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

Abstract

The invention discloses an ACLF immune PCR single molecule detection kit, a detection method and application. The ACLF immune PCR single molecule detection kit comprises the following steps: a) a biotin-labeled capture antibody 8G5; b) a detection antibody 6B4-Oligo which is coupled with oligonucleotide; c) streptavidin magnetic beads; d) a nuclease composition; and e) a specific primer and a probe. According to the invention, an immune PCR-single molecule detection method is adopted, a specially designed oligonucleotide coupling detection antibody is utilized, a nuclease composition is optimized, and PCR reaction conditions are optimized, so that high-sensitivity quantitative detection can be carried out under extremely low sample concentration, background interference in a traditional detection method is avoided, and the detection sensitivity is improved. The specificity and the accuracy of the signal are improved, and a more accurate diagnostic tool is provided for clinic. Compared with the traditional PCR (polymerase chain reaction) and ELISA (enzyme-linked immuno sorbent assay) methods, the kit disclosed by the invention 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] The present invention relates to a kit and method for detecting the level of hepatocyte keratin 18 (CK18) in the serum of patients with acute-on-chronic liver failure (ACLF) based on immuno-PCR single-molecule detection technology, as well as its application in assessing the prognosis of ACLF patients. The present invention further relates to related kits, antibody coupling optimization methods, nucleic acid interference removal technology, and the construction of a prognostic assessment system, belonging to the field of biomedical testing technology. Background Art

[0002] Acute-on-chronic liver failure (ACLF) is characterized by an acute deterioration of chronic liver disease, accompanied by multi-organ failure and a high mortality rate. Common triggers for ACLF include: infection (e.g., spontaneous bacterial peritonitis, sepsis); metabolic decompensation (e.g., diabetic ketoacidosis); cardiovascular events (e.g., ischemic liver injury); drugs / toxins (e.g., acetaminophen overdose); gastrointestinal bleeding or surgical trauma. Patients often present with: jaundice, coagulopathy, ascites, hepatic encephalopathy; and multi-organ failure (renal, circulatory, respiratory). ACLF criteria: Based on the criteria of international societies (e.g., EASL, APASL), cirrhosis is usually required, but it can also occur in patients without cirrhosis. The mortality rate of ACLF is as high as 50% to 90%. Early identification of triggers and active supportive care can improve outcomes.

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

[0004] The clinical application of CK18 faces the following technical bottlenecks: Low-abundance detection: 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: Methods such as ELISA have a narrow linear range, making it difficult to accurately quantify high CK18 concentrations in critically ill patients (such as changes in the M65 / M30 ratio caused by necrosis). Currently, CK18 detection 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 of approximately 10-100 pg / mL) and is easily interfered with by serum matrix effects; Chemiluminescent immunoassay (CLIA): It improves sensitivity by amplifying chemiluminescent signals, but it still cannot meet the needs of accurate detection of extremely low-concentration samples; Flow cytometry combined with fluorescent probes: It can achieve single-cell level analysis, but requires complex pre-processing steps (such as cell separation) and is not suitable for large-scale clinical sample screening. Summary of the Invention

[0005] The primary technical problem to be solved by the present invention is to provide an ACLF immune PCR-single molecule detection reagent.

[0006] Another technical problem to be solved by the present invention is to provide a new use of the above-mentioned detection reagent in the preparation of a kit for detecting CK18 content as a prognosis prediction kit for 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 detection reagent.

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

[0009] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

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

[0011] a) Biotinylated capture antibody 8G5;

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

[0013] c) Streptavidin magnetic beads;

[0014] d) a nuclease composition;

[0015] e) specific primers and probes;

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

[0017] The oligonucleotide-coupled detection antibody 6B4-Oligo is a detection antibody 6B4 coupled with an oligonucleotide shown in SEQ ID No. 1, 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, wherein 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, wherein the 5' end of the probe 2 is connected to a fluorescent group, and the 3' end of the probe 2 is connected to a quenching group BHQ_2;

[0021] The above-mentioned immune PCR-single molecule detection reagent is used in the preparation of a kit for detecting CK18 content as a prognosis prediction kit for patients with acute-on-chronic liver failure.

[0022] A kit for detecting CK18 content as a prognosis prediction kit for patients with acute-on-chronic liver failure comprises the above-mentioned detection reagent.

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

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

[0025] b) adding streptavidin magnetic beads and a nuclease composition, enriching and capturing the antigen-antibody complex by the magnetic beads, and eliminating nonspecific nucleic acid interference by the nuclease composition;

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

[0027] d) performing PCR amplification using specific primers and probes to amplify the oligonucleotide sequence coupled to the detection antibody;

[0028] e) quantitatively analyzing the amplified product using single-molecule detection technology to determine the concentration of CK 18;

[0029] Preferably, the method for preparing the oligonucleotide-coupled detection antibody comprises:

[0030] The antibody and oligonucleotide were coupled via an activation reagent, wherein the oligonucleotide sequence was as shown in SEQ ID No. 1, and the molar ratio of the antibody 6B4 to the oligonucleotide was 3:1.

