Antibody for detecting p-cresol sulfate as well as related product and application thereof

Abstract

The invention discloses an antibody for detecting p-cresol sulfate and related products and application thereof, the antibody can specifically bind to p-cresol sulfate and has high binding activity, the antibody can rapidly and accurately quantify the content of p-cresol sulfate in a sample to be detected through convenient methods such as ELISA and immunochromatography, and the antibody can be applied to the detection of p-cresol sulfate. The clinical early screening requirement on the chronic renal failure patient can be met, the change of the metabolite level in the treatment process can be dynamically tracked, an objective basis is provided for illness state evaluation and treatment scheme adjustment, and the application prospect is wide.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically, it relates to an antibody for detecting p-cresol sulfate and its related products and applications. Background Technology

[0002] Chronic renal failure (CRF), also known as chronic renal insufficiency, refers to chronic, progressive damage to the renal parenchyma caused by various factors, leading to significant kidney atrophy and inability to maintain its basic functions. Clinically, it presents as a syndrome characterized by the retention of metabolic products, water, electrolyte, and acid-base imbalances, and involvement of various systemic systems; it is also called uremia. Given the high incidence, low quality of life, and low life expectancy of CRF, my country is increasingly emphasizing interventions targeting controllable factors in the progression of CRF. In recent years, research on the impact of enterogenic uremic toxins (GDUT) on CRF patients and how to eliminate GDUT has become a hot topic. p-Cresyl sulfate (PCS) is one of the most important toxins in GDUT, and its detection allows for real-time monitoring of the disease.

[0003] However, existing methods for detecting p-cresol sulfate mostly rely on large-scale instruments such as mass spectrometry, which suffer from technical bottlenecks such as complex operation, high cost, and poor accessibility. Detection requires specialized laboratories and is time-consuming, failing to meet the urgent needs of rapid clinical diagnosis. The development of novel antibodies against p-cresol sulfate, based on antigen-antibody specific reactions, can provide a highly specific and sensitive detection tool, promoting its application in rapid clinical diagnosis and disease progression monitoring. Furthermore, it can provide a key tool for exploring the mechanism of action of p-cresol sulfate in disease development and progression, aiding in the discovery of related therapeutic targets, thus possessing dual value in both basic research and clinical translation. Therefore, developing an antibody with good binding activity and specificity to p-cresol sulfate is of significant practical importance. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide an antibody for detecting p-cresol sulfate, as well as related products and applications. This invention is the first to creatively discover and experimentally verify that the antibody has good affinity and specificity for p-cresol sulfate, providing a new idea and strategy for the research and development of p-cresol sulfate-related detection reagents or products, and has good application prospects.

[0005] The present invention achieves the above-mentioned objectives by adopting the following technical solution:

[0006] A first aspect of the present invention provides an antibody against p-cresol sulfate.

[0007] Furthermore, the HCDR1-3 in the heavy chain variable region of the antibody is the HCDR1-3 in the heavy chain variable region as shown in SEQ ID NO:7;

[0008] The LCDR1-3 in the light chain variable region of the antibody is the LCDR1-3 in the light chain variable region as shown in SEQ ID NO:8.

[0009] Furthermore, the amino acid sequences of HCDR1-3 in the heavy chain variable region of the antibody are shown in SEQ ID NO:1-3, respectively;

[0010] The amino acid sequences of LCDR1-3 in the light chain variable region of the antibody are shown in SEQ ID NO:4-6, respectively.

[0011] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7.

[0012] Furthermore, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.

[0013] In some embodiments, antibody sequences corresponding to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having at least 70% homology with the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are also included within the scope of protection of this invention. Herein, at least 70% homology includes at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 86% homology, at least 87% homology, at least 88% homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology.

[0014] In this invention, homology refers to a certain degree of complementarity. It can be partial homology, near-homology, or complete homology. Near-homology refers to a partially complementary sequence that at least partially inhibits hybridization between the same sequence and the target nucleic acid. Inhibition of hybridization between a completely complementary sequence and the target sequence can be tested using hybridization experiments (Southern or Northern blotting, solution hybridization, etc.) under low-stringency conditions. Nearly homologous sequences or hybridization probes will compete for and inhibit the binding of completely homologous sequences to the target sequence under low-stringency conditions. This does not mean that low-stringency conditions allow non-specific binding; low-stringency conditions require that the binding of the two sequences is a specific (selective) interaction.

[0015] In some embodiments, the amino acid sequences corresponding to HCDR1-3 of the present invention are not limited to those shown in SEQ ID NO:1-3, nor are the amino acid sequences corresponding to LCDR1-3 of the present invention limited to those shown in SEQ ID NO:4-6. The amino acid sequences or nucleotide sequences of the antibodies corresponding to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 obtained by defining CDR1, CDR2, and CDR3 in the heavy chain variable region shown in SEQ ID NO:7 and CDR1, CDR2, and CDR3 in the light chain variable region shown in SEQ ID NO:8 using any CDR numbering scheme (existing CDR numbering scheme or new CDR numbering scheme to be generated in the future) are all within the protection scope of the present invention.

[0016] In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to any one or any combination of two or more of the following numbering schemes: IMGT, Chothia, Kabat, Martin (enhanced Chothia), AbM, Aho, and Contact. The antibody sequences corresponding to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 defined in the above manner are also included within the scope of protection of this invention.

[0017] In some embodiments, functional variants of the antibodies described above are also included within the scope of protection of this invention. These functional variants refer to proteins that have significant or marked sequence identity or similarity to the parent antibody (the antibody described in the first aspect of this invention), and that retain the biological activity of the parent antibody. Functional variants encompass, for example, variants of the antibody (parent antibody) described in the first aspect of this invention, which retain the ability to recognize the target antigen (p-cresol sulfate) to a similar, equal, or greater extent than the parent antibody. Referring to the parent antibody, the functional variant may, for example, have at least about 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, or higher homology in amino acid sequence to the parent antibody.

[0018] In some embodiments, the functional variant may, for example, comprise the amino acid sequence of a parent antibody having at least one conserved amino acid substitution. Alternatively or supplementally, the functional variant may comprise the amino acid sequence of a parent antibody having at least one non-conserved amino acid substitution. In this case, the non-conserved amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. The non-conserved amino acid substitution can enhance the biological activity of the functional variant, resulting in increased biological activity of the functional variant compared to the parent antibody.

[0019] In some embodiments, conservative amino acid substitution is known in the art and includes replacing one amino acid having a particular physical and / or chemical property with another amino acid having the same or similar chemical or physical properties.

[0020] In some embodiments, the conserved amino acid substitution may be the substitution of one acidic / negatively charged polar amino acid for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), the substitution of one amino acid with a nonpolar side chain for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), the substitution of one basic / positively charged polar amino acid for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), the substitution of one uncharged amino acid with a polar side chain for another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), the substitution of one amino acid with a β-branched side chain for another amino acid with a β-branched side chain (e.g., He, Thr, and Val), or the substitution of one amino acid with an aromatic side chain for another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr).

[0021] In this invention, p-cresyl sulfate (PCS) is currently the most studied enterogenic uremic toxin. The intestines and liver are important sites for PCS metabolism in vivo. Anaerobic bacteria in the intestines break down dietary proteins into tyrosine, which is further converted into 4-hydroxyphenylacetic acid (4-OH phenylacetic acid). 4-OH phenylacetic acid is decarboxylated by intestinal bacteria such as *Clostridium difficile* to generate p-cresol (PC), the precursor of PCS. Most p-cresol is absorbed through the intestinal mucosa and converted into PCS by sulfotransferase (SULT) in the intestinal epithelium or liver. Under normal renal function, PCS can be completely excreted by the kidneys, and the PCS content in the blood is extremely low. However, in patients with chronic kidney disease (CKD), due to impaired renal excretion function, PCS cannot be adequately excreted by the kidneys, resulting in a significant increase in the PCS concentration in the blood circulation. The large accumulation of PCS molecules in the body is transported into renal tubular cells by renal organic anion transporters, activating the PKC / PI3K-Nox4-ROS signaling pathway, generating a large number of oxygen free radicals, inducing oxidative stress in the kidneys, and causing renal tubular damage and renal interstitial fibrosis.

