Antibody detection kit for PD-1 biological agent and preparation method thereof

Abstract

The present invention discloses an antibody detection kit for PD-1 biological preparations and a preparation method thereof, and relates to the technical field of antibody detection. The kit includes an antibody capture device and an antibody detection device, the antibody capture device includes a solid phase carrier coated with a PD-1 biological preparation, and the antibody detection device is a cellulose membrane for fixing functional fragments or a conjugate coupled with functional fragments; the functional fragments are variable regions, heavy chain variable regions or complementary determining regions of PD-1 biological preparations. The detection kit provided by the present invention has the advantages of high capture efficiency, high accuracy and high sensitivity, can effectively avoid the occurrence of false positives and false negatives, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody detection, and in particular to an antibody detection kit for PD-1 biological preparations and a preparation method thereof. Background Art

[0002] PD-1 (programmed death receptor 1) is an important immunosuppressive molecule, a member of the immunoglobulin superfamily, and a membrane protein with 288 amino acid residues. Immune regulation targeting PD-1 is of great significance in fighting tumors, infections, autoimmune diseases, and organ transplant survival.

[0003] Monoclonal antibody drugs designed to target PD-1 include Sintilimab, Nivolumab, Tislelizumab, Pembrolizumab, Camrelizumab, Avelumab, Atezolizumab and Toripalimab, which are approved by the FDA for the treatment of metastatic melanoma, advanced (metastatic) non-small cell lung cancer, relapsed or refractory classical Hodgkin's lymphoma, locally advanced or metastatic urothelial carcinoma, advanced non-squamous non-small cell lung cancer and other diseases.

[0004] Although clinical practice has proven that such biological agents have good efficacy in treating the above diseases, they are not effective for all patients. Some patients are ineffective from the beginning (primary ineffectiveness), and more patients are effective at the beginning, but become ineffective after multiple uses due to drug resistance (secondary ineffectiveness). Further research and development has found that secondary ineffectiveness is related to the production of antibodies against biological agents in the body. Therefore, the detection of antibodies against biological agents in the blood is an important companion diagnosis.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide an antibody detection kit for PD-1 biological agents and a preparation method to solve the above technical problems.

[0007] Existing anti-PD-1 antibody detection methods have the problem of false positives and false negatives. The inventors have found that the root cause of the false negative problem is that after the double antigen sandwich method captures the anti-PD-1 antibodies in the sample, there is a problem with the binding efficiency with the subsequent detection antibody element, resulting in the failure to detect the positive sample, resulting in a false negative.

[0008] The root cause of the false positive problem is that the sample is interfered by a large amount of irrelevant antibodies, resulting in false positives and other detection distortions.

[0009] The present invention is achieved in that:

[0010] The present invention provides an antibody detection kit for a PD-1 biological agent, which comprises an antibody capture device and an antibody detection device, wherein the antibody capture device comprises a solid phase carrier, and the antibody detection device is a cellulose membrane or a functional fragment with a detectable marker; and a PD-1 biological agent is coated on the solid phase carrier, and a functional fragment without a marker is fixed on the cellulose membrane; the functional fragment is a variable region, a heavy chain variable region or a complementary determining region of the PD-1 biological agent.

[0011] The inventors found that using PD-1 biological agents as capture antigens has the advantage of high capture efficiency, as it is easier to capture anti-antibodies in the sample due to its large spatial contact surface. Using the double antigen sandwich method to detect anti-antibodies can largely avoid a large amount of irrelevant antibody interference, improve detection accuracy, and avoid the occurrence of false positives.

[0012] The antigen-antibody complex formed after capture itself has a large steric hindrance. If the molecular weight of the antibody detection device is large (such as biological preparations), it is difficult to bind to another Fab of the anti-antibody in the complex. The inventors found that the variable region, heavy chain variable region or complementary determining region of the small molecular weight PD-1 biological preparation has the advantage of small steric hindrance and is easier to be bound by the anti-antibody Fab. Using it as an antibody detection device can improve the binding efficiency of the antigen-antibody complex and the detection antibody element, improve the sensitivity of the detection, and avoid the occurrence of false negatives. Achieve the purpose of companion diagnosis of PD-1 biological preparations.

