Antibody for detecting indoxyl sulfate, detection kit and application thereof

Abstract

The invention discloses an antibody for detecting indol sulfate, a detection kit and application of the antibody, and the antibody can be specifically combined with indol sulfate and has high combination activity. The detection kit detects the content of indoxyl sulfate in a to-be-detected sample through a competitive binding reaction of an immunomagnetic bead coated indoxyl sulfate antibody, an antigen in the sample and an alkaline phosphatase labeled indoxyl sulfate antigen, and has important economic value and social significance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to an antibody for detecting indophenol sulfate, a detection kit, and their uses. Background Technology

[0002] Indoxyl sulfate (IS) is a key uremic toxin produced by the metabolism of tryptophan by gut microbiota. Its accumulation in the blood is closely related to the progression of chronic kidney disease (CKD), the risk of cardiovascular complications, and gut microbiota imbalance. As an important biomarker reflecting gut-kidney axis dysfunction, changes in IS levels can provide early indications of the degree of kidney damage and offer important evidence for risk assessment of metabolic-related diseases. Traditional detection methods, such as high-performance liquid chromatography (HPLC), can achieve accurate quantification, but they rely on specialized laboratory equipment, have cumbersome procedures, and long detection cycles, making it difficult to meet the practical needs of rapid screening in primary healthcare, point-of-care testing, or large-scale population health monitoring. Therefore, the development of simple and efficient detection technologies has become an urgent need in clinical and research fields.

[0003] Current detection products for indolesulfonate still face several technical bottlenecks: some methods lack sufficient sensitivity, failing to accurately capture minute fluctuations in trace amounts of indolesulfonate in the early stages of disease or during treatment; specificity is a significant issue, easily cross-reacting with structural analogs such as indoleacetic acid, leading to result bias; simultaneously, interference from the sample matrix (such as proteins and lipids in blood) often reduces detection stability, affecting data reliability. To address these pain points, the development of new detection technologies needs to focus on improving specificity and sensitivity. For example, immunoassay systems based on high-affinity antibodies can reduce cross-reactions through specific antigen-antibody binding; combined with signal amplification technology adapted for small devices, this can reduce dependence on large instruments and achieve rapid quantification. Such innovative solutions will provide more accurate and convenient tools for the early diagnosis, treatment monitoring, and gut microbiota metabolism research of chronic kidney disease, possessing significant clinical application value and social significance. Summary of the Invention

[0004] In order to solve the above-mentioned technical problems existing in the field, the purpose of this invention is to provide an antibody, a detection kit and its use for detecting indophenol sulfate.

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

[0006] In a first aspect, the present invention provides an antibody against indophenol sulfate, wherein 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;

[0007] 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.

[0008] 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;

[0009] 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.

[0010] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7.

[0011] Furthermore, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.

[0012] In some embodiments, antibodies corresponding to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that have at least 70% homology with the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are also included within the scope of protection of this invention.

[0013] Among them, 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 some embodiments, the amino acid sequences corresponding to HCDR1, HCDR2, and HCDR3 of the present invention are not limited to the amino acid sequences described above, nor are the amino acid sequences corresponding to LCDR1, LCDR2, and LCDR3 of the present invention limited to the amino acid sequences described above. 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 as shown in SEQ ID NO:7 and CDR1, CDR2, and CDR3 in the light chain variable region as 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.

[0015] In specific implementation schemes, 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 sequences of the antibodies corresponding to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 defined by the above methods are also included within the scope of protection of this invention.

[0016] Secondly, the present invention provides a nucleic acid molecule encoding the antibody described in the first aspect of the present invention.

[0017] 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. The nucleic acid molecule described in this invention can 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.

[0018] Thirdly, the present invention provides an expression vector comprising the nucleic acid molecule described in the second aspect of the present invention;

[0019] Optionally, the vector is a plasmid, a virus-derived vector, a phage particle, a granule, or an artificial chromosome;

[0020] 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, or baculovirus vector.

[0021] In this invention, there are no particular limitations on the vectors expressing the coding sequences of the antibodies described above, including but not limited to: microbial organisms such as recombinant bacteriophages, bacteria transformed by plasmid or copious DNA expression vectors; yeast transformed by yeast expression vectors; insect cell systems transformed by viral expression vectors (such as baculoviruses); plant cells transformed by 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 the Director™ plasmid from Sigma-Aldrich. For methanogens, useful plasmids include, but are not limited to, pME2001, pMV15, and pMP1.

