PGI monoclonal antibody combination for pepsinogen i detection and application thereof

Abstract

This application provides a PGI monoclonal antibody combination for the detection of pepsinogen I and its application. The PGI monoclonal antibody combination for the detection of pepsinogen I includes a first antibody and a second antibody. The first antibody includes a first light chain variable region and a first heavy chain variable region, and the second antibody includes a second light chain variable region and a second heavy chain variable region. The amino acid sequences of the first light chain variable region include VL-CDR1-3 as shown in SEQ ID NO:1-3; the amino acid sequences of the first heavy chain variable region include VH-CDR1-3 as shown in SEQ ID NO:4-6; the amino acid sequences of the second light chain variable region include VL-CDR1-3 as shown in SEQ ID NO:7-9; and the amino acid sequences of the second heavy chain variable region include VH-CDR1-3 as shown in SEQ ID NO:10-12. This monoclonal antibody combination can improve the sensitivity and accuracy of in vitro detection.

Description

Technical Field

[0001] This application relates to the field of biodetection technology, specifically to a monoclonal antibody combination for the detection of pepsinogen I and its application. Background Technology

[0002] Pepsinogen (PG) is a precursor of pepsin, mainly secreted by chief cells and neck mucous cells of the gastric mucosa. Approximately 1% of pepsinogen enters the bloodstream and remains stable. Based on differences in immunogenicity, it can be divided into two subgroups: PGI (pepsinogen I) and PGII (pepsinogen II). PGI is mainly secreted by chief cells and neck mucous cells of the fundic glands, and its level is closely related to the functional state of the fundic gland mucosa. When the fundic gland mucosa exhibits atrophy, damage, or other lesions, PGI secretion decreases, and serum PGI levels drop significantly. Therefore, PGI is often used as an important biomarker for assessing gastric mucosal function and diagnosing gastric diseases, and is considered a "serological gastroscopy test."

[0003] Currently, PGI testing is widely used for screening and monitoring gastric diseases, but existing testing reagents have several shortcomings: Firstly, some reagents use polyclonal antibodies, which suffer from poor specificity and large batch-to-batch variability, and are prone to cross-reaction with PGII or other proteins, leading to inaccurate test results. Secondly, most existing monoclonal antibodies are derived from mice and are easily interfered with by anti-mouse antibodies in the sample, causing deviations in the test results. In addition, some antibodies have low affinity, resulting in insufficient detection sensitivity and inability to accurately detect PGI levels in low-concentration samples.

[0004] Immunoassay techniques are widely used in pathogen detection due to their high specificity and ease of operation. Existing technologies such as immunochromatography, enzyme-linked immunosorbent assay (ELISA), and chemiluminescent immunoassay can all be used for antigen detection. However, the core of these methods lies in using high-quality antibodies as recognition elements.

[0005] Monoclonal antibodies possess advantages such as high specificity, high affinity, good uniformity, and large-scale production, playing an irreplaceable role in the field of immunoassay. Therefore, preparing rabbit-derived PGI monoclonal antibodies and constructing efficient and accurate detection methods is of great significance for improving the accuracy of PGI detection and expanding its application scenarios, and can promote the development of diagnostic technologies for gastric diseases and the in-depth development of related basic research. Summary of the Invention

[0006] This application provides a PGI monoclonal antibody combination for the detection of pepsinogen I and its application, aiming to solve the technical problems of low detection sensitivity, poor specificity, easy cross-reactivity and poor batch stability of antibodies in the in vitro diagnosis of pepsinogen I infection. It is applicable to a variety of immunoassay methods and can be used to improve the early detection rate and diagnostic accuracy of pepsinogen I infection.

[0007] In a first aspect, this application provides a PGI monoclonal antibody combination for pepsinogen I detection, comprising a first antibody and a second antibody, wherein the first antibody includes a first light chain variable region and a first heavy chain variable region, and the second antibody includes a second light chain variable region and a second heavy chain variable region; wherein...

[0008] The amino acid sequence of the first light chain variable region includes VL-CDR1 shown in SEQ ID NO:1, VL-CDR2 shown in SEQ ID NO:2 and VL-CDR3 shown in SEQ ID NO:3;

[0009] The amino acid sequence of the first heavy chain variable region includes VH-CDR1 shown in SEQ ID NO:4, VH-CDR2 shown in SEQ ID NO:5, and VH-CDR3 shown in SEQ ID NO:6;

[0010] The amino acid sequence of the second light chain variable region includes VL-CDR1 shown in SEQ ID NO:7, VL-CDR2 shown in SEQ ID NO:8, and VL-CDR3 shown in SEQ ID NO:9;

[0011] The amino acid sequence of the variable region of the second heavy chain includes VH-CDR1 shown in SEQ ID NO:10, VH-CDR2 shown in SEQ ID NO:11, and VH-CDR3 shown in SEQ ID NO:12.