[0022] A second aspect of the present invention provides a nucleic acid molecule.

[0023] Furthermore, the nucleic acid molecule encodes the antibody described in the first aspect of the present invention.

[0024] In this invention, the nucleic acid molecule generally refers to any nucleic acid sequence, such as any polynucleotide or polydeoxynucleotide, which can be unmodified RNA or DNA, or modified RNA or DNA. This includes, but is not limited to: single-stranded and double-stranded DNA, DNA including single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA including single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA. It also includes triple-stranded regions containing RNA or DNA, or RNA and DNA. Specifically, it includes mRNA, cDNA, and genomic DNA and any fragments thereof. The polynucleotide includes DNA and RNA containing one or more modified bases, such as tritium-containing bases, or uncommon bases such as inosine. The nucleic acid molecules described in this invention may encompass coding or non-coding sequences. It should be understood that every reference to nucleic acid molecule or similar term herein will include the full-length sequence and any complementary sequences, fragments, variations, derivatives, or variants thereof.

[0025] Those skilled in the art can readily mutate the nucleotide sequence corresponding to the antibody provided in the first aspect of this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that have 80% or more homology to the nucleotide sequence corresponding to the antibody described in this invention, as long as they encode the antibody described in the first aspect of this invention, are all derived from and equivalent to the nucleotide sequence of this invention, and are also included within the scope of protection of this invention.

[0026] A third aspect of the present invention provides a recombinant vector.

[0027] Furthermore, the recombinant vector comprises the nucleic acid molecule described in the second aspect of the present invention;

[0028] Optionally, the vector is a virus-derived vector, plasmid, and / or phage particle;

[0029] Optionally, the vector from which the virus originates may be a lentiviral vector, adenovirus vector, adeno-associated virus vector, retroviral vector, poxvirus vector, herpesvirus vector, and / or baculovirus vector.

[0030] In this invention, there are no particular limitations on the vectors expressing the coding sequences of the antibodies described above. In some embodiments, the vectors include, but are not limited to: bacteria transformed with microbial vectors such as recombinant bacteriophages, plasmids, or copious DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems transformed with viral expression vectors (such as baculoviruses); plant cells transformed with viral expression vectors (such as cauliflower mosaic virus CaMV, tobacco mosaic virus TMV) or bacterial expression vectors (such as Ti, pBR322 plasmids); or animal cell systems. For bacteria, useful plasmids include pET, pRSET, pTrcHis2, and pBAD plasmids from Invitrogen; pET and pCDF plasmids from Novagen; and Director™ plasmids from Sigma-Aldrich. For methanogens, useful plasmids include, but are not limited to, pME2001, pMV15, and pMP1.

[0031] A fourth aspect of the present invention provides a recombinant host cell.

[0032] Furthermore, the recombinant host cell comprises the recombinant vector described in the third aspect of the present invention;

[0033] Optionally, the host cell may be a mammalian cell, plant cell, insect cell, fungal cell, and / or bacterial cell.

[0034] In this invention, there is no particular limitation on the type of host cell; any suitable host cell can be used to encode the DNA sequence encoding the antibody of this invention as described above or the nucleic acid molecule of this invention as described above, including but not limited to: mammalian cells, plant cells, insect cells, fungal cells, or bacterial cells. In some embodiments, the host cell is preferably a mammalian cell.

[0035] In some implementations, the host cells that can be used to express antibodies are well known in the art, and many host cells are available from the American Type Culture Collection (ATCC). These host cells include, but are not limited to: Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells (HeLa), hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, 3T3 cells, HEK-293 cells, and many other cell lines.

[0036] The fifth aspect of the invention provides any of the following products:

[0037] (1) An antibody conjugate, wherein the antibody conjugate is a complex formed by directly or indirectly conjugating an antibody to a detectable marker as described in the first aspect of the present invention;

[0038] (2) A detection reagent comprising the antibody and / or the antibody conjugate described in the first aspect of the present invention;

[0039] (3) A test strip or detection chip, wherein the test strip or detection chip comprises the antibody, the antibody conjugate and / or the detection reagent described in the first aspect of the present invention;

[0040] (4) A p-cresol sulfate detection kit, wherein the detection kit comprises the antibody, the antibody conjugate and / or the detection reagent described in the first aspect of the present invention.

[0041] In some implementations, the detectable marker refers to any substance that can be used to assist the antibody in detecting p-cresol sulfate. Exemplarily, the detectable marker includes, but is not limited to: luciferase, luciferin, alkaline phosphatase, glucose oxidase, protoporphyrin, hematoporphyrin, horseradish peroxidase, β-D-galactosidase, urease, catalase, jellyfish luminescent protein, methylene blue, etc.

[0042] In some implementations, the test strip mainly includes the following core components: a sample pad, a conjugation pad, a nitrocellulose membrane (NC membrane), an absorbent pad, and a base plate. Its detection principle (taking a colloidal gold test strip as an example) is as follows: When the sample contains p-cresol sulfate, as the sample flows through the conjugation pad, the p-cresol sulfate binds to the colloidal gold-labeled anti-p-cresol sulfate antibody, forming an "antigen-labeled antibody" complex. When this complex continues to chromatographically up to the T line, because the antigen analog at the T line has a weaker ability to compete with p-cresol sulfate for antibody binding, it cannot effectively capture the complex, so the T line does not develop color (or the color becomes lighter); while the excess labeled antibody is captured by the secondary antibody at the C line, causing the C line to develop color. By observing the color development of the T line and C line, the presence and content of p-cresol sulfate in the sample can be qualitatively or semi-quantitatively determined (the lighter the T line, the higher the content of p-cresol sulfate in the sample).

[0043] The sample pad is located at the very front of the test strip and is responsible for receiving the sample to be tested (such as urine, serum, plasma, etc.) and pre-treating the sample: (1) filtering impurities, cell debris, etc. in the sample to avoid clogging the subsequent chromatography channel; (2) balancing the pH value and ionic strength of the sample to provide a suitable environment for the binding of antigen (p-cresol sulfate) and antibody; (3) some sample pads are pre-coated with buffer or surfactant to accelerate the dissolution and chromatographic diffusion of the sample.

[0044] Among them, the conjugate pad is adjacent to the sample pad and is the fixed area of ​​the labeled antibody. Its core function is to provide a movable "detection probe": (1) Anti-p-cresol sulfate antibody (i.e. "labeled antibody") pre-coated with colloidal gold (or other labels, such as fluorescent microspheres or latex particles) is dried and fixed in the conjugate pad. When the sample solution flows through it, it will be dissolved and move with the sample in the direction of chromatography; (2) The choice of label directly affects the signal visualization effect: colloidal gold labeling can produce visible bands through the aggregation of red particles, fluorescent microspheres require a fluorescence detector to read the signal, and latex particles can be colored through color or turbidity changes.

[0045] The nitrocellulose membrane serves as the core reaction zone of the test strip. Two key areas are fixed on the membrane to achieve specific antigen-antibody binding and signal determination: (1) Test Line (T line): Coated with an antigen analogue (or p-cresol sulfate conjugated carrier protein), its function is to capture labeled antibodies that have not bound to p-cresol in the sample. When the p-cresol content in the sample is low, the labeled antibody is not completely bound and will move to the T line with the chromatography solvent, binding with the fixed antigen analogue to form a "labeled antibody-antigen analogue" complex, causing the T line to show color; (2) Control Line (C line): Coated with a secondary antibody against the labeled antibody (such as anti-mouse IgG antibody; if the labeled antibody is mouse-derived), its function is to verify the effectiveness of the test strip. Regardless of whether p-cresol is present in the sample, when the labeled antibody flows through the C line, it will bind with the secondary antibody to form a complex, causing the C line to show color (if the C line does not show color, the test strip is invalid).

[0046] The absorbent pad, located at the end of the test strip, is made of a highly absorbent material (such as filter paper). It provides chromatographic kinetics through capillary action, accelerating the flow of the sample solution from the sample pad to the NC membrane and finally to the absorbent pad. At the same time, it absorbs excess sample solution, ensuring the stability of the reaction system.

