Monoclonal antibody combination for detecting human pregnancy-associated plasma protein a and use thereof

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody combination for detecting human pregnancy-associated plasma protein A and application thereof. The combination comprises monoclonal antibodies 4H5 and 1G8, and CDR amino acid sequences of heavy chain and light chain variable regions of the antibodies are shown in SEQ ID NO. 1-SEQ ID NO. 12. The combination can be used to construct a double antibody sandwich ELISA detection system, 4H5 is used as a coating antibody, 1G8 is biotin-labeled, signal amplification is realized by combining biotin-avidin, and the detection sensitivity is significantly improved. The method can effectively distinguish serum samples of patients with acute coronary syndrome and healthy people, and is suitable for rapid and sensitive detection of biomarkers related to cardiovascular diseases in primary clinical practice.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a combination of monoclonal antibodies for detecting human pregnancy-associated plasma protein A and its application. Background Technology

[0002] Pregnancy-associated plasma protein A (PAPP-A) is a zinc-binding metalloproteinase initially discovered in the serum of pregnant women. In normal pregnancies, PAPP-A concentrations gradually increase with gestational age. However, in pregnancies with Down syndrome, PAPP-A concentrations are significantly reduced in early pregnancy. Therefore, PAPP-A is widely recognized as a biomarker for Down syndrome screening in early pregnancy. Besides being produced in the placenta, PAPP-A can also be produced in many non-placental sites. Low concentrations of PAPP-A can be detected in the peripheral blood of men and non-pregnant women. Studies have found that PAPP-A can participate in local inflammatory responses and act on lipid-rich atherosclerotic plaques, ultimately leading to plaque rupture. Therefore, PAPP-A is also considered a marker of atherosclerotic plaque stability and is of great significance for the early differential diagnosis of acute coronary syndrome (ACS).

[0003] PAPP-A exists in the blood of pregnant women as an isotetramer, containing two 200 kDa PAPP-A monomers and two erythrophage major basic protein (ProMBP) precursors, with a molecular weight of approximately 500 kDa. In the circulating blood of non-pregnant women, PAPP-A exists as a homodimer, with a molecular weight of approximately 400 kDa. Therefore, there are structural differences in PAPP-A among different populations. Currently, PAPP-A detection is primarily used for prenatal screening and research on obstetric diseases such as adverse pregnancy outcomes. Methods mainly employ labeled immunoassay and immunochromatography, both of which rely on high-performance monoclonal antibodies. Chemiluminescence immunoassay is highly dependent on equipment and expensive reagents, while immunochromatography, although simple to perform, is difficult to standardize and has a relatively large error margin, especially posing a significant risk of false negatives in low-concentration samples. Enzyme-linked immunosorbent assay (ELISA) is a simple and widely accepted method for both qualitative and quantitative detection. It requires only simple instruments and equipment. Furthermore, when combined with a biotin-avidin amplification system, it can significantly improve the sensitivity of the detection method. It is suitable for detecting low levels of PAPP-A in the serum of non-pregnant patients, such as those with acute coronary syndrome, and is beneficial for its widespread application in clinical settings, especially at the grassroots level.

[0004] Therefore, there is an urgent need to develop a monoclonal antibody that specifically recognizes the PAPP-A antigen in non-pregnant individuals, and to establish an immunoassay method and tool that is easy to operate, highly sensitive, and suitable for clinical applications at the grassroots level. Summary of the Invention

[0005] Given the lack of monoclonal antibodies in the existing technology that can specifically identify free PAPP-A in non-pregnant individuals and possess high sensitivity and good stability, resulting in problems such as low detection sensitivity, poor specificity, and susceptibility to interference in the early screening of cardiovascular diseases such as acute coronary syndrome, this invention provides a monoclonal antibody combination for detecting human pregnancy-associated plasma protein A, which effectively distinguishes serum samples from patients with acute coronary syndrome and healthy individuals, solving the technical problems of insufficient sensitivity and difficulty in standardization of existing detection methods in detecting low concentrations of PAPP-A.

[0006] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0007] In a first aspect, the present invention provides a monoclonal antibody assemblies for detecting human pregnancy-associated plasma protein A, the monoclonal antibody assemblies comprising monoclonal antibody 4H5 and monoclonal antibody 1G8.

[0008] The heavy chain variable region of the monoclonal antibody 4H5 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively.

