A human hsp90 alpha antigen epitope peptide, and a preparation method and application thereof

Abstract

This invention relates to the field of tumor marker detection technology, and discloses a human HSP90α antigenic epitope peptide, its preparation method, and its application. The invention provides the 552-568aa region located on the N-side of the HSP90α C-terminal dimerization interface as an antigenic epitope. This region contains the Q560 key site, which has a fundamental difference in polarity / nonpolarity compared to the corresponding HSP90β site M561. Furthermore, a dual C-terminal epitope pairing strategy is provided, using two epitopes, 552-568aa and 703-717aa, both located in the C-terminal domain, for antibody pairing, avoiding steric hindrance. The kit of this invention has a detection range of 5-200 ng / mL, a limit of detection ≤1.8 ng / mL, and a specificity ≥98.5%, and can be used for the detection of secretory HSP90α in the serum of cancer patients, possessing significant clinical application value.

Description

Technical Field

[0001] This invention relates to the field of tumor marker detection technology, and more specifically, to a human HSP90α antigen epitope peptide, its preparation method, and its application. Background Technology

[0002] Tumor marker testing is a crucial tool for early screening, auxiliary diagnosis, efficacy monitoring, recurrence early warning, and prognostic assessment of clinical tumors, playing an irreplaceable role in the entire cycle of tumor diagnosis and treatment management. However, current tumor detection technologies generally face challenges in practical applications, including insufficient diagnostic efficacy and the difficulty in balancing sensitivity and specificity. Traditional single natural tumor markers typically exhibit low sensitivity in early tumor detection. For example, carcinoembryonic antigen (CEA) alone has only a low sensitivity for early colorectal cancer screening and is susceptible to false positives due to interference from non-malignant diseases such as inflammation and benign tumors. Alpha-fetoprotein (AFP) has a high rate of false negatives in liver cancer screening, and prostate-specific antigen (PSA) suffers from overdiagnosis and false positives, making it difficult to effectively differentiate between benign and malignant prostate diseases. Even with relatively higher sensitivity, circulating tumor DNA (CTB) testing in early-stage or low-shedding tumors can significantly reduce sensitivity and increase the risk of false negatives due to the low frequency of mutated alleles, failing to meet the core clinical need for early diagnosis. Meanwhile, existing technologies have limited capabilities in addressing tumor heterogeneity and achieving dynamic monitoring. Tissue biopsy, as the traditional gold standard, can only reflect information from local sampling sites and cannot fully present the clonal heterogeneity and spatiotemporal evolution characteristics of tumors. It is difficult to achieve multiple samplings and continuous monitoring for tumors in deep organs and metastatic lesions. Although liquid biopsy has made up for the shortcomings of dynamic monitoring to some extent, it is not effective in detecting tumors of the central nervous system and is susceptible to false positive results due to factors such as clonal hematopoiesis.

[0003] In terms of detection procedures and technical applicability, existing technologies also have significant shortcomings. Mass spectrometry-based proteomics detection is susceptible to signal masking by high-abundance proteins in plasma, and the pre-detection pretreatment steps are complex with significant batch-to-batch variations. Liquid biopsy based on high-throughput sequencing is costly and has a long detection cycle, making it difficult to promote in resource-limited areas. PCR amplification-based methods have stringent sample quality requirements, cumbersome pretreatment, and are time-consuming. Furthermore, the lack of standardization between different detection platforms leads to poor comparability of test results, and multi-center clinical validation is challenging, further limiting the large-scale clinical application of these technologies. Regarding early tumor diagnosis, existing biomarkers have a much lower detection rate for stage I tumors than for mid-to-late stage tumors. Most novel biomarkers are still in the clinical research stage, and the proportion of those successfully converted into approved diagnostic kits is low, failing to meet the urgent clinical needs for early tumor screening, diagnosis, and treatment.

[0004] Heat shock protein 90α, a molecular chaperone protein highly associated with tumor development and progression, is specifically highly expressed in various malignant tumors and can be secreted into peripheral blood. Its serum level is closely related to tumor burden, malignancy, metastasis risk, and treatment response. Multiple clinical studies have confirmed that it has excellent diagnostic efficacy in various solid tumors such as liver cancer, colorectal cancer, breast cancer, and lung cancer. It can serve as an important supplement to traditional tumor markers, and it maintains a very high detection rate, especially in patients with alpha-fetoprotein-negative liver cancer. It has the potential for broad-spectrum, high-sensitivity, and high-specificity applications.

[0005] However, existing HSP90 detection reagents and technologies generally use full-length proteins as immunogens to prepare antibodies, which cannot effectively distinguish between HSP90α and HSP90β subtypes. They are also prone to cross-reaction with homologous proteins expressed in normal tissues, resulting in insufficient detection specificity and high background signal. At the same time, they lack specific antigenic epitopes that have been accurately screened and validated, and the antibody affinity and detection stability are difficult to meet the requirements for clinical quantification. Furthermore, the detection sensitivity, linear range, and ease of operation of the kits cannot meet the standards for large-scale clinical screening.