[0012] According to this application, the monoclonal antibody combination contains two rabbit-derived antibodies that can recognize PGI, thereby achieving higher binding affinity and stronger specificity, effectively reducing the risk of false negatives and cross-reactions, and improving the sensitivity and accuracy of in vitro detection.

[0013] Specifically, the first and second antibodies each possess independent light and heavy chain variable regions, and their respective CDR1, CDR2, and CDR3 sequences have been validated through animal immunization, screening, and cloning, demonstrating stability and specific recognition capabilities. By clearly defining the six complementarity-determining regions (CDRs) of the two antibodies at the structural level, the precise localization and reproducibility of the antigen recognition sites are ensured, contributing to maintaining the consistency and stability of antibody function during subsequent recombinant expression.

[0014] Furthermore, the first and second antibodies can work synergistically when binding to the PGI antigen, resulting in a significant enhancement of the antibody combination provided in this application compared to a single antibody in terms of antigen capture, detection signal intensity, and anti-interference ability, thereby effectively improving the sensitivity and accuracy of in vitro detection of pepsinogen I.

[0015] In some embodiments, the amino acid sequence of the first light chain variable region is shown in SEQ ID NO:13; and the amino acid sequence of the first heavy chain variable region is shown in SEQ ID NO:14.

[0016] In some embodiments, the amino acid sequence of the second light chain variable region is shown in SEQ ID NO:15; and the amino acid sequence of the second heavy chain variable region is shown in SEQ ID NO:16.

[0017] In some of the above embodiments, by defining the full-length variable region sequence, rather than just the complementarity-determining region, the natural combination relationship between the frame region (FR) and the CDR can be fully preserved. The frame region plays an important role in maintaining the spatial conformation of the antibody variable region, stabilizing the correct folding of the CDR, and supporting the formation of the antigen-binding surface. Therefore, further defining the full-length variable region sequence based on the CDR sequence can ensure that the antibody maintains a stable tertiary structure and a high level of specific binding ability in recombinant expression and practical applications. Compared with the scheme that relies solely on the CDR sequence definition, this implementation can reduce structural perturbations or affinity reductions caused by frame region differences, ensure batch-to-batch consistency and reproducibility of antibody function, and thus exhibit higher stability and reliability in practical detection applications.

[0018] In some embodiments, the nucleotide sequence of the first light chain variable region is shown in SEQ ID NO:17; and the nucleotide sequence of the first heavy chain variable region is shown in SEQ ID NO:18.

[0019] In some embodiments, the nucleotide sequence of the second light chain variable region is shown in SEQ ID NO:19; and the nucleotide sequence of the second heavy chain variable region is shown in SEQ ID NO:20.

[0020] In some of the above embodiments, by explicitly defining the nucleotide sequence of the antibody's variable region, it can be ensured that the antibody can be stably expressed in different recombinant expression systems and maintain the correct amino acid sequence. Nucleotide-level definition not only covers the translation products of the amino acid sequence but also ensures the consistency between transcription and translation, thereby avoiding amino acid drift caused by codon differences or potential mutations. Compared to defining only at the amino acid level, nucleotide sequence definition provides a more direct technical basis for subsequent plasmid construction, vector assembly, and expression optimization, improving process controllability and production stability.

[0021] Secondly, this application provides the use of the PGI monoclonal antibody according to any embodiment of the first aspect in the preparation of a tool for detecting pepsinogen I.

[0022] According to this application, by applying the above-mentioned antibody combination to in vitro diagnostic tools, a highly sensitive and specific pepsinogen I detection system can be constructed; the antibody combination can simultaneously recognize the PGI antigen during the detection process, achieving multiple capture and signal enhancement, thereby significantly improving the sensitivity and accuracy of the detection; compared with single antibodies, this combination has significant advantages in reducing false negatives, reducing cross-reactivity, and improving the reliability of detection results.

[0023] In some embodiments, the tool for detecting pepsinogen I is used to detect pepsinogen I in an in vitro sample, said in vitro sample including at least one of serum, plasma and whole blood.

