ACLF immune PCR single molecule detection kit, detection method and application

Abstract

The application discloses an ACLF immune PCR single-molecule detection kit, a detection method and application, and comprises the following steps: a) biotin-labeled capture antibody 8G5; b) oligonucleotide-coupled detection antibody 6B4-Oligo; c) streptavidin magnetic beads; d) nuclease composition; e) specific primers and probes. The application adopts an immune PCR-single-molecule detection method, utilizes specially designed oligonucleotide-coupled detection antibodies, optimizes the nuclease composition and optimizes the PCR reaction condition, can carry out high-sensitivity quantitative detection under extremely low sample concentration, avoids background interference in the traditional detection method, improves the specificity and accuracy of the signal, and provides a more accurate diagnosis tool for the clinic. Compared with the traditional PCR and ELISA methods, the application has higher sensitivity, can accurately detect the low concentration of CK18 in the serum of the ACLF patient, and provides an effective tool for prognosis evaluation.

Description

Technical Field

[0001] This invention relates to a kit and detection method for detecting the level of hepatocyte keratin 18 (CK18) in the serum of patients with acute-on-chronic liver failure (ACLF) based on immunoPCR single-molecule detection technology, and its application in assessing the prognosis of ACLF patients. This invention further relates to the construction of related kits, antibody-coupled optimization methods, nucleic acid interference removal technology, and prognostic assessment systems, belonging to the field of biomedical detection technology. Background Technology

[0002] Acute-on-chronic liver failure (ACLF) is an acute exacerbation of chronic liver disease, accompanied by multiple organ failure and high mortality. Common precipitating factors for ACLF include: infections (such as spontaneous bacterial peritonitis, sepsis); metabolic decompensation (such as diabetic ketoacidosis); cardiovascular events (such as ischemic liver injury); drugs / toxins (such as acetaminophen overdose); gastrointestinal bleeding or surgical trauma. Patients often present with: jaundice, coagulation disorders, ascites, hepatic encephalopathy; and multiple organ failure (renal, circulatory, and respiratory systems). ACLF criteria: Based on international academic standards (such as EASL, APASL), it usually requires underlying cirrhosis, but it can also occur in patients without cirrhosis. The mortality rate of ACLF is as high as 50%–90%. Early identification of precipitating factors and aggressive supportive treatment can improve outcomes.

[0003] Hepatic cytokeratin 18 (CK18), as a marker of apoptosis, has been shown to be associated with the occurrence and development of various liver diseases, and has important applications in the early diagnosis and prognostic assessment of diseases such as hepatitis, cirrhosis and liver cancer.

[0004] The clinical application of CK18 faces the following technical bottlenecks: Low abundance detection challenges: In the early or subclinical stages of ACLF, serum CK18 concentrations may be as low as fg / mL, exceeding the sensitivity threshold (usually pg / mL) of traditional detection methods (such as enzyme-linked immunosorbent assay, ELISA); Limited dynamic range: The linear range of methods such as ELISA is narrow, making it difficult to accurately quantify high concentrations of CK18 in critically ill patients (e.g., changes in the M65 / M30 ratio due to necrosis). Currently, the detection of CK18 mainly relies on the following technologies: Enzyme-linked immunosorbent assay (ELISA): Based on the principle of antigen-antibody binding, it is simple to operate but has limited sensitivity (detection limit approximately 10–100 pg / mL) and is easily affected by serum matrix effects; Chemiluminescent immunoassay (CLIA): It improves sensitivity by amplifying the chemiluminescent signal, but still cannot meet the needs for accurate detection of extremely low concentration samples; Flow cytometry combined with fluorescent probes: It can achieve single-cell level analysis, but requires complex pretreatment steps (such as cell separation), making it unsuitable for large-scale clinical sample screening. Summary of the Invention

[0005] The primary technical problem to be solved by this invention is to provide an ACLF immunoPCR single-molecule detection reagent.

[0006] Another technical problem to be solved by the present invention is to provide a new use for the above-mentioned detection reagent in the preparation of a kit for detecting CK18 content as a prognostic prediction of patients with acute-on-chronic liver failure.

[0007] Another technical problem to be solved by the present invention is to provide a method for detecting CK 18 content using the above-mentioned detection reagents.

[0008] Another technical problem to be solved by the present invention is to provide a prognostic assessment system for patients with acute-on-chronic liver failure based on the above method.

[0009] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution:

[0010] An ACLF immunoPCR-single-molecule detection reagent, comprising:

[0011] a) Biotin-labeled capture antibody 8G5;

[0012] b) Oligonucleotide-conjugated detection antibody 6B4-Oligo;

[0013] c) Streptavidin magnetic beads;

[0014] d) Nuclease composition;

[0015] e) Specific primers and probes;

[0016] The nuclease composition consists of a totipotent nuclease, 2% BSA coating solution and salmon sperm DNA, wherein the mass ratio of the totipotent nuclease to salmon sperm DNA is 1:1.

[0017] The oligonucleotide-coupled detection antibody 6B4-Oligo is composed of an oligonucleotide shown in SEQ ID No. 1 coupled to the detection antibody 6B4, wherein the molar ratio of antibody 6B4 to oligonucleotide is 3:1;

[0018] The specific primers are shown in SEQ ID No. 2 and SEQ ID No. 3;

[0019] The probe 1 is shown in SEQ ID No. 4. The 5' end of the probe 1 is connected to a fluorescent group, and the 3' end is connected to a quenching group BHQ_1.

[0020] The probe 2 is shown in SEQ ID No. 5. The 5' end of the probe 2 is connected to a fluorescent group, and the 3' end is connected to a quenching group BHQ_2.

[0021] The above-mentioned application of the immunoPCR-single-molecule detection reagent in the preparation of a kit for detecting CK18 content as a prognostic prediction for patients with acute-on-chronic liver failure.

[0022] A kit for detecting CK18 levels as a prognostic predictor for patients with acute-on-chronic liver failure includes the aforementioned detection reagents.

[0023] A method for detecting CK18 content based on the above-mentioned detection kit includes the following steps:

[0024] a) The biotin-labeled capture antibody 8G5 was bound to the CK18 antigen in the serum sample to be tested to obtain the capture antibody-antigen immune complex.

[0025] b) Add streptavidin magnetic beads and a nuclease composition to capture antigen-antibody complexes by enriching them with magnetic beads and eliminate non-specific nucleic acid interference by the nuclease composition.

[0026] c) Add oligonucleotide-conjugated detection antibody 6B4-Oligo to form a complete immune complex;

[0027] d) PCR amplification using specific primers and probes to amplify oligonucleotide sequences conjugated with detection antibodies;

[0028] e) The amplification products were quantitatively analyzed using single-molecule detection technology to determine the concentration of CK18;

[0029] Preferably, the method for preparing the oligonucleotide-conjugated detection antibody includes:

[0030] The antibody is conjugated to an oligonucleotide using an activating agent, wherein the oligonucleotide sequence is shown in SEQ ID No. 1, and the molar ratio of antibody 6B4 to oligonucleotide is 3:1.

[0031] Preferably, the nuclease composition is used to remove non-specific nucleic acid interference. The composition consists of a totipotent nuclease, a 2% BSA coating solution, and salmon sperm DNA, wherein the mass ratio of the totipotent nuclease to the salmon sperm DNA is 1:1.

[0032] Preferably, the PCR amplification conditions are as follows: 3.125 μL of serum is added to each well, and an amplification program of 20 cycles is used, including pre-denaturation at 95°C for 30 seconds, and 20 cycles of 95°C for 5 seconds and 60°C for 34 seconds.

[0033] Preferably, the single-molecule detection technology includes the following steps:

[0034] a) PCR products were enriched using magnetic beads and separated by centrifugation;

[0035] b) Use a reading plate to capture single-molecule signals, and combine it with a microplate culture shaker for uniform mixing;

[0036] c) Analyze single-molecule signals using software to generate quantitative detection results.

[0037] A prognostic assessment system for patients with acute-on-chronic liver failure based on the above method includes:

[0038] a) CK18 concentration detection module, used to perform immunoPCR-single molecule detection;

[0039] b) Data analysis module, which performs correlation analysis between CK18 concentration and chronic-on-acute liver failure grading and liver function indicators;

[0040] c) Prognostic prediction module, which outputs the patient's adverse prognostic risk assessment results and individualized treatment suggestions.

[0041] The application of CK18 detection in predicting prognosis in patients with acute-on-chronic liver failure.