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

[0032] Preferably, the PCR amplification conditions are: adding 3.125 μL of serum to each well, and using a 20-cycle amplification program, 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 comprises the following steps:

[0034] a) enriching the PCR products using magnetic beads and separating them by centrifugation;

[0035] b) using a reader plate for single-molecule signal capture and combining it with a microplate shaker for uniform mixing;

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

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

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

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

[0040] c) Prognosis prediction module, which outputs the patient's poor prognosis risk assessment results and individualized treatment recommendations.

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

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

[0043] (1) The present invention adopts an immuno-PCR-single molecule detection method, utilizing specially designed oligonucleotide-coupled detection antibodies, optimized nuclease compositions, and optimized PCR reaction conditions. This method enables highly sensitive quantitative detection at extremely low sample concentrations, avoiding background interference in traditional detection methods, improving signal specificity and accuracy, and providing a more accurate diagnostic tool for clinical use. This technology has higher sensitivity than traditional PCR and ELISA methods, can accurately detect low concentrations of CK18 in the serum of ACLF patients, and provides an effective tool for prognostic assessment.

[0044] (2) The present invention adopts a unique method for coupling antibodies and oligonucleotides, and the purity is improved: the purity of the oligonucleotide is significantly improved through HPLC purification, which effectively reduces the interference of impurities on subsequent coupling reactions. Efficiency optimization: 5' terminal amino 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 performance of Ab-oligo conjugates. The use of optimized coating solutions (such as BSA, salmon sperm DNA, mouse serum, etc.) to improve the efficiency of affinity reactions significantly improves the sensitivity and specificity of immune PCR reactions. In particular, the combined effect of BSA + salmon sperm DNA is outstanding in removing nucleic acid contamination and improving enzyme activity, overcoming the problems of nucleic acid interference and non-specific binding in traditional methods.

[0045] (3) The present invention combines a universal nuclease with different coating solutions to form a nuclease composition during the immuno-PCR process, 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 optimization scheme provides a guarantee for efficient, low-background single-molecule detection.

[0046] (4) The present invention determined the optimal experimental conditions (3.125 μL serum per well, 20 PCR cycles) by optimizing the number of PCR amplification cycles and the serum addition concentration, thereby significantly improving the efficiency and specificity of the PCR reaction, reducing nonspecific amplification and PCR inhibition, and further improving the reliability of the test results.

[0047] (5) This invention innovatively combines immuno-PCR with single-molecule detection technology with the detection of CK18 levels in the serum of ACLF patients, providing a new biomarker for the clinical grading and prognostic assessment of ACLF. By comparing CK18 concentrations in patients with different grades (ACLF grade 1, 2, and 3), it was found that CK18 is an independent predictor of poor prognosis, which can provide clinicians with accurate prognostic information and help formulate individualized treatment plans. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a diagram showing the molecular weight of oligonucleotide-coupled detection antibodies in the examples of the present invention;

[0049] Figure 2 This is a diagram verifying the normal operation of the template, primers, and probes in an embodiment of the present invention;

[0050] Figure 3A For the embodiment of the present invention, the primer probe and the paired WO specific map are designed;

[0051] Figure 3B is a graph comparing primer-probe specificity;

[0052] Figure 4 This is a diagram for verifying the specificity of the detection antibody coupled to the oligonucleotide in an embodiment of the present invention;

[0053] Figure 5 This is a diagram showing the verification of the elimination of nonspecific nucleases in the examples of the present invention;

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

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

[0056] Figure 8 This is a diagram evaluating the quenching efficiency 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 showing a study of serum sample dilution concentrations in an embodiment of the present invention;

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

[0060] Figure 12 This is a validation diagram for detecting serum from ACLF patients using the present invention;

[0061] Figure 13 To compare the effectiveness of MELD model in predicting patient prognosis;

[0062] Figure 14 This is a sensitivity-specificity diagram of the CK18 model in the examples of the present invention. DETAILED DESCRIPTION

[0063] The present invention is further described below with reference to specific examples. These examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. Experimental methods in the following examples, where specific conditions are not specified, are generally performed under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred embodiments and materials described herein are for exemplary purposes only.

[0064] The present invention integrates molecular biology, nanotechnology and clinical medicine, and for the first time applies the antibody-linked oligonucleotide immuno-PCR-single molecule detection method to the prognosis assessment of ACLF (acute-on-chronic liver failure). By detecting antibody-coupled oligonucleotides, optimizing the universal nuclease in immuno-PCR to effectively remove non-specific interfering nucleic acids, and optimizing PCR amplification conditions based on the detection system, accurate detection of CK 18 concentrations in serum samples is achieved, thereby achieving prognosis prediction for ACLF patients associated with metabolic-associated fatty liver disease (MASLD). This technology solves the problems of insufficient sensitivity and weak dynamic monitoring capabilities of existing detection methods. It can also reveal the molecular mechanism of ACLF through multi-marker joint testing, providing the clinic with a key basis for early warning, precise stratification and individualized treatment, and has significant clinical application value and market prospects. Specific explanations are as follows:

[0065] Example 1 Design and efficacy verification of the oligonucleotide-coupled detection antibody provided by the present invention 1. Template, oligonucleotide, specific primer and probe sequences that have been verified to work in this example

[0066] Table 1 WO primer probe sequences

[0067]

