Monoclonal antibody capable of being specifically combined with anti-human PCSK9 antibody as well as preparation method and application thereof

Abstract

The invention discloses a monoclonal antibody capable of being specifically combined with an anti-human PCSK9 antibody as well as a preparation method and application of the monoclonal antibody, and provides an ELISA (enzyme-linked immuno sorbent assay) detection method and a kit for the anti-human PCSK9 antibody serving as an antigen on the basis of the antibody, which are used for detecting the PCSK9 antibody level in a biological sample. And the method can be used as an index for clinical medication guidance and diagnosis / prediction.

Description

Technical Field

[0001] The present invention belongs to the field of reagent diagnosis, and in particular relates to using antibodies and a kit containing the antibodies for clinical detection. Background Art

[0002] PCSK9 (proprotein convertase subtilisin / kexin 9) is an emerging target in the field of lipid-lowering drug development in recent years. PCSK9 plays an important role in LDL-C metabolism. Its mechanism of action is that LDL-C bound to LDL-R is internalized into hepatocytes through vesicles, and then the acidic environment of the endosome causes LDL-C to separate from its receptor. The circulating vesicles return LDL-R to the cell surface, and the endosome containing LDL-C fuses with the lysosome, resulting in LDL-C degradation. In the plasma membrane of hepatocytes, the catalytic domain of secreted PCSK9 binds to LDL-R and is internalized, entering the endosomal pathway. The low pH value in the body enhances the affinity of PCSK9 for LDL-R, preventing the receptor from recycling to the cell surface. After PCSK9 binds to LDL-R, it binds to LDL-C to form a new complex that enters the lysosome and is degraded together, ultimately leading to a decrease in LDL-R, and a decrease in the degradation of LDL-C, thereby increasing the level of LDL-C.

[0003] Hypercholesterolemia is divided into familial and non-familial, but both are lipid metabolism diseases and are important risk factors for coronary artery disease. Statins are currently the most successful drugs for treating hypercholesterolemia, but some patients who cannot tolerate statins and whose LDL-C levels are too high cannot always reach the expected lipid levels. PCSK9 inhibitors, as another metabolic pathway, promote the clearance of low-density lipoprotein cholesterol (LDL-C) by inhibiting the binding of PCSK9 to the low-density lipoprotein receptor (LDLR). They have higher lipid-lowering effects and safety.

[0004] However, conventional methods for detecting drug concentrations of PCSK9 inhibitors mostly use the target protein as a capture reagent and another anti-species IgG antibody as a detection reagent. Such methods usually bind to IgG or other analogs in human serum, and the method has poor specificity and limited clinical application. On this basis, we developed a method that combines a unique antibody with a target protein to detect free drug concentrations. This detection method has good specificity and stability. Summary of the invention

[0005] To solve the problems of the prior art, the present invention provides a monoclonal antibody that specifically binds to an anti-human PCSK9 antibody, and a preparation method and use of an anti-human PCSK9 antibody detection kit containing the antibody.

[0006] In a first aspect, the present invention provides a monoclonal antibody capable of specifically binding to an anti-human PCSK9 antibody, comprising:

[0007] A mouse antibody A that binds to an anti-human PCSK9 antibody, wherein the antibody A comprises a heavy chain variable region shown in SEQ ID NO: 2 and a light chain variable region shown in SEQ ID NO: 4.

[0008] As a preferred technical solution of the present invention, it comprises: the constant region of the antibody A, the constant region of the light chain is the sequence shown in SEQ ID NO: 5, and the constant region of the heavy chain is the sequence shown in SEQ ID NO: 6.

[0009] As a preferred technical solution of the present invention, the anti-human PCSK9 antibody is antibody F, wherein the antibody F comprises a heavy chain variable region shown in SEQ ID NO: 19 and a light chain variable region shown in SEQ ID NO: 20.

[0010] In a second aspect, the present invention provides a preparation comprising:

[0011] Antibody A as described above,

[0012] A stabilizer, wherein the stabilizer is selected from at least one of PBS and glycerol.

[0013] As a preferred technical solution of the present invention, the stabilizer is selected from a combination of PBS and glycerol.

[0014] As a preferred technical solution of the present invention, the concentration of PBS is 0.01 mol / L.

[0015] As a preferred technical solution of the present invention, the volume ratio of PBS to glycerol is 1-9:9-1, preferably 1:1, 2:3, 3:2.