[0024] In some of the above embodiments, specifying the sample type facilitates rapid and accurate detection in different clinical scenarios; serum, plasma, and whole blood expand the scope of application. Compared to detection tools limited to a single sample type, the tools described in this embodiment have wider applicability in clinical application.

[0025] In some embodiments, the PGI monoclonal antibody is used in an immunoassay method for detecting pepsinogen I in an in vitro sample.

[0026] In some of the above embodiments, by introducing the antibody combination provided in this application into the immune detection system, the presence of PGI in in vitro samples can be directly detected by utilizing the principle of specific binding between antigen and antibody.

[0027] In some embodiments, the immunoassay method includes at least one of enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, chemiluminescence immunoassay, immunomicrosphere assay, and immunogold assay.

[0028] In some of the above embodiments, different immunoassay methods can utilize the high specificity recognition performance of the antibody combination of this application; enzyme-linked immunosorbent assay (ELISA) is suitable for high-throughput detection in laboratories; immunofluorescence and chemiluminescence methods have higher sensitivity and quantitative detection capabilities; immunomicrosphere methods facilitate multiplexing; and immunogold methods are suitable for rapid and convenient point-of-care testing. Therefore, with the antibody combination of this application, different detection platforms can be flexibly combined to meet the diverse needs of laboratory research and point-of-care testing.

[0029] In some embodiments, the tool for detecting pepsinogen I includes at least one of reagents, kits, test strips, and antibody chips.

[0030] In some of the above embodiments, the antibody combinations provided in this application can be used to prepare different forms of detection products. The reagents or kits are suitable for standardized laboratory testing, the test strips facilitate rapid screening and point-of-care testing, and the antibody chips can achieve high-throughput, automated parallel analysis. Through the expansion of various tool forms, this embodiment can cover a variety of different application scenarios, greatly improving the flexibility and promotional value of testing.

[0031] Compared with the prior art, the beneficial effects of this application are at least as follows:

[0032] 1. The PGI monoclonal antibody combination provided in this application contains two antibodies obtained through animal immunization and selection. By clearly defining the CDR sequences of their respective light chain and heavy chain variable regions, efficient recognition of PGI is achieved, and higher affinity and specificity can be obtained in in vitro detection.

[0033] 2. Using two antibodies together in the same detection system can create a synergistic effect during antigen capture. Compared with single antibody detection methods, this can effectively reduce the risk of false negatives and cross-reactions, thereby improving the sensitivity and accuracy of the test results.

[0034] 3. By further defining the amino acid and nucleotide sequences of the full-length variable region, the structural consistency and functional stability of the antibody in recombinant expression and mass production can be ensured, thereby improving the reproducibility and reliability of the detection tool in clinical and industrial applications. Attached Figure Description

[0035] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0036] Figure 1 This is an SDS-PAGE protein electrophoresis chromatogram of monoclonal antibodies Ab3# and Ab4# in one embodiment of this application. Detailed Implementation

[0037] The various embodiments or implementation schemes in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments.

[0038] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with an embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0039] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0040] In this specification, unless otherwise specified, "parts" refers to "parts by weight".

[0041] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0042] The main process route for preparing monoclonal antibodies in this application is as follows: first, antibodies against PGI are obtained through animal immunization; then, monoclonal antibody cell lines are obtained through hybridoma technology; then, monoclonal antibody sequences are obtained through sequencing; and finally, monoclonal antibody plasmids are constructed through genetic engineering technology, and monoclonal antibodies are expressed using a eukaryotic mammalian expression system.

[0043] I. Experimental Materials:

[0044] Pepsinogen I (PGI): In-house prepared. Point mutations and codon optimization were performed based on publicly available data from GenBank (GenBank accession number: NM_014224.5), and gene synthesis and plasmid construction were commissioned to Nanjing GenScript Biotech Co., Ltd.

[0045] Nickel ion affinity chromatography column, Protein G affinity chromatography column: purchased from GE Healthcare;

[0046] Freund's adjuvant: purchased from SIGMA;

[0047] New Zealand White Rabbit: Purchased from the China National Institutes for Food and Drug Control (Daxing);

[0048] II. Experimental Methods:

[0049] (1) Preparation of PGI:

[0050] A: Protein Expression: The constructed PGI plasmid was added to the culture medium and mixed thoroughly. Then, 4 times the mass of the plasmid transfection reagent PEI was added. After mixing, the mixture was incubated at room temperature in the dark for 10 minutes. Then, while shaking the cell flask, the prepared mixture was added to the 293-F suspension cells (density 2×10⁶). 6 The cells were cultured in a solution of (cells / mL) at 37°C and 5% CO2 for 120 h, and the cell supernatant was collected.