[0042] Compared with the prior art, the present invention has the following technical effects:

[0043] (1) This invention employs an immunoPCR-single-molecule detection method. Utilizing a specially designed oligonucleotide-coupled detection antibody, optimized nuclease composition, and optimized PCR reaction conditions, it enables highly sensitive quantitative detection at extremely low sample concentrations. This avoids background interference in traditional detection methods, improves signal specificity and accuracy, and provides a more precise diagnostic tool for clinical use. Compared to traditional PCR and ELISA methods, this technology has higher sensitivity, accurately detecting low concentrations of CK18 in the serum of ACLF patients, and providing an effective tool for prognostic assessment.

[0044] (2) This invention employs a unique antibody-oligonucleotide conjugation method, resulting in improved purity: HPLC purification significantly enhances the purity of the oligonucleotides, effectively reducing interference from impurities in subsequent conjugation reactions. Efficiency optimization: 5'-terminal amino group modification combined with HPLC purification significantly improves the preparation efficiency of Ab-oligo conjugates. Quality improvement: High-purity oligonucleotides directly improve the stability and functional properties of Ab-oligo conjugates. Optimized coating solutions (such as BSA, salmon sperm DNA, and mouse serum) are used to improve the efficiency of affinity reactions, significantly enhancing the sensitivity and specificity of immunoPCR reactions. In particular, the combination of BSA and salmon sperm DNA demonstrates outstanding performance in removing nucleic acid contamination and improving enzyme activity, overcoming the problems of nucleic acid interference and non-specific binding present in traditional methods.

[0045] (3) In the immunoPCR process, this invention combines a totipotent nuclease with different coating solutions to form a nuclease composition, effectively optimizing the nucleic acid removal effect. Experimental verification shows that the combination of BSA and salmon DNA is the most effective in removing nucleic acid contamination, significantly improving the accuracy and reliability of the experiment. This optimized scheme provides a guarantee for efficient, low-background single-molecule detection.

[0046] (4) By optimizing the number of PCR amplification cycles and the concentration of serum added, the present invention determined the optimal experimental conditions (3.125 μL of serum added to each well, 20 PCR cycles), thereby significantly improving the efficiency and specificity of the PCR reaction, reducing non-specific amplification and PCR inhibition, and further improving the reliability of the detection results.

[0047] (5) This invention innovatively combines immunoPCR with single-molecule detection technology with the detection of CK18 levels in the serum of ACLF patients, providing a novel biomarker for the clinical grading and prognostic assessment of ACLF. By comparing the CK18 concentrations of patients with different grades (ACLF grade 1, 2, and 3), it was found that CK18, as an independent predictor of poor prognosis, can provide clinicians with accurate prognostic information and help develop individualized treatment plans. Attached Figure Description

[0048] Figure 1 This is a diagram illustrating the molecular weight of the oligonucleotide-coupled detection antibody Oligo in an embodiment of the present invention.

[0049] Figure 2 This is a diagram illustrating the normal operation verification of the template, primers, and probe in an embodiment of the present invention.

[0050] Figure 3A In this embodiment of the invention, a specificity map of primers and probes paired with WO was designed;

[0051] Figure 3B To compare primer and probe specificity maps;

[0052] Figure 4 This is a verification diagram for detecting the specificity of antibody-conjugated oligonucleotides in an embodiment of the present invention;

[0053] Figure 5 This is a verification diagram of the elimination of nonspecificity by the pluripotent nuclease in an embodiment of the present invention;

[0054] Figure 6 This is a screening diagram of nuclease compositions in an embodiment of the present invention;

[0055] Figure 7 This is a schematic diagram illustrating the determination of the optimal antibody concentration ratio using the checkerboard method in an embodiment of the present invention.

[0056] Figure 8 This is a graph showing the quenching efficiency evaluation of the BHQ probe in an embodiment of the present invention;

[0057] Figure 9 This is a verification diagram of detection sensitivity in an embodiment of the present invention;

[0058] Figure 10 This is a graph illustrating the study of serum sample dilution concentrations in an embodiment of the present invention;

[0059] Figure 11 This is a graph illustrating the evaluation of PCR amplification cycle number in an embodiment of the present invention;

[0060] Figure 12 To validate the detection of ACLF patient serum using this invention;

[0061] Figure 13 A comparison of the efficacy of the MELD model in predicting patient prognosis;

[0062] Figure 14 This is a sensitivity-specificity plot of the CK18 model in an embodiment of the present invention. Detailed Implementation

[0063] The present invention is further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0064] This invention integrates molecular biology, nanotechnology, and clinical medicine, and for the first time applies an antibody-linked oligonucleotide immunoPCR-single-molecule detection method to the prognostic assessment of ACLF (acute-on-chronic liver failure). By detecting antibody-conjugated oligonucleotides, optimizing the totipotent nuclease in immunoPCR to effectively remove non-specific interfering nucleic acids, and optimizing PCR amplification conditions based on the detection system, it achieves accurate detection of CK 18 concentration in serum samples, thereby enabling prognostic prediction of ACLF patients with metabolic-associated fatty liver disease (MASLD). This technology solves the problems of insufficient sensitivity and weak dynamic monitoring capabilities of existing detection methods, and can also reveal the molecular mechanism of ACLF through multi-marker conjugation, providing key evidence for early warning, precise stratification, and personalized treatment in clinical practice, with significant clinical application value and market prospects. Specific details are as follows:

[0065] Example 1: Design and efficacy verification of the oligonucleotide-coupled detection antibody provided by the present invention. I. The template, oligonucleotide, specific primer, and probe sequences used in this embodiment have been verified to be working.

[0066] Table 1. WO primer and probe sequences

[0067]

[0068] Table 2 Comparative examples: PO primer and probe sequences

[0069]

[0070] Antibody sequence:

[0071] 6B4 heavy chain gene sequence SEQ ID No. 6: 6B4-VH-CH (Mouse IgG1)

[0072] VHGCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGCATGCACAGCTCAGCACTGCTCT

[0073] GTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCCAGGTTCAACTCCAACAAAGCGGCGGCGGA

[0074] CTCGTTCAACCAAAAGGAAGCCTCAAACTCAGCTGCGCCGCCAGCGGATTTACATTCCACAC

[0075] ATTCGCCATGAACTGGGTCAGACAAGCACCCGGAAAAGGCCTCGAATGGGTGGCTAGAATTA

[0076] GAACAAAAACCAACAACTACGCCACCAACTACGCCGACAGCGTGAAGGACAGATTCACCATC

[0077] AGCAGAGACGACAGCCAGAGCATGCTGTTCCTGCAGATGAACAACCTGAAAACTGAAGATAC

[0078] CGCCATGTACTACTGCGTGAGAGGCCACTACGGCAGCAGAAGCCTGTTCGCCTACTGGGGCC

[0079] AGGGCACCCTGGTGACCGTGAGCGCCGCTAAGACCACCCCCCCTTCCGTGTATCCCCTGGCT

[0080] CCTGGATCTGCCGCCCAGACAAACTCCATGGTGACCCTGGGCTGTCTGGTGAAAGGCTATTT

[0081] TCCTGAACCCGTGACCGTGACCTGGAACAGCGGCTCTCTGTCTAGCGGCGTGCATACTTTTC

[0082] CCGCCGTGCTGCAGTCCGACCTGTATACCCTGAGTTCCTCCGTGACCGTGCCCTCTTCCACC

[0083] TGGCCTAGCGAGACCGTGACCTGCAATGTGGCCCACCCCGCTTCCAGCACCAAGGTGGATAA

[0084] GAAAATCGTGCCCCGCGACTGCGGCTGTAAGCCTTGTATCTGCACCGTGCCCGAAGTGAGCT

[0085] CAGTGTTCATTTTCCCCCCCAAGCCCAAAGACGTGCTGACCATCACCCTGACCCCCAAAGTG

[0086] ACCTGCGTGGTGGTGGATATTAGCAAGGATGACCCCGAAGTGCAGTTTTCTTGGTTCGTGGA

[0087] CGACGTGGAAGTGCACACCGCCCAGACCCAGCCTAGAGAGGAGCAGTTCAACTCCACATTCA

[0088] GGAGTGTGAGCGAGCTGCCCATTATGCACCAGGATTGGCTGAACGGGAAGGAGTTCAAATGT

[0089] AGGGTGAACAGCGCCGCCTTCCCCGCTCCTATTGAAAAAACCATCAGCAAGACCAAGGGCAG

[0090] ACCCAAGGCCCCCCAGGTGTACACCATCCCCCCCCCCAAGGAGCAGATGGCCAAGGACAAGG

[0091] TGAGCCTGACCTGCATGATCACCGACTTCTTCCCCGAGGACATCACCGTGGAGTGGCAGTGG

[0092] AACGGCCAGCCCGCCGAGAACTACAAGAACACCCAGCCCATCATGGACACCGACGGCAGCTA

[0093] CTTCGTGTACAGCAAGCTGAACGTGCAGAAGAGCAACTGGGAGGCCGGAAACACCTTCACCT

[0094] GCAGCGTGCTGCACGAGGGCCTGCACAACCACCACACCGAGAAGAGCCTGAGCCACAGCCCC

[0095] GGCAAGTGATTCTAGA

[0096] 6B4 Amino acid sequence SEQ ID No.7:

[0097] MHSSALLCCLVLLTGVRAQVQLQQSGGGLVQPKGSLKLSCAASGFTFHTFAMNWVRQAPGKG

[0098] LEWVARIRTKTNNYATNYADSVKDRFTISRDDSQSMLFLQMNNLKTEDTAMYYCVRGHYGSR

[0099] SLFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSL

[0100] SSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCI

[0101] CTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPRE

[0102] EQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPK

[0103] EQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNW

[0104] EAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0105] 6B4 light chain gene sequence SEQ ID No.8: Signal peptide 6B4-VL-CL (Mouse Kappa) VKGCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGCATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCCAGATCGTCCTCACCCAAAGCCCAGCAATAATGAGCGCCAGCCCCGGAGAAAAGGTCACAATGACCTGCAGCGCCAGCAGCTCAGTCTCATACATGTACTGGTACCAACAAAAGCCCCGGTCCAGCCCAAAACCCTGGATTTACCTGACCAGCAACCTGGCCAGCGGAGTTCCTGCTAGATTCTCCGGTTCCGGCAGCGGAACAAGCTACTCTCTCACCATCAGCAGCATGGAGGCCGAGGATGCTGCTACATACTACTGCCAACAATGGAACAGCAACCCACCTACCTTCGGGGGAGGTACAAAGCTCGAAATCAAGAGAGCAGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTGATTCTAGA

[0106] 6B4 light chain amino acid sequence SEQ ID No.9:

[0107] MHSSALLCCLVLLTGVRAQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWNSNPPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0108]

[0109] 8G5 heavy chain amino acid sequence SEQ ID No.11:

[0110] MHSSALLCCLVLLTGVRAQVQLQQSGPELVKPGASVKISCKTSGYTFTEYSMHWVKQSHGKSLEWIGGINPNNGGTNYNQKFKGKATLTYGQRAYFAYVDRSSRTAYMELRSLTSEDSAVYYCALYGQRAYFAYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0111] 8G5 light chain gene sequence SEQ ID No.12: 8G5-VL-CL(Mouse Kappa)

[0112] VKGCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGCATGCACAGCTCAGCACTGCTCT

[0113] GTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCGACATCGTGATGACCCAGACCCCACTCAGC

[0114] CTGCCTGTTTCTCTGGGAGATCAAGCCAGCATAAGCTGCAGAAGCAGCCAAAGCCTGGTGCA

[0115] CAACAACGGAAACACCTACCTGCACTGGTACCTCCAGAAGCCCGGACAATCCCCAAACCTCC

[0116] TGATCTACAAGGTGAGCAATAGATTCAGCGGCGTGCCCGACAGATTCTCCGGTAGCGGATCA

[0117] GGAACCGACTTCACCCTCAAGATCAGCAGAGTCGAAGCTGAAGATTTAGGTGTGTACTTCTG

[0118] CAGCCAGAGCACCCACGTTCCCTGGACCTTTGGAGGAGGAACTAAGCTGGAGATCAAGAGAG

[0119] CAGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGT

[0120] GCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGAT

[0121] TGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACA

[0122] GCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGC

[0123] TATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAA

[0124] TGAGTGTTGATTCTAGA

[0125] 8G5 light chain amino acid sequence SEQ ID No.13:

[0126] MHSSALLCCLVLLTGVRADIVMTQTPLSLPVSLGDQASISCRSSQSLVHNNGNTYLHWYLQKPGQSPNLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVP WTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0127] I. Oligonucleotide Design Scheme:

[0128] 1. Determine the target sequence

[0129] The first step in designing primers and probes is to identify the nucleic acid sequence to be amplified or detected. In this example, the target sequence is the provided nucleotide sequence:

[0130] SEQ ID No.1

[0131] GGGCGACGATAATTGATATTGGTGGGCAGTCTACACGGCCTGGTTCACATGTTGTTTCTATAGAGGAAGAGATTTCTC

[0132] The sequence is 78 bases long and is the target region for PCR amplification and real-time PCR detection.

[0133] 2. Design primers

[0134] Primers are the core of the PCR reaction, used to specifically amplify target sequences. When designing primers, factors such as specificity, GC content, and melting temperature (Tm value) must be considered.

[0135] 2.1 Forward Primer Design

[0136] SEQ ID No. 2 sequence: ATCTCTTCCTCTATAGAAACAACAT

[0137] Length: 25 bases;

[0138] Location: The primer is partially complementary to the 3' end region of the target sequence (positions 53-77: TTGTTTCTATAGAGGAAGAGATTTCTC), indicating that it is a forward primer that binds to the reverse strand of the template.

[0139] Design principles:

[0140] (1) The Tm value should be between 50 and 60℃ (the specific value needs to be calculated and the Tm value meets the requirements).

[0141] (2) Avoid hairpin structures or primer dimers to ensure amplification efficiency.

[0142] 2.2 Reverse Primer Design

[0143] SEQ ID No. 3 sequence: GGGCGACGATAATTGATATTGG

[0144] Length: 22 bases, within the standard design range;

[0145] Location: The primer perfectly matches the 5' end of the target sequence (positions 1-22: GGGCGACGATAATTGATATTGG), indicating that it is a reverse primer complementary to the forward strand of the template.

[0146] Design principles:

[0147] (1) The GC content is approximately 45%, which is within the ideal range;

[0148] (2) The Tm value is similar to that of the forward primer;

[0149] (3) Ensure specificity and avoid non-specific amplification.

[0150] 3. Design TaqMan probes

[0151] TaqMan probes are used for real-time PCR, detecting the amplification of target sequences via fluorescence signals. The following factors need to be considered when designing probes:

[0152] 3.1 Probe Sequence

[0153] Sequence: TGGGCAGTCTACACG

[0154] Length: 15 bases, allowing for efficient binding;

[0155] Location: Located at positions 25-39 of the target sequence (TGGGCAGTCTACACG), between the forward and reverse primers, ensuring degradation by Taq polymerase during PCR amplification.

[0156] Design principles:

[0157] (1) The Tm value should be 5-10℃ higher than that of the primer (the specific value needs to be calculated, but the Tm value of a 15bp probe is usually between 55-65℃, which meets the requirements);

[0158] (2) It binds to the target sequence with high specificity, avoiding non-specific signals.

[0159] 3.2 Fluorescent groups and quenching groups

[0160] Probe 1: / 5`6-FAM / TGGGCAGTCTACACG / 3BHQ_1 /

[0161] (1) 5' end fluorescent reporter group: 6-FAM (common fluorescent dye);

[0162] (2) 3' quenching group: BHQ_1 (black hole quencher No. 1).

[0163] Probe 2: / 5`Alexa Fluor 647 / TGGGCAGTCTACACG / 3BHQ_2 /

[0164] (1) 5' end fluorescent reporter group: Alexa Fluor 647 (suitable for multiplex detection);

[0165] (2) 3' quenching group: BHQ_2 (Black Hole Quencher No. 2).

[0166] Function: When the probe is intact, the fluorescence is quenched; when the probe is degraded by Taq polymerase, the fluorescence signal is released for real-time detection.

[0167] Oligonucleotide Conjugation Kit Components: (ab218260 Oligonucleotide Conjugation Kit, Abcam subsidiary Expedeon)

[0168] II. Oligonucleotide conjugation detection of antibodies

[0169] 1. Oligonucleotide activation

[0170] Add 100 μL of 100 μM oligonucleotide to the oligonucleotide activation reagent bottle. Mix gently and incubate at room temperature for 30 minutes. Proceed to step 2 during incubation.

[0171] 2. Antibody activation

[0172] Add 100 μL of antibody (concentration 1 mg / mL) to the antibody activation reagent vial. Mix gently and incubate at room temperature for 30 minutes. Proceed to step 3 during incubation.