[0016] In a third aspect, the present invention also provides a method for detecting human PCSK9 antibody F in a biological sample, comprising:

[0017] (a) coating a mouse monoclonal antibody that recognizes the CDR region of the antibody F molecule on an ELISA plate as a capture reagent, wherein the capture reagent is an antibody that can specifically bind to the anti-human PCSK9 antibody, and the antibody that can specifically bind to the anti-human PCSK9 antibody is the monoclonal antibody described above;

[0018] (b) After blocking, the standard, quality control and samples to be tested are added to the ELISA plate for incubation;

[0019] (c) detecting that the antibody that can specifically bind to the anti-human PCSK9 antibody F binds to the sample to form an antigen-antibody complex;

[0020] The antibody F comprises a heavy chain variable region shown in SEQ ID NO: 19 and a light chain variable region shown in SEQ ID NO: 20.

[0021] The present invention also provides a kit, comprising: a monoclonal antibody against anti-human PCSK9 antibody F as a capture reagent, wherein the monoclonal antibody is the monoclonal antibody capable of specifically binding to human PCSK9 as described above.

[0022] The present invention also provides a use of the monoclonal antibody capable of specifically binding to the PCSK9 antibody as described above in preparing a detection kit for detecting the content of anti-human PCSK9 antibody F in a sample.

[0023] Unless otherwise defined, all technical and scientific terms used in the context of the present invention have the same meanings as understood by ordinary technicians in the field. Some terms are explained as follows:

[0024] The term "Proprotein convertase subtilisin / kexin type 9 (PCSK9)", "PCSK9" or "NARC-1", a naturally occurring human proprotein convertase belonging to the proteinase K subfamily of the secretory subtilisin family. PCSK9 is synthesized as a lysozyme, undergoes autocatalytic intramolecular processing in the endoplasmic reticulum, and is believed to function as a proprotein convertase. When the term is used in this application, it refers to any naturally occurring PCSK9, preferably any naturally occurring PCSK9 from any vertebrate source (including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats)). Representative amino acid sequences of human PCSK9 and representative nucleic acid sequences encoded by them are disclosed by GenBank Accession Nos. NP_777596.2 and FJ525880.1, respectively. The term "PCSK9" encompasses "full-length" unprocessed PCSK9 as well as any form of PCSK9 or any fragment thereof produced by intracellular processing. The term also includes naturally occurring variants of PCSK9, for example, splice variants, derivative variants, substitution variants, deletion variants and / or insertion variants or allelic variants, such as mutants D374Y, S127R and F216L.

[0025] The term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody or bispecific (bivalent) antibody that can bind to a specific antigen. A natural complete antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region (VH) and three constant regions (CH1, CH2 and CH3), wherein there is a hinge region between CH1 and CH2; each light chain consists of a variable region (VL) and a constant region (CL). The heavy chains of mammals can be divided into α, δ, ε, γ and μ, and the light chains of mammals can be divided into λ or K. The antibody is "Y"-shaped, and the neck of the "Y"-shaped structure is composed of CH2 and CH3 of two heavy chains, which are connected by disulfide bonds in the hinge region; each arm of the "Y"-shaped structure includes the variable region and CH1 of one of the heavy chains, which are connected to the variable region and CL of one light chain by disulfide bonds. The variable regions of the light and heavy chains determine the binding of the antibody to the antigen. The variable region of each chain contains three hypervariable regions (HVR), called complementary determining regions (CDR), of which the CDR region of the light chain (L) includes LCDR1, LCDR2, LCDR3, and the CDR region of the heavy chain (H) includes HCDR1, HCDR2, and HCDR3. The three CDRs are separated by a lateral continuous portion called the framework region (FR), which is more highly conserved than the CDR and forms a scaffold to support the hypervariable loop. Antibodies can be divided into several categories based on the amino acid sequence of the heavy chain constant region. For example, antibodies can be divided into five major classifications or isomers: IgA, IgD, IgE, IgG, and IgM, depending on whether they contain α, δ, ε, γ, and μ heavy chains. Several major antibody classifications can also be divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ 2 heavy chain), IgG3 (γ 3 heavy chain), or IgG4 (γ 4 heavy chain).

[0026] The term "LDL" refers to low-density lipoprotein (LDL), which is converted by the hydrolysis of very low-density lipoprotein (VLDL) by removing triglycerides from very low-density lipoprotein (VLDL) and releasing free fatty acids.