[0051] B: Protein purification: Nickel ion affinity chromatography was used for purification. After sample loading, the column was washed with washing buffer (50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0) until the UV absorbance returned to baseline. The target protein was then eluted with elution buffer (50 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, pH 8.0), and the elution peak was collected. The collected target protein in the elution peak was dialyzed in 0.02 mol / L PBS (pH 7.4) using a dialysis bag (molecular weight cutoff of 10 kDa). The dialysate was changed 3 times. After dialysis, the protein concentration was measured and stored at -80℃ for later use.

[0052] (2) Animal immunization:

[0053] A: Mix the self-made immunogen with Freund's adjuvant in equal volumes to a suitable volume and emulsify it completely. Immunize the New Zealand white rabbits by subcutaneous injection at multiple points. Each New Zealand white rabbit is injected with 200 μg of immunogen, once every two weeks, for a total of 10 New Zealand white rabbits.

[0054] B: After 10 immunizations, one week after the last immunization, blood was collected from the ear vein of the New Zealand white rabbits. The serum was collected by centrifugation and tested using the ELISA method (plate coated with PGI). The OD values ​​of different New Zealand white rabbit serums after different dilutions are shown in Table 1.

[0055] Table 1

[0056]

[0057] According to Table 1, rabbits #2, #4, #6, and #7 had low antibody titers and were eliminated; among rabbits #1, #3, #5, #8, #9, and #10, rabbit #5 had the best antibody titer and was selected for monoclonal cell preparation.

[0058] (3) Preparation of monoclonal antibodies:

[0059] A: Screening of hybridoma cells

[0060] a) The spleens of the New Zealand white rabbits obtained from the screening were processed, and the isolated spleen cells were fused with myeloma cells. The fused cells were screened and plated for culture using the limiting dilution method.

[0061] b) Screening and culturing monoclonal cell wells, and detecting the supernatant after cell culture by ELISA. The four monoclonal cell wells with the highest OD values ​​in the cell culture plate were selected as target hybridoma cells (1#, 2#, 3#, 4#) for culture and amplification, which were used for cryopreservation of cell seeds and purification and verification of monoclonal antibodies.

[0062] B: Preparation of monoclonal antibodies

[0063] a) Isolation of antibody variable region genes from hybridoma cells using RT-PCR: After homogenizing the optimal hybridoma cells, add cell lysis buffer for RNA extraction, precipitate the RNA from the aqueous phase with isopropanol, wash the precipitated RNA after centrifugation to remove impurities, resuspend and reverse transcribe to obtain cDNA.

[0064] b) Using existing New Zealand white rabbit-specific primers (synthesized by Genscript Biotech Co., Ltd.), PCR gene amplification was performed. Hybridoma cell cDNA was used as a template to amplify the variable regions of the heavy and light chains of the antibody. A 50 μL system contained 5 μL cDNA, HotStar Taq Plus enzyme, dNTPs, and 0.5 μM specific primers. PCR amplification was performed under the following conditions: pre-denaturation, 94℃, 5 min; amplification, 94℃, 30 s, 55℃, 30 s, 72℃, 50 s, 35 cycles; annealing, 72℃, 7 min. The obtained PCR product was identified by 1% agarose gel electrophoresis, the target fragment was recovered, and the sample was sent for sequencing to obtain the antibody gene sequence.

[0065] c) Constructing expression vectors for monoclonal antibodies:

[0066] Based on the antibody gene sequence obtained from step b, an expression vector for monoclonal antibodies was constructed. Homologous recombination arms were added to both ends of the variable region gene of the antibody heavy chain and the variable region gene of the light chain using homologous recombination primers. The expression plasmid containing the constant regions of the rabbit antibody heavy and light chains IgG1 was linearized using a double enzyme to generate homologous recombination arms. The variable region gene fragment with added homologous recombination arms and the linearized plasmid were ligated together by homologous recombination to form a complete expression vector, pCDNA3.4. The recombination product was transformed into TOP10 E. coli competent cells to amplify the plasmid. Through the above experimental operations, paired pCDNA3.4-antibody heavy chain plasmid and pCDNA3.4-antibody light chain plasmid were obtained.