[0009] The light chain variable region of the monoclonal antibody 4H5 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively.

[0010] The heavy chain variable region of the monoclonal antibody 1G8 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively.

[0011] The light chain variable region of the monoclonal antibody 1G8 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

[0012] In some embodiments, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 4H5 is shown in SEQ ID NO. 13; the amino acid sequence of the light chain variable region of the monoclonal antibody 4H5 is shown in SEQ ID NO. 14.

[0013] In some embodiments, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1G8 is shown in SEQ ID NO. 15; the amino acid sequence of the light chain variable region of the monoclonal antibody 1G8 is shown in SEQ ID NO. 16.

[0014] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 4H5 is shown in SEQ ID NO. 17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 4H5 is shown in SEQ ID NO. 18.

[0015] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1G8 is shown in SEQ ID NO. 19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1G8 is shown in SEQ ID NO. 20.

[0016] Secondly, the present invention provides the application of the above-described monoclonal antibody combination in the preparation of a tool for detecting human pregnancy-associated plasma protein A.

[0017] In some embodiments, the tool includes reagents, kits, test strips, and antibody chips.

[0018] In some embodiments, the kit includes a double-antibody sandwich ELISA kit.

[0019] In some embodiments, the kit includes a biotin-avidin amplified double antibody sandwich ELISA kit.

[0020] In some embodiments, the kit is coated with the monoclonal antibody 4H5 and labeled with the monoclonal antibody 1G8.

[0021] Beneficial effects:

[0022] This application provides a monoclonal antibody combination for detecting pregnancy-associated plasma protein A (PAPP-A). The combination comprises monoclonal antibody 4H5 and monoclonal antibody 1G8, which specifically recognize PAPP-A. The complementarity-determining regions (CDRs) of the heavy and light chain variable regions of monoclonal antibodies 4H5 and 1G8 are clearly defined (as shown in SEQ ID NO. 1-12, respectively), ensuring high affinity and specific binding ability of the antibodies to the target antigen. This antibody combination effectively avoids cross-reactivity and significantly improves detection accuracy. It is suitable for constructing a double-antibody sandwich ELISA detection system. Combined with biotin-avidin signal amplification technology, the detection sensitivity can reach 0.2 ng / mL, significantly distinguishing serum samples from patients with acute coronary syndrome from those from healthy individuals. This provides a high-performance, easy-to-use, and suitable immunological tool for the clinical detection of PAPP-A in the early diagnosis of cardiovascular diseases, suitable for promotion at the grassroots level. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The image shows the identification results of the purified monoclonal antibody.

[0025] Figure 2 This is a sensitivity identification diagram for PAPP-A recombinant protein;

[0026] Figure 3 Images showing the results of ACS and normal serum tests;

[0027] Figure 4 This is a diagram illustrating the binding activity of paired monoclonal antibodies. Detailed Implementation

[0028] The embodiments of this application will be further described in detail below with reference to the accompanying drawings and examples. The detailed description of the following embodiments and the accompanying drawings are used to illustrate the principles of this application by way of example, but should not be used to limit the scope of this application. This application can be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

[0029] These embodiments are provided to make the application thorough and complete, and to fully express the scope of the application to those skilled in the art. It should be noted that, unless otherwise specifically stated, the relative arrangement of components and steps, material composition, numerical expressions, and values ​​illustrated in these embodiments should be interpreted as merely exemplary and not as limiting.

[0030] The recombinant human homodimer PAPP-A can be efficiently expressed using a mammalian cell expression system, and its bioactivity is consistent with that of endogenous proteins present in atherosclerotic plaques. Therefore, it can be used as an immunogen for monoclonal antibody screening. This invention utilizes hybridoma technology to screen for specific mouse monoclonal antibodies against PAPP-A and obtains a monoclonal antibody combination suitable for sandwich-paired detection of PAPP-A. Validation has shown that it has excellent detection sensitivity for recombinant PAPP-A, effectively distinguishing between serum from patients with acute coronary syndrome and normal healthy adults. This combination is suitable for rapid and sensitive detection of cardiovascular disease-related biomarkers in primary care settings, providing a raw material basis for the detection of PAPP-A and the diagnosis of related diseases.

[0031] The double-antibody sandwich ELISA detection technology of the present invention belongs to the in vitro immunoassay method and is used to detect the content of PAPP-A in a sample. It does not include the diagnosis or treatment of diseases.