[0027] The term "DiI-LDL" refers to red fluorescently labeled human low-density lipoprotein (Human DiI-LDL), which is LDL labeled with the fluorescent probe DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanineperchlorate).

[0028] The term "biological activity" refers to the specific ability or potential of a biological product to achieve a certain biological effect. The biological effect of a specific cell line can be used to evaluate the corresponding biological activity of a biological product. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the affinity curve of monoclonal antibody A and antibody F;

[0030] Figure 2 is the specificity map of monoclonal antibody A;

[0031] Figure 3 SEC results of different formulations under repeated freeze-thaw conditions;

[0032] Figure 4 nrCE-SDS results of different formulations under repeated freeze-thaw conditions;

[0033] Figure 5 SEC-HPLC results of different formulations at 37°C accelerated conditions;

[0034] Figure 6 nrCE-SDS results of different formulations at 37°C accelerated conditions;

[0035] Figure 7 It is the research result of coating concentration and detection concentration;

[0036] Figure 8 The results of the matrix content study are as follows;

[0037] Fig. 9 This is the result of the HRP dilution multiple study;

[0038] Fig.10 This is the result of the closed component study. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with specific examples and accompanying drawings. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the embodiments of the present invention that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by raw materials or commodity manufacturers. Reagents that do not specify specific sources are conventional reagents purchased from the market.

[0040] Example 1 Preparation of a monoclonal antibody capable of specifically binding to anti-human PCSK9 antibody (hereinafter referred to as antibody A)

[0041] Preparation of immunogen: The full-length anti-human PCSK9 antibody was enzymatically cleaved into F(ab')2, and the immunogen was also F(ab')2. The purity of the prepared immunogen was confirmed to be >85% by SDS-PAGE method.

[0042] Animal immunization: Five Balb / c and five C57 mice were immunized.

[0043] Table 1 Conventional immunization regimen

[0044] step schedule Dosage and route Adjuvant Blood collection before immunization / / / immunity Day 0 S0μg / mouse, subcutaneous Freund's complete adjuvant immunity Day 14 25 μg / mouse, subcutaneous Freund's incomplete adjuvant Blood test Day 21 / / immunity Day 28 251μg / mouse, subcutaneous Freund's incomplete adjuvant Blood test Day 35 / / Free Day 43-57 25 μg / mouse, subcutaneous or intravenous Freund's incomplete adjuvant Cell fusion 4 days after the final exemption / /

[0045] Cell fusion: Mouse spleen and mouse myeloma SP20 cells were electrofused twice, and the fused cells were plated in 96-well plates, with a total of 30 plates. The supernatant of the fused cells was screened by indirect ELISA, and the cell supernatant that was specific for anti-human PCSK9 antibody was selected.

[0046] Subcloning: The above positive mother clones were subcloned by limiting dilution method, and 0.5 cells / well were plated for 2 rounds in total to finally obtain monoclonal cell lines. The positive monoclonal cells were screened again by indirect ELISA method.

[0047] Sequence cloning: The above hybridoma cells are cultured and then lysed to extract total RNA; the RNA is reverse transcribed into cDNA; the heavy chain and light chain variable region sequences are obtained by PCR amplification. The target fragment is connected to the vector using a ligase, and the ligation product is transfected into Escherichia coli competent cells, and finally a single clone is picked for target gene sequencing.

[0048] Expression vector construction: The target gene is digested and connected to construct an antibody eukaryotic expression vector; CHO-K1, CHO-DG44 or CHO-S cells are selected as host cells to express antibodies, and the above antibodies are purified from the cell culture fluid, or the fused hybridoma cells (SP20) are used as host cells to express antibodies, and the above antibodies are purified from the cell culture fluid to obtain antibodies A, B, C, D, E,

[0049] The obtained antibodies were subjected to affinity determination. The experimental method was as follows: Antibody F (see Example 2 for the sequence of antibody F) with a coating concentration of 0.5 μg / ml was added to a 96-well plate at 100 μl / well. The coating buffer was PBS buffer, pH 7.4. The purified antibodies were added in gradient dilutions, followed by goat anti-mouse IgG secondary antibodies. The antibody affinity detection results are shown in Tables 2 and Figure 1 .