[0006] Existing patent CN105669834B discloses two HSP90α epitopes: - Epitope 1: 200-215aa (YPRDRLDP RPGS PSEAS) - Epitope 2: 697-716aa (YRGI DD PTA DDTSA ATVTE). Through in-depth research, the inventors have discovered that the epitopes selected in the above-mentioned prior art have the following inherent technical defects: Defect 1: Insufficient subtype specificity. The 200-215 amino acids are located in the intermediate linker region, showing over 90% sequence homology between HSP90α and HSP90β, but lacking key subtype-differentiating amino acid sites. Antibodies prepared based on this epitope generally have a cross-reactivity rate >3% against HSP90β, resulting in a high false positive rate in clinical applications.

[0007] Defect 2: Poor conformational stability of 697-716aa in serum, located near the C-terminal flexible region, makes it susceptible to the influence of proteolytic enzymes in serum. After clinical samples were placed at 4°C for 7 days, the detection signal decreased by approximately 17%, affecting the reliability of the test results.

[0008] Defect 3: Spatial hindrance of antibody pairing. Existing technologies employ a cross-domain pairing strategy of "200-215aa (intermediate domain) + 697-716aa (C-terminus)". In the three-dimensional structure of the HSP90α dimer, the actual distance between the two epitopes is only about 28 Å, resulting in significant spatial hindrance during antibody binding and affecting detection sensitivity.

[0009] Defect 4: Inability to distinguish between secreted and intracellular HSP90α. The epitopes selected by the existing technology cannot distinguish between HSP90α secreted by tumor cells (which has clinical diagnostic value) and intracellular HSP90α released by cell lysis (which has no diagnostic value), resulting in insufficient clinical specificity of the test results.

[0010] This invention addresses the aforementioned technical biases and defects of existing technologies by discovering a novel C-terminal epitope region (552-568aa) and constructing a dual C-terminal epitope pairing strategy, thus overcoming the long-standing technical problems of existing technologies. Summary of the Invention

[0011] In view of this, the present invention proposes a human HSP90α antigenic epitope peptide, its preparation method and application, aiming to solve at least one of the current background art problems.

[0012] This invention proposes a human HSP90α antigenic epitope peptide, wherein the human HSP90α antigenic epitope peptide is a polypeptide, and its amino acid sequence is as shown in SEQ ID NO.1; The SEQ ID NO.1 is: Leu-Pro-Glu-Asp-Glu-Glu-Glu-Lys-Lys-Lys-Gln-Glu-Glu-Lys-Lys-Thr-Lys-Cys; The epitope peptide contains glutamine at position 560 (Q560), which is a key amino acid residue that distinguishes the HSP90α and HSP90β subtypes. The 561st position of HSP90β corresponding to Q560 is methionine (M561). The polar / nonpolar difference between Q560 and M561 determines the specific recognition of HSP90α by the epitope peptide. The epitope peptide is located on the N side of the HSP90α C-terminal dimerization interface, and the epitope is fully exposed on the molecular surface in the HSP90α dimer state. The cross-reactivity rate of the epitope peptide with HSP90β is less than 1.5%.

[0013] The present invention also provides a method for preparing the human HSP90α antigenic epitope peptide according to claim 1, comprising the following steps: (1) Two specific antigenic epitope regions were obtained by analyzing the human HSP90α amino acid sequence through bioinformatics analysis; (2) The amino acid sequence of human HSP90α protein from position 552 to 568 was taken as the backbone of epitope peptide 1, and the amino acid sequence of human HSP90α protein from position 703 to 717 was taken as the backbone of epitope peptide 2. (3) Add a cysteine ​​residue Cys to the N-terminus of both the epitope peptide 1 backbone and the epitope peptide 2 backbone; (4) The antigenic epitope peptide shown in SEQ ID NO.1 was synthesized by solid-phase chemical synthesis based on the skeleton of epitope peptide 1 and epitope peptide 2.

[0014] The present invention also provides a recombinant antigen, which is prepared by coupling the antigen epitope peptide described in the above technical solution with a carrier protein.

[0015] Preferably, the carrier protein is KLH, BSA, or OVA.

[0016] This invention also provides a monoclonal antibody specific to HSP90α. The antibody specifically binds to the epitope peptide of claim 1; The complementarity-determining region (CDR3) of the antibody contains an amino acid sequence that specifically interacts with the continuous charged region (Glu-Glu-Glu-Lys-Lys) at positions 558-562 of the epitope peptide. The antibody does not recognize the HSP90β protein, and its cross-reactivity with HSP90β is less than 1%. The antibody preferentially recognizes secretory HSP90α in serum, and its recognition activity for secretory HSP90α is higher than that for recombinantly expressed intracellular HSP90α.

[0017] Preferably, the antibody is a monoclonal antibody or a polyclonal antibody.

[0018] The present invention also provides a double-antibody sandwich ELISA kit for detecting secretory HSP90α in serum, comprising a first antibody and a second antibody, wherein the first antibody recognizes the 552-568aa epitope as described in claim 1, and the second antibody recognizes the 703-717aa epitope at the C-terminus of HSP90α. The two epitopes are approximately 45 Å apart in the three-dimensional structure of HSP90α, and there is no steric hindrance when the antibody binds. The kit employs a dual C-terminal epitope pairing strategy. The kit has a detection range of 5-200 ng / mL for serum samples, with a detection limit of ≤2 ng / mL. The kit has a specificity of ≥98.5% in distinguishing between HSP90α and HSP90β.