[0068] Table 2 Comparative Example PO primer 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] 1. Oligonucleotide design plan:

[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 nucleotide sequence provided:

[0130] SEQ ID No.1

[0131] GGGCGACGATAATTGATATTGGTGGGCAGTCTACACGGCCTGGTTCACATGTTGTTTCTATAGAGGAAGAGATTTCTC

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

[0133] 2. Primer Design

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

[0135] 2.1 Forward primer design

[0136] SEQ ID No. 2 sequence: ATCTCTTCCTCTATAGAAACAACAT

[0137] Length: 25 bases;

[0138] Position: This primer is partially complementary to the 3' end region of the target sequence (positions 53 to 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°C (the specific calculation is required 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 conventional design range;

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

[0146] Design principles:

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

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

[0149] (3) Ensure specificity and avoid nonspecific amplification.

[0150] 3. Design of TaqMan Probes

[0151] TaqMan probes are used in real-time PCR to detect the amplification of target sequences through fluorescence signals. The following factors should be considered when designing probes:

[0152] 3.1 Probe sequence

[0153] Sequence: TGGGCAGTCTACACG

[0154] Length: 15 bases, for effective binding;

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

[0156] Design principles:

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

[0158] (2) Highly specific binding to the target sequence, avoiding nonspecific signals.

[0159] 3.2 Fluorophores and Quenchers

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

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

[0162] (2) 3' end 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' end 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 fluorescent signal is released for real-time detection.

[0167] Oligonucleotide coupling kit components: (ab218260 oligonucleotide coupling kit, Expedeon, a subsidiary of Abcam)

[0168] 2. Oligonucleotide-coupled detection antibodies

[0169] 1. Oligonucleotide Activation

[0170] Add 100 μL of 100 μM oligonucleotide to the Oligonucleotide Activation Reagent vial. Mix gently and incubate at room temperature for 30 minutes. During this incubation, proceed to Step 2.

[0171] 2. Antibody Activation

[0172] Add 100 μL of antibody (1 mg / mL) to the Antibody Activation Reagent vial. Mix gently and incubate at room temperature for 30 minutes. During this incubation, proceed to step 3.

[0173] 3. Desalting of Activation Reagents

[0174] 3.1 Vertically secure each separation column. First, open the upper cap. Then, open the lower cap and allow the storage liquid to flow through the separation column. Discard the liquid flowing out of the separation column.

[0175] 3.2 Add 3 mL of wash buffer to the top of the separation column and allow the liquid to flow out under gravity to balance each separation column. Discard the outflowing liquid. Repeat the above steps four times.

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

[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. Wait until the liquid is completely absorbed before proceeding to the next step. Collect the outflowing liquid and let it stand until you confirm successful coupling.

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

[0179] 3.6 Collect the eluate from the bottom of the separation column. This eluate (300 μL) contains the activated oligonucleotide or antibody and is ready for coupling.

[0180] 4. Preparation of Purified Oligonucleotide-Conjugated Antibodies

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

[0182] 4.1 Add 300 μL of activated antibody to the appropriate amount of activated oligonucleotide and wash buffer as shown in the table below.

[0183] Table 3

[0184]

[0185] Note: The ratio of antibody to oligonucleotide is an average, as a large number of labeled antibodies will be 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 antibody is now ready for use. If necessary, the conjugated antibody can be purified to remove any unconjugated oligonucleotide (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 visualize the precipitate, at least 50 μg of antibody should be used.

[0191] 5.1 Place the test tube containing the purification reagent in warm water (no higher than 40°C) and heat for 10 minutes and mix thoroughly. If the sample is not completely dissolved, place the sample in a benchtop microcentrifuge and vortex at the maximum recommended speed of 13,000 × g for 1 minute. Remove the supernatant and set aside.

[0192] 5.2 Add an equal volume of purification reagent to the antibody / oligonucleotide mixture, mix thoroughly, 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 15,000 × g for 5 minutes in a tabletop microcentrifuge.

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

[0195] 5.5 Carefully remove the supernatant and let it stand until effective precipitation is confirmed.

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

[0197] 5.7 To remove as much free oligonucleotide as possible, a second purification should be performed on the same conjugated antibody.

[0198] 5.8 Oligonucleotide-conjugated antibodies are now available.

[0199] 3. Verification of oligonucleotide design results

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

[0201] like Figure 2 As shown in FIG, the template, primer and probe designed by the present invention can work normally. Figure 3A As shown, the present invention has developed a highly specific detection system. By designing specific primers and probes, binding experiments were conducted with different nucleotides, CK18 antigens, fCK18 antigens, and 8G5 antibodies to evaluate its specificity and binding efficiency. First, specific primers and probes were designed, and binding experiments were conducted with different nucleotides, CK18 antigens, fCK18 antigens, and 8G5 antibodies. Figure 3A The results showed that the designed primers and probes specifically bound only to the paired WO nucleotides and did not cross-react with other nucleotides or antigens, demonstrating high specificity. This result provides a reliable basis for subsequent immunoassays and ensures the specificity and sensitivity of the detection system.