[0047] The base plate provides physical support for the entire test strip and is usually an inert plastic plate (such as a PVC plate). The sample pad, conjugate pad, NC membrane, and absorbent pad are pasted on it in sequence to ensure that the components are tightly connected and the chromatography channel is unobstructed, so as to avoid affecting the detection effect due to component displacement.

[0048] In some implementations, the detection chip is based on the principle of antibody-antigen specific binding and achieves high-sensitivity, automated detection through microfluidics or biosensing technology. The detection chip includes the following core components: a substrate material, a microfluidic channel system, an antibody immobilization layer, a signal detection module, a signal processing and readout system, and a quality control system.

[0049] The substrate material is selected from silicon-based chips, glass chips, or polymer chips (such as PDMS and PMMA).

[0050] The microfluidic channel system includes an inlet, a microchannel network, a mixing zone, and a reaction chamber.

[0051] The antibody immobilization layer can be used to immobilize antibodies via physical adsorption, chemical cross-linking, or a biotin-avidin system. Physical adsorption directly adsorbs antibodies onto the substrate surface using van der Waals forces or electrostatic interactions. Chemical cross-linking introduces active groups (such as amino or aldehyde groups) onto the substrate surface using silanizing agents (e.g., APTES) or bifunctional cross-linking agents (e.g., glutaraldehyde), which covalently bind to the amino or thiol groups of the antibody, maintaining its spatial conformation and activity. The biotin-avidin system immobilizes biotinylated antibodies onto a pre-coated biotin substrate using avidin (e.g., streptavidin).

[0052] The signal detection module includes optical detection (fluorescence detection, colorimetric detection), electrochemical detection (amperometric sensor, potentiometric sensor), or surface plasmon resonance (SPR).

[0053] The signal processing and readout system includes an integrated circuit (ASIC), a wireless transmission module, and a display screen.

[0054] The quality control system includes internal parameter points, micro-valve and pump systems.

[0055] In some implementations, the test kit includes immunomagnetic beads, enzyme-labeled antigens, and calibrators.

[0056] Furthermore, the immunomagnetic beads are obtained by coating the antibody with biotin-streptavidin, the enzyme-labeled antigen is alkaline phosphatase-labeled p-cresol sulfate antigen, and the calibrator is a p-cresol sulfate standard solution.

[0057] In this invention, the p-cresol sulfate detection kit is based on the competitive immunoassay principle. It uses a biotin-streptavidin system to coat p-cresol sulfate antibodies onto the surface of immunomagnetic beads, while simultaneously employing alkaline phosphatase-labeled p-cresol sulfate antigen. During detection, p-cresol sulfate in the sample competes with the alkaline phosphatase-labeled p-cresol sulfate antigen for binding to the antibodies on the magnetic beads. After washing to remove unbound enzyme-labeled antigen, a substrate is added. The signal intensity generated by the alkaline phosphatase-catalyzed substrate (inversely proportional to the p-cresol sulfate concentration in the sample) is used to calculate the concentration of p-cresol sulfate in the sample by combining the reaction curve fitted by a 4PLC (calibrated using high and low concentration calibrators in the kit).

[0058] In some embodiments, the p-cresol sulfuric acid test kit further includes the following auxiliary reagents and consumables to ensure the integrity and stability of the detection process:

[0059] Substrate solution: It is paired with alkaline phosphatase in enzyme-labeled antigen, such as chemiluminescent substrate (AMPPD) or colorimetric substrate (p-NPP), and is the core reagent for generating a detectable signal. In actual detection process, the formulation can be designed according to the catalytic characteristics of the enzyme.

[0060] Washing solution: Used to remove unbound free components (such as unbound enzyme-labeled antigens, sample impurities, etc.) during the detection process. It is usually a solution containing buffer and surfactant to maintain appropriate pH and ionic strength and reduce non-specific binding.

[0061] Sample diluent: If the concentration of p-cresol in the sample is too high and exceeds the detection range, it can be used to dilute the sample to the linear range. The components are mostly buffers that match the sample matrix (such as PBS containing fetal bovine serum) to avoid matrix effects.

[0062] Blocking solution: Used in the preparation or detection of immunomagnetic beads, containing bovine serum albumin (BSA) or casein, etc., to block non-specific binding sites on the surface of magnetic beads or reaction containers and reduce background signal.

[0063] Reaction buffer: Maintains the pH stability of the detection system (e.g., 0.02M PBS, pH 7.4), provides a suitable environment for antigen-antibody reactions and enzyme-catalyzed reactions, and may contain preservatives (e.g., sodium azide) to extend the shelf life of the reagents.

[0064] Instructions and quality control documents: including detailed operating procedures, precautions, calibration curve parameters, quality control standards, etc., to ensure the standardization of testing operations and the traceability of results.

[0065] Consumables: such as reaction tubes, pipette tips, detection plates, etc., adapted to the reaction system of the reagent kit to avoid cross-contamination of samples.

[0066] The sixth aspect of the present invention provides any of the following methods:

[0067] (1) A method for producing the antibody according to the first aspect of the present invention, the method comprising the following steps: culturing the recombinant host cell according to the fourth aspect of the present invention, and isolating the antibody according to the first aspect of the present invention from the recombinant host cell culture product;

[0068] (2) A method for preparing the recombinant host cell according to the fourth aspect of the present invention, the method comprising the following steps: introducing the recombinant vector according to the third aspect of the present invention into a host cell to obtain the recombinant host cell according to the fourth aspect of the present invention;

[0069] (3) A method for detecting p-cresol sulfate in a test sample for non-diagnostic and non-therapeutic purposes, the method comprising the following steps: contacting the test sample with the antibody described in the first aspect of the present invention, the antibody conjugate described in the fifth aspect of the present invention, the detection reagent, the test strip or the detection chip to detect the formation of antigen-antibody immune complexes;

[0070] (4) A method for preparing a p-cresol sulfuric acid detection kit, the preparation method comprising the following steps:

[0071] 1) Immunomagnetic beads were obtained by coating the antibodies with biotin-streptavidin magnetic beads;

[0072] 2) Alkaline phosphatase-labeled p-cresol sulfate antigen was obtained by labeling p-cresol sulfate with alkaline phosphatase.

[0073] Further, step 1) includes the following steps: ① desalting the p-cresol sulfate antibody; ② mixing the desalted antibody with Sulfo-NHS-Biotin at a molar ratio of 0.1-5:10-30, reacting at room temperature for 10-60 min to obtain biotinylated antibody; ③ desalting the biotinylated antibody to remove free biotin; ④ mixing the streptavidin magnetic bead solution with the biotinylated antibody obtained in step ③, reacting at room temperature for 10-60 min, adding blocking solution and blocking for 10-60 min, and removing the free biotinylated antibody by magnetic separation to obtain the immunomagnetic beads;

[0074] Optionally, the molar ratio of the desalted antibody to Sulfo-NHS-Biotin is 1:20; optionally, the room temperature reaction time is 30 min; optionally, the blocking time is 30 min.

[0075] In this invention, Sulfo-NHS-Biotin (sulfonyl-N-hydroxysuccinimide-biotin) is a commonly used water-soluble biotinylation reagent, mainly used to covalently link biotin molecules to amino-containing (-NH2) biomolecules (such as antibodies, proteins, peptides, etc.) to form stable biotinylated complexes. Its molecular structure contains three key parts: a biotin group (providing a specific binding site for streptavidin), an N-hydroxysuccinimide (NHS) ester group (as an activating group, capable of amidation with amino groups), and a sulfonic acid group (-SO3). - (To impart water solubility to the reagent, avoid aggregation in aqueous solution, and reduce non-specific binding), the present invention does not have any particular restrictions on the source of the Sulfo-NHS-Biotin, which can be obtained by those skilled in the art through conventional purchasing channels.

[0076] In a specific embodiment of the present invention, the Sulfo-NHS-Biotin is used to biotinylate the p-cresol sulfate antibody (specifically, the antibody and Sulfo-biotin are mixed and reacted at a molar ratio of 1:20). The NHS ester forms a stable amide bond with the amino group of the lysine residue of the antibody, so that the biotin molecule is directionally linked to the antibody without affecting the antigen-binding activity of the antibody.