[0019] The present invention also provides the application of the antigen epitope peptide described in the above technical solution in the preparation of in vitro diagnostic reagents for tumors.

[0020] Preferably, the tumor is at least one of liver cancer, lung cancer, colorectal cancer, and breast cancer.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention has higher diagnostic accuracy. By screening specific antigenic epitopes, the present invention can obtain antibodies that have a greater ability to recognize tumor-associated HSP90.

[0022] (2) SP90α and HSP90β have high sequence homology, but their functions differ. Through epitope screening, the kit obtained in this invention can specifically identify tumor-associated HSP90 subtypes and reduce false positives caused by benign lesions.

[0023] (3) The kit of the present invention has diagnostic value in a variety of malignant tumors such as liver cancer, lung cancer, colorectal cancer, and breast cancer, and can realize the detection of multiple cancers in one kit.

[0024] (3) The kit of the present invention is prepared by double antibody sandwich ELISA method, which is simple to operate, has a short detection time (usually 3-4.5 hours), and is suitable for a variety of biological samples such as serum, plasma, tissue homogenate, and cell lysate, making it convenient for clinical collection.

[0025] (5) Compared with the prior art, the 552-568aa epitopes selected in this invention have achieved several technical effects, specifically: Order-of-magnitude improvement in subtype specificity - Existing technology (Anqun Biotechnology patent): Cross-reactivity rate to HSP90β is approximately 3.2-3.8% - Epitope of this invention: Cross-reactivity rate to HSP90β <1.5% - Improvement magnitude: Specificity is increased by more than double; The epitope of this invention contains the Q560 key site, which has a fundamental difference in polarity / nonpolarity from M561 of HSP90β. This discovery was obtained unexpectedly by the inventors through a large number of site-directed mutagenesis experiments and could not be predicted through conventional sequence alignment; Serum stability is significantly improved - Existing epitope: 83-86% recovery rate after 7 days at 4℃ - Epitope of this invention: >94% recovery rate after 7 days at 4℃ - Improvement: Stability is improved by approximately 10 percentage points; Improved detection sensitivity - Existing technology pairing: Limit of detection 2.9-3.2 ng / mL - This invention's dual C-terminal pairing: Limit of detection 1.8 ng / mL - Improvement: Sensitivity increased by approximately 40%; Preferred recognition of secretory HSP90α: The 552-568aa epitopes of this invention exhibit a specific open conformation in secretory HSP90α, while being partially masked in intracellular HSP90α. Therefore, the antibody of this invention exhibits more than 30% higher recognition activity for secretory HSP90α than for intracellular HSP90α, and has significant clinical diagnostic value. Attached Figure Description

[0026] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 A schematic diagram of the composition of the human HSP90α double antibody sandwich ELISA detection kit; Figure 2 The graph shows the diagnostic performance of the human HSP90α double antibody sandwich ELISA kit on four types of solid tumors. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0028] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] This invention provides a human HSP90α antigenic epitope peptide, wherein the human HSP90α antigenic epitope peptide is a polypeptide, and its amino acid sequence is as shown in SEQ ID NO.1; The SEQ ID NO.1 is: Leu-Pro-Glu-Asp-Glu-Glu-Glu-Lys-Lys-Lys-Gln-Glu-Glu-Lys-Lys-Thr-Lys-Cys; The epitope peptide contains glutamine at position 560 (Q560), which is a key amino acid residue that distinguishes the HSP90α and HSP90β subtypes. The 561st position of HSP90β corresponding to Q560 is methionine (M561). The polar / nonpolar difference between Q560 and M561 determines the specific recognition of HSP90α by the epitope peptide. The epitope peptide is located on the N side of the HSP90α C-terminal dimerization interface, and the epitope is fully exposed on the molecular surface in the HSP90α dimer state. The cross-reactivity rate of the epitope peptide with HSP90β is less than 1.5%.

[0033] This invention also provides a method for preparing the human HSP90α antigenic epitope peptide described in the above technical solution, comprising the following steps: (1) Two specific antigenic epitope regions were obtained by analyzing the human HSP90α amino acid sequence through bioinformatics analysis; Specifically, the amino acid sequences of HSP90α and HSP90β are analyzed using bioinformatics methods such as online databases (e.g., uniport, MIMI) and bioinformatics software (e.g., DNAStar, IMGT) to predict potential antigenic epitope regions. (2) The amino acid sequence of human HSP90α protein from position 552 to 568 was taken as the backbone of epitope peptide 1, and the amino acid sequence of human HSP90α protein from position 703 to 717 was taken as the backbone of epitope peptide 2. (3) Add a cysteine ​​residue Cys to the N-terminus of both the epitope peptide 1 backbone and the epitope peptide 2 backbone; (4) The antigenic epitope peptide shown in SEQ ID NO.1 was synthesized by solid-phase chemical synthesis based on the skeleton of epitope peptide 1 and epitope peptide 2.

[0034] The present invention also provides a recombinant antigen, which is prepared by coupling the antigen epitope peptide described in the above technical solution with a carrier protein.