[0202] Table 2 shows the oligonucleotide sequences and primers used in the early stages of the present invention, which are described as comparative examples below:

[0203] 1. Oligonucleotide chain:

[0204] SEQ ID No. 14 (F chain), length: 87 nt, 5' modification: amino group (NH2) + 12C carbon chain spacer (C12), which facilitates the fixation of the oligo chain to a solid phase support (chip, magnetic beads, microplate, etc.) by methods such as NHS-esterification and reduces steric hindrance.

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

[0206] 2. Primer Design

[0207] The target product is approximately 67 bp in length (the distance from the R primer binding site to 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 to 87 of the F strand (corresponding to the 3' end of the double-stranded template). GC content: ≈45%; predicted T m :≈58℃;

[0209] Design principles:

[0210] T m Between 50 and 60°C, and the reverse primer ΔT m ≤2°C; no more than three consecutive identical bases at the 3' end to avoid nonspecific extension; check for self-dimers / inter-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 to 39 of the F strand (corresponding to the 5' end 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°C); GC content of 40-60% to facilitate specific binding; avoid 3' end complementarity to reduce dimers / hairpins.

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

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

[0216] Design principles:

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

[0218] 4. Results: 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 annealing, interfering with primer / probe binding. As a result, primer and probe specificity for the comparison sequence is poor.

[0219] IV. Verification of Antibody-Conjugated Nucleotide Specificity

[0220] Methods: To systematically evaluate the targeted recognition capabilities of antibody-nucleotide conjugates, a multidimensional cross-reactivity detection system was constructed. CK18, SARS-CoV-2 S protein, cardiac troponin I, and hepatitis B core antigen (HBcAg) were selected as detection targets. The conjugated antibodies were incubated with the above antigens at 37°C for 30 minutes, 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, demonstrating that the conjugated antibody is highly specific for CK18 protein. These results provide a reliable foundation for subsequent immunoassays and ensure 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 should be above 2.5 mg / mL for optimal results. Otherwise, concentration should be performed before the experiment to increase the concentration. 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 the 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:

[0228] 2.1 Add the sample to an ultrafiltration tube, add 200-150 μL PBS, centrifuge at 12,000 g, 4°C for 10 min, and discard the filtrate; add PBS again, centrifuge at 12,000 g, 4°C for 10 min; after centrifugation, remove the inner core of the ultrafiltration tube, invert it into a clean outer tube, centrifuge at 4,000 g, 4°C for 2 min, and collect the sample after replacing the buffer.

[0229] 2.2 Conjugate preparation

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

[0231] (2) Pipette 2.5 μL of biotin labeling 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 hour.

[0232] (3) Add an appropriate amount of PBS to the reaction solution of 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 12,000 g for 10 min.

[0233] (4) Discard the filtrate, add an appropriate amount of PBS (to a total volume of 500 μL) to the purification column, and centrifuge at 4°C and 12,000 g for 10 min.

[0234] (5) Remove the purification column and invert it into a clean test tube. Centrifuge at 4°C 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 Conjugate Storage

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

[0238] Example 3 Development of Nuclease Compositions

[0239] The present invention provides a method for optimizing the effect of a omnipotent nuclease. The method screens out the best nuclease composition that can significantly improve the effect of the omnipotent nuclease by combining different coating solutions (BSA, salmon sperm DNA, mouse serum, goat serum) with the omnipotent nuclease.

[0240] The method of the present invention uses BeyoZonase to treat a 10-fold diluted CK18 antigen standard to efficiently remove nucleic acids, which is particularly suitable for scenarios in the field of biotechnology where precise control of nucleic acid background is required.

[0241] In the prior art, CK18 antigen standards are commonly used in immunology or molecular biology research, but residual nucleic acids may remain during their preparation, affecting the accuracy of experimental results. Universal nucleases (such as BeyoZonase) are widely used for nucleic acid removal, but their effectiveness needs to be further improved by optimizing experimental conditions. The present invention verifies the nucleic acid removal efficacy of BeyoZonase in a specific concentration of CK18 antigen standard by systematically designing an experimental method and comparing it with a normal control group.

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

[0243] 1. Universal Nuclease Treatment Method and Results

[0244] 1. Materials

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

[0246] BeyoZonase: concentration is 25 U / well.

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

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

[0249] 2. Experimental Group Setup

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

[0251] Normal control group: 10-fold diluted CK18 antigen standard (concentration ranges from 1 to 10,000 pg / mL) without BeyoZonase added, used as a benchmark for nucleic acid background levels.

[0252] 3. Experimental Procedure

[0253] 3.1 Dilution of CK18 antigen standard

[0254] A CK18 antigen standard with an initial concentration of 10-100,000 pg / mL was diluted 10-fold using PBS to prepare dilutions with concentrations of 1, 10, 100, 1,000, and 10,000 pg / mL, respectively.

[0255] The diluted standards were added to the experimental wells, with a volume of 100 μL per well.

[0256] 3.2 Universal nuclease treatment

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

[0258] The normal control group was not added with BeyoZonase, and only an equal volume of PBS was added as blank treatment.

[0259] 3.2.1 Reaction conditions

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

[0261] 3.2.2 Nucleic Acid Testing

[0262] After the reaction is completed, the reaction products in each well are collected.

[0263] The residual nucleic acid content was detected by fluorescence quantitative PCR.