[0032] Example 1

[0033] 1. Screening of PAPP-A monoclonal antibodies

[0034] 1.1 Mouse Immunization

[0035] Human PAPP-A is a recombinantly expressed protein in mammalian cells, a product of Hytest (CAT#8PA1), containing 82-1627aa of human PAPP-A (UniProtKB #Q13219), exhibiting a homodimeric conformation with a His tag at the C-terminus. The immunomodulatory activity of recombinant dPAPP-A is similar to that of endogenous proteins found in atherosclerotic plaques, thus it can be used as an immunizing or screening antigen. Mice were immunized with recombinant PAPP-A protein, and a His-tagged recombinant protein obtained from the same expression system was used as a reverse screening antigen for monoclonal antibody selection. Specifically, purified recombinant PAPP-A protein was mixed with an equal volume of Freund's complete adjuvant (total volume 200 μL) and subcutaneously injected at multiple sites into 6-week-old female BALB / c mice at a dose of 30 μg / mouse. At weeks 2, 4, and 6, booster immunizations were administered subcutaneously at multiple sites with the same dose mixed with an equal volume of Freund's incomplete adjuvant. Seven days after the final immunization, mouse serum was collected to detect antibody titers. Mice with high titers were selected and given a booster immunization of 20 μg of recombinant PAPP-A protein via intraperitoneal pulse. Three days later, the spleens of these mice were harvested for hybridoma cell preparation.

[0036] 1.2 Screening of hybridoma cells

[0037] All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in logarithmic growth phase and then cultured in HAT medium for selection. When the fused cells reached halfway up the bottom of the well, clones that reacted positively with recombinant PAPP-A protein were selected by indirect ELISA. Since the immunogen contained a His tag, background components needed to be screened to identify specific cell lines targeting PAPP-A protein. Positive cells were cloned to a monoclonal state using limiting dilution, and then the cell lines were expanded and cryopreserved.

[0038] Indirect ELISA method for screening positive clones:

[0039] PAPP-A recombinant protein and other recombinant proteins expressed in mammalian cells (CSFV / E2, His tag, Suzhou Nearshore, DRA227) were coated in microplates (coating buffer: sodium carbonate 1.59 g, sodium bicarbonate 2.93 g, diluted to 1 L of pure water, pH 9.6) at a concentration of 1 μg / mL, and incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 150 μL of 3% sucrose + 2% BSA per well, and incubated at 37°C for 2 hours. The plates were then washed once with PBST (PBS containing 0.05% Tween-20, pH 7.4) and patted dry. 50 μL of cell culture supernatant was added, and the plates were incubated at 37°C for 30 min. Discard the liquid from the wells, wash the plate four times with PBST, blot dry, and add 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (Solepro, diluted 5000 times with PBS). Incubate at 37°C for 30 min, wash four more times, blot dry, and add 50 μL / well of TMB chromogenic buffer for incubation at room temperature for 10 min. Finally, add 50 μL of TMB stop solution (Beijing Meike Wande, 1001SA) to stop the reaction. Measure the OD450nm value using a microplate reader. The screening results are shown in Table 1. Select positive cell lines that react with PAPP-A recombinant protein but not with the control recombinant protein for subsequent experiments.

[0040] Table 1: Screening results of monoclonal antibodies

[0041]

[0042] After the selected hybridoma cell lines were expanded and cultured, 0.2 ml (containing 2.5 × 10⁻⁶ cells) was injected intraperitoneally. 6 Female BALB / c mice (cells) were used to collect ascites fluid approximately 10 days later, when the mice’s abdomens were noticeably swollen.

[0043] 2. Purification of monoclonal antibodies

[0044] Centrifuge the ascites fluid at 12000 rpm for 10 minutes, collect 1 ml of the supernatant, dilute it 10-fold with binding buffer (20 mM PB, 150 mM NaCl, pH 7.4), filter it through a 0.22 μm filter, and pump the filtered sample slowly into a Protein L purification column equilibrated with binding buffer using a peristaltic pump. Connect the column to a protein purification instrument, wash with binding buffer for 5-10 column volumes until the UV absorption peak flattens, then elute with elution buffer (0.1 M glycine, pH 2.7), collect the elution peak, adjust the collected sample to neutral with 1 M Tris-HCl (pH 9), and transfer it to a dialysis bag (MW: 8000-14000). Dialyze the sample in 20 mM PBS (pH 7.4) at 2-8 °C for 16 hours. Transfer the liquid from the dialysis bag to a centrifuge tube and centrifuge at 12000 rpm for 5 minutes. The supernatant is the purified monoclonal antibody.