[0173] 3. Desalting with activating reagent

[0174] 3.1 Secure each separator column vertically. First, open the top cap. Then, open the bottom cap to allow the stored liquid to pass through the separator column. Discard the liquid flowing out of the separator column.

[0175] 3.2 Add 3 mL of wash buffer to the top of the separation column, allowing the liquid to flow out under gravity to equilibrate each column. Discard the outflowing liquid. Repeat the above steps 4 times.

[0176] 3.3 Following the activation steps in steps 1 and 2, after incubating the sample for 30 minutes, add 100 μL of the activated oligonucleotide or antibody from the top of the separation column, and wait for the liquid to be completely absorbed by the separation column. Collect the effluent and let it stand until successful coupling is confirmed.

[0177] 3.4 Add 550 μL of wash buffer to the top of the separation column. Push the activated reagent to the bottom of the separation column. Allow the liquid to be completely absorbed before proceeding to the next step. Collect the eluent and let it stand until you confirm successful coupling.

[0178] 3.5 Place a clean microcentrifuge tube below the separation column. Add 300 μL of wash buffer from the top of the separation column.

[0179] 3.6 Collect the eluent from the bottom of the separation column. This eluent (300 μL) contains activated oligonucleotides or antibodies and can be used for conjugation at any time.

[0180] 4. Preparation of purified oligonucleotide-conjugated antibodies

[0181] This kit (ab218260 Oligonucleotide Conjugation Kit, an Abcam subsidiary, Expedeon) can be used to generate a series of oligonucleotide-conjugated antibodies with different ratios of antibody to oligonucleotide. Simply refer to Table 1 to add different amounts of oligonucleotide to the antibody. The optimal ratio depends on the specific experiment using this conjugate and may need to be determined experimentally.

[0182] 4.1 Add 300 μL of activated antibody to an appropriate amount of activated oligonucleotides and washing buffer, as shown in the table below.

[0183] Table 3

[0184]

[0185] Note: The antibody to oligonucleotide ratio is an average, as a large number of labeled antibodies are produced after the conjugation reaction. The number of oligonucleotides conjugated to each antibody will not be exactly the same.

[0186] 4.2 Mix well and incubate at room temperature for 1 hour.

[0187] 4.3 The conjugated antibodies are now available for use. They can also be purified as needed to remove any unconjugated oligonucleotides (see step 5 for details).

[0188] 4.4 Unused activated oligonucleotides can be stored at -20°C.

[0189] 5. Purification of conjugated antibodies

[0190] Note: To clearly observe the precipitation, at least 50 μg of antibody is required.

[0191] 5.1 Place the test tube containing the purification reagent in warm water (not exceeding 40℃) and heat for 10 minutes, then mix well. If the sample is not completely dissolved, place the sample in a benchtop microcentrifuge and vortex at the maximum recommended speed of 13000×g for 1 minute, then collect the supernatant for later use.

[0192] 5.2 Add an equal volume of purification reagent to the antibody / oligonucleotide mixture, mix well, and incubate at room temperature or on ice for 20 minutes. For example, add 600 μL of purification reagent to 600 μL of antibody / oligonucleotide mixture.

[0193] 5.3 Centrifuge at 15000×g for 5 minutes in a benchtop microcentrifuge.

[0194] 5.4 Remove the sample from the centrifuge, being careful not to disturb the small amount of precipitate at the bottom of the tube. If no precipitate is observed, add an additional 1 / 10 volume of purification reagent, mix well, and incubate on ice for 10 minutes, then centrifuge. If no precipitate is observed when using non-antibody proteins, add half the volume of purification reagent described in 5.2, mix well, and incubate on ice for 10 minutes, then centrifuge. For example: if 600 μL of purification reagent was added in 5.2, then add an additional 300 μL of purification reagent.

[0195] 5.5 Carefully remove the supernatant and let stand until a valid precipitate is confirmed to have formed.

[0196] 5.6 Add 100 μL of antibody suspension buffer to the precipitate and mix well.

[0197] 5.7 To remove as many free oligonucleotides as possible, the same conjugated antibody should be purified twice.

[0198] 5.8 Oligonucleotide conjugate antibodies are now available.

[0199] III. Validation of Oligonucleotide Design Results

[0200] like Figure 1 As shown, a highly specific nucleotide is linked to the detection antibody 6B4 via amino-terminal coupling. Amino-terminal coupling is a commonly used method that introduces an active group at the amino terminus of a nucleotide or antibody to achieve covalent binding with other molecules. This method has advantages such as simple operation and high coupling efficiency. To verify the coupling efficiency between the nucleotide and the detection antibody 6B4, SDS-PAGE analysis was performed. The SDS-PAGE results showed that the target nucleotide and the 6B4 antibody were successfully and efficiently coupled. The molecular weights of the coupled nucleotide and the detection antibody 6B4 were significantly increased, and no obvious free antibody residue was observed, confirming the molecular integrity of the coupling product. The coupling reaction between the nucleotide and the detection antibody 6B4 was successful and highly efficient, laying a solid foundation for subsequent immunoassays.

[0201] like Figure 2 As shown, the template, primers, and probes designed in this invention function normally. Figure 3A As shown, this invention develops a highly specific detection system. By designing specific primers and probes, binding experiments were conducted with different nucleotides, CK18 antigen, fCK18 antigen, and 8G5 antibody to evaluate its specificity and binding efficiency. First, specific primers and probes were designed, and binding experiments were performed with different nucleotides, CK18 antigen, fCK18 antigen, and 8G5 antibody. For example... Figure 3A The results showed that the designed primers and probes specifically bound only to the paired WO nucleotides, without cross-reacting with other nucleotides or antigens, demonstrating high specificity. This result provides a reliable basis for subsequent immunoassays, ensuring the specificity and sensitivity of the detection system.

[0202] Table 2 shows the oligonucleotide sequences and primers used in the early stages of the research and development of this invention, illustrated below as a comparative example:

[0203] 1. Oligonucleotide chain:

[0204] SEQ ID No. 14 (F chain), length: 87nt, 5' modification: amino (NH2) + 12C carbon chain spacer (C12), which facilitates the fixation of oligochains onto solid supports (chips, magnetic beads, microplates, etc.) by methods such as NHS-esterification and reduces steric hindrance.

[0205] SEQ ID No. 15 (R strand), length: 79 nt, unmodified, used to anneal with F strand to generate double-stranded DNA, which can be used as a qPCR standard or for amplification and detection after immobilization.

[0206] 2. Design primers

[0207] The target product is approximately 67 bp in length (the distance between the R primer binding site and the F primer binding site).

[0208] 2.1 Forward primer (SEQ ID No. 16), length: 22 nt. Position: complementary to the sequence at positions 66-87 of the F strand (corresponding to the 3' end region of the double-stranded template). GC content: ≈45%; predicted T m ≈58℃;

[0209] Design principles:

[0210] T m Between 50 and 60°C, with the reverse primer ΔT m ≤2℃; no more than 3 consecutive identical bases at the 3' end to avoid non-specific extension; check for self-dimers / tie-dimers and hairpin structures to ensure amplification efficiency.

[0211] 2.2 Reverse primer (SEQ ID No. 17); Length: 19 nt; Position: Completely complementary to positions 21-39 of the F strand (corresponding to the 5' end region of the double-stranded template). GC content: ≈58%; Predicted T m ≈60℃.

[0212] Design principles:

[0213] With forward primer T m Matching (ΔT) m ≤2℃); GC content 40-60%, which helps with specific binding; avoid 3' end complementarity to reduce dimer / hairpin.

[0214] 3. Design of the TaqMan probe (SEQ ID No. 18) sequence: 20 nt; Location: entirely between the two primers, corresponding to positions 41-60 of the double-stranded template. GC content: ≈75%; Predicted T m ≈70℃ (8-10℃ higher than primers)

[0215] Labeling: 5' end: fluorophore FAM ( / 56-FAM / ); 3' end: quencher BHQ1 ( / 3BHQ_1 / )

[0216] Design principles:

[0217] probe T m The temperature should be 8–10°C higher than the primer temperature to ensure preferential binding during the annealing stage; the first 5' base should avoid being G to reduce fluorophore self-quenching; check for internal hairpins or dimers to ensure FRET efficiency and fluorescence release; the probe and primer should not overlap to prevent interference during amplification.