[0077] In this invention, the streptavidin magnetic beads are functional reagents formed by immobilizing streptavidin on the surface of magnetic microspheres using chemical or physical methods. They combine the high specific binding capacity of streptavidin with the magnetic response characteristics of magnetic beads. Their core structure includes a magnetic core (such as iron oxide particles, which endow the magnetic beads with the ability to rapidly separate under an applied magnetic field) and surface-modified streptavidin. This invention does not impose any particular restrictions on the source of the streptavidin magnetic beads; those skilled in the art can obtain them through conventional purchasing channels.

[0078] In a specific embodiment of the present invention, streptavidin magnetic beads are a key carrier for immobilizing biotinylated antibodies: through the specific interaction between biotin and streptavidin, biotinylated p-cresol sulfate antibodies are efficiently anchored on the surface of the magnetic beads to form immunomagnetic beads; during the detection process, the magnetic properties of the magnetic beads can be used to quickly separate the conjugate from the free components (such as unbound enzyme-labeled antigens), simplifying the washing steps. At the same time, the glycosylation-free properties of streptavidin can reduce non-specific binding, lower background signals, and significantly improve the specificity and efficiency of detection, making it a core material for constructing a stable and efficient immunoassay system.

[0079] Furthermore, step 2) includes the following steps:

[0080] ① Desalting alkaline phosphatase; ② Mixing the desalted alkaline phosphatase with SMCC at a molar ratio of 0.1-5:5-20, reacting at room temperature for 10-60 min, desalting to remove free SMCC, and obtaining activated alkaline phosphatase; ③ Desalting p-cresol sulfate antigen; ④ Mixing the desalted p-cresol sulfate antigen with 2-IT at a molar ratio of 0.1-5:5-25, reacting at room temperature for 10-60 min, desalting to remove free 2-IT, and obtaining activated antigen; ⑤ Mixing the activated antigen with activated alkaline phosphatase at a molar ratio of 1-10:0.1-5, stirring at room temperature for 10-60 min, and obtaining the enzyme-labeled antigen;

[0081] Optionally, the molar ratio of the desalted alkaline phosphatase to SMCC is 1:10; optionally, the molar ratio of the desalted p-cresol sulfate antigen to 2-IT is 1:15; optionally, the molar ratio of the activated antigen to the activated alkaline phosphatase is 5:1; optionally, the room temperature reaction time is 30 min.

[0082] In this invention, the alkaline phosphatase (ALP) is a phosphatase widely found in living organisms that catalyzes the hydrolysis of various phosphate ester compounds, removing phosphate groups to generate inorganic phosphate and corresponding alcohols, phenols, and other products. This invention does not impose any particular limitation on the source of the alkaline phosphatase; those skilled in the art can obtain it through conventional purchasing channels.

[0083] In a specific embodiment of the present invention, alkaline phosphatase, as a key enzyme marker, is chemically coupled with p-cresol sulfate antigen to form an "enzyme-labeled antigen": In a competitive detection system, the enzyme-labeled antigen and p-cresol sulfate in the sample compete to bind to antibodies on immunomagnetic beads. The bound enzyme-labeled antigen can catalyze specific substrates (such as p-NPP, AMPPD, etc.) to produce color changes, fluorescence, or chemiluminescence signals. The signal intensity is inversely proportional to the concentration of p-cresol sulfate in the sample, thereby achieving quantitative detection of the target analyte.

[0084] In this invention, the SMCC (N-succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester) is a commonly used heterobifunctional crosslinking agent. Its molecular structure contains two reactive groups: N-hydroxysuccinimide ester (NHS ester) and a maleimide group. The NHS ester can undergo amidation with amino-containing molecules (such as lysine residues in proteins) under neutral pH conditions, while the maleimide group can specifically bind with thiol-containing molecules (such as proteins treated with reducing agents) to form thioether bonds under pH 6.5-7.5 conditions. This invention does not impose any particular limitation on the source of the SMCC; those skilled in the art can obtain it through conventional purchasing channels.

[0085] In a specific embodiment of the present invention, the SMCC is used to activate alkaline phosphatase (by reacting with the amino group of the enzyme), providing a reaction site for subsequent coupling with activated antigens containing thiol groups, thereby achieving directional cross-linking between the enzyme and the antigen. Furthermore, its cyclohexane structure can reduce steric hindrance and ensure the biological activity of the enzyme and the antigen after cross-linking, making it a key reagent for constructing enzyme-labeled antigens.

[0086] In this invention, 2-IT (2-iminothiacyclopentane) is a thiolating agent that reacts with amino-containing molecules (such as lysine residues of proteins and peptides) to introduce free thiol groups (-SH) onto the molecular surface. The reaction is characterized by high specificity, mild conditions (typically carried out in a neutral pH buffer), and the introduced thiol groups exhibit good stability, making them suitable for subsequent crosslinking with molecules containing maleimide, iodoacetyl, or other similar groups. This invention does not impose any particular restrictions on the source of the 2-IT; those skilled in the art can obtain it through conventional purchasing channels.

[0087] In a specific embodiment of the present invention, 2-IT is used to activate the p-cresol sulfate antigen (by introducing a thiol group through a reaction with the amino group of the antigen), enabling it to specifically bind to alkaline phosphatase (containing a maleimide group) activated by SMCC, thereby achieving efficient coupling between the antigen and the enzyme. It is an important reagent for conferring antigen reactivity in the preparation of enzyme-labeled antigens.

[0088] (5) A method for detecting p-cresol sulfate, wherein the test kit, test strip or test chip as described above is used to detect the sample to be tested;

[0089] Optionally, the detection method includes the following steps: sample processing, adding the immunomagnetic beads and enzyme-labeled antigen, incubation, washing, and detection.

[0090] In a specific embodiment of the present invention, the detection method includes the following steps: Sample processing: 50 μL of the sample to be tested is injected into the reaction system; Reagent addition: 30 μL of immunomagnetic beads and 30 μL of enzyme-labeled antigen are added, wherein the concentration of immunomagnetic beads is 0.15 mg / mL and the concentration of enzyme-labeled antigen is 1 μg / mL; Incubation: The mixed system is incubated for 420 s; Washing: After injecting the washing solution, centrifuge for 2 s, let stand for 1 s, then drain the washing solution for 10 s, let stand for 1 s, and repeat the washing once; Detection: Add substrate, centrifuge and inject the sample to detect the signal value, and calculate the concentration of p-cresol sulfate in the sample based on the reaction curve fitted by 4PLC. The reaction curve is calibrated using the high-concentration calibrator and low-concentration calibrator provided with the kit.

[0091] The reaction curve fitted by the 4PLC is traceable to the measurement program selected by the kit and is pre-built into the detection system.

[0092] This invention does not impose any particular limitation on the specific type of the test sample. The test sample is derived from clinical samples of subjects in need, including but not limited to: cells, tissues, and body fluids, such as skin; mucous membranes; blood; blood derivatives, such as serum; extracted bile; tissues obtained through biopsy or surgery, including, for example, unfixed, frozen, formalin-fixed, and / or paraffin-embedded tissues; tears; surface cleaning solutions; urine; sputum; cerebrospinal fluid; prostatic fluid; pus; bone marrow aspirate; middle ear effusion; bronchoalveolar lavage fluid; sputum or saliva. Any test sample required for the detection of p-cresol sulfate will fall within the scope of protection of this invention.

[0093] In some implementations, the recombinant vector can be introduced into host cells using techniques well known in the art. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses. For bacterial cells, suitable techniques may include calcium chloride conversion, electroporation, and transfection using bacteriophages. After introduction, nucleic acid expression may be induced or permitted, for example, by culturing host cells under conditions of gene expression.

[0094] Furthermore, the present invention also provides a method for detecting p-cresol sulfate or a method for real-time monitoring of the condition of patients with chronic renal failure. The method includes the following steps: contacting the antibody described in the first aspect of the present invention, the antibody-drug conjugate described in the fifth aspect of the present invention, the detection reagent, the test strip and / or the detection chip with a test sample derived from the subject, detecting whether p-cresol sulfate is present in the test sample derived from the subject or the level of p-cresol sulfate content in the test sample, thereby real-time monitoring of the condition of patients with chronic renal failure.