[0013] In an optional embodiment, the functional fragments generally have the same binding specificity as the antibody from which they are derived. The detection antigen of the variable region (Fv) is: a Fv (variable region) in which the heavy chain variable region and the light chain variable region are connected by a connecting peptide or a disulfide bond to maintain the stability of the Fv. The complementary determining region includes CDR1, CDR2 and CDR3 in the heavy chain complementary determining region and CDR1, CDR2 and CDR3 in the light chain complementary determining region. It is easy for a person skilled in the art to understand based on the contents described in the present invention that the functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, a person skilled in the art can easily obtain the above functional fragments.

[0014] The functional fragments of the above antibodies can also be synthesized by recombinant genetic techniques known to those skilled in the art (such as recombinant expression by gene recombination) or by, for example, an automatic peptide synthesizer, such as the automatic peptide synthesizer sold by Applied BioSystems.

[0015] In a preferred embodiment of the present invention, the PD-1 biological agent is selected from a monoclonal antibody drug of PD-1.

[0016] In an optional embodiment, the monoclonal antibody drugs for PD-1 include but are not limited to Sintilimab, Nivolumab, Tislelizumab, Pembrolizumab, Camrelizumab, Avelumab, Atezolizumab or Toripalimab.

[0017] In an optional embodiment, the solid phase carrier is selected from fluorescent microspheres, latex microspheres, resin microspheres, magnetic microspheres, colloidal gold particles, quantum dots, microporous plates or microporous membranes. The colloidal gold is a colloidal solution obtained by reducing tetrachloroauric acid with a reducing agent. In other embodiments, the colloidal gold can also be other colloidal substances, such as colloidal carbon, colloidal silver or colloidal selenium.

[0018] In an optional embodiment, the quantum dots are core-shell quantum dots, such as quantum dots formed by ZnS / CdSe or ZnS / CdTe. In addition, in other embodiments, as needed, the quantum dots can be adjusted to quantum dots formed by a single compound, such as cadmium selenide (CdSe), zinc sulfide (ZnS), cadmium telluride (CdTe), cadmium sulfide (CdS), zinc selenide (ZnSe), indium phosphide (InP) or indium arsenide (InAs), or a nanocrystal or semiconductor nanocrystal composed of a layer of ZnS or CdS wrapped on a CdSe core.

[0019] In an optional embodiment, the PD-1 biological agent coated on the solid phase carrier is modified with avidin or biotin, and the modified avidin or biotin is beneficial to further improve the amplification of the detection signal and improve the sensitivity of the detection. It should be noted that in an optional embodiment, the PD-1 biological agent may also be modified without avidin or biotin as needed.

[0020] In an optional embodiment, the solid phase carrier is coated with the full length of the PD-1 biological preparation. Using the full length of the PD-1 biological preparation as a capture antigen, due to the large spatial contact surface, it is easier to capture the anti-PD-1 biological preparation antibody (i.e., anti-antibody) in the sample, which has the advantage of high capture efficiency. By coating the full length of the PD-1 biological preparation on the capture device, the occurrence of false positives can be better avoided.

[0021] In an optional embodiment, the fluorescent microspheres are selected from time-resolved fluorescent microspheres or fluorescent microspheres containing fluorescein, and the magnetic microspheres are selected from magnetic beads. In an optional embodiment, the fluorescent microspheres containing fluorescein have a fluorescence excitation wavelength of 300-520 nm, an emission wavelength of 340-630 nm, and a microsphere diameter range of 100 nm-1000 nm.

[0022] Fluorescent microspheres containing fluorescein include, but are not limited to, blue fluorescent microspheres, green fluorescent microspheres, red fluorescent microspheres, or green and red fluorescent microspheres.

[0023] The time-resolved fluorescent substance is one of lanthanide elements, a combination of lanthanide elements and latex, and a chelate of lanthanide elements; the lanthanide element can be any one of europium, terbium, samarium or dysprosium.

[0024] Detectable markers refer to a class of substances that have properties that can be directly observed by the naked eye or detected or detected by instruments, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0025] In an alternative embodiment, the detectable label includes but is not limited to fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent agents or nanoparticle labels.

[0026] In an optional embodiment, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, Cy3.23, Cy3.24, Cy3.25, Cy3.26, Cy3.27, Cy3.28, Cy3.29, Cy3.30, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), pre-chlorophyll protein (preCP), etc.).