[0045] The purified monoclonal antibody was used to identify the antibody-antigen binding reaction using the indirect ELISA method described above. The antibody was diluted to 1 μg / ml, and the binding reaction with PAPP-A recombinant protein and other recombinant proteins expressed in mammalian cells (CSFV / E2, His tag, Suzhou Nearshore, DRA227) was detected. Results are shown below. Figure 1 And Table 2.

[0046] Table 2: Purification results of monoclonal antibodies

[0047]

[0048] 3. Double-antibody sandwich ELISA method for identifying paired antibodies

[0049] HRP labeling of antibodies:

[0050] HRP-labeled monoclonal antibodies were diluted with carbonate-coupled buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6) to a final concentration of 2 mg / mL. 2 mg HRP was dissolved in 0.5 mL of ultrapure water and thoroughly mixed with 0.5 mL of 0.06 M sodium periodate solution. Then, 0.5 mL of the diluted antibody solution was added to the tube containing HRP, and the mixture was pipetted and incubated at room temperature for 1 h, with periodic mixing during incubation. The labeling reaction was terminated by adding 50 μL of 5 mg / mL sodium borohydride and mixing for 15 min. Finally, the labeled antibody was dialyzed overnight in 0.01 M PBS, pH 7.4 buffer. Glycerol was added at a 1:1 volume ratio, and the mixture was aliquoted and stored at -20 °C.

[0051] Screening for paired monoclonal antibodies:

[0052] The purified monoclonal antibody was diluted to a concentration of 1 μg / mL with coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6), 50 μL / well, and coated overnight at 4°C. The coating buffer was discarded the next day, and the plate was blocked with 3% sucrose and 2% BSA, 150 μL / well, and incubated at 37°C for 2 h. The blocking buffer was discarded. The test antigen (PAPP-A) and control antigen (CSFV / E2) were diluted with PBS at 50 ng / mL and added to the microplate, 50 μL / well, and incubated at 37°C for 35 min. The plate was washed 4 times with PBST washing buffer. 50 μL / well of HRP-labeled monoclonal antibody diluted 1000 times with PBS was added and incubated at 37°C for 35 min. The plate was washed 4 more times, patted dry, and 50 μL / well of TMB chromogenic buffer was added. The plate was incubated at room temperature for 10 min. Finally, 50 μL of TMB chromogenic buffer was added. The reaction was terminated using TMB stop solution, and the OD450nm value was measured using a microplate reader. The pairing condition with the highest P / N value was selected for testing in the biotin-avidin amplification system. The results are shown in Table 3.

[0053] Table 3: Screening results of double antibody sandwich pairing.

[0054]

[0055] According to the data in Table 3, when monoclonal antibody 4H5 is used as the coating antibody and HRP-labeled monoclonal antibody 1G8 is paired, the P / N value is the largest when detecting antigen. This pairing is used to debug the biotin-avidin amplification system.

[0056] 4. Biotin conjugation with antibodies:

[0057] Biotin can specifically bind to avidin or biotin monoclonal antibodies, exhibiting multi-stage amplification. The binding is highly stable and specific, significantly improving the sensitivity of detection methods and minimizing non-specific binding of reagents in practical applications. Therefore, utilizing a biotin-avidin amplification system can effectively enhance the detection performance of antibody pairs.

[0058] The molar ratio of biotin (Thermo, EZ-Link NHS Biotin, 20217) to antibody was 20:1. First, 2.0 mg of activated biotin was dissolved in 360 μL of ultrapure water to prepare a 10 mM biotin solution. 2 mg of antibody was then reacted with 26.6 μL of 10 mM biotin at room temperature with shaking for 3 h (the reaction volume was controlled at approximately 2 mL). Afterward, the biotin-antibody mixture was dialyzed against 0.01 M PBS (pH 7.4) to remove excess free biotin. The antibody concentration was measured after dialyzing, and an equal volume of glycerol was added. The mixture was then stored at -20 °C. The final labeled antibody concentration was approximately 0.5 mg / mL. The biotin-conjugated monoclonal antibody used in this experiment was the previously screened optimal monoclonal antibody 1G8. This antibody, after biotin labeling, was used to construct a biotin-avidin signal amplification system.