[0035] In this invention, the carrier protein is KLH, BSA, or OVA.

[0036] This invention also provides a monoclonal antibody specific to HSP90α. The antibody specifically binds to the epitope peptide described in the above technical solution; The complementarity-determining region (CDR3) of the antibody contains an amino acid sequence that specifically interacts with the continuous charged region (Glu-Glu-Glu-Lys-Lys) at positions 558-562 of the epitope peptide. The antibody does not recognize the HSP90β protein, and its cross-reactivity with HSP90β is less than 1%. The antibody preferentially recognizes secretory HSP90α in serum, and its recognition activity for secretory HSP90α is higher than that for recombinantly expressed intracellular HSP90α.

[0037] In this invention, the antibody is a monoclonal antibody or a polyclonal antibody.

[0038] The specific method for preparing the monoclonal antibody is as follows: The above-mentioned immunogen was thoroughly mixed with an equal volume of Freund's complete adjuvant and administered to Balb / c mice via intraperitoneal injection at a dose of 50 μg antigen per mouse. Serum titers were measured 4 weeks later, and mice with good immunoreactivity were selected for booster immunization: the antigen was thoroughly mixed with an equal volume of incomplete Freund's adjuvant, and administered via intraperitoneal injection at a dose of 50 μg antigen per mouse. Three days later, spleen cells were fused with Sp2 / 0 myeloma cells using standard methods mediated by 50% PEG (MW4000), and cultured selectively in HAT conditioned medium. After fusion, the cells were incubated at 37°C in a CO2 incubator for 9–11 days, after which large cell clones appeared in the wells. At day 11, a differential selection strategy was employed: ELISA screening was performed using the full-length HSP90 protein and different epitope peptides to obtain positive hybridoma cell lines targeting specific epitopes. The wells with initially positive results were subjected to four clonal culture cycles using limiting dilution (to allow the selected cells to proliferate rapidly), followed by cell expansion, cryopreservation, and ascites preparation.

[0039] Balb / c mice were treated with 0.5 ml of phenazine per mouse, and one week later, they were intraperitoneally inoculated with 2 × 10⁶ hybridoma cells. 6 Ascites fluid was collected from each animal after 10 days. The titer of the monoclonal antibody prepared using the HSP90α antigen was determined by indirect ELISA, and the results showed that the titer of the monoclonal antibody reached over 1:32000. Monoclonal antibodies targeting different epitopes were prepared and named α1 (recognizing epitope 1) and α2 (recognizing epitope 2).

[0040] The specific method for preparing the polyclonal antibody is as follows: Three-month-old New Zealand white rabbits weighing approximately 2 kg were selected as immunization animals. 50 μg of the prepared HSP90α antigens (1) and (2) (immunogens) were mixed with an equal volume of rabbit eight-week rapid adjuvant and injected subcutaneously at multiple points in the rabbit's leg. Booster immunizations were administered on days 21 and 42 in the same manner. (The adjuvant and antigen were prepared and used immediately each time, and the injection sites were the same as for the initial immunization.) From days 52 to 56, a small amount of blood was collected for ELISA testing; the antibody titer should be within the range of 1:10000-1:1000000, after which a large amount of blood could be collected. Ten days after the final booster immunization, blood was collected from the heart, and serum was separated.

[0041] The titer of polyclonal antibody (1) prepared using HSP90α antigen (1) was determined by indirect ELISA, and the results showed that the antibody titer reached over 1:32000. The titer of polyclonal antibody (2) prepared using HSP90α antigen (2) was also determined using the same method, and its titer also reached over 1:32000. After ammonium sulfate precipitation, the antibody was purified by affinity chromatography using a Protein G column, concentrated by ultrafiltration centrifuge tubes, aliquoted, lyophilized, and stored at -20°C.

[0042] The present invention also provides a double-antibody sandwich ELISA kit for detecting secretory HSP90α in serum, comprising a first antibody and a second antibody, wherein the first antibody recognizes the 552-568aa epitope as described in claim 1, and the second antibody recognizes the 703-717aa epitope at the C-terminus of HSP90α. The two epitopes are approximately 45 Å apart in the three-dimensional structure of HSP90α, and there is no steric hindrance when the antibody binds. The kit employs a dual C-terminal epitope pairing strategy. The kit has a detection range of 5-200 ng / mL for serum samples, with a detection limit of ≤2 ng / mL. The kit has a specificity of ≥98.5% in distinguishing between HSP90α and HSP90β.

[0043] Specifically, the preferred method for preparing the reagent kit of the present invention is as follows: Capture antibodies from the selected antibody pairs were pre-coated onto 96-well microplates, dried, and sealed. Detection antibodies were HRP-labeled and prepared to working concentrations. Calibrators and quality control samples were aliquoted according to a gradient. Other components were assembled into a kit according to the instructions.

[0044] More preferably, the principle of use of the above reagent kit is as follows: Add the serum sample to be tested to the wells of the ELISA plate coated with capture antibody α1. If HSP90 is present in the sample, HSP90 will specifically bind to the capture antibody through its epitope 1. After incubation, wash to remove unbound substances. HRP-labeled detection antibody α2 is added. The detection antibody binds to the captured HSP90 by recognizing epitope 2 on HSP90, forming a sandwich complex of "capture antibody-HSP90-detection antibody". After incubation, the antibody is washed to remove unbound detection antibody.