[0027] In an alternative embodiment, the enzyme that catalyzes the color development of the substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxidase.

[0028] In an alternative embodiment, the radioisotope is selected from 212 Bi, 131 I. 111 In, 90 Y. 186 Re, 211 At 125 I. 188 Re, 153 Sm, 213 Bi, 32 P. 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn、 97 Such as 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y. 88 Y. 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu and 18 F;

[0029] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, or peroxyoxalates and their derivatives.

[0030] In an alternative embodiment, the nanoparticle-based marker is selected from nanoparticles and colloids.

[0031] In an optional embodiment, the colloid is selected from colloidal metals, dispersed dyes, dye-labeled microspheres and latex; in an optional embodiment, the colloidal metal includes but is not limited to colloidal gold, colloidal silver, colloidal carbon or colloidal selenium.

[0032] In an alternative embodiment, the nanoparticles are selected from organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles or rare earth complex nanoparticles.

[0033] In an optional embodiment, a quality control antibody is also fixed on the cellulose membrane as a quality control T line, and the functional fragment and the quality control antibody are arranged at intervals;

[0034] In an alternative embodiment, the cellulose membrane is selected from cellulose acetate membrane or nitrocellulose membrane.

[0035] In an optional embodiment, the variable region (Fv) comprises a heavy chain variable region and a light chain variable region connected by a disulfide bond or a connecting peptide. The heavy chain variable region and the light chain variable region of sintilimab are shown in SEQ ID NO.1-2 respectively:

[0036] SEQ ID NO.1:

[0037] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGLIIPMFDTAGYAQKFQGRVAITVDESTSTAYMELSSLRSEDTAVYYCARAEHSSTGTFDYWGQGTLVTVSS.

[0038] SEQ ID NO.2:

[0039] DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLISAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANHLPFTFGGGTKVEIKRTV.

[0040] The complementarity determining regions of sintilimab include CDR1-VH1, CDR2-VH2, CDR3-VH3, CDR1-VL1, CDR2-VL2, and CDR3-VL3 connected by disulfide bonds or connecting peptides:

[0041] CDR1-H1: SYAIS;

[0042] CDR2-H2: LIIPMFDTAGYAQKFQG;

[0043] CDR3-H3:AEHSSTGTFDY;

[0044] CDR1-VL1:RASQGISSWLA;

[0045] CDR2-VL2:AASSLQS;

[0046] CDR3-VL3:QQANHLPFT.

[0047] The heavy chain variable region and light chain variable region of nivolumab are shown in SEQ ID NO.3-4 respectively:

[0048] SEQ ID NO.3:

[0049] QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS

[0050] SEQ ID NO.4:

[0051] STGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTV.

[0052] The complementarity determining regions of nivolumab include CDR1-VH1, CDR2-VH2, CDR3-VH3, CDR1-VL1, CDR2-VL2, and CDR3-VL3, which are connected by disulfide bonds or linkers:

[0053] CDR1-VH1: NSGMH;

[0054] CDR2-VH2: VIWYDGSKRYYADSVKG;

[0055] CDR3-VH3: NDDY;

[0056] CDR1-VL1:RASQSVSSYLA;

[0057] CDR2-VL2:DASNRAT;

[0058] CDR3-VL3:QQSSNWPRT.

[0059] The heavy chain variable region and light chain variable region of tislelizumab are shown in SEQ ID NO.5-6 respectively:

[0060] SEQ ID NO.5:

[0061] QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVHWIRQPPGKGLEWIGVIYADGSTNYNPSLKSRVTISKDTSKNQVSLKLSSVTAADTAVYYCARAYGNYWYIDVWGQGTTVTVSS.

[0062] SEQ ID NO.6:

[0063] DIVMTQSPDSLAVSLGERATINCKSSESVSNDVAWYQQKPGQPPKLLINYAFHRFTGVPDRFSGSGYGTDFTLTISSLQAEDVAVYYCHQAYSSPYTFGQGTKLEIKRTV.

[0064] The complementarity determining regions of tislelizumab include CDR1-VH1, CDR2 –VH2, CDR3–VH3, CD1R-VL1, CDR2 –VL2, and CDR3–VL3, which are connected by disulfide bonds or linkers:

[0065] CDR1-VH1:SYGVH;

[0066] CDR2-VH2: VIYADGSTNYNPSLKS;

[0067] CDR3-VH3:AYGNYWYIDV;

[0068] CDR1-VL1: KSSESVSNDVA;

[0069] CDR2-VL2: YAFHRFT;

[0070] CDR3-VL3:HQAYSSPYT.