[0059] 5. Establishment and optimization of the biotin-avidin amplified ELISA system

[0060] The monoclonal antibody combinations selected above were coated and biotin-conjugated respectively. The most suitable reaction conditions for the scale-up system were determined by exploring the concentrations of the coating antibody, the biotin-conjugated antibody, and the dilution of HRP-labeled streptavidin. The monoclonal antibody 4H5 was coated in microplates (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6) at concentration gradients of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, 50 μL / well, and incubated overnight at 4°C. The plates were washed once with washing buffer (PBST, PBS containing 0.05% Tween-20), blotted dry, and blocked with 3% BSA and 2% sucrose, 150 μL per well, incubated at 37°C for 2 hours, blotted dry, and then dried for later use. The PAPP-A recombinant protein was diluted to a concentration of 10 ng / mL with PBS and added 50 μL to the microplates coated with the monoclonal antibody. At the same time, the recombinant protein CSFV / E2 was diluted to 10 ng / mL as a negative control. The plates were incubated at 37°C for 30 min. Discard the liquid from the wells, wash the plate four times with PBST, and blot dry. Dilute biotin-conjugated monoclonal antibody 1G8 with PBS to concentrations of 1 μg / mL, 2 μg / mL, and 4 μg / mL, and add 50 μL / well to each well of the microplate. Incubate at 37°C for 30 min. Discard the liquid from the wells, wash the plate four times with PBST, blot dry, and add 50 μL / well of HRP-polymerized streptavidin (BIOSYNTH, 65R-S105PHRP, diluted 10000, 20000, and 40000 times with PBS). Incubate at 37°C for 30 min, wash four more times, blot dry, and add 50 μL / well of TMB chromogenic buffer. Incubate at room temperature for 10 min. Finally, add stop solution to terminate the reaction and measure the OD450nm value using a microplate reader. The optimal reaction conditions were selected based on the coating concentration, biotin-conjugated monoclonal antibody concentration, and dilution of HRP-labeled streptavidin, which showed the most significant difference between positive and negative results. The optimization results are shown in Table 4.

[0061] Table 4. Optimization results of the biotin-avidin amplified ELISA system

[0062]

[0063] Table 4 shows that the optimal reaction conditions were: 4H5 monoclonal antibody coating concentration of 1 μg / ml, biotin-labeled monoclonal antibody 1G8 dilution of 1 μg / ml, and HRP-streptavidin dilution of 20,000-fold.

[0064] 6. Sensitivity and specificity identification of biotin-avidin amplified ELISA detection method

[0065] To determine the optimal coating concentration, biotin monoclonal antibody concentration, HRP-avidin dilution, and other reaction conditions, following the above detection steps, PAPP-A recombinant protein was first serially diluted with PBS buffer to obtain concentrations of 20 ng / mL, 2 ng / mL, 0.2 ng / mL, 0.02 ng / mL, and 0 ng / mL (blank control). Simultaneously, CSFV / E2 recombinant protein was used as a negative control at the same dilution. 50 μL of each diluted protein concentration was then used for detection.

[0066] See Figure 2 , Figure 2 PAPP-A is the recombinant PAPP-A protein, and CSFV / E2 is the recombinant CSFV / E2 protein. Using optimized reaction conditions: monoclonal antibody 4H5 coating concentration of 1 μg / ml, biotin-labeled monoclonal antibody 1G8 dilution of 1 μg / ml, and HRP-streptavidin dilution of 20,000-fold, this system showed a positive reaction even when the PAPP-A recombinant protein was diluted to 0.2 ng / ml, but did not react with the CSFV / E2 recombinant protein, demonstrating the good specificity and high sensitivity of this amplified system.

[0067] 7. Application of serum sample testing

[0068] Using the established ELISA amplification system, serum samples from 15 patients diagnosed with acute coronary syndrome (ACS) and 15 healthy adults (normal) were tested. Serum samples from pregnant women were excluded. To reduce matrix effects, the serum samples were diluted 10-fold with 0.01M PBS (pH 7.4) before testing. The results are as follows: Figure 3 Table 5: Using the established ELISA amplification system to test 30 serum samples, the serum test results of patients with acute coronary syndrome (ACS) were significantly different from those of healthy adults (normal).