[0067] d) Expression and purification of monoclonal antibodies:

[0068] The paired monoclonal antibody heavy and light chain expression plasmids obtained in step c were added to the culture medium at a 1:1 ratio. After thorough mixing, PEI transfection reagent (4 times the mass of DNA) was added. After mixing, the mixture was incubated at room temperature in the dark for 10 minutes. Then, the plasmids were added to 293T cells while shaking the cell culture flask. The cells were then incubated in a 5% CO2 incubator at 37°C with shaking for 5 days. The cell culture supernatant was then collected and purified using affinity purification (Protein A) to obtain the monoclonal antibody. The specific steps are as follows:

[0069] (1) Centrifuge the supernatant of the expressed antibody at 10000×g at room temperature for 30 min to remove the precipitate;

[0070] (2) Wash the affinity purification column containing Protein A thoroughly with 10 volumes of binding buffer;

[0071] (3) Pass the expression supernatant through the purification column at a flow rate of 5 mL / min;

[0072] (4) Wash the purification column thoroughly with 20 times the column volume of binding buffer;

[0073] (5) Elute the purification column with 0.1M citrate buffer (pH=3.0-3.5) until the elution peak drops to equilibrium, and adjust the pH to 7.0 with 1M Tris-HCl buffer (pH=9.0).

[0074] (6) The purified monoclonal antibody was concentrated using a concentrated centrifuge column, PBS was used as the antibody preservation buffer, and finally the concentration of the concentrated antibody was determined using an ultra-micro UV spectrophotometer.

[0075] Monoclonal antibodies Ab1#, Ab2#, Ab3#, and Ab4# were thus obtained.

[0076] III. Testing Section

[0077] 1. Screening of monoclonal antibodies:

[0078] a) Coat the microplate with PGI (0.1 μg / well). Dilute the four antibodies (Ab1#, Ab2#, Ab3#, Ab4#) at concentrations of 100, 10, 1, and 0 ng / mL, respectively. Add the above dilutions to the microplate and incubate at 37°C for 1 h. After washing the plate, add the enzyme-labeled secondary antibody dilution (5000-fold dilution) and incubate at 37°C for 0.5 h. Add TMB for color development for 15 min and then stop the reaction. Measure the absorbance (OD) at wavelengths of 450 / 620 nm. The results are shown in Table 2.

[0079] Table 2

[0080]

[0081] Based on the analysis of Table 2, the monoclonal antibodies Ab1#, Ab2#, Ab3#, and Ab4# showed good recognition effects on different PGIs.

[0082] b) Confirmation of antibody pairing combinations

[0083] Serum samples for clinical testing were collected from relevant hospitals, including 30 positive and 30 negative cases. Alternative monoclonal antibodies were combined using a sandwich method (forming a coated antibody-antigen-detection antibody complex). Fluorescent immunochromatography (coating conditions: 0.02M PBS buffer, coating concentration 1.5 mg / mL, drying temperature 53℃, drying time 13 h; labeling conditions: 0.02M PBS buffer, antibody to fluorescent microspheres (particle size approximately 200 nm) mass ratio 1.5:1, labeling 12 h; sample loading volume 100 μL, results read 15 min after loading) was used to detect the above 60 samples and the critical sample (PG I diluted with purified water to a concentration of 70 ng / mL). The ratio (I) of the sample T / C value to the critical sample T / C value was calculated, and this parameter was used as the critical value (I≥1 for positive, I<1 for negative). Antibody combinations with high positive-negative concordance rates and large sample gradients were screened. The results are shown in Table 3.

[0084] Table 3

[0085]

[0086] As shown in Table 3, the positive and negative concordance rates of samples coated with Ab1# antibody and tested with Ab4# antibody, and coated with Ab3# antibody and tested with Ab4# antibody, were good.

[0087] c) Verification of cross-reactivity of monoclonal antibodies:

[0088] Two pairs of candidate monoclonal antibodies were combined using a sandwich method (forming a coated antibody-antigen-detection antibody complex). Different concentrations of hemoglobin, trypsinogen I, trypsinogen II, and pepsinogen II were detected using fluorescence immunochromatography to observe the anti-interference ability of different antibodies against these easily cross-reactive substances. The cross-reactivity is shown in Table 4.

[0089] Table 4

[0090]

[0091] As shown in Table 4, the results indicate that the obtained monoclonal antibody showed the best detection effect on Ab3# antibody-coated + Ab4# antibody, with no cross-reactivity. Therefore, monoclonal antibodies Ab3# and Ab4# were selected for the detection of pepsinogen I.