[0095] In this invention, the subject refers to any animal, preferably a mammal, including but not limited to: humans, higher primates, domestic and farm animals, as well as zoo animals, racing animals, or pets, such as horses, pigs, cattle, dogs, cats, and ferrets. In a specific embodiment of this invention, the subject is preferably a human.

[0096] The seventh aspect of the present invention provides for any of the following applications:

[0097] (1) The use of the antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the recombinant vector described in the third aspect of the present invention, and / or the recombinant host cell described in the fourth aspect of the present invention in the preparation of an antibody conjugate for the detection of p-cresol sulfate;

[0098] (2) The use of the antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the recombinant vector described in the third aspect of the present invention, the recombinant host cell described in the fourth aspect of the present invention, and / or the antibody conjugate described in the fifth aspect of the present invention in the preparation of a detection reagent for detecting p-cresol sulfate;

[0099] (3) The application of the antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the recombinant vector described in the third aspect of the present invention, the recombinant host cell described in the fourth aspect of the present invention, the antibody conjugate and / or detection reagent described in the fifth aspect of the present invention in the preparation of a test strip or detection chip for detecting p-cresol sulfate;

[0100] (4) The use of the antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the recombinant vector described in the third aspect of the present invention, the recombinant host cell described in the fourth aspect of the present invention, the antibody conjugate described in the fifth aspect of the present invention, the detection reagent, the test strip and / or the detection chip in the non-diagnostic and non-therapeutic target detection of p-cresol sulfate;

[0101] (5) The use of the antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the recombinant vector described in the third aspect of the present invention, the recombinant host cell described in the fourth aspect of the present invention, the antibody conjugate described in the fifth aspect of the present invention, the detection reagent, the test strip and / or the detection chip in the preparation of monitoring products or diagnostic products for real-time monitoring of the condition of patients with chronic renal failure.

[0102] (6) The use of the antibody conjugate, detection reagent, test strip, detection chip and / or detection kit described in the fifth aspect of the present invention in the detection of p-cresol sulfate.

[0103] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0104] This invention discloses a novel antibody for detecting p-cresol sulfate. The antibody can specifically bind to p-cresol sulfate and has high binding activity. The antibody can be used to quickly and accurately quantify the content of p-cresol sulfate in the sample through convenient methods such as ELISA and immunochromatography. It can not only meet the clinical needs for early screening of patients with chronic renal failure, but also dynamically track changes in metabolite levels during treatment, providing objective evidence for disease assessment and treatment plan adjustment, and has broad application prospects. Attached Figure Description

[0105] Figure 1 Correlation between tandem mass spectrometry results and chemiluminescence results;

[0106] Figure 2 Correlation comparison fitting graph. Detailed Implementation

[0107] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents.

[0108] The drugs, reagents, raw materials, and experimental consumables used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying particular conditions in this invention are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following examples are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0109] Example 1: Detection of the specificity of anti-p-cresol sulfate antibody against p-cresol sulfate.

[0110] 1. Experimental Methods

[0111] In previous studies, this application obtained an anti-p-cresol sulfate antibody through screening. The sequence information of the antibody is as follows:

[0112] Heavy chain variable region HCDR1 amino acid sequence: ALSGGISL (SEQ ID NO:1);

[0113] Heavy chain variable region HCDR2 amino acid sequence: LIESGGLR (SEQ ID NO:2);

[0114] Heavy chain variable region HCDR3 amino acid sequence: HLSCAARKYT (SEQ ID NO:3);

[0115] The amino acid sequence of the light chain variable region LCDR1: YYPSDFEGQKITY (SEQ ID NO:4);

[0116] The amino acid sequence of the light chain variable region LCDR2: ESSVSK (SEQ ID NO:5);

[0117] The amino acid sequence of the light chain variable region LCDR3: LVSFTAVYDSWA (SEQ ID NO:6);

[0118] Heavy chain variable region amino acid sequence: VKMLEVQLVVALSGGISLRVPLIESGGLRRGLYYPGLLKLFD CKHLSCAARKYTFSSKAPTLYAMQPGFSYAPGEYFEWVGISGSGQDFF (SEQ ID NO:7);

[0119] The amino acid sequence of the light chain variable region is: VSTYYGSSFDWKGRVYSRDLASWVRNRGADENTLYYPSD FEGQKITYYTGSMQMCARYDTVQWVSIGTNDQESSVSKLEPGFSLLYPDFSGLSALASAA WSSFTINPVDQNVDWRAEDSQDLVSFTAVYDSWAAVIFGVAGIDNSLYTGS (SEQ ID NO:8).

[0120] The specific detection methods are as follows:

[0121] Sample source: 20 clinical serum samples were collected from the Department of Nephrology, First Affiliated Hospital of Dalian Medical University. These samples were from patients with chronic renal failure.

[0122] The content of p-cresol sulfate in the above samples was determined by tandem mass spectrometry.

[0123] The above-mentioned antibodies were coated with magnetic beads using conventional methods, and alkaline phosphatase was labeled with p-cresol sulfate to form a chemiluminescent reagent kit to test the p-cresol sulfate content in the above samples.

[0124] The detection principle of the chemiluminescence reagent kit is as follows: Immunomagnetic beads are coated with p-cresol sulfate antibody through a biotin-streptavidin system, and p-cresol sulfate antigen is labeled with alkaline phosphatase. The antigen in the sample competes with the alkaline phosphatase-labeled antigen for binding to the antibody-coated magnetic beads. Unbound alkaline phosphatase-labeled antigen is washed away, and then the substrate is added to read the signal.

[0125] The chemiluminescence reagent kit consists of immunomagnetic beads, enzyme-labeled antibodies, and two concentration calibrators. The preparation process mainly includes the preparation of immunomagnetic beads and the preparation of enzyme-labeled antigens.

[0126] The preparation process of immunomagnetic beads is as follows:

[0127] (1) Antibody treatment (desalting): Centrifuge the Zeba desalting column at 1500g for 2 min, discard the waste liquid, and wash three times each time with the corresponding amount of 0.02M PBS (pH 7.4) at 1500g for 2 min, discard the waste liquid. Load the sample and recover the centrifuged liquid.

[0128] (2) Antibody binding to Sulfo-Biotin (Sulfo-NHS-Biotin): The antibody and Sulfo-Biotin were mixed in a certain ratio (molar ratio 1:20) and reacted in a shaker at room temperature for 30 min.

[0129] (3) Removal of free biotin (desalting): Centrifuge the Zeba desalting column at 1500g for 2 min, discard the waste liquid, and wash three times with the corresponding amount of 0.02M PBS (pH 7.4) at 1500g for 2 min each time, discarding the waste liquid. Load the sample and recover the centrifuged liquid.

[0130] (4) Binding of biotinylated antibody to streptavidin magnetic beads: Take 100 mL of streptavidin magnetic bead stock solution and place it in a 500 mL glass bottle. Add 200 mL of storage buffer and place the glass bottle on a magnet for 15 min. Discard the supernatant. Wash twice with 300 mL of storage buffer and resuspend in 300 mL of storage buffer.

[0131] Add a certain amount of antibody and react in a shaker at room temperature for 30 minutes. Add blocking buffer and block for 30 minutes.

[0132] (5) Removal of free biotinylated antibodies (magnetic separation)

[0133] Place the glass bottle on a magnet for 15 minutes, then discard the supernatant. Wash three times with 300 mL of storage buffer; resuspend in 300 mL of storage buffer.

[0134] The preparation process of enzyme-labeled antigen is as follows:

[0135] (1) Alkaline phosphatase treatment (desalination)

[0136] Centrifuge the Zeba desalting column at 1500g for 2 minutes, discard the waste liquid, and then wash three times with the corresponding amount of 0.02M PBS (pH 7.4) at 1500g for 2 minutes each time, discarding the waste liquid. Load the sample and recover the centrifuged liquid.

[0137] (2) Alkaline phosphatase binds to (activates) SMCC (N-succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester).

[0138] Alkaline phosphatase and SMCC were mixed in a certain ratio (molar ratio 1:10) and reacted in a shaker at room temperature for 30 minutes.