[0071] The heavy chain variable region and light chain variable region of pembrolizumab are shown in SEQ ID NO.7-8 respectively:

[0072] SEQ ID NO.7:

[0073] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS.

[0074] SEQ ID NO.8:

[0075] EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTV.

[0076] The complementarity determining regions of pembrolizumab include CDR1-VH1, CDR2 –VH2, CDR3–VH3, CDR1-VL1, CDR2 –VL2, and CDR3–VL3, which are connected by disulfide bonds or linkers:

[0077] CDR1-VH1: NYYMY;

[0078] CDR2-VH2: GINPSNGGTNFNEKFKN;

[0079] CDR3-VH3: RDYRFDMGFDY;

[0080] CDR1-VL1: RASKGVSTSGYSYLH;

[0081] CDR2-VL2:LASYLES;

[0082] CDR3-VL3:QHSRDLPLT.

[0083] The heavy chain variable region and light chain variable region of Avelumab are shown in SEQ ID NOs. 9-10, respectively:

[0084] SEQ ID NO.9:

[0085] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSS.

[0086] SEQ ID NO.10:

[0087] QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQP.

[0088] The complementarity determining regions of avelumab include CDR1-VH1, CDR2 –VH2, CDR3–VH3, CDR1-VL1, CDR2 –VL2, and CDR3–VL3 connected by disulfide bonds or linkers:

[0089] CDR1-VH1:SYIMM;

[0090] CDR2-VH2:SIYPSGGITFYADTVKG;

[0091] CDR3-VH3: IKLGTVTTVDY;

[0092] CDR1-VL1:TGTSSDVGGYNYVS;

[0093] CDR2-VL2:DVSNRPS;

[0094] CDR3-VL3:SSYTSSSTRV.

[0095] The heavy chain variable region and light chain variable region of Atezolizumab are shown in SEQ ID NO.11-12 respectively:

[0096] SEQ ID NO.11:

[0097] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA.

[0098] SEQ ID NO.12:

[0099] DIQMTQSPSSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVA.

[0100] The complementarity determining regions of atezolizumab include CDR1-VH1, CDR2-VH2, CDR3-VH3, CDR1-VL1, CDR2-VL2, and CDR3-VL3 connected by disulfide bonds or linkers:

[0101] CDR1-VH1: DSWIH;

[0102] CDR2-VH2:WISPYGGSTYYADSVKG;

[0103] CDR3-VH3: RHWPGGFDY;

[0104] CDR1-VL1:RASQDVSTAVA;

[0105] CDR2-VL2: SASFLYS;

[0106] CDR3-VL3:QQYLYHPAT.

[0107] The heavy chain variable region and light chain variable region of Teplizumab are shown in SEQ ID NO.13-14 respectively:

[0108] SEQ ID NO.13:

[0109] QGQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPIHGLEWIGVIESETGGTAYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREGITTVATTYYWYFDVWGQGTTVTVSS.

[0110] SEQ ID NO.14:

[0111] DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIKRTV.

[0112] The complementarity determining regions of toripalimab include CDR1-VH1, CDR2 –VH2, CDR3–VH3, CDR1-VL1, CDR2 –VL2, and CDR3–VL3 connected by disulfide bonds or linkers:

[0113] CDR1-VH1: DYEMH;

[0114] CDR2-VH2: VIESETGGTAYNQKFKG;

[0115] CDR3-VH3:EGITTVATTYYWYFDV;

[0116] CDR1-VL1: RSSQSIVHSNGNTYLE;

[0117] CDR2-VL2:KVSNRFS;

[0118] CDR3-VL3: FQGSHVPLT.

[0119] It should be noted that, in other embodiments, the amino acid sequence of the variable region, heavy chain variable region or complementary determining region of the PD-1 biological agent provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the above-mentioned corresponding variable region, heavy chain variable region or complementary determining region.

[0120] In an optional embodiment, the antibody detection kit for PD-1 biological agents is a quantitative detection kit or a qualitative detection kit.