[0218] 4. Results: such as Figure 3B As shown, continuous high-GC regions such as “GCCGCCCGCGTCGAAGATG…” in the nucleotide template (SEQ ID 14) are prone to forming hairpins or local structures between complementary strands during the annealing stage, interfering with primer / probe binding. Therefore, the primers and probes of the contrast sequence have poor specificity.

[0219] IV. Validation of antibody-coupled nucleotide specificity

[0220] Methods: To systematically evaluate the targeting recognition ability of antibody-nucleotide conjugates, this invention constructs a multi-dimensional cross-reactivity detection system. CK18, SARS-CoV-2S protein, cardiac troponin I, and hepatitis B core antigen (HBcAg) were selected as detection targets. The conjugate antibodies were co-incubated with the above antigens at 37°C for 30 min, followed by detection using the Tapman qPCR system.

[0221] like Figure 4 As shown, the nucleotide-conjugated detection antibody 6B4 only produced a significant amplification signal with the target CK18 protein, while the Ct values ​​for non-target antigens (SARS-CoV-2 RBD, cTnI, HBC) were all greater than 35, indicating that the conjugated antibody has high specificity for the CK18 protein. These results provide a reliable basis for subsequent immunoassays, ensuring the specificity and sensitivity of the detection system.

[0222] Example 2: Preparation of Biotin-labeled Capture Antibody Biotin-8G5

[0223] 1. Sample Preparation: The initial concentration of the sample to be labeled should be higher than 2 mg / mL. It is recommended that the protein concentration reach 2.5 mg / mL or higher for optimal results; otherwise, concentration is required before the experiment. The components (or storage buffer) of the sample to be labeled should meet the following requirements:

[0224] (1) It does not contain amino components, otherwise it will affect the coupling effect.

[0225] (2) A small amount of BSA will not affect the coupling effect, but it is recommended to use PBS as a buffer.

[0226] (3) If the sample contains substances that may interfere with labeling, it is recommended to replace the buffer with PBS.

[0227] 2. The specific method is as follows:

[0228] 2.1 Add the sample to the ultrafiltration tube, add 200-150 μL of PBS, centrifuge at 12000g, 4℃ for 10 min, and discard the filtrate; add PBS again, centrifuge at 12000g, 4℃ for 10 min; after centrifugation, remove the inner core of the ultrafiltration tube, invert it into a clean outer tube, centrifuge at 4000g, 4℃ for 2 min, and collect the sample after replacing the buffer.

[0229] 2.2 Preparation of Couplings

[0230] (1) Add 5 μL of biotin-labeled solution to the antibody 8G5 (100 μg) solution to be labeled, and mix gently with a pipette.

[0231] (2) Pipette 2.5 μL of biotin-labeled solution into the reaction solution of step (1), add deionized water to 150 μL, mix gently, and let stand at 37°C in the dark for 1 h.

[0232] (3) Add an appropriate amount of PBS to the reaction solution in step (2) (make up to a total volume of 500 μL), mix gently, transfer the solution to a purification column, and centrifuge at 4°C and 12000g for 10 min.

[0233] (4) Discard the filtrate, add an appropriate amount of PBS (to bring the total volume to 500 μL) to the purification column, and centrifuge at 4°C and 12000g for 10 min.

[0234] (5) Take out the purification column and invert it into a clean test tube. Centrifuge at 4℃ and 4000g for 2 minutes. The solution in the test tube is the coupling product.

[0235] Note: Steps (3) / (4) / (5) are purification steps.

[0236] 2.3 Storage of Couplings

[0237] The conjugate is stable for one month at 4°C in the dark. For long-term storage, please add an equal volume of glycerol, dispense into smaller containers, and store at -20°C in the dark, avoiding repeated freeze-thaw cycles.

[0238] Example 3: Development of Nuclease Composition

[0239] This invention provides a method for optimizing the effect of totipotent nucleases. The method involves using different coating solutions (BSA, salmon sperm DNA, mouse serum, goat serum) to bind to the totipotent nuclease, and then screening for the optimal nuclease composition that can significantly improve the effect of the totipotent nuclease.

[0240] This invention provides a method for efficiently removing nucleic acids by treating a 10-fold diluted CK18 antigen standard with a beyozoase, which is particularly suitable for scenarios in the biotechnology field that require precise control of the nucleic acid background.

[0241] In existing technologies, CK18 antigen standards are commonly used in immunological or molecular biology research, but residual nucleic acids may remain during their preparation, affecting the accuracy of experimental results. Totipotent nucleases (such as BeyoZonase) are widely used for nucleic acid removal; however, their effectiveness needs further improvement through optimization of experimental conditions. This invention uses a systematically designed experimental method to verify the nucleic acid removal efficacy of BeyoZonase in specific concentrations of CK18 antigen standards and compares it with a normal control group.

[0242] This invention provides a method for significantly reducing nucleic acid residue by treating 10-fold diluted CK18 antigen standards (concentration range 10–100,000 pg / mL) with BeyoZonase. The effectiveness of this method is demonstrated by comparison with a normal control group.

[0243] I. Treatment methods and results of totipotent nuclease

[0244] 1. Materials

[0245] CK18 antigen standard: initial concentration range 10-100000 pg / mL, used after 10-fold dilution.

[0246] BeyoZonase: 25 U / well.

[0247] Dilution buffer: Phosphate-buffered saline (PBS).

[0248] Nucleic acid detection reagents, fluorescent quantitative PCR reagents.

[0249] 2. Experimental group setup

[0250] Experimental group: CK18 antigen standard diluted 10 times (concentrations ranging from 1 to 10,000 pg / mL), 20 μg BeyoZonase was added to each well.

[0251] Normal control group: CK18 antigen standards diluted 10-fold (concentrations ranging from 1 to 10,000 pg / mL), without the addition of BeyoZonase, were used as a baseline for nucleic acid background levels.

[0252] 3. Experimental Procedure

[0253] 3.1 Dilution of CK18 antigen standard

[0254] Take CK18 antigen standard with an initial concentration of 10-100000 pg / mL and perform 10-fold serial dilutions with PBS to prepare dilutions with concentrations of 1, 10, 100, 1000, and 10000 pg / mL.

[0255] The diluted standard was added to each well, with a volume of 100 μL per well.

[0256] 3.2 Treatment with omnipotent nuclease

[0257] Add 25 U of BeyoZonase to each well in the experimental group and mix gently.

[0258] The normal control group did not receive BeyoZonase, but only received an equal volume of PBS as a blank treatment.

[0259] 3.2.1 Reaction conditions

[0260] The experimental and control groups were placed in a constant temperature environment of 37°C for reaction. The reaction time was set to 30 minutes, and the enzymes were washed three times with DPBST to ensure enzyme activity and reaction consistency.

[0261] 3.2.2 Nucleic acid testing

[0262] After the reaction is complete, collect the reaction products from each well.

[0263] The residual nucleic acid content was detected by real-time PCR.

[0264] 3.2.3 Real-time PCR: Using nucleic acid-specific primers and fluorescent probes, the Ct value of residual nucleic acid is measured, and the nucleic acid concentration is calculated.

[0265] 4. Experimental Results

[0266] Experimental group: After treatment with 20 μg BeyoZonase, the residual nonspecific nucleic acid content in CK18 antigen standards in the concentration range of 10–10,000 pg / mL was significantly reduced, and the results were more stable.

[0267] Normal control group: The non-specific nucleic acid level in the CK18 antigen standard without the addition of BeyoZonase remained high, with a large fluctuation range and a low Ct value, indicating that the non-specific nucleic acid was not degraded.

[0268] 5. Conclusion

[0269] like Figure 5 As shown, during the optimization of the real-time quantitative PCR detection system, the pluripotent nuclease exhibited excellent non-specific interference elimination ability under different concentrations of CK18 antigen (1-100000 pg / ml). Compared with the control group, the experimental group showed significant improvements in both system stability and non-specific interference elimination efficiency, confirming that the enzyme possesses broad-spectrum anti-interference properties.

[0270] This experiment demonstrates that adding 20 μg of BeyoZonase to each well of a 10-fold diluted CK18 antigen standard (10-100,000 pg / mL) efficiently removes nucleic acids, showing a significant advantage over the normal control group. This method is simple to operate and highly effective, providing a reliable technical solution for nucleic acid removal from CK18 antigen standards.