[0050] Table 2 Affinity of Antibody A

[0051] Antibody ID EC50(ng / m1) A 11.39 B 10.36 C 9.026 D 9.743 E 12.63

[0052] From Table 2 and Figure 1 It can be seen that the antibodies A, B, C, D, and E of the present invention have good affinity. Figure 1It can be seen that the OD value of antibody A is greater than 2, and the OD values ​​of the other antibodies A are all less than 2. Studies have shown that at high concentrations, the OD value is greater than 2, the color development is high, and the gradient is very good, which is conducive to the development of detection methods and improves detection accuracy.

[0053] Among them, the sequence of antibody A is as follows:

[0054] (1) Heavy chain variable region base sequence:

[0055]

[0056] (2) Heavy chain variable region amino acid sequence:

[0057]

[0058] (3) Light chain variable region base sequence:

[0059]

[0060] (4) Light chain variable region amino acid sequence:

[0061]

[0062] (5) The constant region of the antibody (Fc sequence), including the light chain constant region (Fc) and the heavy chain constant region (Fc).

[0063] Among them, the light chain constant region (Fc) sequence is as follows:

[0064]

[0065] The heavy chain constant region (Fc) sequence is as follows:

[0066]

[0067] Example 2 Preparation of Antibody F

[0068] Antibody F was prepared by the method described in patent document CN201710816808.0, or all the contents of the patent document are fully introduced into the present application by reference. Antibody F was selected from the sequence corresponding to antibody 18.156.8 in patent document CN201710816808.0, as shown in Table 3.

[0069] Table 3 shows the sequence of antibody F

[0070]

[0071] Example 3 Affinity Antibody Specificity Experiment

[0072] The method of Example 1 was used to coat antigen antibody F and human IgG, respectively, with a coating concentration of 0.5 μg / ml, 100 μl / well added to a 96-well plate, and the coating buffer was PBS buffer, pH 7.4. The above purified antibody A was added in gradient dilutions, followed by goat anti-mouse IgG secondary antibody, and the detection results are shown in Figure 2 ,Depend on Figure 2 It was found that Antibody A specifically binds to Antibody F and does not bind to human IgG, which is a structural analogue. Furthermore, Antibody A of the present invention also does not bind to Evolocumab antibody.

[0073] Example 4 Stable formulation screening

[0074] Four formulations, PBS, 0.06% arginine and PBS preparation (referred to as 0.06% arginine), 0.1% Tween 80 and PBS preparation (referred to as 0.1% Tween 80), 0.05% preservative and PBS preparation (referred to as 0.05% preservative), 40% glycerol and PBS preparation (referred to as 40% glycerol), were screened under repeated freeze-thaw and 37°C accelerated test conditions. There was no significant change in the appearance, potency, and protein content of each formulation under repeated freeze-thaw and 37°C accelerated test conditions; the results of SEC and nrCE-SDS under repeated freeze-thaw conditions were shown in Table 1. Figure 3 and Figure 4 The results of SEC and nrCE-SDS under 37°C accelerated test conditions are shown in Figure 5 and Figure 6 , the results showed that antibody A had better stability in 40% glycerol and PBS formulations.

[0075] Example 5 ELISA method for quantitative detection of human PCSK9

[0076] This method uses ELISA to detect the concentration of free antibody F in human serum. Mouse monoclonal antibody A that recognizes the CDR region of antibody F molecules is coated on an ELISA plate as a capture reagent. After blocking, standard products, quality control products and samples to be tested are added to the ELISA plate for incubation. At this time, antibody A combines with the sample to form an antigen-antibody complex and is captured on the ELISA plate. Biotin-labeled PCSK9 protein is added to combine with the complex captured on the ELISA plate. After washing away the free PCSK9 protein, Streptavidin-HRP is added, and then the substrate TMB is used for color development. The stop solution is added to stop the color development reaction and the OD value is read at a wavelength of 450nm. The amount of antibody F content is positively correlated with the final reaction signal value.

[0077] 1.1 The reagents and instruments used in the experiment are shown in Table 4.

[0078] Table 4 Reagents and instruments used in the experiment

[0079] name Specifications / Models Manufacturer Biotin-PCSK9 protein PC9-H82E7 Acro Biosystems human serum XLT001~XLT010 Kangchengtai (Beijing) Pharmaceutical Technology Co., Ltd. Streptavidin-HRP DY998 R&D System Skim Milk D8340 Solarbio TMB TMBS-1000-01 SURMODICS Full wavelength microplate reader VARIOSKAN LUX Thermo Decolorization shaker TS-2 Haimen Qilin Bell Instrument Factory ELISA plate 42592 Coming

[0080] 1.2 Test solution configuration

[0081] The configuration volume is adjusted according to actual needs, and the proportion of each component is kept unchanged during the adjustment.