[0121] In an optional embodiment, the antibody detection kit for PD-1 biological agents is a fluorescent immunochromatographic detection kit, a colloidal gold immunochromatographic detection kit, a quantum dot fluorescent immunochromatographic detection kit, an enzyme-linked immunosorbent assay kit, or a chemiluminescent detection kit;

[0122] In an optional embodiment, the fluorescent immunochromatographic detection kit is a time-resolved fluorescent immunochromatographic detection kit.

[0123] The present invention also provides a method for preparing an antibody detection kit for a PD-1 biological agent, which comprises: fixing the full length of the PD-1 biological agent on a solid phase carrier as an antibody capture device, and binding or fixing a functional fragment without a marker on a cellulose membrane or marking the functional fragment with a marker as an antibody detection device.

[0124] In an optional embodiment, when the solid phase carrier is selected from a microplate, before fixing the full-length PD-1 biological agent on the microplate, streptavidin is coated on the microplate, and then the full-length PD-1 biological agent modified with biotin is incubated with the microplate coated with streptavidin.

[0125] The present invention has the following beneficial effects:

[0126] The present invention uses PD-1 biological agents as capture antigens. Due to the large spatial contact surface, it is easier to capture anti-antibodies in the sample, and has the advantage of high capture efficiency. The detection of anti-antibodies can be achieved by using the double antigen sandwich method, which can greatly avoid a large amount of irrelevant antibody interference, improve detection accuracy, and avoid the occurrence of false positives.

[0127] The antigen-antibody complex formed after capture itself has a large steric hindrance. The inventors found that the variable region, heavy chain variable region or complementary determining region of the small molecular weight PD-1 biological agent has the advantage of small steric hindrance and is easier to be bound by anti-antibody Fab. Using it as an antibody detection device can improve the binding efficiency of the antigen-antibody complex and the detection antibody element, improve the sensitivity of the detection, and avoid the occurrence of false negatives. Achieve the purpose of companion diagnosis of PD-1 biological drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0128] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0129] Figure 1 It is a device diagram of the anti-sintilimab antibody time-resolved fluorescent immunochromatographic test paper of the present invention;

[0130] Figure 2 This is a detection principle diagram of the anti-nivolumab antibody enzyme-linked immunosorbent assay kit of the present invention;

[0131] Figure 3 This is a detection principle diagram of the anti-atezolizumab antibody chemiluminescence assay kit of the present invention;

[0132] Figure 4 It is a regression graph of the calibration curve of the anti-sintilimab antibody time-resolved fluorescent immunochromatography kit of the present invention;

[0133] Figure 5 is a calibration curve regression graph of the anti-nivolumab antibody enzyme-linked immunosorbent assay kit of the present invention;

[0134] Figure 6 is a calibration curve regression diagram of the anti-atezolizumab antibody chemiluminescence assay kit of the present invention;

[0135] Reference numerals: 1 - sample pad; 2 - binding pad; 3 - nitrocellulose membrane; 4 - detection line; 5 - quality control line; 6 - water-absorbing membrane; 7 - back substrate. DETAILED DESCRIPTION

[0136] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0137] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0138] The working principle of the kit provided by the present invention is as follows: 1) A fixed solid phase carrier of a PD-1 biological preparation is used to capture anti-antibodies in the sample to be tested. Since the molecular spatial action surface of the PD-1 biological preparation is large, the anti-antibodies in the sample can be efficiently captured to form an antigen-antibody complex; 2) The variable region, heavy chain variable region or complementary determining region of the PD-1 biological preparation is used as a detection antigen. Due to its small molecular weight, it can effectively overcome steric hindrance and bind to another Fab of the antibody in the antigen-antibody complex to form an antigen-antibody-antigen complex. The complex is quantitatively or qualitatively detected using a well-known color development method.

[0139] Example 1

[0140] This embodiment provides an anti-sintilimab antibody time-resolved fluorescent immunochromatography quantitative detection kit and a preparation method thereof.

[0141] Reference Figure 1 As shown, the test paper is provided with a nitrocellulose membrane 3, a sample pad 1, a conjugation pad 2 and a water-absorbing membrane 6 on a back substrate 7, the sample pad 1 is stacked on one end of the conjugation pad 2, and the other end of the conjugation pad 2 and the water-absorbing membrane 6 are respectively stacked on both ends of the nitrocellulose membrane 3; the conjugation pad 2 is coated with sindilimab labeled with time-resolved fluorescent microspheres and sheep anti-chicken IgY antibody 2 labeled with time-resolved fluorescent microspheres; the nitrocellulose membrane 3 is provided with a detection line 4 (T line) and a quality control line 5 (C line), the detection line 4 (T line) is coated with the variable region Fv of sindilimab, and the quality control line 5 (C line) is coated with chicken IgY antibody.