[0045] Add TMB substrate solution, HRP catalyzes TMB color development, and the color intensity is directly proportional to the HSP90 concentration in the sample; add stop solution to terminate the reaction, and measure absorbance (OD value) at 450 nm wavelength.

[0046] A standard curve is plotted based on the calibrator concentration and its corresponding OD value. The concentration of HSP90 in the sample is then calculated by substituting the sample OD value into the standard curve.

[0047] Based on the above-mentioned operating principle of the reagent kit, its preferred method of use is: (1) Sample preparation: Collect fasting venous blood, separate serum (or plasma), and store at 4℃ for later use; (2) Sample addition: Add calibrators, quality control samples and test samples to the corresponding enzyme-labeled wells, 100 μL per well; (3) Incubation: Incubate at 37℃ for 60 minutes; (4) Washing: Discard the liquid in the hole, add washing solution and wash 5 times, then pat dry; (5) Add detection antibody: Add 100 μL of HRP-labeled detection antibody to each well; (6) Incubation: Incubate at 37℃ for 30 minutes; (7) Washing: Same as step 4; (8) Color development: Add 100 μL of TMB substrate solution to each well and develop color at 37°C in the dark for 15 minutes; (9) Termination: Add 50 μL of termination solution to each well; (10) Measurement: OD value was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm; (11) Calculation: Plot the standard curve and calculate the sample concentration.

[0048] The present invention also provides the application of the antigen epitope peptide described in the above technical solution in the preparation of in vitro diagnostic reagents for tumors.

[0049] In this invention, the tumor is at least one of liver cancer, lung cancer, colorectal cancer, and breast cancer.

[0050] Example 1: Screening and Synthesis of Human HSP90α Antigenic Epitope Peptides (1) The full-length amino acid sequences of human HSP90α (P07900) and HSP90β (P08238) were obtained from the UniProt database. B cell epitopes were predicted using IEDB, DNAStar and IMGT software. Based on hydrophilicity, surface accessibility, antigen index and sequence differences, two HSP90α-specific epitope regions were screened.

[0051] (2) The 552nd to 568th amino acids of the HSP90α protein were taken as the backbone of epitope peptide 1, and the 703rd to 717th amino acids were taken as the backbone of epitope peptide 2.

[0052] (3) Cysteine ​​(Cys) was added to the N-terminus of both peptide backbones, and the peptides were synthesized by Fmoc solid-phase chemical synthesis method. After purification by HPLC and identification by mass spectrometry, the purity was >95%, and the antigenic epitope peptides shown in SEQ ID NO.1 and SEQ ID NO.2 were obtained.

[0053] Example 2 Preparation of recombinant HSP90α antigen (1) Take the epitope peptides SEQ ID NO.1 and SEQ ID NO.2 obtained in Example 1 respectively, and couple them with KLH carrier protein by glutaraldehyde crosslinking method.

[0054] (2) The cross-linked product was purified by dialysis and identified by SDS-PAGE and indirect ELISA to confirm that the epitope peptide was successfully coupled with KLH.

[0055] (3) Obtain recombinant HSP90α antigen, which can be used for subsequent animal immunization.

[0056] Example 3: Preparation of specific monoclonal antibodies and polyclonal antibodies Monoclonal antibodies were prepared by emulsifying the recombinant antigen obtained in Example 2 with Freund's complete adjuvant and immunizing Balb / c mice intraperitoneally at a dose of 50 μg / mouse. A booster immunization was performed 4 weeks later, and spleen cells were fused with Sp2 / 0 myeloma cells 3 days later, followed by screening on HAT medium. Positive clones were screened using indirect ELISA, with the corresponding epitope peptide as the coating antigen and BSA as a control. Cloning was performed after four limiting dilutions to obtain hybridoma cell lines that stably secreted specific antibodies. Cells were seeded into the peritoneal cavity of mice pretreated with phytane, and ascites fluid was collected. The ascites fluid was purified by ammonium sulfate precipitation and affinity purification with Protein G to obtain α1 and α2 monoclonal antibodies with a titer ≥1:32000, specifically binding only to the corresponding epitope peptides.

[0057] Polyclonal antibody preparation: New Zealand white rabbits were immunized with recombinant antigen. Booster immunizations were performed on days 21 and 42 after the initial immunization. Blood was collected from the heart 10 days after the final immunization, and serum was separated. High-specificity polyclonal antibodies with a titer ≥1:32000 were obtained after Protein G affinity purification, suitable for constructing detection systems.

[0058] Example 4 Antibody pairing screening and establishment of a bispecific antibody sandwich system α1 and α2 monoclonal antibodies were cross-paired and used as capture antibodies to coat ELISA plates, while another antibody was HRP-labeled as the detection antibody. Using recombinant HSP90α protein as the detection target, the coating concentration, incubation time, and washing conditions were optimized to screen for the optimal pairing: α1 as the capture antibody and HRP-α2 as the detection antibody. These two antibodies recognize different epitopes, have no spatial interference, and can form a stable sandwich complex.

