ ID NO: 5, the first complementarity-determining region is the amino acid region from position 28 to 34, the second complementarity-determining region is the amino acid region from position 52 to 58, and the third complementarity-determining region is the amino acid region from position 97 to 105; specifically, SEQ ID NO: 5: MAEVQIVESGGGLVNAGESLRLSCAAS TTIFDRL MGWYRNAPGKQRELAAF ISPSGR T TYADSVKGRFTISRDNSVQTVYLQMDSLKPEDSAIYFC YYRLNPDQN YWGQGTQVTVSS

[0031] As shown in SEQ ID NO: 6, the first complementarity-determining region is the amino acid region from position 28 to 34, the second complementarity-determining region is the amino acid region from position 52 to 58, and the third complementarity-determining region is the amino acid region from position 97 to 106. Specifically, SEQ ID NO: 6: MAEVQLVESGGGLVQAGESLRLTCAAS TSAFDRL MGWYRQAPGKQRELAAF ITPTGRT TYADSVKGRFTISRDNSVNTVYLQMDSLKPEDTAIYFC YYRLNPDNQY WGQGTQVTVSS

[0032] (1) Antibody ELISA screening method:

[0033] Reagents used: Coating buffer: 50mM carbonate buffer, pH 9.6; Antibody dilution buffer: 20mM PBS pH 7.4 + 0.2% BSA; Washing buffer: 20mM PBS pH 7.4 + 0.05% Tween-20; Blocking buffer: 50mM PBS pH 9.6 + 2.0% BSA; Chromogenic buffer: TMB-hydrogen peroxide urea solution; Stop solution: 2mol / L H2SO4; Enzyme-labeled secondary antibody rabbit anti-camel crosslinked HRP.

[0034] CRP antigen was diluted to 1 μg / ml and coated in 96-well plates at 100 µL per well, incubated overnight at 4°C. The next day, the plates were washed three times with washing buffer and patted dry. Blocking buffer was added at 120 µL per well, incubated at 37°C for 1 hour, and patted dry. Diluted CRP antibody was added at 100 µL per well, incubated at 37°C for 1 hour, washed three times with washing buffer, and patted dry. Secondary antibody labeled with rabbit anti-camel crosslinked HRP was added at 100 µL per well, incubated at 37°C for 1 hour, washed three times with washing buffer, and patted dry. Chromogenic solutions A and B were mixed 1:1 and added to each well at 100 µL, incubated for 10 minutes. Stop solution was added at 50 µL per well. OD values ​​were read at 450 nm using a microplate reader. Results are shown in Table 2.

[0035] Table 2. Results of ELISA detection of recombinant CRP-VHH antibody

[0036]

[0037] As can be seen from Table 2, CRP-VHH antibodies Seq2, Seq15, Seq33, Seq36, Seq68, and Seq81 have absorbance values, indicating that their recombinant antibodies have potency.

[0038] Example 2: Application directions of the above-mentioned antibodies

[0039] The purified CRP-VHH monoclonal antibody was crosslinked onto magnetic microspheres (Fe3O4 nanoparticles, 1μm self-produced), latex microspheres (150nm latex carboxyl microspheres, self-produced), and coated onto ELISA plates (corning, catalog number: 42592) using coating solution. Chemiluminescence, crosslinked latex microspheres, and ELISA detection were then performed.

[0040] (1) ELISA kit direction

[0041] Kit components: Raw materials: antibody mAb135-HRP (screening mouse monoclonal antibody labeled with HRP), CRP-VHH; chromogenic solution A: TMB, chromogenic solution B: H2O2; blocking solution: skim milk powder; washing solution: 1XPBST; stop solution: 2mol / L concentrated sulfuric acid.

[0042] Pairing and antibody application method: Dilute CRP-VHH antibody with coating buffer (50mM carbonate buffer, pH 9.6) and coat 96-well plates with 100µL per well, incubate overnight at 4℃; wash 3 times with washing buffer the next day and blot dry; add blocking buffer, 120µL per well, incubate at 37℃ for 1h and blot dry; add diluted CRP antigen, 100µL / well, incubate at 37℃ for 1h and wash 3 times with washing buffer and blot dry; add labeled mAb135-HRP, 100µL per well, incubate at 37℃ for 1h and wash 3 times with washing buffer and blot dry; mix chromogenic solutions A and B 1:1 and add 100µL to each well, incubate for 10min; add stop solution, 50µL / well; read the OD value at 450nm on a microplate reader.

[0043] The experimental results are shown in Tables 3 and 4. Figure 2 By establishing a standard curve, it can be seen that the screened CRP-VHH has antibody titers, with Seq33 and Seq81 showing higher titers, making them suitable for application in ELISA kits. The standard curve is constructed with RD concentration as the ordinate and OD value as the abscissa, resulting in the equation y = 4.482x. 2 +5.6224x, R 2=0.9997, and each CRP-VHH test with an OD value above 0.8 is considered to have antibody titer.