[0022] Fourthly, the present invention provides a host cell comprising the expression vector described in the third aspect of the present invention;

[0023] Optionally, the host cell may be a mammalian cell, plant cell, insect cell, fungal cell, and / or bacterial cell.

[0024] 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.

[0025] Fifthly, the present invention provides any of the following products:

[0026] (1) An antibody derivative, wherein the antibody derivative is an antibody derivative obtained by conjugation or conjugation of an antibody with a diagnostic agent as described above;

[0027] (2) A detection reagent comprising the antibody and / or the antibody derivative as described above;

[0028] (3) An indophenol sulfate detection product, the detection product comprising the antibody as described above, the antibody derivative and / or the detection reagent;

[0029] Optionally, the detection product is a test kit, a test strip, or a detection chip;

[0030] Optionally, the test kit includes immunomagnetic beads, enzyme-labeled antigens, and calibrators;

[0031] Optionally, the immunomagnetic beads are obtained by coating the antibodies with biotin-streptavidin.

[0032] Optionally, the enzyme-labeled antigen is alkaline phosphatase-labeled indophenol sulfate antigen;

[0033] Optionally, the calibrator is an indophenol sulfate standard solution.

[0034] In this invention, the indolesulfonate detection kit is based on the competitive immunoassay principle. Indolesulfonate antibodies are coated onto the surface of immunomagnetic beads using a biotin-streptavidin system, while indolesulfonate antigen is labeled with alkaline phosphatase. During detection, indolesulfonate in the sample competes with the alkaline phosphatase-labeled indolesulfonate 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 concentration of indolesulfonate in the sample) is used to calculate the concentration of indolesulfonate in the sample by combining the reaction curve fitted by 4PLC (calibrated using high and low concentration calibrators in the kit).

[0035] In some embodiments, the indophenol sulfate detection kit further includes the following auxiliary reagents and consumables to ensure the integrity and stability of the detection process: substrate solution (such as chemiluminescent substrate (AMPPD) or colorimetric substrate (p-NPP)), washing solution (such as a solution containing buffer and surfactant), sample diluent (such as PBS containing fetal bovine serum), blocking solution, reaction buffer, instruction manual and quality control documents, and consumables.

[0036] In some embodiments, the diagnostic agents include, but are not limited to: bioluminescent agents, chemiluminescent agents, paramagnetic ions, radionuclides, enzymes, and photosensitizing diagnostic agents. Any reagent capable of being conjugated or coupled to the antibody described above for the detection of indophenol sulfate is within the scope of this invention.

[0037] In some embodiments, the antibody can be recovered and purified from recombinant cell cultures using methods known in the art, including but not limited to: ammonium sulfate or ethanol precipitation, acid extraction, protein A affinity chromatography, protein G affinity chromatography, anion or cation exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High-performance liquid chromatography (HPLC) can be used for purification.

[0038] The antibodies described in this invention include naturally purified products, products synthesized by chemical methods, and products generated from prokaryotic and eukaryotic hosts through recombinant technology. These eukaryotic hosts include, but are not limited to, yeast, higher plants, insects, and mammals. The antibodies described in this invention can be glycosylated or non-glycosylated. Such methods are described in many standard laboratory manuals.

[0039] Sixthly, the present invention provides any of the following methods:

[0040] (1) A method for preparing the antibody according to the first aspect of the present invention, the method comprising the following steps: culturing the host cells as described above, and separating and recovering the antibody;

[0041] (2) A method for preparing the host cell according to the fourth aspect of the present invention, the method comprising the following steps: introducing the expression vector as described above into the host cell to obtain the host cell as described above;

[0042] (3) A method for detecting indophenol sulfate for non-diagnostic and non-therapeutic purposes, the method comprising the steps of: contacting the sample to be tested with the antibody, antibody derivative, detection reagent or detection product as described above, and detecting the formation of antigen-antibody immune complexes;

[0043] (4) A method for preparing the indolesulfate detection kit described in the fifth aspect of the present invention, the method comprising the following steps: obtaining immunomagnetic beads by coating the antibody with biotin-streptavidin magnetic beads; obtaining alkaline phosphatase-labeled indolesulfate antigen by labeling indolesulfate with alkaline phosphatase.