[0092] 2. Characterization of monoclonal antibodies Ab3# and Ab4#

[0093] a) Monoclonal antibody Ab3#:

[0094] Light chain:

[0095] The nucleotide sequence of the light chain variable region is as SEQ ID Shown in NO.17: GATGTCGTGATGACCCAGACCGCCAGCCCAGTCTCAGCCGCCGTCGGAAGCACCGTGACCATCAGTTGTAGGGCTAGCCAGTCTGTGAGCAGCTCCTACCTGGCATGGTACCAGCAAAAGCCTGGACAGGCCCCTCGGCTCCTGATCTATGGGGCCTC TTCCCGGGCCACCGGGATCCCTGACCGCTTTTTCTGGCTCCGGCTCCGGAACTGATTTCACTCTGACAATCTCTCGCCTGGAGCCCGAGGACTTCGCCGTCTATTACTGCCAGCAGTACGGGTCAAGTCTGTTCACCTTCGGCGGCGGCACCAAACTCGAGATCAAG;

[0096] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.13: DVVMTQTASPVSAAVGSTVTISCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSLFTFGGGTKLEIK;

[0097] The amino acid sequence of the complementarity-determining region VL-CDR1 of the light chain variable region is shown in SEQ ID NO:1: RASQSVSSSYLA;

[0098] The amino acid sequence of the complementarity-determining region VL-CDR2 of the light chain variable region is shown in SEQ ID NO:2: GASSRAT;

[0099] The amino acid sequence of the complementarity-determining region VL-CDR3 of the light chain variable region is shown in SEQ ID NO:3: QQYGSSLFT.

[0100] Heavy chain:

[0101] The nucleotide sequence of the heavy chain variable region is as SEQ ID Shown in NO.18: CAGGAGAGCCTGGAAGAATCCAGAGGCGGACTGATTAAGCCCGGCGGGACACTGACCCTGACCTGCACCGCCAGCGGCTTCACCATTTCCAGCAAGGACGACTACTGGAGCTGGATCCGCCAGCCTCCGGGCAAGGGCCTGGAATGGATCGGCTACATCTACTACTCCGGCAGTACCTACT ACAACCCTAGCCTCAAGTCCAGAGTGACCATCCTGGTGGATACATCCAAGAACCAGTTTAGCCTGAAGCTGAGCAGCGTGACCGCAGCCGACACCGCCGTGTATTACTGCGCCAGGGTGCGCGTGAGGGGCGTGATGTCATATGCCATGGATGTGTGGGGCCAGGGAACACTGGTGACCGTGAGCAGC;

[0102] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.14: QESLEESRGGLIKPGGTLTLTCTASGFTISSKDDYWSWIRQPPGKGLEWIGYIYYSGSTYYNPSLKSRVTILVDTSKNQFSLKLSSVTAADTAVYYCARVRVRGVMSYAMDVWGQGTLVTVSS;

[0103] The amino acid sequence of the complementarity-determining region VH-CDR1 of the heavy chain variable region is shown in SEQ ID NO:4: SKDDYWS;

[0104] The amino acid sequence of the complementarity-determining region VH-CDR2 of the heavy chain variable region is shown in SEQ ID NO:5: YIYYSGSTYYNPSLKS;

[0105] The amino acid sequence of the complementarity-determining region VH-CDR3 of the heavy chain variable region is shown in SEQ ID NO:6: VRVRGVMSYAMDV.

[0106] b) Monoclonal antibody Ab4#:

[0107] Light chain:

[0108] The nucleotide sequence of the light chain variable region is as SEQ ID Shown in NO.19: GACGTGGTGATGACCCAGTCACCTTTTTCTCTGTCTGCCTCCGTGGGCGATCGGGTGTCCATTAACTGTCAGGCCGGCCAGGGAGTGGGGTCAAGCCTTAACTGGTATCAGCAGAAGCCCGGCAGGGCCCCTAAGCTGCTGGTGCATGGCG CCTCCAATGTGCAGAGGGGCGTGCCCTCCCGCTTCTCCGGCTCTGGCTTCCACACAACCTTTACCATTACCATTCCAGCCTGCAGCCCGACGACGTGGCCACCTACTTCTGTGAGGTGTTCCAGTGGTTTGGCGGCGGCACTAAACTGGAGATCAAG;

[0109] The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.15: DVVMTQSPFSLSASVGDRVSINCQAGQGVGSSLNWYQQKPGRAPKLLVHGASNVQRGVPSRFSGSGFHTTFTITISSLQPDDVATYFCEVFQWFGGGTKLEIK;