[0139] (3) Removal of free SMCC (desalination)

[0140] Centrifuge the Zeba desalting column at 1500g for 2 minutes, discard the waste liquid, and then wash three times with the corresponding amount of 0.02M PBS (pH 7.4) at 1500g for 2 minutes each time, discarding the waste liquid. Load the sample and recover the centrifuged liquid.

[0141] (4) Antigen treatment (desalting)

[0142] Centrifuge the Zeba desalting column at 1500g for 2 minutes, discard the waste liquid, and then wash three times with the corresponding amount of 0.02M PBS (pH 7.4) at 1500g for 2 minutes each time, discarding the waste liquid. Load the sample and recover the centrifuged liquid.

[0143] (5) The antigen binds to 2-IT (2-iminothiacyclopentane) (activation).

[0144] The antigen and 2-IT were mixed in a certain ratio (molar ratio 1:15) and reacted in a shaker at room temperature for 30 minutes.

[0145] (6) Removal of free 2-IT (desalination)

[0146] Centrifuge the Zeba desalting column at 1500g for 2 minutes, discard the waste liquid, and then wash three times with the corresponding amount of 0.02M PBS (pH 7.4) at 1500g for 2 minutes each time, discarding the waste liquid. Load the sample and recover the centrifuged liquid.

[0147] (7) Activation of antigen binding to alkaline phosphatase

[0148] Activated alkaline phosphatase was added to the activated antigen at a ratio of 5 mol: 1 mol. The mixture was added dropwise while stirring, and the reaction was carried out at room temperature on a shaker for 30 min.

[0149] The correlation between the measured values ​​and the results of tandem mass spectrometry was compared to verify the specificity of the antibody in detecting p-cresyl sulfate (PCS).

[0150] 2. Experimental Results

[0151] The experimental results are shown in Table 1 and Figure 1 As shown in Table 1, the results indicate that the chemiluminescence detection method based on the above antibody maintains high consistency with tandem mass spectrometry at different concentration levels, further demonstrating its specificity. That is, the antibody can specifically recognize p-cresol sulfate, is not significantly interfered with by other substances, and stably produces detection results consistent with the reference method. Figure 1 The results show that the R of the fitted line 2 =0.9913, indicating a high linear correlation between the results of the chemiluminescence detection method based on the above antibody and the results of the tandem mass spectrometry detection method. This means that the chemiluminescence detection method based on the above antibody can accurately reflect the true content of p-cresol sulfate in the sample, unaffected by interference from other substances. All these results demonstrate that the above antibody has high specificity for p-cresol sulfate, specifically binding to it and producing detection results highly consistent with accurate methods.

[0152] Table 1. Concentration values ​​of p-cresol sulfate in 20 clinical samples determined by tandem mass spectrometry and chemiluminescence immunoassay systems.

[0153]

[0154] Example 2: Detection of the affinity of anti-p-cresol sulfate antibody for p-cresol sulfate.

[0155] 1. Experimental Materials and Instruments

[0156] Antibody: The anti-p-cresol sulfate antibody described in Example 1.

[0157] Hapten: p-cresol sulfate.

[0158] Microdialysis system: CMA / 20 probe (membrane length 4 mm, molecular weight cutoff 10 kDa), perfusion solution: phosphate buffer (PBS, pH 7.4).

[0159] FI-CL system: flow injection apparatus + fully automated chemiluminescence immunoassay analyzer MI600, chemiluminescence reagent: APS-5.

[0160] 2. Experimental Methods

[0161] The affinity of anti-p-cresol sulfate antibodies for p-cresol sulfate was detected using a microdialysis-chemiluminescence immunoassay (MD-FI-CL). The specific experimental method is as follows:

[0162] (1) Antibody-hapten incubation

[0163] 1) Prepare gradient concentration hapten solutions

[0164] p-Cresol sulfate concentrations: 0, 0.1, 0.5, 1, 5, 10, 50, 100 nM (diluted with PBS containing 0.1% BSA).

[0165] 2) Incubation reaction

[0166] Take a fixed concentration of antibody (10 nM) and mix it with an equal volume of each gradient hapten (final volume 200 μL).

[0167] Incubate at 37°C with shaking for 30 minutes.

[0168] (2) Microdialysis separation of free haptens

[0169] 1) Microdialysis probe setup: flow rate: 2 μL / min, perfusion solution: PBS (pH 7.4).

[0170] 2) Sample dialysis: Inject the incubated sample into the microdialysis sample cell. Collect the dialysate (containing free hapten) for 30 minutes (60 μL per sample).

[0171] (3) Chemiluminescence detection of free hapten

[0172] Record the peak intensity of chemiluminescence (unit: RLU, relative luminescence unit).

[0173] 3. Experimental results

[0174] The test results are shown in Table 2. The results show that the anti-p-cresol sulfate antibody described in Example 1 still maintains a high binding rate (>78%) at low concentrations, indicating that it has a strong binding ability to p-cresol sulfate.

[0175] The affinity parameters are shown in Table 3. The results show that the anti-p-cresol sulfate antibody described in Example 1 has a good affinity for p-cresol sulfate.

[0176] The above results indicate that the anti-p-cresol sulfate antibody described in Example 1 possesses both strong binding ability and high affinity. It can bind to the target antigen (p-cresol sulfate) efficiently, stably, and tightly. Combined with the experimental results of Example 1, it is evident that it also has high specificity and can be used for the rapid and accurate detection of p-cresol sulfate.

[0177] Table 2 Detection Results

[0178] Total PCS added (nM) Measured amount of free PCS (nM) Antibody binding rate (%) 0.01 0.002 80.0 0.1 0.025 75.0 1 0.18 82.0 10 1.73 82.7 100 17.77 82.2

[0179] Table 3 Affinity parameters

[0180] parameter result unit Affinity constant (Ka) <![CDATA[2.7×10 8 ]]> M-1 Dissociation constant (Kd) <![CDATA[3.7×10 -9 ]]> M(3.7nM) <![CDATA[Linear fitting R 2 > 0.993

[0181] Example 3: A p-cresol sulfuric acid detection kit and its preparation method

[0182] 1. Detection principle of the kit

[0183] Immunomagnetic beads were coated with p-cresol sulfate antibody using a biotin-streptavidin system. P-cresol sulfate antigen was labeled with alkaline phosphatase. The antigen in the sample competed with the alkaline phosphatase-labeled antigen for binding to the antibody-coated beads. Unbound alkaline phosphatase-labeled antigen was removed by washing, and then substrate was added to read the signal. Concentration was calculated using a pre-built curve.

[0184] 2. Composition and preparation process of the reagent kit

[0185] The chemiluminescence reagent kit consists of immunomagnetic beads, enzyme-labeled antibodies, and two concentration calibrators. The preparation process mainly includes the preparation of immunomagnetic beads and the preparation of enzyme-labeled antigens.

[0186] (1) The preparation process of immunomagnetic beads is as follows:

[0187] 1) Antibody treatment (desalting): Centrifuge the Zeba desalting column at 1500g for 2 minutes, discard the waste liquid, and wash three times each time with the corresponding amount of 0.02M PBS (pH 7.4) at 1500g for 2 minutes, discarding the waste liquid. Load the sample and recover the centrifuged liquid.

[0188] 2) Antibody binding to Sulfo-Biotin (Sulfo-NHS-Biotin): The antibody and Sulfo-Biotin were mixed in a certain ratio (molar ratio 1:20) and reacted in a shaker at room temperature for 30 min.

[0189] 3) Removal of free biotin (desalting): Centrifuge the Zeba desalting column at 1500g for 2 min, discard the waste liquid, and wash three times each time with the corresponding amount of 0.02M PBS (pH 7.4) at 1500g for 2 min, discarding the waste liquid. Load the sample and recover the centrifuged liquid.

[0190] 4) Binding of biotinylated antibody to streptavidin magnetic beads: Take 100 mL of streptavidin magnetic bead stock solution and place it in a 500 mL glass bottle. Add 200 mL of storage buffer, place the glass bottle on a magnet for 15 min, and discard the supernatant. Wash twice with 300 mL of storage buffer; resuspend in 300 mL of storage buffer.

[0191] Add a certain amount of antibody and react in a shaker at room temperature for 30 minutes. Add blocking buffer and block for 30 minutes.