[0044] In some embodiments, the present invention does not particularly limit the test sample, which is derived from clinical samples of subjects in need, including but not limited to: cells, tissues, body fluids, such as: skin; mucous membranes; blood; blood derivatives, such as serum; extracted bile; tissues obtained from biopsy or surgery, including, for example, unfixed, frozen, formalin-fixed and / or paraffin-embedded tissues; tears; breast milk; skin flakes; surface cleaning solutions; urine; sputum; cerebrospinal fluid; prostatic fluid; pus; bone marrow aspirate; middle ear effusion; bronchoalveolar lavage fluid; sputum or saliva.

[0045] In some implementations, the subjects include, but are not limited to, humans, birds, and non-human mammals. For example, the birds include, but are not limited to, chickens, ducks, geese, quails, wild geese, pigeons, peacocks, and partridges, and the non-human mammals include, but are not limited to, pigs, cats, dogs, minks, foxes, seals, sea lions, and rats.

[0046] In a seventh aspect, the present invention provides applications in any of the following aspects:

[0047] (1) The use of the antibodies, nucleic acid molecules, expression vectors and / or host cells as described above in the preparation of antibody derivatives for the detection of indophenol sulfate;

[0048] (2) The use of the antibodies, nucleic acid molecules, expression vectors, host cells and / or antibody derivatives as described above in the preparation of a detection reagent for the detection of indophenol sulfate;

[0049] (3) The use of the antibodies, nucleic acid molecules, expression vectors, host cells, antibody derivatives and / or detection reagents as described above in the preparation of detection products for detecting indophenol sulfate;

[0050] (4) The use of the antibodies, nucleic acid molecules, expression vectors, host cells, antibody derivatives, detection reagents and / or detection products as described above in the detection of indophenol sulfate for non-diagnostic and non-therapeutic purposes;

[0051] (5) The use of the antibodies, nucleic acid molecules, expression vectors, host cells, antibody derivatives, detection reagents and / or detection products as described above in the preparation of monitoring or diagnostic products for real-time monitoring of the condition of patients with chronic renal failure.

[0052] (6) The use of antibody derivatives, detection reagents or detection products as described above in the detection of indophenol sulfate;

[0053] Optionally, the detection product is a test kit, a test strip, or a detection chip.

[0054] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0055] This invention discloses a novel antibody that specifically binds to indolesulfonate, exhibiting high binding activity for indolesulfonate. Furthermore, this invention provides a highly specific, sensitive, and cost-effective indolesulfonate detection kit. This kit detects the indolesulfonate content in the sample through a competitive binding reaction between the indolesulfonate antibody coated with immunomagnetic beads and the antigen and alkaline phosphatase-labeled indolesulfonate antigen in the sample. This kit has significant economic and social value. Attached Figure Description

[0056] Figure 1 Correlation between tandem mass spectrometry results and chemiluminescence results;

[0057] Figure 2 Correlation comparison fitting graph. Detailed Implementation

[0058] 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.

[0059] 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.

[0060] Example 1: Detection of the specificity of anti-indophenol sulfate antibody against indophenol sulfate.

[0061] 1. Experimental Methods

[0062] In previous studies, this application obtained an anti-indophenol sulfate antibody through screening. The sequence information of the antibody is as follows:

[0063] Heavy chain variable region HCDR1 amino acid sequence: KKPTYSMHWV (SEQ ID NO:1);

[0064] The amino acid sequence of the heavy chain variable region HCDR2: MGWIQRLTTTYAK (SEQ ID NO:2);

[0065] Heavy chain variable region HCDR3 amino acid sequence: AVNYVARV (SEQ ID NO:3);

[0066] The amino acid sequence of the light chain variable region LCDR1: GMVKNMKSVKL (SEQ ID NO:4);

[0067] The amino acid sequence of the light chain variable region LCDR2: TCAYATDFKE (SEQ ID NO:5);

[0068] The amino acid sequence of the light chain variable region LCDR3 is: HDTWGLYWGQFAG (SEQ ID NO:6);

[0069] Heavy chain variable region amino acid sequence: QLVVQASGKVAEVGASASVSCKGYKKPTYSMHWVRDPGQ GRWNTYMGWIQRLTTTYAKDFMAKFTGYRDEPKRTNRLFTDQSTFTDDTAVNYVARVRD HAALWFAYWEQGTLVGVVTVSS (SEQ ID NO:7);

[0070] The amino acid sequence of the light chain variable region is: QSRLVQVQAESGMVKNMKSVKLRCKAVSSDSGLRYQYYL MHAPGYQIWAAVQGLEYYVRWWNGYGIGPSKDTCAYATDFKETRFTPNIRYAEENARVR TLDHDTWGLYWGQFAGGSGTLS (SEQ ID NO:8).