[0271] II. Research and Conclusions on the Combination of Different Coating Solution Combinations with Totipotent Nucleases

[0272] In the experiment, 25 U of BeyoZonase (totipotent nuclease) was added to each well to test the effect of different coating solutions on the totipotent nuclease effect. The final results showed that the combination of BSA and salmon DNA significantly improved the effect of the totipotent nuclease and achieved the best nucleic acid removal effect.

[0273] This experiment aimed to explore methods for optimizing nucleic acid removal efficiency by comparing the effects of different coating solution combinations on beyozoase. To this end, the experiment included four experimental groups and a normal control group to systematically evaluate the influence of each coating solution combination on the performance of beyozoase.

[0274] 1. Experimental setup: In the experiment, different combinations of coating solutions were used to compare with totipotent nucleases.

[0275] Group 1: A combination of BSA (bovine serum albumin) coating solution and salmon sperm DNA.

[0276] Group 2: Salmon sperm DNA was used as the coating medium only.

[0277] Group 3: Mouse serum was used as the coating solution.

[0278] Group 4: Goat serum was used as the coating solution.

[0279] Normal group: Control group without the addition of totipotent nuclease.

[0280] 2. Experimental Procedure

[0281] 2.1 Coating treatment: The coating solution of each experimental group was added to the detection well.

[0282] 2.2 Treatment with totipotent nuclease: Add 20 μg BeyoZonase to each well in groups 1 to 4.

[0283] 2.3 Reaction conditions: All experimental and control groups were reacted under the same conditions to ensure the comparability of results.

[0284] 2.4 Nucleic acid detection: After the reaction, the standard nucleic acid detection method, real-time PCR, was used to assess the effect of removing nonspecific substances.

[0285] 3. Experimental Results

[0286] Experimental results show that different coating solutions have significantly different effects on nucleic acid removal by totipotent nucleases:

[0287] Group 1 (BSA + salmon DNA): This combination significantly enhances the nucleic acid degradation ability of the totipotent nuclease, resulting in the lowest residual nucleic acid content and achieving the best removal effect.

[0288] Group 2 (Salmon DNA): The nucleic acid removal effect was less than that of Group 1, showing moderate performance.

[0289] Group 3 (mouse serum) and Group 4 (goat serum): The nucleic acid removal effect was significantly worse than that of Group 1, and the degradation efficiency was lower.

[0290] 4. Conclusion

[0291] Experiments have shown that the combination of BSA and salmon sperm DNA coating solution with BeyoZonase yields the best results, superior to other single or combined coating solutions. This discovery provides a new approach for optimizing nuclease processing, possessing significant application value and patent protection potential.

[0292] The results showed that the optimal combination of adding 2% BSA, 0.1 μL of totipotent nuclease (250 U / μL), and 2.5 μL of salmon sperm DNA (10 mg / ml) per well was significantly better than other coating solutions in removing nucleic acid contamination. This combination effectively enhanced the role of totipotent nuclease in the experiment, reduced nucleic acid interference, and improved the accuracy and reliability of the experiment. Figure 5 BeyoZonase demonstrates excellent effectiveness in eliminating nonspecificity. Figure 6 The results showed that BSA + salmon sperm DNA exhibited the best blocking effect. The two-component blocking system (5% BSA + 0.1 mg / mL salmon sperm DNA) had significant advantages over single blocking agents (BSA, salmon sperm DNA) or serum-based blocking agents (mouse serum, goat serum). qPCR data showed that the BSA-DNA composite blocking system not only achieved the lowest CT value but also had the largest CT difference compared to the blank control group, a statistically significant difference (p < 0.001). These results indicate that BSA and salmon sperm DNA can effectively reduce non-specific adsorption through a synergistic mechanism of steric hindrance and charge neutralization. This finding provides a new optimization strategy for the selection of blocking agents in multi-component detection systems.

[0293] Example 4: Based on the oligonucleotide-coupled detection antibody, biotin-labeled capture antibody, and optimization provided by this invention. Optimization of the method for immunoPCR detection of CK18 expression using nuclease composition.

[0294] I. Optimization of the testing system

[0295] This invention relates to a method for optimizing a dual-antibody detection system, specifically by systematically determining the optimal concentration ratio of capture antibody and detection antibody through checkerboard titration combined with quantitative real-time PCR verification.

[0296] Dual-antibody detection systems are widely used in biotechnology and clinical diagnostics, and their detection sensitivity and specificity are highly dependent on the selection of antibody concentrations. However, traditional optimization methods often rely on empirical adjustments, making it difficult to comprehensively assess the interaction between the concentrations of capture and detection antibodies, thus limiting optimization efficiency and accuracy. This invention proposes a systematic optimization strategy based on checkerboard titration and validation using quantitative real-time PCR to overcome the shortcomings of existing technologies.

[0297] This invention provides a method for optimizing a dual-antibody detection system, specifically including the following steps:

[0298] 1. Design of checkerboard titration method:

[0299] Antibody concentration gradients were detected: 100ng, 10ng, 1ng, 0.1ng.

[0300] Capture antibody concentration gradient: 400ng, 200ng, 100ng, 50ng.

[0301] By using a checkerboard arrangement, 16 antibody concentration combinations of 4×4 were constructed, covering a multidimensional parameter space.

[0302] 2. Detection signal acquisition: Perform double antibody detection experiments for each concentration combination and record the corresponding detection signals.

[0303] 3. Validation by quantitative real-time PCR: Quantitative real-time PCR technology was used to quantitatively evaluate the detection effect of each concentration combination and obtain objective data.

[0304] 4. Determination of the optimal ratio: Based on a comprehensive analysis of the results of quantitative real-time PCR, the optimal combination of capture antibody and detection antibody concentrations with both high sensitivity and specificity was selected.

[0305] 5. Technological advantages

[0306] Systematic: The checkerboard titration method comprehensively explores antibody concentration combinations, avoiding the omission of key ratios.

[0307] Accuracy: Quantitative real-time PCR provides highly sensitive quantitative verification, ensuring the scientific validity of optimal ratios.

[0308] High efficiency: The optimal ratio can be quickly determined through limited experiments, significantly shortening the optimization cycle. 6. Specific Implementation Examples

[0310] In the specific implementation, the detection antibody concentrations were set to 100ng, 10ng, 1ng, and 0.1ng, and the capture antibody concentrations were set to 400ng, 200ng, 100ng, and 50ng, resulting in 16 concentration combinations. After performing the dual-antibody detection experiment and acquiring the signals, quantitative analysis was performed using real-time PCR. Figure 7 As shown, this invention systematically optimizes a dual-antibody detection system based on a checkerboard titration method, determining the optimal combination through three-dimensional parameter space modeling (antibody concentration gradient: 0.01-400 ng / well). As illustrated, the system exhibits optimal detection efficiency when the capture antibody (8G5 clone) coating concentration is 100 ng / well and the detection antibody (6B4 clone) working concentration is 0.1 ng / well. Quantitative analysis shows that the cycle threshold (CT) of the blank control group is significantly higher than 32.1 under this combination, while the CT value of the positive control group remains stable at 25.6, with a CT difference of 6.5 cycles (p < 0.001). Cost-benefit analysis reveals that compared to the suboptimal combination (400 ng / well capture antibody + 0.1 ng / well detection antibody), the unit detection cost of the optimized system is reduced by 75%.

[0311] II. Probe Design and Enzyme Treatment Optimization

[0312] 1. Design a fluorescent probe with BHQ labeled at the 3' end of the primer.

[0313] 2. Divide the probes into three groups:

[0314] Untreated group (control group);

[0315] DNA enzyme treatment group;

[0316] Totipotent nuclease treatment group.

[0317] 3. DNase treatment: Use DNase and react at 37°C for 30 minutes.

[0318] 4. Totipotent nuclease treatment: Use a totipotent nuclease and react at 37°C for 30 minutes.

[0319] 5. Fluorescence signal detection and analysis

[0320] 5.1 The fluorescence signal intensity of each probe group was detected using a single-molecule diagnostic platform.

[0321] 5.2 Data processing: The fluorescence signal was quantified by quantitative real-time PCR, and the average fluorescence intensity of each group was calculated.

[0322] 5.3 Statistical analysis: The t-test was used to compare the differences in fluorescence signals between the enzyme-treated group and the untreated group. The results showed that p < 0.0001, indicating that the difference was significant.

[0323] Experimental results showed that the fluorescence signal in the enzyme-treated group was significantly higher than that in the untreated group, further verifying the excellent performance of the BHQ probe in quenching non-specific signals.