[0110] The amino acid sequence of the complementarity-determining region VL-CDR1 of the light chain variable region is shown in SEQ ID NO:7: QAGQGVGSSLN;

[0111] The amino acid sequence of the complementarity-determining region VL-CDR2 of the light chain variable region is shown in SEQ ID NO:8: GASNVQR;

[0112] The amino acid sequence of the complementarity-determining region VL-CDR3 of the light chain variable region is shown in SEQ ID NO:9: EVFQW;

[0113] Heavy chain:

[0114] The nucleotide sequence of the heavy chain variable region is as SEQ ID NO.20 shows: CAGCAGCTGAAAGAATCCGGCGGCGGCCTGGTGAAGCCCGGAGGATCCTTGAAACTGTGTTGTAAGGCCTCCGGCGGCAGCATCAATAGCGGCCACTACGCCATGCACTGGGTGAGGCAGGCTCCAGGCAAGGGCCTGGAGTGGGTGAGCGTGATCTACTCCGGCGGGGCCACCTCCTA CTATCTGGCCAGCCTGAAGGGCAGATTCACAATCAGTCGGGATGACAGCAAGAATACCCTGCATCTGCAGATCAACTCACTCCGCGGCGAGGACACCGCCATCTATTACTGTGCTAAGGACCTGAGTACCGGAACAACCGGAGCCGCCGACTCCTGGGGCCAGGGAACCCTGGTCACGGTGTCTTCT;

[0115] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.16: QQLKESGGGLVKPGGSLKLCCKASGGSINSGHYAMHWVRQAPGKGLEWVSVIYSGGATSYYLASLKGRFTISRDDSKNTLHLQINSLRGEDTAIYYCAKDLSTGTTGAADSWGQGTLVTVSS;

[0116] The amino acid sequence of the complementarity-determining region VH-CDR1 of the heavy chain variable region is shown in SEQ ID NO:10: SGHYAMH;

[0117] The amino acid sequence of the complementarity-determining region VH-CDR2 of the heavy chain variable region is shown in SEQ ID NO:11: VIYSGGATSYYLASLKG;

[0118] The amino acid sequence of the complementarity-determining region VH-CDR3 of the heavy chain variable region is shown in SEQ ID NO:12: DLSTGTTGAADS;

[0119] c) Molecular weight determination of monoclonal antibodies Ab3# and Ab4#

[0120] Its SDS-PAGE protein electrophoresis chromatogram is as follows: Figure 1 As shown, according to Figure 1 Lane M is the marker, lane 1 is the monoclonal antibody Ab3#, and lane 2 is the monoclonal antibody Ab4#. Both monoclonal antibodies are reduced to two parts, one with a molecular weight of 50kDa and the other with a molecular weight of 25kDa.

[0121] 3. Development and validation of a pepsinogen I detection kit based on the obtained anti-PGI monoclonal antibody pairs (monoclonal antibody Ab3# and monoclonal antibody Ab4#).

[0122] Brief description of the reagent kit development process:

[0123] Ab3# antibody was used as the coating antibody to coat the NC membrane, and Ab4# antibody was used as the detection antibody after labeling fluorescent microspheres. A series of optimization studies were conducted on the coating conditions of Ab3# antibody (coating buffer, coating concentration, drying temperature and time, etc.), the labeling conditions of Ab4# antibody (antibody to fluorescent microsphere ratio, labeling buffer, labeling time, etc.), and the sample loading volume. After the kit was finalized, its performance (sensitivity, repeatability, stability, etc.) was also studied.

[0124] The coating conditions were determined as follows: 0.02M PBS buffer, coating concentration 1.5 mg / mL, drying temperature 53℃, and drying time 13 h.

[0125] Labeling conditions: 0.02M PBS buffer, antibody to fluorescent microspheres (particle size about 200 nm) mass ratio of 1.5:1, labeling for 12 h; sample loading volume of 100 μL, results read 15 min after loading.

[0126] 1) Sensitivity:

[0127] Samples were diluted to eight concentration levels (200.0, 100.0, 50.0, 25.0, 10.0, 5.0, 2.5, and 1 ng / mL) using a pepsinogen I assay kit (fluorescent immunochromatography). The analytical sensitivity was determined when the T / C value of the sample was greater than 2.0 compared to the T / C value at concentration 0. The corresponding concentration value was considered the analytical sensitivity. The results are shown in Table 5.