[0059] Example 5: Validation of the reagent kit's analytical performance A schematic diagram of the composition of the human HSP90α double antibody sandwich ELISA detection kit described in this invention is shown below. Figure 1 As shown; (1) A standard curve was established using HSP90α standards (0, 10, 25, 50, 100 ng / mL), and the double-antibody sandwich ELISA method was used for detection. The regression equation was y = 0.0084x + 0.086, R0 2 =0.994.

[0060] Twenty zero-concentration standard samples were tested repeatedly for 10 times. The standard deviation (SD) was calculated. The limit of detection (LOD) was calculated as 1.5 ng / mL based on 3 times the SD; the limit of quantitation (LOQ) was calculated as 5.0 ng / mL based on 10 times the SD. The results of the linear range validation are shown in Table 1. Table 1. Validation Results of Linear Range

[0061] The results showed that this kit exhibited good linearity in the concentration range of 5–200 ng / mL (R0). 2 >0.99).

[0062] (2) Quality control samples at three concentration levels—low (15 ng / mL), medium (50 ng / mL), and high (150 ng / mL)—were tested 20 times each, and the coefficient of variation (CV%) was calculated. The results are shown in Table 2. Table 2 Intra-batch precision results

[0063] As shown in Table 2, the kit exhibits good intra-batch repeatability and high stability for low, medium, and high concentration detection.

[0064] (3) Using three different batches of reagent kits, the quality control samples at the above three concentration levels were tested 10 times each, and the inter-batch variation coefficient was calculated.

[0065] Table 3. Inter-batch precision results

[0066] Table 3 shows that the kits have small differences between different batches, and the test results are reliable and have good batch stability.

[0067] (4) Take five serum samples from healthy individuals with known concentrations (baseline concentrations of 28.3, 35.6, 42.1, 56.8, and 68.4 ng / mL, respectively), add low, medium, and high concentrations of HSP90α standard, and determine the recovery rate. The recovery rate results are shown in Table 4: Table 4 Recovery Rate Results

[0068] Based on Table 4, the recovery rate ranges from 88.6% to 106.2%, with an average recovery rate of 98.3%. The kit exhibits high spiked recovery and can accurately determine the concentration of HSP90α in serum.

[0069] (5) The following potential cross-reactive substances were detected using this kit, and their specificity was evaluated: The cross-reactivity results are shown in Table 5: Table 5 Cross-reactivity results

[0070] Note: Cross-reactivity rate (%) = (Detection value of cross-reactant / Detection value of HSP90α at the same concentration) × 100% As mentioned above, antibodies traditionally prepared using full-length HSP90α protein immunization typically exhibit a cross-reactivity rate >30% with HSP90β due to the presence of multiple conserved regions on the protein surface. This invention employs an epitope peptide screening strategy to prepare antibodies targeting only the unique epitope regions of HSP90α (positions 552-568 and 703-717), successfully controlling the HSP90β cross-reactivity rate below 1.5%. This effectively reduces false positive results caused by HSP90β cross-reactivity and improves detection specificity.

[0071] (6) Take 3 batches of reagent kits and test them at 0, 3, 6, 9 and 12 months respectively to evaluate each performance index.

[0072] Table 6. Results of stability during storage at 2~8℃

[0073] As shown in Table 6, the performance indicators of the kit remained within acceptable range after being stored at 2-8℃ for 12 months.

[0074] (7) Store the kit in a 37°C incubator and take samples for testing on days 1, 3 and 7. 7 days of storage at 37°C is equivalent to about 6 months of storage at 2~8°C (estimated according to the Arrhenius formula).

[0075] Table 7 Test Results

[0076] Based on Table 7, it can be seen that the reagent kit has good thermal stability and reliable performance under transportation and short-term high temperature conditions.

[0077] Application Example 1 A multicenter, retrospective case-control study was conducted, including 481 participants, comprising healthy controls, patients with various solid tumors, and patients with benign lesions. All samples were collected from inpatients or outpatients at five tertiary hospitals in China between March 2019 and June 2021. Sample grouping is shown in Table 8; lung cancer pathological classification is shown in Table 9; and benign lesion grouping is shown in Table 10. Table 8 Sample Grouping Table

[0078] Table 9. Pathological Classification of Lung Cancer

[0079] Table 10 Grouping of Benign Lesions

[0080] (1) Determination of critical value The cutoff value was determined using serum HSP90α levels in 120 healthy controls. The HSP90α levels in the healthy control group were normally distributed, with a mean of 56.8 ng / mL and a standard deviation of 13.35 ng / mL. Following internationally recommended methods (mean + 2 SD), the cutoff value for serum HSP90α was determined to be 83.5 ng / mL.

[0081] Distribution characteristics of HSP90α in healthy control group: • Mean: 56.8 ng / mL • Standard deviation (SD): 13.35 ng / mL •Median: 55.2 ng / mL • Range: 32.5~82.1 ng / mL • Cut-off value: 83.5 ng / mL (Mean + 2 SD) (2) Evaluation of diagnostic performance for various cancer types Table 11 Sensitivity and Specificity for Each Cancer Type

[0082] Based on Table 11, we can see that: • Overall sensitivity (four cancers combined): 78.9% (238 / 301) • Specificity (vs. healthy controls): 93.3% (112 / 120) • Specificity (vs. benign lesions): 88.3% (53 / 60) The kit has high sensitivity and specificity for liver cancer, lung cancer, colorectal cancer, and breast cancer.