[0324] 6. Conclusion

[0325] like Figure 8 As shown, the quenching efficiency of the BHQ 1 probe was evaluated by treating the probes designed in the table with DNase and totipotent nuclease, followed by detection of fluorescence signal intensity using a single-molecule diagnostic platform. Experimental results showed that the fluorescence signal of the DNase- and totipotent nuclease-treated groups was significantly enhanced compared to the untreated group (p < 0.0001). This significant difference confirms that the BHQ 3' end-labeled probe has a good quenching effect, effectively eliminating non-specific signal interference and ensuring experimental accuracy. This method is highly innovative and practical, providing a scientific basis for probe optimization in molecular biology detection.

[0326] Example 5: Optimization of serum addition concentration and PCR amplification cycle number

[0327] 1. Experimental Objective: The objective of this experiment is to determine the optimal combination of serum concentration and PCR amplification cycle number to improve the effectiveness of PCR experiments. This invention provides a method for optimizing PCR experimental conditions, ultimately determining 3.125 μL of serum per well and 20 PCR cycles as the optimal experimental conditions.

[0328] 2. Experimental Methods: In this experiment, we tested the effect of different combinations of serum concentration and PCR cycle number on the PCR amplification effect. Specific experimental groups included:

[0329] 2.1 Serum Concentration Series: 1.5625 μL, 3.125 μL, 6.25 μL, 12 μL, and 25 μL of serum were added to each well, respectively. Normal Group: Control group without serum.

[0330] 2.2 PCR cycle number series: 5, 10, 15 and 20 PCR cycles were tested in the experiment.

[0331] In each experimental group, we adjusted the serum concentration and PCR cycle number separately, and determined the optimal experimental conditions by observing the quantity, specificity and amplification efficiency of the PCR products.

[0332] 3. Experimental Results:

[0333] like Figure 10 As shown, the experimental results indicate that the optimal dilution was determined by performing a 2-fold serial dilution of serum samples (dilution range from 25 μL to 1.5625 μL) in this invention. The results show that a 3.125 μL serum sample exhibited the best performance in the detection.

[0334] like Figure 11 As shown, this invention systematically evaluated the impact of PCR cycle number on detection performance using a single-molecule detection platform, and ultimately determined 20 PCR cycles as the optimal condition. Under this condition, PCR amplification is optimal, fully utilizing serum components while ensuring efficient PCR reaction, avoiding non-specific amplification or PCR inhibition caused by excessive cycle number.

[0335] Example 6: Single-molecule detection method based on immunoPCR provided by the present invention

[0336] 1. Materials

[0337] Primary antibody buffer: PBS + 1% BSA; Secondary antibody buffer: DPBS + 1% BSA; Wash buffer: DPBST; Premix Ex Taq TM (Probe qPCR): Takara,rr390; BeyoZonase TMSuper nuclease (≥99%, with His-tag): Beyotime, D7126-25KU; Dynabeads TM MyOne TM Streptavidin T1: Invitrogen TM , 65601.

[0338] 2. Experimental Procedure

[0339] 2.1 Binding of 100 ng / well 8G5-Biotin (100 μl) + different concentrations of CK18 antigen (100 μL);

[0340] 2.2 Add 1 μl of streptavidin magnetic beads, 0.1 μl of 2% BSA, 0.1 μl of BeyoZonase (250 U / μL), and 2.5 μl of salmon sperm DNA (10 mg / ml) to each well and incubate at 37°C for 30 minutes.

[0341] 2.3 Washing procedure: Wash 3 times with DPBST (0.05% Tween-20in PBS); inject 300μL of pre-cooled (4℃) washing solution into each well, use a magnetic separator for 2 minutes, pour off the supernatant and repeat the operation, and air dry at room temperature for 5 minutes after the last wash.

[0342] 2.4 Add 0.1 ng / well (100 μl) of oligonucleotide-conjugated 6B4 detection antibody and incubate at 37°C for 45 minutes;

[0343] 2.5 Washing procedure: Five washes were performed using DPBST buffer (0.05% Tween-20in PBS): 300 μL of pre-cooled (4°C) washing buffer was injected into each well, the mixture was magnetically separated for 2 minutes, the supernatant was poured off and the process was repeated, and the mixture was air-dried at room temperature for 5 minutes after the last wash.

[0344] 2.6 Prepare the PCR mix, see Table 4;

[0345] Table 4

[0346]

[0347] 2.7 Add 20 μL of mix to each well. Add the corresponding reagents to 1.5 mL EP tubes according to the table above to prepare a total volume of 20 μL of mixture. Add this mixture to a 96-well plate, with three replicates per sample. Cover with the 96-well plate PCR amplification membrane and centrifuge at 3000 rpm for 5 min. Perform amplification on a PCR instrument using the following program: Stage 1 pre-denaturation: 95℃, 30 s; Stage 2 cycling reaction: 95℃, 5 s; 60℃, 34 s; 20 cycles; Stage 3: Store at 12℃.

[0348] 2.10 Add 20 μL to each well of the 384 reading plate, gently mix the PCR amplified product using an eight-slot mixture, and add 2 μL to each well. Signal detection: Read the signal values ​​using the SMCXPRO single-molecule diagnostic platform.

[0349] Method and steps:

[0350] (1) Fix the board base to SMCxPRO TM Add 20 μL of PBS to each well at the bottom of the reading plate;

[0351] (2) Place the PCR plate on a spherical Mag plate for 2 minutes to allow the beads to accumulate on one side of the well;

[0352] (3) Use a pipette to transfer 2 μL of the 384-well PCR plate to each well. Ensure that each well has beads on its side;

[0353] (4) The cover plate 2 is sealed with an aluminum glue plate sealing machine to ensure good adhesion between the cover plate and the plate and prevent leakage and cross-contamination;

[0354] (5) Place tablet 2 (on the tablet stand) into Jitterbug. TM In a microplate culture shaker, set to 7 shakes for 1 minute (1500 rpm);

[0355] (6) Centrifuge at 1100×g for 1 minute.

[0356] (7) On the machine: Set up the program: In the SMC software interface, select the new 384 name; Enter EXperiaiment in Create → Unknow: Each group consists of three wells, named according to the corresponding sample loading order; Click Eject, put the 384 board into the slot → Run → Start the program to start running; After the program ends, take out the 384 board;

[0357] like Figure 9 As shown, using CK18 antigen as a standard, this invention constructed standard curves with concentrations ranging from 0.01 pg / ml to 1,000,000 pg / ml, covering eight logarithmic levels. To ensure the accuracy of data analysis, a four-parameter logarithmic (4PL) model was used for statistical fitting, resulting in the following fitting equation: Y = 479.9 + 18799 / 1 + (x / 8639) -0.2805 Goodness of fit R 2 The accuracy reached 0.9992, demonstrating extremely high fitting precision, with a detection sensitivity as low as 10 fg / ml.

[0358] Example 7: Detection of CK18 levels and their content in ACLF patients using the immunoPCR-single-molecule detection method provided by this invention. Relationship with prognosis

[0359] This invention uses an immunoPCR-single-molecule detection method to detect CK18 levels in the serum of ACLF patients. Through comprehensive analysis of serum levels and other blood indicators from patients with different ACLF grades (grades 1, 2, and 3), the relationship between CK18 and ACLF patient prognosis is revealed. The study found that the concentration of CK18 in ACLF patients is closely related to patient prognosis, and CK18 is an independent predictor of poor prognosis in ACLF patients.

[0360] 1. Experimental Methods:

[0361] 1.1 Study subjects: Patients meeting the diagnostic criteria for ACLF were selected. ACLF patients admitted to Beijing You'an Hospital, affiliated with Capital Medical University, in 2022-2023, who experienced one or more of the following acute events in acute decompensation: ascites, hepatic encephalopathy, gastrointestinal bleeding, or bacterial infection (such as spontaneous bacterial peritonitis). Organ Failure: The function of the following six organ systems was assessed according to the CLIF-SOFA score to determine the presence of organ failure: Liver: bilirubin ≥12 mg / dL; Kidney: creatinine ≥2 mg / dL, or requiring renal replacement therapy; Brain: Grade III-IV hepatic encephalopathy; Coagulation: International Normalized Ratio (INR) ≥2.5; Circulation: Mean Arterial Pressure <70 mmHg, or requiring vasoactive drug support; Respiration: PaO2 / FiO2 ≤200, or SpO2 / FiO2 ≤214; ACLF Classification: Classified according to the number and type of organ failure: Grade 1: Single kidney failure, or single non-renal organ failure with renal insufficiency (creatinine 1.5–1.9 mg / dL) and / or mild cerebral dysfunction (Grade I-II hepatic encephalopathy). Grade 2: Two organ failures. Grade 3: Three or more organ failures. Patients were clinically classified into ACLF Grade 1, 2, and 3 groups and compared with a normal healthy control group.