[0071] The specific detection methods are as follows:

[0072] 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.

[0073] The content of indophenol sulfate in the above samples was determined by tandem mass spectrometry.

[0074] The above-mentioned antibodies were coated with magnetic beads using conventional methods, and alkaline phosphatase was labeled with indophenol sulfate to form a chemiluminescent reagent kit to test the content of indophenol sulfate in the above samples.

[0075] The detection principle of the chemiluminescence reagent kit is as follows: Immunomagnetic beads are coated with indolesulfonate antibody through a biotin-streptavidin system, and indolesulfonate 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 removed by washing, and then the substrate is added to read the signal.

[0076] 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.

[0077] The preparation process of immunomagnetic beads is as follows:

[0078] (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.

[0079] (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.

[0080] (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.

[0081] (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.

[0082] 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.

[0083] (5) Removal of free biotinylated antibodies (magnetic separation)

[0084] 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.

[0085] The preparation process of enzyme-labeled antigen is as follows:

[0086] (1) Alkaline phosphatase treatment (desalting)

[0087] 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.

[0088] (2) Alkaline phosphatase binds to (activates) SMCC (N-succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester).

[0089] 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.

[0090] (3) Removal of free SMCC (desalination)

[0091] 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.

[0092] (4) Antigen treatment (desalting)

[0093] 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.

[0094] (5) The antigen binds to 2-IT (2-iminothiacyclopentane) (activation).

[0095] 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.

[0096] (6) Removal of free 2-IT (desalination)

[0097] 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.

[0098] (7) Activation of antigen binding to alkaline phosphatase

[0099] 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.

[0100] The correlation between the measured values ​​and the results of tandem mass spectrometry was compared to verify the specificity of the antibody in detecting indoxyl sulfate (IS).

[0101] 2. Experimental Results

[0102] 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 indophenol 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 =0.9922, 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 indophenol sulfate in the sample, unaffected by interference from other substances. All these results demonstrate that the above antibody has high specificity for indophenol sulfate, can specifically bind to indophenol sulfate, and produces detection results highly consistent with accurate methods.

[0103] Table 1. Concentration values ​​of indolesulfonate in 20 clinical samples determined by tandem mass spectrometry and chemiluminescence immunoassay systems.

[0104]

[0105] Example 2: Detection of the affinity of the anti-indophenol sulfate antibody for indophenol sulfate.

[0106] 1. Experimental Materials and Instruments

[0107] Antibody: The anti-indophenol sulfate antibody described in Example 1.

[0108] Hapten: Indophenol sulfate.

[0109] Microdialysis system: CMA / 20 probe (membrane length 4 mm, molecular weight cutoff 10 kDa), perfusion solution: phosphate buffer (PBS, pH 7.4).

[0110] FI-CL system: flow injection apparatus + fully automated chemiluminescence immunoassay analyzer MI600, chemiluminescence reagent: APS-5.

[0111] 2. Experimental methods

[0112] The affinity of anti-indophenol sulfate antibody for indophenol sulfate was detected using a microdialysis-chemiluminescence immunoassay (MD-FI-CL). The specific experimental method is as follows:

[0113] (1) Antibody-hapten incubation

[0114] 1) Prepare gradient concentration hapten solutions

[0115] Indophenol sulfate concentrations: 0.1, 10, 20, 50, 100 nM (diluted with PBS containing 0.1% BSA).

[0116] 2) Incubation reaction

[0117] Take a fixed concentration of antibody (10 nM) and mix it with an equal volume of each gradient hapten (final volume 200 μL).

[0118] Incubate at 37°C with shaking for 30 minutes.

[0119] (2) Microdialysis separation of free haptens

[0120] 1) Microdialysis probe setup: flow rate: 2 μL / min, perfusion solution: PBS (pH 7.4).