[0264] 3.2.3 Fluorescence quantitative PCR: Use nucleic acid-specific primers and fluorescent probes to determine the Ct value of residual nucleic acid and calculate the nucleic acid concentration.

[0265] 4. Experimental Results

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

[0267] Normal control group: The nonspecific nucleic acid level in the CK18 antigen standard without BeyoZonase remained high, the results fluctuated widely, and the Ct value was low, proving that the nonspecific nucleic acid was not degraded.

[0268] 5. Conclusion

[0269] like Figure 5 As shown in the results, during the optimization of the real-time fluorescence quantitative PCR detection system, the universal nuclease demonstrated excellent nonspecific interference elimination capabilities at varying CK18 antigen concentrations (1-100,000 pg / ml). Compared to the control group, the experimental group showed significant improvements in both system stability and nonspecific interference elimination efficiency, confirming the enzyme's broad-spectrum anti-interference properties.

[0270] This experiment demonstrated that the addition of 20 μg of BeyoZonase per well to a 10-fold dilution of a CK18 antigen standard (10-100,000 pg / mL) effectively removed nucleic acids, demonstrating significant superiority compared to 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] 2. Research and conclusions on the combination of different coating solutions and universal nucleases

[0272] In the experiment, 25U BeyoZonase (universal nuclease) was added to each well to test the effects of different coating solution combinations on the effectiveness of the universal nuclease. The final results showed that the combination of BSA + salmon sperm DNA can significantly enhance the effect of the universal nuclease and achieve the best effect of nucleic acid removal.

[0273] This study aimed to explore methods to optimize nucleic acid removal efficiency by comparing the effects of different coating solution combinations with BeyoZonase. To this end, the following four experimental groups and a normal control group were designed to systematically evaluate the effects of various coating solution combinations on BeyoZonase performance.

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

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

[0276] Group 2: Only salmon sperm DNA was used as the coating fluid.

[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 addition of universal nuclease.

[0280] 2. Experimental Procedure

[0281] 2.1 Coating treatment: Add the coating solution of each experimental group into the detection wells respectively.

[0282] 2.2 Treatment with universal nuclease: 20 μg BeyoZonase (universal nuclease) was added to each experimental well of 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 the results.

[0284] 2.4 Nucleic Acid Detection: After the reaction is completed, standard nucleic acid detection method fluorescence quantitative PCR is used to evaluate the removal of non-specific effects.

[0285] 3. Experimental Results

[0286] The experimental results show that different coating solution combinations have significant differences in the nucleic acid removal effect of universal nuclease:

[0287] Group 1 (BSA + salmon sperm DNA): This combination significantly enhanced the nucleic acid degradation ability of the universal nuclease, with the lowest residual nucleic acid content and achieved the best removal effect.

[0288] Group 2 (Salmon Sperm DNA): The nucleic acid removal effect was inferior to that of Group 1, and the performance was moderate.

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

[0290] 4. Conclusion

[0291] Experiments have shown that a coating solution combining BSA and salmon sperm DNA with BeyoZonase is the most effective, outperforming other single or combined coating solutions. This discovery provides new insights into optimizing nuclease treatment processes and has significant application value and patent protection potential.

[0292] The results showed that the combination of 2% BSA, 0.1 μL of universal nuclease (250 U / μL), and 2.5 μL of salmon sperm DNA (10 mg / ml) per well was the most effective, significantly outperforming other coating solution combinations in removing nucleic acid contamination. This combination effectively enhances the effectiveness of the universal nuclease in experiments, reduces nucleic acid interference, and improves experimental accuracy and reliability. Figure 5 It showed that BeyoZonase had a good effect in eliminating nonspecific effects. Figure 6 It shows that BSA + salmon sperm DNA has the best effect. The two-component blocking system (5% BSA + 0.1 mg / mL salmon sperm DNA) has significant advantages over single blocking agents (BSA, salmon sperm DNA) or serum blocking agents (mouse serum, goat serum). qPCR detection data show that the BSA-DNA composite blocking system not only obtains the lowest CT value, but also has the largest CT difference with the blank control group, and the difference is statistically significant (p < 0.001). The above results show that BSA and salmon sperm DNA can effectively reduce nonspecific adsorption through the synergistic mechanism of steric hindrance and charge neutralization. This finding provides a new optimization strategy for the selection of blocking agents for multi-component detection systems.

[0293] Example 4 Based on the oligonucleotide-coupled detection antibody, biotin-labeled capture antibody and optimization provided by the present invention Optimization of the method for immuno-PCR detection of CK18 expression based on a nuclease composition

[0294] 1. Optimization of the detection system

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

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

[0297] The present invention provides a method for optimizing a dual antibody detection system, which specifically comprises the following steps:

[0298] 1. Checkerboard titration design:

[0299] Detection antibody concentration gradient: 100ng, 10ng, 1ng, 0.1ng.

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

[0301] Through chessboard arrangement, a total of 16 4×4 antibody concentration combinations were constructed, covering the multi-dimensional parameter space.

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

[0303] 3. Fluorescence quantitative PCR verification: Fluorescence quantitative PCR technology is used to quantitatively evaluate the detection effect under various concentration combinations and obtain objective data.