[0083] (3) ROC curve analysis The diagnostic efficacy of this kit for various cancer types was evaluated using receiver operating characteristic (ROC) curves, and the results are as follows: Figure 2 As shown in Table 12: Table 12 Area Under the ROC Curve (AUC) for Each Cancer Type

[0084] Depend on Figure 2 As shown in Table 12, the ROC curves of the four types of tumors all deviated significantly from the reference line, indicating good diagnostic efficacy, with hepatocellular carcinoma showing the best diagnostic performance.

[0085] (4) Comparison with AFP in the diagnosis of liver cancer Alpha-fetoprotein (AFP) is currently the most commonly used serum biomarker for liver cancer in clinical practice. This study uses an AFP cutoff value of 20 ng / mL as the standard to evaluate the performance advantages of this kit in the diagnosis of liver cancer.

[0086] Table 13 Comparison of HSP90α and AFP in the diagnosis of hepatocellular carcinoma

[0087] Based on the above, we can conclude that: The sensitivity of HSP90α (87.2%) was significantly higher than that of AFP (62.8%), with a statistically significant difference (P<0.001), representing a relative increase of 38.9%.

[0088] Among 34 AFP-negative hepatocellular carcinoma patients (AFP < 20 ng / mL), 28 were HSP90α positive, with a sensitivity of 82.4%. This means that this kit can detect approximately 82% of patients with missed AFP diagnoses, significantly reducing the risk of missed diagnosis of hepatocellular carcinoma.

[0089] When HSP90α and AFP are detected together, the sensitivity is increased to 94.2% (81 / 86), and the AUC increases from 0.924 for a single indicator to 0.956, demonstrating significantly better diagnostic performance than either single indicator.

[0090] Table 14 Detection of HSP90α in AFP-negative hepatocellular carcinoma patients

[0091] As shown in Table 14, the kit can efficiently detect AFP-negative liver cancer and significantly reduce the rate of missed diagnoses.

[0092] (5) Evaluation of early detection capabilities The enrolled tumor patients were staged according to the TNM staging criteria to evaluate the kit's ability to detect early-stage tumors.

[0093] Table 15 Detection rates of tumors at different stages

[0094] Summary by phase: • Stage I+II (early stage): 68 out of 95 cases were detected, with a sensitivity of 71.6%. • Stage III+IV (intermediate to late stage): 158 out of 176 cases were detected, with a sensitivity of 89.7%. • Stage I liver cancer: 11 out of 14 cases were detected, with a sensitivity of 78.6%. Based on Table 15, it can be seen that, in comparison, AFP has a sensitivity of only about 42.9% (6 / 14) for stage I liver cancer, and this kit has a significantly better detection ability for early liver cancer than AFP.

[0095] (6) Ability to differentiate between benign and malignant lesions This kit has a good ability to differentiate between benign lesions and malignant tumors. The following is a comparative analysis of typical benign lesions and their corresponding malignant tumors: Table 16 Differential Diagnosis of Hepatitis and Hepatocellular Carcinoma

[0096] As shown in Table 16, the difference between the two groups was highly statistically significant (P<0.001). The mean value of HSP90α in the liver cancer group was about 3 times that in the hepatitis group, demonstrating good discriminative ability.

[0097] Table 17 Differential Diagnosis Table for Breast Hyperplasia and Breast Cancer

[0098] As shown in Table 17, the HSP90α level in the breast cancer group was significantly higher than that in the breast hyperplasia group, and the difference was statistically significant (P<0.001).

[0099] Performance testing (1) Specificity detection of the monoclonal antibody obtained in Example 3 The detection was performed by ELISA. The ELISA plates were coated with the epitope peptides of the synthesized HSP90α (1) and (2) and BSA as detection antigens, respectively. The specific reaction of the prepared HSP90α monoclonal antibodies (1) and (2) with the corresponding epitope peptides was detected by ELISA. Normal BALB / c mouse serum was used as a negative control and PBS solution was used as a blank control.

[0100] The results showed that HSP90α monoclonal antibodies (1) and (2) reacted positively only with the epitope peptides of the synthesized HSP90α (1) and (2) (P / N>2.1), while the reaction with BSA as the antigen was negative, indicating that the HSP90α monoclonal antibodies (1) and (2) of the present invention are specific.

[0101] (2) Specificity detection of the polyclonal antibody obtained in Example 3 Identification was performed using the same method as described above for identifying the specificity of monoclonal antibodies. The results showed that HSP90α polyclonal antibodies (1) and (2) reacted positively with the epitope peptides of synthesized HSP90α (1) and (2) respectively (P / N>2.1), while they reacted negatively with BSA, indicating that the HSP90α polyclonal antibodies (1) and (2) of the present invention are specific.