[0121] 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).

[0122] (3) Chemiluminescence detection of free hapten

[0123] Record the peak intensity of chemiluminescence (unit: RLU, relative luminescence unit).

[0124] 3. Experimental Results

[0125] The test results are shown in Table 2. The results show that the anti-indophenol sulfate antibody described in Example 1 still maintains a high binding rate (>85%) at low concentrations, indicating that it has a strong binding ability to indophenol sulfate.

[0126] The affinity parameters are shown in Table 3. The results show that the anti-indophenol sulfate antibody described in Example 1 has a good affinity for indophenol sulfate.

[0127] The above results indicate that the anti-indolesulfate antibody described in Example 1 possesses both strong binding ability and high affinity. It can bind to the target antigen (indolesulfate) 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 indolesulfate.

[0128] Table 2 Detection Results

[0129]

[0130]

[0131] Table 3 Affinity parameters

[0132] parameter result unit Affinity constant (Ka) <![CDATA[3.82×10 8 ]]> M-1 Dissociation constant (Kd) <![CDATA[2.94×10 -9 ]]> M(2.9nM) <![CDATA[Linear fitting R 2 > 0.995

[0133] Example 3: A reagent kit for detecting indophenol sulfate and its preparation method

[0134] 1. Detection principle of the kit

[0135] Immunomagnetic beads were coated with indolesulfonate antibody using a biotin-streptavidin system. Indolesulfonate 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.

[0136] 2. Composition and preparation process of the reagent kit

[0137] 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.

[0138] (1) The preparation process of immunomagnetic beads is as follows:

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 5) Removal of free biotinylated antibodies (magnetic separation)

[0145] 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.

[0146] (2) The preparation process of enzyme-labeled antigen is as follows:

[0147] 1) Alkaline phosphatase treatment (desalination)

[0148] 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.

[0149] 2) Alkaline phosphatase binds to (activates) SMCC (N-succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester).

[0150] 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.

[0151] 3) Removal of free SMCC (desalination)

[0152] 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.

[0153] 4) Antigen treatment (desalting)

[0154] 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.

[0155] 5) The antigen binds to 2-IT (2-iminothiacyclopentane) (activation).

[0156] 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.

[0157] 6) Removal of free 2-IT (desalination)

[0158] 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.

[0159] 7) Activated antigen binds to alkaline phosphatase

[0160] 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.

[0161] (3) The preparation process of the calibrator is as follows:

[0162] Indophenol sulfate was diluted with fetal bovine serum to two concentrations, and values ​​were assigned to them. 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.

[0163] (4) The reaction system is shown in Table 4 below:

[0164] Table 4 Reaction System

[0165]

[0166]

[0167] (5) Result Settlement

[0168] Reaction curve: Curve fitting was performed using a 4PLC (four-parameter logic curve) and traced back to the selected measurement program. A built-in reaction curve can be used initially, and then calibrated using the high and low concentration calibrators provided with the kit during application. Example 4: Verification of the detection effect of the indophenol sulfate detection kit described in Example 3.

[0169] 1. Blank limit test of the indophenol sulfate detection kit

[0170] (1) Test method

[0171] The content of indolesulfonate in 20 samples was determined using the indolesulfonate detection kit described in Example 3.

[0172] The 20 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.

[0173] (2) Test Results

[0174] The test results are shown in Table 5. The results show that the blank limit of the kit for indophenol sulfate is ≤0.02 ng / mL. This result indicates that the kit has excellent detection sensitivity. Even if the content of indophenol sulfate in the sample is as low as 0.02 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 indophenol sulfate.

[0175] Table 5. Blank Limit Test Results

[0176]

[0177]

[0178] 2. Accuracy test of the indophenol sulfate detection kit

[0179] (1) Test method

[0180] A high-concentration indophenol sulfate sample A 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 indophenol sulfate detection kit described in Example 3, and each was measured 3 times.

[0181] (2) Test Results

[0182] The test results are shown in Tables 6 and 7. The results show that the recovery rate of indophenol sulfate sample (A) added to serum sample B was 103.99%, indicating that the kit's ability to quantify the target analyte in complex serum matrices is stable and minimally affected by matrix interference. The deviations of accuracy control ZQ1 were -1.37%, 0.09%, and -1.59%, and the deviations of accuracy control ZQ2 were -2.18%, -1.39%, and -1.89%, indicating that the kit has high detection accuracy.