[0304] 4. Determine the optimal ratio: Comprehensively analyze the results of fluorescent quantitative PCR to screen for the optimal concentration combination of capture antibody and detection antibody with both high sensitivity and specificity.

[0305] 5.Technological advantages

[0306] Systematic: Checkerboard titration method comprehensively explores antibody concentration combinations to avoid missing key ratios.

[0307] Accuracy: Fluorescence quantitative PCR provides highly sensitive quantitative verification to ensure the scientific nature of the optimal ratio.

[0308] High efficiency: Rapidly locate the optimal ratio through limited experiments, significantly shortening the optimization cycle. 6. Specific Examples

[0310] In the specific implementation, the detection antibody concentration was set to 100ng, 10ng, 1ng, 0.1ng, and the capture antibody concentration was set to 400ng, 200ng, 100ng, 50ng, forming a total of 16 concentration combinations. After implementing the double antibody detection experiment and collecting the signal, quantitative analysis was performed by fluorescent quantitative PCR. Figure 7 As shown, the present invention systematically optimizes the dual antibody detection system based on the checkerboard titration method, and determines the optimal ratio combination through three-dimensional parameter space modeling (antibody concentration gradient: 0.01-400 ng / well). As shown in the figure, 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, the system exhibits optimal detection efficiency. Quantitative analysis showed that the threshold cycle number (Cycle Threshold, CT) of the blank control group under this combination was significantly higher than 32.1, while the CT value of the positive control group was stable at 25.6, and the CT difference was 6.5 cycles (p < 0.001). Through cost-effectiveness evaluation, it was found that the unit detection cost of the optimized system was reduced by 75% compared with the suboptimal combination (capture antibody 400 ng / well + detection antibody 0.1 ng / well).

[0311] 2. 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] DNase-treated group;

[0316] Universal nuclease treatment group.

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

[0318] 4. Nuclease treatment: Use universal nuclease and react at 37°C for 30 minutes.

[0319] 5. Fluorescence Signal Detection and Analysis

[0320] 5.1 Use the single-molecule diagnostic platform to detect the fluorescence signal intensity of each group of probes.

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

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

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

[0324] 6. Conclusion

[0325] like Figure 8 As shown, to evaluate the quenching efficiency of the BHQ 1 probe, the probes designed in the table were treated with DNA enzyme and universal nuclease, and the fluorescence signal intensity was subsequently measured using a single-molecule diagnostic platform. The experimental results showed that the fluorescence signal in the DNA enzyme and universal nuclease-treated groups was significantly enhanced compared to the untreated group (p < 0.0001). This significant difference confirms that the BHQ labeled at the 3' end of the probe has a good quenching effect, effectively eliminating nonspecific signal interference and ensuring the accuracy of the experiment. This method is highly innovative and practical, providing a scientific basis for probe optimization in molecular biology assays.

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

[0327] 1. Experimental Purpose: The purpose of this experiment is to determine the optimal combination by optimizing the serum concentration and the number of PCR amplification cycles, thereby improving PCR performance. This paper provides a method for optimizing PCR experimental conditions, ultimately determining the optimal experimental conditions as adding 3.125 μL of serum per well and using 20 PCR cycles.

[0328] 2. Experimental Methods: In this experiment, we tested the effects of different combinations of serum concentrations and PCR cycle numbers on PCR amplification. The 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. Normal group: a control group without serum addition.

[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 respectively and determined the optimal experimental conditions by observing the quantity, specificity and amplification efficiency of PCR products.

[0332] 3. Experimental results:

[0333] like Figure 10 As shown, the experimental results show that in the present invention, the serum sample was serially diluted 2-fold (dilution range: 25 μL to 1.5625 μL) to determine the optimal dilution. The results show that 3.125 μL of serum sample showed the best performance in the detection.

[0334] like Figure 11 As shown, the present invention systematically evaluated the effect of PCR cycle number on detection performance using a single-molecule detection platform and ultimately determined that 20 PCR cycles were the optimal condition. Under these conditions, PCR amplification was optimal, fully utilizing the components in serum while ensuring efficient PCR reactions and avoiding nonspecific amplification or PCR inhibition caused by excessive cycles.

[0335] Example 6: Single-molecule detection method based on immune PCR 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): Bio-Tech, D7126-25KU; Dynabeads TM MyOne TM Streptavidin T1: Invitrogen TM , 65601.

[0338] 2. Experimental Procedure

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

[0340] 2.2 Add 1 μl of streptavidin magnetic beads, 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° for 30 minutes.

[0341] 2.3 Washing procedure: Wash three times with DPBST (0.05% Tween-20 in PBS); inject 300 μL of pre-cooled (4°C) washing solution into each well, adsorb on a magnetic separation stand for 2 minutes, pour off the supernatant and repeat the operation. After the last wash, dry at room temperature for 5 minutes.

[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 Second washing procedure: Use DPBST buffer (0.05% Tween-20 in PBS) to wash five times: inject 300 μL of pre-cooled (4°C) washing solution into each well, adsorb on the magnetic separation stand for 2 minutes, pour off the supernatant and repeat the operation. After the last wash, dry at room temperature for 5 minutes.