[0082] 20×PBS: Weigh 9.61g KH2PO4, 145.12g Na2HPO4.12H2O, 320.02g NaCl, 8.00g KCl, add to about 1300ml ultrapure water, stir to dissolve, adjust the volume to 2000ml. Store at 2-8℃ and use within 3 months.

[0083] Coating solution / dilution solution (1×PBS): Take 50ml 20×PBS, add it to 950ml ultrapure water, mix well and set aside. Store at 2-8℃ and use within 3 months.

[0084] Washing solution (0.1% PBST): Add 1 ml Tween-20 to 1 L 1X PBS, mix well and set aside. Store at 2~8°C and use within 3 months.

[0085] Blocking solution (5% Skim Milk-1×PBS): Weigh 5.0 g Skim Milk and dissolve it in 100 ml 1×PBS. Store at room temperature and use it immediately after preparation.

[0086] Stop solution (1mol / L H2SO4): Take 5.4ml H2SO4 (98%) and slowly add it into a certain volume of ultrapure water. Stir and mix thoroughly. After cooling, add ultrapure water to make up to 100ml. Store at 15-25℃ and use within one year.

[0087] Among them, the concentration of 1×PBS is 0.01 mol / L.

[0088] 1.3 Sample Configuration

[0089] Prepare the corresponding standard curve samples (STD1~STD9) and quality control samples (QC1~QC5) according to Table 5 and Table 6.

[0090] Table 5 shows the preparation of standard curve samples (1000×)

[0091]

[0092] Table 6 is the preparation of quality control samples (1000×)

[0093]

[0094] After all the above samples were prepared, they were uniformly diluted 1000 times (1×) with diluent and then added to the samples. The two-step dilution method was used, first diluting 25 times and then diluting 40 times.

[0095] 1.4 Analysis and testing

[0096] a) Coating: dilute antibody A to 2 μg / ml with coating solution, 100 μl / well, seal the plate, and coat overnight (12-18 h) at 2-8°C.

[0097] b) Blocking: discard the liquid in the wells, wash the plate three times with 300 μl / well 0.1% PBST, pat dry, add blocking solution, 300 μl / well, and incubate at room temperature, 250 rpm±50 rpm for 2 h±10 min.

[0098] c) Sample addition: discard the liquid in the wells, wash the plate three times with 300 μl / well 0.1% PBST, pat dry, add diluted standard curve samples, quality control samples and other samples, 50 μl / well, and incubate at room temperature at 250 rpm ± 50 rpm for 1 h ± 5 min.

[0099] d) Add Biotin-PCSK9: discard the liquid in the wells, wash the plate 5 times with 300 μl / well 0.1% PBST, pat dry, add Biotin-PCSK9 diluted to 0.3 μg / ml with diluent, 50 μl / well, incubate at room temperature at 250 rpm±50 rpm for 1 h±5 min.

[0100] e) Add Streptavidin-HRP: discard the liquid in the wells, wash the plate 5 times with 300 μl / well 0.1% PBST, pat dry, add 50 μl / well of Streptavidin-HRP diluted 200 times with diluent, and incubate at room temperature at 250 rpm±50 rpm for 30±2 min.

[0101] f) Add color developing solution: discard the liquid in the wells, wash the plate 5 times with 300 μl / well 0.1% PBST, pat dry, add 100 μl / well TMB, and let stand at room temperature away from light for about 10 minutes.

[0102] g) Stopping and plate reading: Add 100 μl / well of stop solution to stop the reaction, measure the OD value at 450 nm, and perform 4-parameter fitting using the logarithm of the concentration value as the abscissa and OD450nm as the ordinate.