[0069] Table 5: PAPP-A detection results (OD) of serum samples from 15 ACS patients and 15 healthy individuals. 450 (nm value)

[0070]

[0071] In detection scenarios specifically targeting acute coronary syndrome (ACS), the interference of pregnancy on test results should be excluded. Because serum PAPP-A levels in pregnant women are significantly higher than in non-pregnant individuals, and normal pregnancy itself can lead to a substantial increase in PAPP-A concentration, far exceeding the increase in ACS patients, using PAPP-A as a biomarker for ACS in this case would easily result in the signal being masked by the high background value associated with pregnancy, leading to distorted interpretation or loss of clinical significance. Therefore, PAPP-A is not suitable as an auxiliary diagnostic marker for ACS in pregnant individuals. This detection method and the antibody combination used are specifically designed to identify free PAPP-A in the circulation of non-pregnant individuals. It is suitable for cardiovascular disease risk assessment in non-pregnant populations and for the in vitro detection and analysis of relevant biomarker levels. In clinical applications, pregnant samples must be explicitly excluded to ensure the specificity and accuracy of the test.

[0072] The 15 serum samples selected from ACS patients were all diagnosed based on a comprehensive diagnostic criteria including clinical symptoms, electrocardiogram changes, and myocardial injury markers. They included patients with unstable angina and myocardial infarction, whose common pathological basis was the rupture of unstable atherosclerotic plaques.

[0073] 8. Identification of binding activity of paired monoclonal antibodies

[0074] To verify the binding ability of the monoclonal antibodies in this combination, the binding activity with the recombinant PAPP-A protein was again identified using an indirect ELISA method. The results showed that the antibody still had a significant signal even at a concentration of 1 ng / mL, further demonstrating its high affinity and good detection performance. Specifically, based on the screening of potential paired antibodies using a double-antibody sandwich ELISA, the selected paired monoclonal antibodies and other murine-derived unrelated monoclonal antibodies were serially diluted (concentrations of 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, and 100 pg / mL, respectively) using the aforementioned indirect ELISA method to evaluate their binding activity with the recombinant PAPP-A protein. GFAP murine monoclonal antibody (Hytest, GFAP81cc) was used as a negative control to exclude the influence of non-specific binding. Results are shown below. Figure 4 . Figure 4 The Ctrl key represents the negative control GFAP mouse monoclonal antibody (Hytest, GFAP81cc).

[0075] 9. Variable region gene sequence of monoclonal antibodies

[0076] Total RNA was extracted from hybridoma cells using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using RandomPrimers. Universal primers for the variable region of mouse antibodies were designed, and the VH and VL genes were amplified by two rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the primers for the third round of PCR. The PCR products were purified by gel extraction and ligated into the pUC19 vector, transformed into TOP10 strain, and single colonies were picked and sequenced after culturing at 37°C for 14 h to obtain the gene sequences of the light and heavy chains of the monoclonal antibody.

[0077] The specific sequence is as follows:

[0078] Coated monoclonal antibody 4H5:

[0079] Light chain variable region nucleotide sequence:

[0080] The nucleotide sequence encoding the variable region of the 4H5 light chain of the monoclonal antibody is shown in SEQ ID NO.18:

[0081] GATGTCCAGATGATTCAGTCTCCAGCACTCATGGCTGCATCTCCAGGGGAGAAGGTCCCCATCACCTGCAGTGTCAGCTCAAATATAAGTTCCAGCTACTTGCACTGGTACCAGCAGAAGTCAGGAATCTCCCCCAAATCCTGGATTTATGGCACATCCAACCTGGC TTCTGGAGTCCCTGCTCGTTTCAGTGGCAGTGGATCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGTGGAGTAGTTCCCCATATACGTTCGGAGGGGGGACCAAGCTGGAAATCAAACGTACGGTG.

[0082] Light chain variable region amino acid sequence:

[0083] The amino acid sequence of the variable region of the light chain of monoclonal antibody 4H5 is shown in SEQ ID NO.14:

[0084] DVQMIQSPALMAASPGEKVPITCSVSSNISSSYLHWYQQKSGISPKSWIYGTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSSPYTFGGGTKLEIKRTV.

[0085] Light chain CDR area annotation:

[0086] The CDR-L1 amino acid sequence of the complementarity-determining region of the light chain variable region of monoclonal antibody 4H5 is shown in SEQ ID NO. 4:

[0087] CDR-L1: SVSSNISSSYLH;

[0088] The CDR-L2 amino acid sequence of the complementarity-determining region of the light chain variable region of monoclonal antibody 4H5 is shown in SEQ ID NO. 5:

[0089] CDR-L2: GTSNLAS;

[0090] The CDR-L3 amino acid sequence of the complementarity-determining region of the light chain variable region of monoclonal antibody 4H5 is shown in SEQ ID NO. 6:

[0091] CDR-L3: QQWSSSPYT.

[0092] Heavy chain variable region nucleotide sequence:

[0093] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 4H5 is shown in SEQ ID NO.17:

[0094] CAGGGTGCAGCTGCAGCAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCGGTTTCAGTAGCTATGACATGTCTTGGGTTCGCCAGACTCCGGGGAAGAGACTGGAGTGGGTCGCTTCCATTATTGGTGGTGGTACTTACACCTACTA TCAAGACAGTGTGAAGGGCCGAATCACCATCTCCAGAGACAATGCCAGGAGTACACTGTATCTTCAATTGAGCGGTCTGAGGTCTGAGGACACGGCCTTGTATTACTGTGCAAGACAGGACTTTAAGTATGCCTGGTTTGATTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA.

[0095] Heavy chain variable region amino acid sequence:

[0096] The amino acid sequence of the heavy chain variable region of monoclonal antibody 4H5 is shown in SEQ ID NO.13:

[0097] QVQLQQSGGGLVKPGGSLKLSCAASGFGFSSYDMSWVRQTPGKRLEWVASIIGGGTYTYYQDSVKGRITISRDNARSTLYLQLSGLRSEDTALYYCARQDFKYAWFDYWGQGTLVTVSS.

[0098] Heavy chain CDR region annotation:

[0099] The CDR-H1 amino acid sequence of the complementarity-determining region of the heavy chain variable region of monoclonal antibody 4H5 is shown in SEQ ID NO.1:

[0100] CDR-H1: SYDMS;

[0101] The CDR-H2 amino acid sequence of the complementarity-determining region of the heavy chain variable region of monoclonal antibody 4H5 is shown in SEQ ID NO.2:

[0102] CDR-H2: SIIGGGTYTYYQDSVKG;

[0103] The CDR-H3 amino acid sequence of the complementarity-determining region of the heavy chain variable region of monoclonal antibody 4H5 is shown in SEQ ID NO.3:

[0104] CDR-H3: QDFKYAWFDY.

[0105] Labeled monoclonal antibody 1G8

[0106] Light chain variable region nucleotide sequence:

[0107] The nucleotide sequence encoding the variable region of the light chain of the monoclonal antibody 1G8 is shown in SEQ ID NO.20:

[0108] GACATTTGTGCTGACCCAATCTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTCTACACAGTAATGGAAACACCTATTTCAATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAA CCGAATTTCTGAGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTATACATGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAAATAAAACGTACGGTG.

[0109] Light chain variable region amino acid sequence:

[0110] The amino acid sequence of the variable region of the light chain of monoclonal antibody 1G8 is shown in SEQ ID NO.16:

[0111] DIVLTQSPLSLPVSLGDQASISCRSSQSLLHSNGNTYFNWYLQKPGQSPKLLIYKVSNRISEVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSIHVPLTFGAGTKLEIKRTV.

[0112] Light chain CDR area annotation:

[0113] The amino acid sequence of the complementarity-determining region (CDR-L1) of the light chain variable region of monoclonal antibody 1G8 is shown in SEQ ID NO. 10:

[0114] CDR-L1: RSSQSLLHSNGNTYFN;

[0115] The amino acid sequence of the complementarity-determining region (CDR-L2) of the light chain variable region of monoclonal antibody 1G8 is shown in SEQ ID NO. 11:

[0116] CDR-L2: KVSNRIS;

[0117] The amino acid sequence of the complementarity-determining region CDR-L3 of the light chain variable region of monoclonal antibody 1G8 is shown in SEQ ID NO. 12:

[0118] CDR-L3: SQSIHVPLT.