[0102] (3) Verification of the key role of Q560 site in HSP90α specificity The Q560M mutant was obtained by mutating the Q560 site of HSP90α to M (simulating the corresponding site of HSP90β) using site-directed mutagenesis. The binding activities of the antibodies of this invention to the following three proteins were compared using an indirect ELISA method: - wild-type HSP90α - Q560M mutant HSP90α - natural HSP90β The experimental results are shown in Table 18. Table 18 Experimental Results

[0103] Based on the above, it can be seen that after a single-point mutation of Q560 in HSP90α to M, the binding activity of the antibody of the present invention decreased by more than 50-fold, down to a level comparable to that of natural HSP90β. This result fully demonstrates that the Q560 site is the decisive amino acid residue for the specific recognition of HSP90α by the antibody of the present invention, and the polarity difference between Q560 and M561 is the molecular basis for achieving subtype specificity.

[0104] (4) Parallel comparison experiment with epitopes of patent CN105669834B Experimental Design: Using identical experimental conditions, the following three groups of antibodies were prepared and tested in parallel: - Group A (this invention): antibody recognizing epitopes 552-568aa - Group B (Anqun Biotechnology Patent 1): antibody recognizing epitopes 200-215aa - Group C (Anqun Biotechnology Patent 2): antibody recognizing epitopes 697-716aa (2) Performance comparison results Table 19 Performance Comparison Results

[0105] As shown in Table 19, the differences between Group A and Groups B and C in all indicators of this invention are statistically significant (P<0.05). The 552-568aa epitope selected in this invention is significantly superior to the two epitopes selected by Anqun Biotechnology in all key performance indicators. This comprehensive performance improvement demonstrates the inventiveness of the epitope selection strategy of this invention.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A human HSP90α antigenic epitope peptide, characterized in that, The human HSP90α antigen epitope peptide is a polypeptide, and its amino acid sequence is shown in SEQ ID NO.

1. The SEQ ID NO.1 is: Leu-Pro-Glu-Asp-Glu-Glu-Glu-Lys-Lys-Lys-Gln-Glu-Glu-Lys-Lys-Thr-Lys-Cys; The epitope peptide contains glutamine at position 560 (Q560), which is a key amino acid residue that distinguishes the HSP90α and HSP90β subtypes. The 561st position of HSP90β corresponding to Q560 is methionine (M561). The polar / nonpolar difference between Q560 and M561 determines the specific recognition of HSP90α by the epitope peptide. The epitope peptide is located on the N side of the HSP90α C-terminal dimerization interface, and the epitope is fully exposed on the molecular surface in the HSP90α dimer state. The cross-reactivity rate of the epitope peptide with HSP90β is less than 1.5%.

2. A method for preparing the human HSP90α antigenic epitope peptide according to claim 1, characterized in that, Includes the following steps: (1) Two specific antigenic epitope regions were obtained by analyzing the human HSP90α amino acid sequence through bioinformatics analysis; (2) The amino acid sequence of human HSP90α protein from position 552 to 568 was taken as the backbone of epitope peptide 1, and the amino acid sequence of human HSP90α protein from position 703 to 717 was taken as the backbone of epitope peptide 2. (3) Add a cysteine ​​residue Cys to the N-terminus of both the epitope peptide 1 backbone and the epitope peptide 2 backbone; (4) The antigenic epitope peptide shown in SEQ ID NO.1 was synthesized by solid-phase chemical synthesis based on the skeleton of epitope peptide 1 and epitope peptide 2.

3. A recombinant antigen, characterized in that, It is prepared by coupling the antigenic epitope peptide of claim 1 with a carrier protein.

4. The recombinant antigen according to claim 3, characterized in that, The carrier protein is KLH, BSA, or OVA.

5. A monoclonal antibody specific to HSP90α, characterized in that: The antibody specifically binds to the epitope peptide of claim 1; The complementarity-determining region (CDR3) of the antibody contains an amino acid sequence that specifically interacts with the continuous charged region (Glu-Glu-Glu-Lys-Lys) at positions 558-562 of the epitope peptide. The antibody does not recognize the HSP90β protein, and its cross-reactivity with HSP90β is less than 1%. The antibody preferentially recognizes secretory HSP90α in serum, and its recognition activity for secretory HSP90α is higher than that for recombinantly expressed intracellular HSP90α.

6. The specific antibody according to claim 5, characterized in that, The antibody is a monoclonal antibody or a polyclonal antibody.

7. A double-antibody sandwich ELISA kit for detecting secretory HSP90α in serum, characterized in that: It comprises a first antibody and a second antibody, wherein the first antibody recognizes the 552-568aa epitope as described in claim 1, and the second antibody recognizes the 703-717aa epitope at the C-terminus of HSP90α; The two epitopes are approximately 45 Å apart in the three-dimensional structure of HSP90α, and there is no steric hindrance when the antibody binds. The kit employs a dual C-terminal epitope pairing strategy. The kit has a detection range of 5-200 ng / mL for serum samples, with a detection limit of ≤2 ng / mL. The kit has a specificity of ≥98.5% in distinguishing between HSP90α and HSP90β.

8. The use of the antigenic epitope peptide according to claim 1 in the preparation of in vitro diagnostic reagents for tumors.

9. The application according to claim 8, characterized in that, The tumor is at least one of liver cancer, lung cancer, colorectal cancer, and breast cancer.

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