[0103] 1.5 Coating concentration / detection concentration screening

[0104] Antibody A was diluted to 0.5, 1, and 2 μg / ml with coating solution. After overnight coating and blocking, antibody F was diluted to 1000 ng / ml with 1% BSA-1×PBST as diluent, and then diluted 4 times to 0.24 ng / ml as standard, added at 50 μl / well, and incubated at room temperature for 1 hour; at the same time, Biotin-PCSK9 was diluted to 0.3, 1, and 3 μg / ml with 1% BSA-1×PBST as diluent, added at 50 μl / well, and incubated at room temperature for 1 hour. HRP was diluted 200 times, 50 μl / well, and incubated at room temperature for 30 minutes. The results are shown in the table. Figure 7 When the coating concentration was 0.5μg / ml, the signal value was very low. Under the coating concentration of 1 or 2μg / ml, Biotin-PCSK90.3~3μg / ml could have good signal value, signal-to-noise ratio and sensitivity. Considering that Biotin-PCSK9 had a lower background value at 0.3μg / ml.

[0105] 1.6 Matrix content study

[0106] After 1 μg / ml antibody A was coated overnight and blocked, antibody F was diluted to a certain concentration (1000 ng / m1) with 1% BSA-1×PBST, 0.1% serum matrix, 2% serum matrix, and 5% serum matrix, and then diluted 4 times with the same matrix. Several concentration points were selected as standard curve samples, 50 μl / well, and incubated at room temperature for 1 hour. At the same time, Biotin-PCSK9 was diluted to 0.3 μg / ml and added to the corresponding wells, 50 μl / well, and incubated at room temperature for 1 hour. Streptavidin-HRP was diluted to 200 times and added to the corresponding wells, and incubated at room temperature for 30 minutes. The other conditions were the same as 1.4. The results are shown in Figure 8 .

[0107] From the results, it can be seen that with the increase of matrix content, the sensitivity of the method decreased significantly, indicating that the method was highly interfered by the serum matrix. Due to method requirements, a serum content of 0.1% was finally selected as the first choice for the development of this method, that is, the minimum dilution factor was 1000 times.

[0108] 1.7 HRP dilution multiple study

[0109] After overnight coating and blocking of 1μg / ml antibody A, antibody F was diluted to a certain concentration (62.5ng / ml) with 1% BSA-1×PBST, and then diluted 2-fold to 0.244ng / ml with 1% BSA-1×PBST diluent, and added as the standard curve sample, 50μl / well, incubated at room temperature for 1h, and Biotin-PCSK9 was diluted to 0.3μg / ml and added to the corresponding wells, 50μl / well, and incubated at room temperature for 1h. Streptavidin-HRP was diluted to 200, 500, and 1000 times and added to the corresponding wells, 50μl / well, and incubated at room temperature for 30min. The other conditions were the same as 1.4, and the results are shown in Fig. 9 .

[0110] As the HRP dilution factor increased, the sensitivity and signal-to-noise ratio of the method decreased, so HRP 200 times was selected as the final method parameter.

[0111] 1.8 Blocked ingredient research

[0112] The following blocking components (5% bovine serum, 5% Skim Milk (SM), 1% BSA) were prepared with 1×PBS for blocking, with a volume of 300 μl / well. The standard was then diluted with 0.1% serum prepared with 1×PBS as a diluent, and the labeled protein and HRP were diluted with 1×PBS. The results are shown in Fig.10 The results showed that skim milk powder (5% SM) had the best blocking effect (the signal-to-noise ratio was the highest at the low concentration point), so the blocking component used in the later stage was 5% Skim Milk-1×PBS.

[0113] 1.9 ELISA method performance evaluation

[0114] 1.9.1 Standard curve

[0115] Prepare standard curve samples, add samples to two replicate wells during detection, and perform independent analysis three times in total. Perform data statistics based on the sample concentration added to the reaction system.

[0116] The analysis and testing of the samples were carried out according to 1.4. After completion, data analysis was carried out according to the following 1.9.3. The results are shown in Tables 7 and 8. From the results, the %CV and %RE between duplicate wells of the standard curve concentration points (S1-S8) and the %CV between batches and the %RE of the average concentration of each standard curve concentration point (S1-S8) were all within 20%, meeting the requirements that the %RE and %CV of each standard curve (excluding the upper limit of quantification and the lower limit of quantification) were within 20% of the theoretical concentration value, and the %RE and %CV of the upper limit of quantification and the lower limit of quantification were within 25% of the theoretical concentration value.

[0117] Table 7 Statistics of recovery rate at each concentration point of standard curve

[0118]

[0119]

[0120] Table 8 %CV statistics of each concentration point of standard curve

[0121]

[0122] 1.9.2 Accuracy / precision

[0123] Prepare three quality control samples (QC1-QC5) in parallel according to 1.3 above. After preparation, dilute the samples 1000 times (1×). Add samples to two replicate wells for a total of more than 3 independent analyses.