[0044] Table 3. Results of the CRP-VHH Antibody Kit Detection Standard Curve

[0045]

[0046] Table 4. Results of Antibody Titer Screening Sequences

[0047]

[0048] (2) Application directions of chemiluminescence reagent kits

[0049] Kit components:

[0050] Raw material R1: antibody mAb135-HRP, six kinds of CRP-VHH obtained in Example 1 coupled with magnetic microspheres, CRP antigen standard; colorimetric solutions A and B; washing solution: TBST.

[0051] Chemiluminescence assay procedure: Remove the coated plate containing CRP-VHH monoclonal antibody. Use serially diluted CRP antigen standard, adding 20 µL of standard to each well, followed by 0.0 µL of mAb135-HRP. Incubate at 37°C for 1 h. Wash 5 times with washing buffer and pat dry with absorbent paper. Add 50 µL each of chromogenic solution A and solution B to each well. Incubate at room temperature in the dark for 20 min before detecting the results using a chemiluminescence analyzer.

[0052] The test results are shown in Table 5 and Figure 3 By establishing a standard curve for detection, it can be seen that the screened CRP-VHH has antibody titer and can be applied to chemiluminescence reagent kits.

[0053] Table 5. Results of CRP-VHH Antibody Detection by Chemiluminescence Assay

[0054]

[0055] (3) Application directions of latex reagent kits

[0056] The kit components are as follows: Reaction solution 1: 150nm latex carboxyl microspheres crosslinked with the six CRP-VHH antibodies obtained in Example 1; Reaction solution 2: 150nm latex carboxyl microspheres crosslinked with mAb135; CRP antigen standard; Buffer: 10mM Tris buffer.

[0057] The detection method of immunoturbidimetric assay: Add 240 µL of the prepared reaction solution 1 to the reaction, and use serial dilution of the standard to add 3 µL to each well. Incubate at 37 °C for 5 min. Add 240 µL of reaction solution 2, mix well, and detect the results using a biochemical analyzer after 5 min.

[0058] The experimental results are shown in Table 6 and Figure 4 .

[0059] Table 6. Detection Results of CRP-VHH Antibody-Conjugated Latex Microspheres

[0060]

[0061] Comparison of clinical outcomes:

[0062] Clinical samples were tested using the company's selected normal CRP mouse monoclonal antibody ELISA kit and camel-derived CRP-VHH monoclonal antibody (selected Seq2 sequence antibody) ELISA kit (a total of 50 serum samples were used for testing; the criteria for judgment were clinically diagnosed normal serum and samples with abnormal CRP detection). The results are shown in Table 7. The two groups showed that the selected CRP-VHH camel-derived monoclonal antibody and the normal CRP mouse monoclonal antibody had the same detection results, and the detection results were consistent with the clinical results, with high accuracy.

[0063] Table 7 Comparison of results of normal mouse monoclonal antibody and CRP-VHH ELISA detection

[0064]

[0065] Based on chemiluminescence, latex turbidimetry, and ELISA detection, it was determined that all six selected camel-derived antibodies could be used for the above applications. (The text then abruptly shifts to a seemingly unrelated topic: CRP-VHH and antibody F.) C The complete antibody sequence composed of the fragments still has the above-mentioned uses (that is, CRP-VHH plus the Fc end forms a complete antibody sequence, and the complete sequence still maintains the application direction of CRP-VHH and can be used in the development of tumor drugs).

[0066] Without causing conflict, those skilled in the art can freely combine and use the above-mentioned additional technical features.

[0067] The above description is only a preferred embodiment of the present invention. Any technical solution that achieves the purpose of the present invention by essentially the same means is within the protection scope of the present invention.

Claims

1. A CRP camel-derived single-domain antibody, characterized in that, It includes a framework region and a complementarity-determining region, wherein the complementarity-determining region has a first complementarity-determining region, a second complementarity-determining region and a third complementarity-determining region; the first complementarity-determining region is the amino acid region of positions 28-34 on the amino acid sequence SEQ ID NO:3, the second complementarity-determining region is the amino acid region of positions 52-58 on the amino acid sequence SEQ ID NO:3, and the third complementarity-determining region is the amino acid region of positions 97-106 on the amino acid sequence SEQ ID NO:

3.

2. An ELISA kit, characterized in that, It contains the CRP camel-derived single-domain antibody as described in claim 1.

3. A chemiluminescence reagent kit, characterized in that, It contains the CRP camel-derived single-domain antibody as described in claim 1.

4. A latex reagent kit, characterized in that, It contains the CRP camel-derived single-domain antibody as described in claim 1.

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