[0362] 1.2 Sample collection: Serum samples were collected from each group of patients, and other blood indicators (such as liver function, kidney function, white blood cell count, C-reactive protein, etc.) were tested.

[0363] 1.3 CK18 Detection: The CK18 content in the serum of each group was quantitatively analyzed using the immunoPCR-single-molecule detection kit provided in this invention. The experimental procedure was the same as in Example 6.

[0364] 1.4 Data Analysis: By analyzing the correlation between CK18 levels in each group and other clinical indicators, and combining this with the patients' clinical prognostic data, the independence of CK18 in predicting the prognosis of ACLF patients was evaluated.

[0365] 2. Experimental Results:

[0366] like Figure 12As shown in the table below, serum CK18 levels in patients with different grades of ACLF were measured using an immunoPCR-single-molecule detection method. ACLF patients were divided into three pathological stages based on the CLIF-C Organ Failure Score (CLIF-C OF). The results show that the CK18 level in the ACLF grade 3 group was significantly higher than that in the ACLF grade 1 and 2 groups. With the progression of the disease, serum CK18 levels showed a significant increasing trend, reflecting the gradual worsening of liver damage. This finding not only confirms the sensitivity of CK18 as a biomarker of liver injury but also reveals the close relationship between its level changes and the pathological progression of ACLF. Further statistical analysis showed that CK18 can serve as an independent predictor of poor prognosis in ACLF patients, and its concentration level is closely related to patient survival and liver function recovery.

[0367] Table 5 shows that CK18 can serve as an independent predictive biomarker for poor prognosis in ACLF patients.

[0368]

[0369]

[0370] Example 8: Comparison of the performance evaluation of the present invention with the MELD score performance evaluation

[0371] I. MELD Scoring and Performance Evaluation

[0372] 1. Data Acquisition:

[0373] Collect patients' serum creatinine (Cr, unit: mg / dL), total bilirubin (TBIL), international normalized ratio (INR), and etiological information.

[0374] 2. Scoring Calculation:

[0375] MELD score = 9.6 × ln(Cr) + 3.8 × ln(TBIL) + 11.2 ×

[0376] ln(INR) + 6.4 × (etiology), where Cr is in mg / dL

[104] .

[0377] 3. Performance Analysis:

[0378] Receiver operating characteristic (ROC) analysis showed that the AUC of the MELD model was 0.60 (95% CI: 0.44–0.76).

[0379] The accuracy was 0.63 (95% CI: 0.49–0.76), the sensitivity was 0.79 (95% CI: 0.63–0.94), and the specificity was 0.46 (95% CI: 0.27–0.65).

[0380] PPV was 0.61 (95% CI: 0.45-0.77), and NPV was 0.67 (95% CI: 0.45-0.88).

[0381] II. Construction and Performance Evaluation of the CK18 Model Provided by This Invention

[0382] 1. CK18 testing:

[0383] The level of CK18 in the patient's serum was determined by single-molecule detection combined with antibody-coupled DNA immunoPCR.

[0384] 2. Model Construction:

[0385] Performance analysis of the prognostic assessment model based on CK18 levels: The AUC of the CK18 model was 0.95 (95% CI: 0.89–1.00). Accuracy was 0.91 (95% CI: 0.82–0.97), sensitivity was 0.89 (95% CI: 0.78–0.99), and specificity was 0.94 (95% CI: 0.86–1.00). PPV was 0.94 (95% CI: 0.86–1.00), and NPV was 0.89 (95% CI: 0.79–0.99).

[0386] The CK18 model outperforms the MELD score in predictive performance, such as... Figure 13 As shown, the MELD model had an AUC of 0.60 (95% CI: 0.44–0.76), indicating relatively limited predictive power. The model's accuracy was 0.63 (95% CI: 0.49–0.76), sensitivity was 0.79 (95% CI: 0.63–0.94), and specificity was 0.46 (95% CI: 0.27–0.65). PPV and NPV were 0.61 (95% CI: 0.45–0.77) and 0.67 (95% CI: 0.45–0.88), respectively. While the MELD model demonstrated good sensitivity and the ability to identify positive cases, its low specificity could lead to a high false-positive rate, limiting its overall reliability.

[0387] like Figure 14As shown, the CK18 model of this invention exhibits excellent predictive performance, with an area under the curve (AUC) of 0.95 (95% CI: 0.89–1.00), demonstrating extremely high discriminative ability. The overall accuracy of the model is 0.91 (95% CI: 0.82–0.97), sensitivity is 0.89 (95% CI: 0.78–0.99), and specificity is 0.94 (95% CI: 0.86–1.00). The positive predictive value (PPV) and negative predictive value (NPV) are 0.94 (95% CI: 0.86–1.00) and 0.89 (95% CI: 0.79–0.99), respectively. These indicators demonstrate that the CK18 model can accurately identify positive cases while effectively excluding negative cases, showing significant clinical application value.

[0388] 5. Conclusion

[0389] This invention, through a systematic comparison of the efficacy of the MELD score and the CK18 model, demonstrates that the CK18 model has a significant advantage in predicting the prognosis of liver diseases, with its predictive efficacy far exceeding that of the traditional MELD score. This method provides clinicians with a more accurate and reliable prognostic assessment tool and has broad application prospects.

Claims

1. An ACLF immune PCR-single molecule detection reagent, characterized in that include: a) Biotin-labeled capture antibody 8G5; b) Oligonucleotide-conjugated detection antibody 6B4-Oligo; c) Streptavidin magnetic beads; d) Nuclease composition; e) Specific primer pairs, probe 1, and probe 2; The oligonucleotide-conjugated detection antibody 6B4-Oligo is an detection antibody 6B4 conjugated with the oligonucleotide shown in SEQ ID No. 1; The specific primer pairs are shown in SEQ ID No. 2 and SEQ ID No. 3; The probe 1 is shown in SEQ ID No.

4. The 5' end of the probe 1 is connected to a fluorescent group, and the 3' end is connected to a quenching group BHQ_1. The probe 2 is shown in SEQ ID No.

5. The 5' end of the probe 2 is connected to a fluorescent group, and the 3' end is connected to a quenching group BHQ_2. The nuclease composition consists of a totipotent nuclease, 2% BSA coating solution, and salmon sperm DNA. The detection antibody 6B4 has a heavy chain amino acid sequence as shown in SEQ ID No. 7 and a light chain amino acid sequence as shown in SEQ ID No. 9; the capture antibody 8G5 has a heavy chain amino acid sequence as shown in SEQ ID No. 11 and a light chain amino acid sequence as shown in SEQ ID No.

13. 2.The ACLF immunization PCR-single molecule detection reagent of claim 1, wherein The molar ratio of antibody 6B4 to oligonucleotide is 3:

1.

3. The use of the ACLF immunoPCR-single-molecule detection reagent according to claim 1 or 2 in the preparation of a kit for detecting CK18 content as a prognostic prediction for patients with acute-on-chronic liver failure.

4. A kit for detecting the content of CK18 as a prognostic test for patients with acute-on-chronic liver failure, characterized by Includes the detection reagent as described in claim 1 or 2.

5. A prognosis evaluation system for patients with acute-on-chronic liver failure based on the method for detecting the content of CK18 by using the kit according to claim 4, characterized in that include: a) CK18 concentration detection module, used to perform immunoPCR-single molecule detection; b) Data analysis module, which performs correlation analysis between CK18 concentration and chronic-on-acute liver failure grading and liver function indicators; c) Prognostic prediction module, which outputs the patient's adverse prognostic risk assessment results and individualized treatment recommendations; The method for detecting CK18 content using the kit described in claim 4 includes the following steps: a) Bind the biotin-labeled capture antibody 8G5 to the CK18 antigen in the serum sample to be tested; b) Add streptavidin magnetic beads and a nuclease composition to enrich antigen-antibody complexes with magnetic beads and eliminate non-specific nucleic acid interference with the nuclease composition. c) Add the oligonucleotide-conjugated detection antibody 6B4-Oligo to form a complete immune complex; d) PCR amplification was performed using specific primer pairs and probes to amplify oligonucleotide sequences conjugated with detection antibodies; e) The amplification products were quantitatively analyzed using single-molecule detection technology to determine the concentration of CK18.

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