[0128] Table 5

[0129]

[0130] According to Table 5, the minimum concentration corresponding to a T / C value greater than 2.0 for the sample T / C value / 0 concentration is 10 ng / mL. Therefore, the present invention provides a pepsinogen I detection kit (fluorescent immunochromatography) with a sensitivity of 10 ng / mL for the detection of pepsinogen I.

[0131] (ii) Repeatability:

[0132] The PGI levels in samples 1 (PGI concentration of 25 ng / mL) and 2 (PGI concentration of 75 ng / mL) were determined using a pepsinogen I assay kit (fluorescent immunochromatography). The test results are shown in Table 6.

[0133] Table 6

[0134]

[0135] As shown in Table 6, the pepsinogen I detection kit (fluorescent immunochromatography) exhibits good repeatability, with CV values ​​all below 10%.

[0136] (iii) Stability:

[0137] The pepsinogen I assay kit (fluorescent immunochromatography) was stored at 2–8°C. The kits were removed at 0, 3, 6, 9, and 12 months post-production, and the PGI in sample 1 (PGI concentration 25 ng / mL) and sample 2 (PGI concentration 75 ng / mL) was measured. The test results are shown in Table 7.

[0138] Table 7

[0139]

[0140] As can be seen from Table 7, the pepsinogen I detection kit (fluorescent immunochromatographic assay) has good stability.

[0141] Finally, it should be noted that the above text has described the various embodiments of the present invention in detail. To avoid obscuring the concept of the present invention, 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. The above embodiments are only used to illustrate the technical solutions of this application, and not to limit them;

[0142] Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A monoclonal antibody combination for the detection of pepsinogen I, characterized in that, It includes a first antibody and a second antibody, wherein the first antibody includes a first light chain variable region and a first heavy chain variable region, and the second antibody includes a second light chain variable region and a second heavy chain variable region; wherein, The amino acid sequence of the first light chain variable region includes VL-CDR1 shown in SEQ ID NO:1, VL-CDR2 shown in SEQ ID NO:2 and VL-CDR3 shown in SEQ ID NO:3; The amino acid sequence of the first heavy chain variable region includes VH-CDR1 shown in SEQ ID NO:4, VH-CDR2 shown in SEQ ID NO:5, and VH-CDR3 shown in SEQ ID NO:6; The amino acid sequence of the second light chain variable region includes VL-CDR1 shown in SEQ ID NO:7, VL-CDR2 shown in SEQ ID NO:8, and VL-CDR3 shown in SEQ ID NO:9; The amino acid sequence of the variable region of the second heavy chain includes VH-CDR1 shown in SEQ ID NO:10, VH-CDR2 shown in SEQ ID NO:11, and VH-CDR3 shown in SEQ ID NO:

12.

2. The monoclonal antibody combination for pepsinogen I detection according to claim 1, characterized in that, The amino acid sequence of the first light chain variable region is shown in SEQ ID NO:13; the amino acid sequence of the first heavy chain variable region is shown in SEQ ID NO:

14.

3. The monoclonal antibody combination for pepsinogen I detection according to claim 1, characterized in that, The amino acid sequence of the second light chain variable region is shown in SEQ ID NO:15; the amino acid sequence of the second heavy chain variable region is shown in SEQ ID NO:

16.

4. The monoclonal antibody combination for pepsinogen I detection according to claim 1, characterized in that, The nucleotide sequence of the first light chain variable region is shown in SEQ ID NO:17; the nucleotide sequence of the first heavy chain variable region is shown in SEQ ID NO:

18.

5. The monoclonal antibody combination for pepsinogen I detection according to claim 1, characterized in that, The nucleotide sequence of the second light chain variable region is shown in SEQ ID NO:19; the nucleotide sequence of the second heavy chain variable region is shown in SEQ ID NO:

20.

6. The use of the monoclonal antibody combination for pepsinogen I detection according to any one of claims 1 to 5 in the preparation of a tool for detecting pepsinogen I.

7. The application according to claim 6, characterized in that, The tool for detecting pepsinogen I is used to detect pepsinogen I in in vitro samples, wherein the in vitro samples include at least one of serum, plasma, and whole blood.

8. The application according to claim 7, characterized in that, The monoclonal antibody combination used for pepsinogen I detection is used in an immunoassay method for detecting pepsinogen I in in vitro samples.

9. The application according to claim 8, characterized in that, The immunoassay method includes at least one of enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, chemiluminescence immunoassay, immunomicrosphere assay, and immunogold assay.

10. The application according to any one of claims 6 to 9, characterized in that, The tool for detecting pepsinogen I includes at least one of reagents, kits, test strips, and antibody chips.

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