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

[0345] Table 4

[0346]

[0347] 2.7 Add 20 μL of mix to each well. Add the corresponding reagents according to the table above to a 1.5 mL EP tube to a total volume of 20 μL. Add the mixture to a 96-well plate, with three replicates per sample. Cover the wells with a 96-well PCR amplification membrane and centrifuge at 3000 rpm for 5 minutes. Amplify in a PCR instrument using the following protocol: Stage 1: Initial denaturation at 95°C for 30 seconds; Stage 2: Cycling reaction at 95°C for 5 seconds and 60°C for 34 seconds for 20 cycles; Stage 3: Store at 12°C.

[0348] 2.10 Add 20 μL of PCR amplification product to each well of a 384-well plate. Gently mix the PCR amplification product using an eight-well plate reader, and then add 2 μL of PCR amplification product to each well. Signal detection: Read the signal using the single-molecule diagnostic platform SMCXPRO.

[0349] Method steps:

[0350] (1) Fix the plate holder on SMCxPRO TM At the bottom of the reading plate, add 20 μL PBS to each well;

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

[0352] (3) Use a pipette to transfer 2 μL of solution from each well of the PCR plate to the corresponding wells of the 384-well plate. Ensure that there are beads on the side of each well.

[0353] (4) The cover plate 2 is sealed with an aluminum sheet sealing machine to ensure that the cover plate is well bonded to the plate to prevent leakage and cross contamination;

[0354] (5) Place Tablet 2 (on the Tablet Stand) into the Jitterbug TM Incubate the microplate in a shaker. Set the shaker to 7 for 1 minute (1500 rpm).

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

[0356] (7) On the machine: Set the program: In the SMC software interface, select a new 384 name; Create, enter EXperiaiment → Unknown: every three wells are a Group, named according to the corresponding loading order; Click Eject, put the 384 plate into the slot → Run → Start the program to start running; after the program is completed, remove the 384 plate;

[0357] like Figure 9 As shown, using CK18 antigen as a standard, the present invention constructed a standard curve with a concentration range of 0.01 pg / ml to 1,000,000 pg / ml, covering 8 logarithmic levels. To ensure the accuracy of data analysis, a four-parameter logistic (4PL) model was used for statistical fitting, and the following fitting equation was obtained: Y = 479.9 + 18799 / 1 + (x / 8639) -0.2805 , goodness of fit R 2 It reached 0.9992, showing extremely high fitting accuracy and detection sensitivity as low as 10fg / ml.

[0358] Example 7 Detection of CK18 content in ACLF patients and its Relationship with prognosis

[0359] This study used immuno-PCR-single-molecule detection to measure CK18 levels in the serum of ACLF patients. Furthermore, a comprehensive analysis of serum levels and other blood markers from patients with different ACLF grades (grades 1, 2, and 3) revealed the relationship between CK18 and ACLF patient prognosis. The study found that CK18 concentration in ACLF patients was closely correlated with patient prognosis, and that CK18 was an independent predictor of poor prognosis in ACLF patients.

[0360] 1. Experimental methods:

[0361] 1.1 Subjects: Patients meeting the diagnostic criteria for ACLF and admitted to Beijing You'an Hospital, Capital Medical University, between 2022 and 2023 with acute decompensation and one or more of the following acute events: ascites, hepatic encephalopathy, gastrointestinal bleeding, and bacterial infection (e.g., spontaneous bacterial peritonitis) were enrolled. Organ failure: The CLIF-SOFA score was used to assess the function of the following six major organ systems and determine the presence of organ failure: Liver: bilirubin ≥12 mg / dL; Kidney: creatinine ≥2 mg / dL or need for renal replacement therapy; Brain: Grade III-IV hepatic encephalopathy; Coagulation: International Normalized Ratio (INR) ≥2.5; Circulation: Mean arterial pressure <70 mmHg or need for vasopressor support; Respiration: PaO2 / FiO2 ≤200 or SpO2 / FiO2 ≤214. ACLF grade: Grade 1: Single renal 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 divided into ACLF grade 1, 2, and 3 groups according to clinical grade and compared with a healthy control group.

[0362] 1.2 Sample collection: Serum samples were collected from patients in each group, 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 immuno-PCR-single molecule detection kit provided by the present invention. The experimental steps were the same as those in Example 6.

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

[0365] 2. Experimental results:

[0366] like Figure 12As shown in the table below, CK18 levels in the serum of patients with different grades of ACLF were measured using an immuno-PCR-single molecule detection method. ACLF patients were divided into three pathological stages based on the CLIF-C organ failure score (CLIF-C OF). As shown, the CK18 level in the ACLF grade 3 group was significantly higher than that in the ACLF grade 1 and 2 groups. As the disease worsened, the serum CK18 level showed a significant increasing trend, reflecting the gradual worsening of liver damage. This finding not only confirmed the sensitivity of CK18 as a biomarker of liver damage, but also revealed the close correlation between changes in its level 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 correlated with the patient's survival and liver function recovery.

[0367] Table 5 CK18 can be used as an independent predictive marker for poor prognosis in ACLF patients

[0368]

[0369]

[0370] Example 8 Comparison of efficacy evaluation of the present invention and efficacy evaluation using MELD score

[0371] 1. MELD score and efficacy evaluation

[0372] 1. Data collection:

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

[0374] 2. Rating calculation:

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

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

[104] .