[0142] The variable region (Fv) of sintilimab comprises a sintilimab heavy chain variable region and a sintilimab light chain variable region connected by a disulfide bond, wherein the sequence of the sintilimab heavy chain variable region is as follows:

[0143] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGLIIPMFDTAGYAQKFQGRVAITVDESTSTAYMELSSLRSEDTAVYYCARAEHSSTGTFDYWGQGTLVTVSS.

[0144] The sequence of the light chain variable region of sintilimab is as follows:

[0145] DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLISAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANHLPFTFGGGTKVEIKRTV.

[0146] The preparation method is as follows:

[0147] 1) Time-resolved fluorescent microsphere labeling

[0148] Take 500μL of time-resolved fluorescent microspheres (300nm) (1% stock solution), centrifuge at 13000rpm, 4℃ for 10min, and discard the supernatant. Add 10mg / ml EDC, react for 15min; centrifuge, discard the supernatant, add 1000μL of borate buffer (20mM, pH8.0), mix by ultrasound for 10 seconds; add 100μg of labeled antibody (ustekinumab or goat anti-chicken IgY) (labeling concentration 100μg / mL), react at room temperature for 2h; centrifuge, discard the supernatant, add blocking solution, react for 1h; centrifuge, discard the supernatant, add 1000μL of borate buffer, mix by ultrasound, label well, and store at 4℃ for use.

[0149] 2) Processing of sample pad and conjugate pad

[0150] The polyester membrane was pre-blocked by soaking in a buffer solution containing a surfactant (formula: 20mM pH 8.0 BB, containing 2% BSA, 0.5% S9 and 2% trehalose), and dried at 37°C overnight to prepare a binding pad; the prepared binding pad was taken and mixed with sintilimab labeled with time-resolved fluorescent microspheres and goat anti-chicken IgY antibody using a Gold Standard HM3035 gold spray film instrument, and then sprayed onto the pre-treated binding pad with a width of 1 cm at 3μL / cm, and dried at 37°C for 3h to prepare a specific binding pad.

[0151] The glass cellulose membrane was pre-blocked by soaking in a buffer solution containing a surfactant (formula: 100 mM pH 7.4 PB, containing 2% BSA and 0.5% S9), dried at 37°C overnight, and cut into 30*1.5 cm to prepare a sample pad.

[0152] 3) Coating of nitrocellulose membrane (NC membrane)

[0153] The variable region of sintilimab was synthesized by an amino acid synthesis method. It was verified that the synthetic polypeptide coated on an ELISA plate could react with anti-sintilimab antibodies.

[0154] The NC membrane was attached to the designated position of the backing substrate, and the heavy chain variable region of sintilimab was diluted to 1.5 mg / mL with 50 mM pH 7.4 phosphate buffer for preparing T lines; the chicken IgY antibody was diluted to 0.5 mg / mL with 50 mM pH 7.4 phosphate buffer for preparing C lines; the two diluted antibodies were evenly streaked onto the NC membrane using a gold standard HM3035 gold spray film streaker at a streaking volume of 1 μL / cm to prepare T lines and C lines; the streaked NC membrane was placed in a 37°C drying oven to dry overnight.

[0155] 4) Assembly

[0156] The conjugate pad of step 2) is stacked on one end of the nitrocellulose membrane obtained in step 3), and the absorbent membrane is fixedly stacked on the other end of the nitrocellulose membrane. Finally, the sample pad obtained in step 2) is stacked on the other end of the conjugate pad, and each strip is cut into 4 mm width using a film cutter and loaded into the chromatography strip shell to obtain the finished product.

[0157] 5) Detection

[0158] Dilute the serum 10 times, take 90 μL and add it to the sample well. After reacting for 10 minutes, place it in the fluorescence reader for detection. Compare it with the value of the standard or reference product to determine the content of anti-antibodies in the sample to be tested.