[0192] 5) Removal of free biotinylated antibodies (magnetic separation)

[0193] Place the glass bottle on a magnet for 15 minutes, then discard the supernatant. Wash three times with 300 mL of storage buffer; resuspend in 300 mL of storage buffer.

[0194] (2) The preparation process of enzyme-labeled antigen is as follows:

[0195] 1) Alkaline phosphatase treatment (desalination)

[0196] Centrifuge the Zeba desalting column at 1500g for 2 minutes, discard the waste liquid, and then wash three times with the corresponding amount of 0.02M PBS (pH 7.4) at 1500g for 2 minutes each time, discarding the waste liquid. Load the sample and recover the centrifuged liquid.

[0197] 2) Alkaline phosphatase binds to (activates) SMCC (N-succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester).

[0198] Alkaline phosphatase and SMCC were mixed in a certain ratio (molar ratio 1:10) and reacted in a shaker at room temperature for 30 minutes.

[0199] 3) Removal of free SMCC (desalination)

[0200] Centrifuge the Zeba desalting column at 1500g for 2 minutes, discard the waste liquid, and then wash three times with the corresponding amount of 0.02M PBS (pH 7.4) at 1500g for 2 minutes each time, discarding the waste liquid. Load the sample and recover the centrifuged liquid.

[0201] 4) Antigen treatment (desalting)

[0202] Centrifuge the Zeba desalting column at 1500g for 2 minutes, discard the waste liquid, and then wash three times with the corresponding amount of 0.02M PBS (pH 7.4) at 1500g for 2 minutes each time, discarding the waste liquid. Load the sample and recover the centrifuged liquid.

[0203] 5) The antigen binds to 2-IT (2-iminothiacyclopentane) (activation).

[0204] The antigen and 2-IT were mixed in a certain ratio (molar ratio 1:15) and reacted in a shaker at room temperature for 30 minutes.

[0205] 6) Removal of free 2-IT (desalination)

[0206] Centrifuge the Zeba desalting column at 1500g for 2 minutes, discard the waste liquid, and then wash three times with the corresponding amount of 0.02M PBS (pH 7.4) at 1500g for 2 minutes each time, discarding the waste liquid. Load the sample and recover the centrifuged liquid.

[0207] 7) Activated antigen binds to alkaline phosphatase

[0208] Activated alkaline phosphatase was added to the activated antigen at a ratio of 5 mol: 1 mol. The mixture was added dropwise while stirring, and the reaction was carried out at room temperature on a shaker for 30 min.

[0209] (3) The preparation process of the calibrator is as follows:

[0210] Two concentrations (100 ng / mL and 500 ng / mL) of p-cresol sulfate were diluted with fetal bovine serum and assigned values. The specific assignment method is as follows: each point on the main curve was measured three times, and the signal-dose four-parameter fitting was performed to obtain a four-parameter equation. Then, the two concentration calibrators were measured three times each in the morning and afternoon, and the concentration values ​​were calculated under the above curve.

[0211] (4) The reaction system is shown in Table 4 below:

[0212] Table 4 Reaction System

[0213]

[0214]

[0215] (5) Result Settlement

[0216] Reaction curve: Curve fitting is performed using 4PLC (four-parameter logic curve) and traced back to the selected measurement program of the product. The reaction curve can be built in first, and then calibrated using the high and low concentration calibrators provided with the reagent kit during application.

[0217] Example 4: Verification of the detection effect of the sulfuric acid-p-cresol detection kit described in Example 3

[0218] 1. Limit of detection test for p-cresol sulfuric acid detection kit

[0219] (1) Test method

[0220] The p-cresol sulfate content in 5 samples was determined using the p-cresol sulfate detection kit described in Example 3.

[0221] The five samples were clinical serum samples collected from the Department of Nephrology, First Affiliated Hospital of Dalian Medical University. These samples originated from patients with chronic renal failure.

[0222] (2) Test Results

[0223] The test results are shown in Table 5. The results show that the detection limit of the kit for p-cresol sulfate is ≤0.30 ng / mL. This result indicates that the kit has excellent detection sensitivity. Even if the content of p-cresol sulfate in the sample is as low as 0.30 ng / mL or below, it can still be detected stably. It can effectively avoid false negatives due to insufficient sensitivity, providing reliable detection guarantee for early disease screening, disease progression monitoring and other scenarios. It also proves the practical value of the kit in the detection of p-cresol sulfate.

[0224] Table 5 Results of detection limit test

[0225]

[0226]

[0227] 2. Accuracy test of the p-cresol sulfuric acid detection kit

[0228] (1) Test method

[0229] A high-concentration p-cresol sulfate sample A with a concentration of approximately 500 ng / mL was added to a low-concentration sample B (the volume of A added did not exceed 10% of the total volume (A+B)). Serum sample B and the mixed sample (A+B) were measured using the p-cresol sulfate detection kit described in Example 3, and each was measured 3 times.

[0230] (2) Test Results

[0231] The test results are shown in Table 6. The results show that the recovery rate of p-cresol sulfate sample (A) added to serum sample B was 107.39%, indicating that the kit has a stable ability to quantify the target in complex serum matrices and is less affected by matrix interference.

[0232] Table 6 Accuracy Test Results

[0233]

[0234] 3. Linearity test of the p-cresol sulfuric acid detection kit

[0235] (1) Test method

[0236] The p-cresol sulfate content in linear samples 1-11 was determined using the p-cresol sulfate detection kit described in Example 3.

[0237] The linear samples 1-11 were low-concentration samples (24.64 ng / mL) and high-concentration samples (1109.34 ng / mL) mixed in the following ratios: 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10.

[0238] Based on the test data, the linear results are evaluated, and the linear regression equations and linear correlation coefficients for the linear samples 1-11, 1-10, 1-9, and 1-8 are calculated respectively.

[0239] (2) Test Results

[0240] The test results are shown in Tables 7-10. Table 8 shows that the linear correlation coefficient (r) for the concentration range of 24.64-1109.34 ng / mL is greater than 0.9900. Table 10 shows that the linear correlation coefficient (r) for the concentration range of 27.57-1059.66 ng / mL is also greater than 0.9900. The results indicate that the kit exhibits excellent linear performance over a wide concentration range: for 11 linear samples covering concentrations of 24.64-1109.34 ng / mL, the correlation coefficient (r) of the linear regression equation reached 0.9992. Even with a gradual reduction in the concentration range, the correlation coefficient remained stable at 0.9989 or higher, indicating that within the set core detection range and a wider concentration range, the linear fit between the measured and theoretical concentrations of the samples is extremely high. The dose-response relationship shows a stable linear trend with no significant deviation, demonstrating that the kit can accurately quantify the content of p-cresol sulfate over a large concentration range and can meet the detection needs of samples at different concentration levels (from low to high concentrations).

[0241] Table 7 Test results of linear establishment

[0242]

[0243] Table 8 Evaluation of the results of linear establishment

[0244]

[0245]

[0246] Table 9 Test results of linear verification

[0247]

[0248] Table 10 Evaluation of the results of linear validation

[0249] Linear regression equation Linear correlation coefficient (r) Standard requirements Whether it meets the standards y = 1.0098x - 13.305 0.9995 r≥0.9900 Comply with standards

[0250] 4. Repeatability test of the p-cresol sulfuric acid detection kit

[0251] (1) Test method

[0252] The p-cresol sulfate content in samples with concentrations of (200±40) ng / mL and (500±100) ng / mL was determined using the p-cresol sulfate detection kit described in Example 3, with each test repeated 10 times.

[0253] The samples were clinical serum samples collected from the Department of Nephrology, First Affiliated Hospital of Dalian Medical University. The samples were derived from patients with chronic renal failure.

[0254] (2) Test Results

[0255] The test results are shown in Table 11. The results show that the coefficient of variation (CV) for repeatable sample CF1 is 1.01%, and the CV for repeatable sample CF2 is 0.91%, with the coefficient of variation (CV) of the obtained results not exceeding 8.0%. This indicates that under the same experimental conditions, when the kit is used to detect different concentrations of sulfuric acid and p-cresol samples multiple times, the results are highly consistent with minimal random error, and the kit can reliably and stably reproduce accurate detection data, meaning that the kit has excellent repeatability.