[0183] Table 6 Accuracy Test Results

[0184]

[0185]

[0186] Table 7. Accuracy and Correctness Control Item Test Results

[0187]

[0188] 3. Linearity test of the indophenol sulfate detection kit

[0189] (1) Test method

[0190] The content of indophenol sulfate in linear samples 1-11 was determined using the indophenol sulfate detection kit described in Example 3.

[0191] 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.

[0192] (2) Test Results

[0193] The test results are shown in Tables 8-11. Table 9 shows that the linear correlation coefficient r for the range of 0.003-421.931 ng / mL is greater than 0.9900. Table 11 shows that the linear correlation coefficient r for the range of 0.004-421.811 ng / mL is also greater than 0.9900. The results indicate that the kit exhibits excellent linear performance over a wide concentration range. Within the set core detection interval and a wider concentration range, the linear fit between the measured and theoretical concentrations is extremely high, and the dose-response relationship shows a stable linear trend without significant deviation. This demonstrates that the kit can accurately quantify the content of indophenol sulfate over a large concentration range and can meet the detection needs of samples at different concentration levels (from low to high concentrations).

[0194] Table 8 Test results of linear establishment

[0195]

[0196]

[0197] Table 9 Evaluation of the results of linear establishment

[0198]

[0199] Table 10 Test results of linear verification

[0200]

[0201]

[0202] Table 11 Evaluation of the results of linear validation

[0203] Linear regression equation Linear correlation coefficient (r) Standard requirements Does it meet the standards? y = 0.9945x + 7.8111 0.9992 r≥0.9900 Meets standards

[0204] 4. Repeatability test of the indophenol sulfate detection kit

[0205] (1) Test method

[0206] The indolesulfonate content in samples with concentrations of (15±3) ng / mL and (100±20) ng / mL was determined using the indolesulfonate detection kit described in Example 3, with each test repeated 10 times.

[0207] 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.

[0208] (2) Test Results

[0209] The test results are shown in Table 12. The results show that the coefficient of variation (CV) for repeatable sample CF1 was 1.83%, and for repeatable sample CF2 it was 1.10%, with the coefficient of variation (CV) of the results not exceeding 8.0%. This indicates that under the same experimental conditions, when the kit is used to detect different concentrations of indophenol sulfate samples multiple times, the results are highly consistent with minimal random error, and the kit can reliably and stably reproduce accurate detection data, demonstrating excellent repeatability.

[0210] Table 12 Results of Repeatability Tests

[0211]

[0212]

[0213] 5. Inter-batch variation test of the indophenol sulfate detection kit

[0214] (1) Test method

[0215] The indolesulfonate assay kit described in Example 3 was used to determine the indolesulfonate content in samples with concentrations of (15±3) ng / mL and (100±20) ng / mL, respectively, in three batches. Each batch was tested 10 times to obtain the corresponding inter-batch coefficient of variation (CV).

[0216] 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.

[0217] (2) Test Results

[0218] The test results are shown in Table 13. The results show that the coefficient of variation (CV) for repeatability sample CF1 was 1.93%, and for repeatability sample CF2 it was 1.85%, 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 of indophenol 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.

[0219] Table 13 Test Results of Inter-batch Difference

[0220]

[0221]

[0222] 6. Specificity test of the indophenol sulfate detection kit

[0223] (1) Test method

[0224] Specificity tests were performed using nonspecific neuronal enolase (NNE), cytokeratin 19 fragment (CYFRA21-1), and squamous cell carcinoma antigen (SCCA).

[0225] (2) Test Results

[0226] The test results are shown in Table 14. The results show that the specificity test results are all ≤0.02ng / mL, indicating that the kit has high specificity.

[0227] Table 14 Specificity Test Results

[0228]

[0229]

[0230] 7. Interference test of the indophenol sulfate detection kit

[0231] (1) Test method

[0232] 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).

[0233] (2) Test Results

[0234] The test results are shown in Table 15. The results show that when the samples contain triglycerides (≤2000mg / dL), bilirubin (≤72mg / dL), total protein (≤10g / dL), biotin (≤1000000ng / mL), rheumatoid factor (≤1000IU / mL), and human anti-mouse antibody (≤50ng / mL), the relative deviation of the test is within ±10%, indicating that the kit has excellent anti-interference ability.