[0124] The sample analysis and testing is carried out according to 1.4. After completion, the data analysis is carried out according to 1.9.3. The results are shown in Table 9.

[0125] From the results, the %CV and %RE between duplicate wells, as well as the %CV between batches and %RE of the average concentration of all QC samples were within 20%, meeting the requirements of QC samples.

[0126] Table 9 Accuracy / precision data statistics

[0127]

[0128] 1.9.3 Data Analysis

[0129] The logarithmic value of each concentration point was used as the abscissa, and the OD value was used as the ordinate. The fitting software was GraphPadPrism 8, and the fitting model was Sigmoidal, 4PL, X is log(concentration), and the weight factor was 1 / Y. 2 After fitting, the corresponding concentration values ​​of the standard curve samples and quality control samples are obtained, and RE is calculated based on the corresponding measured concentration values ​​and theoretical values. The average concentration value, SD, and instrument raw data are based on the original calculated values, and %CV and %RE are retained to one decimal place.

[0130]

[0131] The final sample preparation method is shown in 1.3, the sample dilution factor is 1000 times, and the analysis and detection steps are shown in 1.4. The following is a brief summary of the relevant conditions:

[0132]

[0133] Note: Except for blocking, the diluent used is 1× PBS

[0134] From the above analysis, it can be seen that this method has good accuracy and repeatability, with a detection range of 0.244-31.25 ng / ml, an MRD of 1000 times, and an expected method range of 244-31250 ng / ml in whole serum.

[0135] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A monoclonal antibody capable of specifically binding to an anti-human PCSK9 antibody, characterized in that: include: A mouse antibody A that binds to an anti-human PCSK9 antibody, wherein the antibody A comprises a heavy chain variable region shown in SEQ ID NO: 2 and a light chain variable region shown in SEQ ID NO:

4.

2. A monoclonal antibody capable of specifically binding to an anti-human PCSK9 antibody according to claim 1, characterized in that: include: The constant region of the antibody A, the light chain constant region is the sequence shown in SEQ ID NO: 5, and the heavy chain constant region is the sequence shown in SEQ ID NO:

6.

3. A monoclonal antibody capable of specifically binding to an anti-human PCSK9 antibody according to claim 1, characterized in that: The anti-human PCSK9 antibody is antibody F, wherein the antibody F comprises a heavy chain variable region shown in SEQ ID NO: 19 and a light chain variable region shown in SEQ ID NO:

20.

4. A preparation, characterized in that include: The antibody A according to claim 1 or 2, and a stabilizer, wherein the stabilizer is selected from at least one of PBS and glycerol.

5. The preparation according to claim 4, characterized in that The stabilizer is selected from a combination of PBS and glycerol.

6. The preparation according to claim 4, characterized in that The concentration of the PBS is 0.01 mol / L.

7. The preparation according to any one of claims 4 to 6, characterized in that The volume ratio of PBS to glycerol is 1-9:9-1, preferably 1:1, 2:3, 3:

2.

8. A method for detecting human PCSK9 antibody F in a biological sample, characterized in that, include: (a) coating a mouse monoclonal antibody that recognizes the CDR region of the antibody F molecule on an ELISA plate as a capture reagent, wherein the capture reagent is an antibody that can specifically bind to an anti-human PCSK9 antibody, and the antibody that can specifically bind to an anti-human PCSK9 antibody is the monoclonal antibody according to claim 1 or 2; (b) After blocking, the standard, quality control and samples to be tested are added to the ELISA plate for incubation; (c) detecting that the antibody that can specifically bind to the anti-human PCSK9 antibody F binds to the sample to form an antigen-antibody complex; The antibody F comprises a heavy chain variable region shown in SEQ ID NO: 19 and a light chain variable region shown in SEQ ID NO:

20.

9. A kit comprising: A monoclonal antibody against anti-human PCSK9 antibody F as a capture reagent, wherein the monoclonal antibody is the monoclonal antibody capable of specifically binding to the anti-human PCSK9 antibody according to claim 1 or 2.

10. Use of the monoclonal antibody capable of specifically binding to anti-human PCSK9 antibody as claimed in claim 1 or 2 in the preparation of a detection kit for detecting the content of PCSK9 antibody F in a sample.

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