[0119] Heavy chain variable region nucleotide sequence:

[0120] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1G8 is shown in SEQ ID NO.19:

[0121] CAGGTTCAGCTGCAACAGTCTGGACCTGAGCTAGTGAAGACTGGGGCTTCAGTGAAGATGTCCTGTAAGGCCTCTGGTTACTTATTCACTGGCTACTACATGCACTGGGTCAGACAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATATTAGTTGTTACGATGGTGCTACTAGTTATAA CCAGAAGTTCAAGGACAAGGCCACATTTTCTGTAGACGCATCCTCCAGCACAGTCTATATGCAGTTCAACAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCCCCCGAGGGTTTGCGACGGGGGAGCTATGGTTTGGACTACTGGGGTCAAGGAACCTCAGTCACTGTCTCTGCA.

[0122] Heavy chain variable region amino acid sequence:

[0123] The amino acid sequence of the heavy chain variable region of monoclonal antibody 1G8 is shown in SEQ ID NO.15:

[0124] QVQLQQSGPELVKTGASVKMSCKASGYLFTGYYMHWVRQSHGKSLEWIGYISCYDGATSYNQKFKDKATFSVDASSSTVYMQFNSLTSEDSAVYYCAPEGLRRGSYGLDYWGQGTSVTVSA.

[0125] Heavy chain CDR region annotation:

[0126] The amino acid sequence of the complementarity-determining region CDR-H1 of the heavy chain variable region of monoclonal antibody 1G8 is shown in SEQ ID NO.7;

[0127] CDR-H1; GYYMH;

[0128] The amino acid sequence of the complementarity-determining region CDR-H2 of the heavy chain variable region of monoclonal antibody 1G8 is shown in SEQ ID NO.8;

[0129] CDR-H2: YISCYDGATSYNQKFKD;

[0130] The amino acid sequence of the complementarity-determining region CDR-H3 of the heavy chain variable region of monoclonal antibody 1G8 is shown in SEQ ID NO.9;

[0131] CDR-H3: EGRRGSYGLDY.

[0132] The embodiments of this application have now been described in detail. To avoid obscuring the concept of this application, some details known in the art have not been described. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein based on the above description.

[0133] While specific embodiments of this application have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art should understand that modifications can be made to the above embodiments or equivalent substitutions can be made to some technical features without departing from the scope and spirit of this application.

Claims

1. A monoclonal antibody combination for detecting human pregnancy-associated plasma protein A, characterized in that, The monoclonal antibody combination includes monoclonal antibody 4H5 and monoclonal antibody 1G8. The heavy chain variable region of the monoclonal antibody 4H5 includes three complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.1, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.

3. The light chain variable region of the monoclonal antibody 4H5 includes three complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, the amino acid sequence of CDR-L1 is shown in SEQ ID NO.4, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.5 and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

6. The heavy chain variable region of the monoclonal antibody 1G8 includes three complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.7, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.8, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.

9. The light chain variable region of the monoclonal antibody 1G8 includes three complementarity-determining regions, CDR-L1, CDR-L2, and CDR-L3. The amino acid sequence of CDR-L1 is shown in SEQ ID NO.10, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.11, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

12.

2. The monoclonal antibody combination for detecting human pregnancy-associated plasma protein A according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 4H5 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 4H5 is shown in SEQ ID NO.

14.

3. The monoclonal antibody combination for detecting human pregnancy-associated plasma protein A according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1G8 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 1G8 is shown in SEQ ID NO.

16.

4. The monoclonal antibody combination for detecting human pregnancy-associated plasma protein A according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 4H5 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 4H5 is shown in SEQ ID NO.

18.

5. The monoclonal antibody combination for detecting human pregnancy-associated plasma protein A according to claim 4, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1G8 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1G8 is shown in SEQ ID NO.

20.

6. The use of the monoclonal antibody combination according to claim 1 in the preparation of a tool for detecting human pregnancy-associated plasma protein A.

7. The application according to claim 6, characterized in that, The tools include reagents, kits, test strips, and antibody chips.

8. The application according to claim 7, characterized in that, The kit includes a double-antibody sandwich ELISA kit.

9. The application according to claim 8, characterized in that, The kit includes a biotin-avidin amplified double antibody sandwich ELISA kit.

10. The application according to claim 9, characterized in that, The kit is coated with the monoclonal antibody 4H5 and labeled with the monoclonal antibody 1G8.

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