[0235] Table 15 Test results of interference experiment

[0236]

[0237]

[0238]

[0239] 8. Correlation test of the indophenol sulfate detection kit

[0240] (1) Test method

[0241] Clinical samples were selected and simultaneously tested using this test kit and the comparison system, and correlation analysis was performed.

[0242] (2) Test Results

[0243] The test results are shown in Table 16. The results show that the correlation coefficient r = 0.9938 and the slope is 1.0632. 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 indophenol sulfate in the sample and have good consistency with the mature comparison method, further verifying the accuracy and reliability of the kit's detection results.

[0244] Table 16. Test results of correlation comparison

[0245]

[0246]

Claims

1. An antibody against indophenol 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 encoding the antibody according to any one of claims 1-4.

6. An expression vector comprising the nucleic acid molecule of claim 5; Optionally, the vector is a plasmid, a virus-derived vector, a phage particle, a granule, or an artificial chromosome; 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, or baculovirus vector.

7. A host cell comprising the expression vector of 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: (1) An antibody derivative, wherein the antibody derivative is an antibody derivative obtained by conjugation or conjugation of the antibody according to any one of claims 1-4 with a diagnostic agent; (2) A detection reagent comprising the antibody and / or the antibody derivative of any one of claims 1-4; (3) An indophenol sulfate detection product, wherein the detection product comprises the antibody, the antibody derivative and / or the detection reagent as described in any one of claims 1-4; Optionally, the detection product is a test kit, a test strip, or a detection chip; 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 indophenol sulfate antigen; Optionally, the calibrator is an indophenol sulfate standard solution.

9. Any of the following methods: (1) A method for preparing the antibody according to any one of claims 1-4, the method comprising the following steps: culturing the host cell according to claim 7, and separating and recovering the antibody according to any one of claims 1-4; (2) A method for preparing the host cell of claim 7, the method comprising the following steps: introducing the expression vector of claim 6 into the host cell to obtain the host cell of claim 7; (3) A method for detecting indophenol sulfate for non-diagnostic and non-therapeutic purposes, the method comprising the following steps: contacting the sample to be tested with the antibody of any one of claims 1-4, the antibody derivative of claim 8, the detection reagent or the detection product, and detecting the formation of antigen-antibody immune complexes; (4) A method for preparing the indolesulfate detection kit of claim 8, the method comprising the following steps: obtaining immunomagnetic beads by coating the antibody with biotin-streptavidin magnetic beads; obtaining alkaline phosphatase-labeled indolesulfate antigen by labeling indolesulfate with alkaline phosphatase.

10. Applications in any of the following aspects: (1) The use of the antibody of any one of claims 1-4, the nucleic acid molecule of claim 5, the expression vector of claim 6, and / or the host cell of claim 7 in the preparation of antibody derivatives for the detection of indophenol sulfate; (2) The use of the antibody of any one of claims 1-4, the nucleic acid molecule of claim 5, the expression vector of claim 6, the host cell of claim 7, and / or the antibody derivative of claim 8 in the preparation of a detection reagent for detecting indophenol sulfate; (3) The use of the antibody of any one of claims 1-4, the nucleic acid molecule of claim 5, the expression vector of claim 6, the host cell of claim 7, the antibody derivative of claim 8, and / or the detection reagent in the preparation of a detection product for detecting indophenol sulfate; (4) The use of the antibody of any one of claims 1-4, the nucleic acid molecule of claim 5, the expression vector of claim 6, the host cell of claim 7, the antibody derivative of claim 8, the detection reagent and / or detection product in the detection of indophenol sulfate for non-diagnostic and non-therapeutic purposes; (5) The use of any one of the antibodies of claims 1-4, the nucleic acid molecule of claim 5, the expression vector of claim 6, the host cell of claim 7, the antibody derivative of claim 8, the detection reagent and / or the detection product in the preparation of a monitoring product or diagnostic product for real-time monitoring of the condition of patients with chronic renal failure; (6) The use of the antibody derivative, detection reagent or detection product as described in claim 8 in the detection of indophenol sulfate; Optionally, the detection product is a test kit, a test strip, or a detection chip.

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