[0377] 3. Performance Analysis:

[0378] Receiver operating characteristic (ROC) curve 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] The PPV was 0.61 (95% CI: 0.45-0.77), and the NPV was 0.67 (95% CI: 0.45-0.88).

[0381] 2. Construction and efficacy evaluation of the CK18 model provided by the present invention

[0382] 1. CK18 test:

[0383] CK18 levels in patient serum were measured by single-molecule detection combined with antibody-coupled DNA immunoPCR.

[0384] 2. Model construction:

[0385] Efficacy analysis of the prognostic evaluation model established 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 predictive efficacy of the CK18 model is better than that of the MELD score, such as Figure 13 As shown, the AUC value of the MELD model was 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). The PPV and NPV were 0.61 (95% CI: 0.45-0.77) and 0.67 (95% CI: 0.45-0.88), respectively. The MELD model performed well in terms of sensitivity, demonstrating its ability to identify positive cases, but its low specificity may result in a higher false positive rate, limiting its overall reliability.

[0387] like Figure 14As shown, the CK18 model of the present invention exhibited excellent predictive performance, with an area under the curve (AUC) of 0.95 (95% CI: 0.89-1.00), demonstrating extremely high discriminatory power. The overall accuracy of the model 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). The positive predictive value (PPV) and negative predictive value (NPV) were 0.94 (95% CI: 0.86-1.00) and 0.89 (95% CI: 0.79-0.99), respectively. These indicators indicate that the CK18 model can effectively exclude negative cases while accurately identifying positive cases, demonstrating significant clinical application value.

[0388] 5. Conclusion

[0389] This study systematically compared the effectiveness of the MELD score and the CK18 model, confirming that the CK18 model has significant advantages in predicting liver disease prognosis, far exceeding the traditional MELD score. This method provides a more accurate and reliable prognostic assessment tool in clinical practice and has broad application prospects.

Claims

1. An ACLF immune PCR-single molecule detection reagent, characterized in that include: a) Biotinylated capture antibody 8G5; b) oligonucleotide-conjugated detection antibody 6B4-Oligo; c) Streptavidin magnetic beads; d) a nuclease composition; e) a specific primer pair, probe 1 and probe 2; The oligonucleotide-coupled detection antibody 6B4-Oligo is a detection antibody 6B4 coupled with an oligonucleotide shown in SEQ ID No. 1; The specific primers are shown in SEQ ID No. 2 and SEQ ID No. 3; The probe 1 is shown in SEQ ID No. 4, wherein 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, wherein the 5' end of the probe 2 is connected to a fluorescent group, and the 3' end of the probe 2 is connected to a quenching group BHQ_2; The nuclease composition consists of universal nuclease, 2% BSA coating solution and salmon sperm DNA.

2. The ACLF immune PCR-single molecule detection reagent according to claim 1, characterized in that: The molar ratio of the antibody 6B4 to the oligonucleotide was 3:

1.

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

4. A kit for detecting CK 18 content as a prognosis prediction kit for patients with acute-on-chronic liver failure, characterized in that The method comprises the detection reagent according to claim 1 or 2.

5. A method for detecting CK18 content based on the kit according to claim 4, characterized in that The following steps are involved: a) binding the biotin-labeled capture antibody 8G5 to the CK18 antigen in the serum sample to be tested; b) adding streptavidin magnetic beads and a nuclease composition, enriching the antigen-antibody complex by the magnetic beads, and eliminating nonspecific nucleic acid interference by the nuclease composition; c) adding oligonucleotide-coupled detection antibody 6B4-Oligo to form a complete immune complex; d) performing PCR amplification using specific primers and probes to amplify the oligonucleotide sequence coupled to the detection antibody; e) Quantitatively analyze the amplified product using single molecule detection technology to determine the concentration of CK 18.

6. The method according to claim 5, wherein The preparation method of the oligonucleotide-coupled detection antibody comprises: The antibody and oligonucleotide were coupled via an activation reagent, wherein the oligonucleotide sequence was as shown in SEQ ID No. 1, and the molar ratio of the antibody 6B4 to the oligonucleotide was 3:

1.

7. The method according to claim 5, wherein The PCR amplification conditions were as follows: 3.125 μL of serum was added to each well, and a 20-cycle amplification program was 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.

8. The method according to claim 5, wherein The single molecule detection technique comprises the following steps: a) enriching the PCR products using magnetic beads and separating them by centrifugation; b) using a reader plate for single-molecule signal capture and combining it with a microplate shaker for uniform mixing; c) Analyze single-molecule signals through software to generate quantitative detection results.

9. A prognosis assessment system for patients with acute-on-chronic liver failure based on the method of claim 5, characterized in that include: a) CK18 concentration detection module, used to perform immuno-PCR-single molecule detection; b) Data analysis module, which performs correlation analysis between CK18 concentration, acute-on-chronic liver failure grade, and liver function indicators; c) Prognosis prediction module, which outputs the patient's poor prognosis risk assessment results and individualized treatment recommendations.

10. Use of a kit for detecting CK18 content in predicting the prognosis of patients with acute-on-chronic liver failure, characterized in that: The kit is the kit according to claim 4.

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