[0256] Table 11 Repeatability Test Results

[0257]

[0258]

[0259] 5. Inter-batch variation test of the p-cresol sulfuric acid detection kit

[0260] (1) Test method

[0261] The p-cresol sulfate content in samples with concentrations of (200±40) ng / mL and (500±100) ng / mL were determined using the p-cresol sulfate detection kit described in Example 3 in three batches. Each batch was tested 10 times, and the corresponding inter-batch coefficient of variation (CV) was obtained.

[0262] The samples were clinical serum samples collected from the Department of Nephrology, First Affiliated Hospital of Dalian Medical University. The samples were derived from patients with chronic renal failure.

[0263] (2) Test Results

[0264] The test results are shown in Table 12. The results show that the coefficient of variation (CV) for repeatability sample CF1 was 1.41%, and for repeatability sample CF2 it was 1.25%, with the inter-batch coefficient of variation (CV) not exceeding 10.0%. This indicates that even during testing across different batches, the kit maintains a high degree of consistency in the detection results for p-cresol sulfate samples, with minimal fluctuations in the detection values ​​between the two samples. This excellent inter-batch repeatability ensures the comparability of test results across different batches and avoids result deviations caused by batch differences.

[0265] Table 12 Test Results of Inter-batch Difference

[0266]

[0267] 6. Interference test of the sulfuric acid-p-cresol detection kit

[0268] (1) Test method

[0269] Add the interfering agent to the sample according to the concentrations in the table below. Repeat the measurement three times for each sample and calculate the deviation from the control sample (interfering agent concentration is 0).

[0270] (2) Test Results

[0271] The test results are shown in Table 13. The results show that when the relative deviations of the samples for triglycerides (≤2000mg / dL), bilirubin (≤13mg / dL), hemoglobin (≤0.56g / dL), total protein (≤12g / dL), biotin (≤1000000ng / mL), rheumatoid factor (≤600IU / mL), and human anti-mouse antibody (≤50ng / mL) are within ±10.0%, it indicates that the kit has excellent anti-interference ability.

[0272] Table 13 Test results of interference experiment

[0273]

[0274]

[0275]

[0276] 7. Correlation test of the p-cresol sulfuric acid detection kit

[0277] (1) Test method

[0278] Forty clinical samples were selected and simultaneously measured using this test kit and the comparison system (tandem mass spectrometry PCS measurement system), and correlation analysis was performed.

[0279] (2) Test Results

[0280] The test results are shown in Table 14. As shown in Table 16, the correlation coefficient r = 0.9805 and the slope is 0.9757. The detection results of the kit and the comparison method are highly consistent, indicating that the detection results of the kit can reliably reflect the true content of p-cresol sulfate in the sample and have good consistency with the mature comparison method (tandem mass spectrometry), further verifying the accuracy and reliability of the kit's detection results.

[0281] Table 14. Test results of correlation comparison

[0282]

[0283]

Claims

1. An antibody against p-cresol sulfate, characterized in that, The HCDR1-3 in the heavy chain variable region of the antibody is the HCDR1-3 in the heavy chain variable region as shown in SEQ ID NO:7; The LCDR1-3 in the light chain variable region of the antibody is the LCDR1-3 in the light chain variable region as shown in SEQ ID NO:

8.

2. The antibody according to claim 1, characterized in that, The amino acid sequences of HCDR1-3 in the heavy chain variable region of the antibody are shown in SEQ ID NO:1-3, respectively. The amino acid sequences of LCDR1-3 in the light chain variable region of the antibody are shown in SEQ ID NO:4-6, respectively.

3. The antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

7.

4. The antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody according to any one of claims 1-4.

6. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid molecule of claim 5; Optionally, the vector is a virus-derived vector, plasmid, and / or phage particle; Optionally, the vector from which the virus originates may be a lentiviral vector, adenovirus vector, adeno-associated virus vector, retroviral vector, poxvirus vector, herpesvirus vector, and / or baculovirus vector.

7. A recombinant host cell, characterized in that, The recombinant host cell comprises the recombinant vector according to claim 6; Optionally, the host cell may be a mammalian cell, plant cell, insect cell, fungal cell, and / or bacterial cell.

8. Any of the following products, characterized in that, The products include: (1) An antibody conjugate, wherein the antibody conjugate is a complex formed by directly or indirectly conjugating an antibody to a detectable marker as described in any one of claims 1-4; (2) A detection reagent comprising the antibody and / or the antibody conjugate according to any one of claims 1-4; (3) A test strip or test chip, wherein the test strip or test chip comprises the antibody, the antibody conjugate and / or the test reagent as described in any one of claims 1-4; (4) A p-cresol sulfate detection kit, wherein the detection kit comprises the antibody, the antibody conjugate and / or the detection reagent as described in any one of claims 1-4; Optionally, the test kit includes immunomagnetic beads, enzyme-labeled antigens, and calibrators; Optionally, the immunomagnetic beads are obtained by coating the antibodies with biotin-streptavidin. Optionally, the enzyme-labeled antigen is alkaline phosphatase-labeled p-cresol sulfate antigen; Optionally, the calibrator is a p-cresol sulfate standard solution.

9. The method described below, characterized in that, The method includes: (1) A method for producing the antibody according to any one of claims 1-4, the method comprising the steps of: culturing the recombinant host cell according to claim 7, and isolating the antibody according to any one of claims 1-4 from the recombinant host cell culture product; (2) A method for preparing the recombinant host cell of claim 7, the method comprising the following steps: introducing the recombinant vector of claim 6 into a host cell to obtain the recombinant host cell of claim 7; (3) A method for detecting p-cresol sulfate in a test sample for non-diagnostic and non-therapeutic purposes, the method comprising the following steps: contacting the test sample with the antibody of any one of claims 1-4, the antibody conjugate of claim 8, the detection reagent, the test strip or the detection chip to detect the formation of antigen-antibody immune complexes; (4) A method for preparing the p-cresol sulfuric acid detection kit as described in claim 8, the method comprising the following steps: 1) Immunomagnetic beads were obtained by coating the antibodies with biotin-streptavidin magnetic beads; 2) Alkaline phosphatase-labeled p-cresol sulfate antigen was obtained by labeling p-cresol sulfate with alkaline phosphatase; (5) A method for detecting p-cresol sulfuric acid, characterized in that the test kit, test strip or test chip described in claim 8 is used to detect the sample to be tested; Optionally, the detection method includes the following steps: sample processing, adding the immunomagnetic beads and enzyme-labeled antigen, incubation, washing, and detection.

10. The following application, characterized in that, The applications include: (1) The use of the antibody of any one of claims 1-4, the nucleic acid molecule of claim 5, the recombinant vector of claim 6, and / or the recombinant host cell of claim 7 in the preparation of an antibody conjugate for the detection of p-cresol sulfate; (2) The use of the antibody of any one of claims 1-4, the nucleic acid molecule of claim 5, the recombinant vector of claim 6, the recombinant host cell of claim 7, and / or the antibody conjugate of claim 8 in the preparation of a detection reagent for detecting p-cresol sulfate; (3) The use of the antibody of any one of claims 1-4, the nucleic acid molecule of claim 5, the recombinant vector of claim 6, the recombinant host cell of claim 7, the antibody conjugate and / or detection reagent of claim 8 in the preparation of a test strip or detection chip for detecting p-cresol sulfate; (4) The use of the antibody of any one of claims 1-4, the nucleic acid molecule of claim 5, the recombinant vector of claim 6, the recombinant host cell of claim 7, the antibody conjugate of claim 8, the detection reagent, the test strip and / or the detection chip in non-diagnostic and non-therapeutic purposes for the detection of p-cresol sulfate; (5) The use of any one of the antibodies of claims 1-4, the nucleic acid molecules of claim 5, the recombinant vectors of claim 6, the recombinant host cells of claim 7, the antibody conjugates of claim 8, the detection reagents, the test strips and / or the detection chips in the preparation of monitoring products or diagnostic products for real-time monitoring of the condition of patients with chronic renal failure; (6) The use of the antibody conjugate, detection reagent, detection kit, test strip or detection chip as described in claim 8 in the detection of p-cresol sulfate.

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