[0159] In the present embodiment, the time-resolved fluorescent latex microspheres are europium-containing latex microspheres, that is, the time-resolved fluorescent material is a combination of the lanthanide element europium and latex; as needed, the time-resolved fluorescent material can be adjusted to one of the lanthanide elements, a combination of lanthanide elements and latex, and a chelate of lanthanide elements; the lanthanide element can be one of europium, terbium, samarium or dysprosium.

[0160] Example 2

[0161] This embodiment provides a method for preparing an anti-nivolumab antibody enzyme-linked immunosorbent assay kit. Figure 2 shown.

[0162] The kit includes an ELISA plate, biotinylated nivolumab, anti-nivolumab antibody calibrators, anti-nivolumab antibody quality control products, enzyme conjugates, cleaning solutions, and stop solutions. The enzyme conjugates are horseradish peroxidase (HRP)-labeled complementary determining regions (CDRs) of nivolumab. The ELISA plate is coated with streptavidin.

[0163] The complementarity determining regions (CDRs) of nivolumab include CDR1-VH1, CDR2 –VH2, CDR3–VH3, CDR-VL1, CDR2 –VL2, and CDR3–VL3 connected by a linker peptide:

[0164] CDR1-VH1: NSGMH;

[0165] CDR2-VH2: VIWYDGSKRYYADSVKG;

[0166] CDR3-VH3: NDDY;

[0167] CDR1-VL1:RASQSVSSYLA;

[0168] CDR2-VL2:DASNRAT;

[0169] CDR3-VL3:QQSSNWPRT.

[0170] The connecting peptide is selected from GGGGSGGGGSGGGGS.

[0171] The preparation method is as follows:

[0172] 1) Biotinylation of Nivolumab

[0173] Take 1 mg of nivolumab and place it in a 5 mL centrifuge tube. Add 50 μL of 10 mg / mL activated biotin (Sulfo-NHS-LC-Bintin). React at room temperature for 1 hour. Take out the reaction solution and dialyze it against 0.02M PBS overnight. Take out the biotinylated nivolumab, add 0.03% Proclin300 for preservation, and store at 4 degrees for later use.

[0174] 2) HRP-labeled nivolumab complementarity determining regions (CDRs)

[0175] The complementary determining regions (CDRs) of nivolumab were synthesized by an amino acid synthesis method, and it was verified that the synthetic polypeptide package could react with anti-nivolumab antibodies.

[0176] Weigh 5 mg of HRP into a 5 mL centrifuge tube, add 1 mL of pure water to dissolve, and add 1 mL of NaIO 4 Solution (10 mg / mL, prepared immediately before use), react at 4 degrees in the dark for 30 minutes. Add 0.02 mL of ethylene glycol and react at room temperature for 15 minutes. Take out the above solution after dialyzing. Add 1 mg of nivolumab complementary determining region (CDRs) to the above activated solution, react at room temperature in the dark for 2 hours, add 0.2 mL of NaBH4 solution (5 mg / mL, prepared immediately before use), mix well, and dialyze with 0.05 M CB overnight. Take out the labeled enzyme-labeled antibody, add an equal volume of glycerol, and store at -20°C for later use.

[0177] 3) Coating ELISA plate

[0178] Dilute streptavidin to 1 μg / mL in CB buffer, coat the microplate with 100 μL per well, and incubate at 37°C for 2 hours. Aspirate the coating solution, and use 200 μL per well of blocking buffer containing 2% BSA, incubate at 37°C for 2 hours, tap off the blocking solution, and dry at 37°C for later use.

[0179] 4) Coating with biotinylated nivolumab

[0180] Dilute biotinylated nivolumab to 1 μg / mL with neutral phosphate buffer, add 100 μL to each well, and incubate at 37°C for 2 hours. Wash the plate 3 times, add 200 μL of blocking buffer containing 2% BSA to each well, incubate at 37°C for 2 hours, tap off the blocking solution, and dry at 37°C for later use.

[0181] 5) Dilute the serum sample to be tested 100 times, add 100 μL to each well of the microplate, incubate at 37 degrees for 2 hours, and wash the plate 4 times.

[0182] 6) Add HRP-labeled nivolumab complementary determining regions (CDRs), incubate for 1 hour, and wash the plate 4 times

[0183] 7) Add TMB, the substrate of HRP enzyme, to the microplate and incubate for 15 minutes. Add HCL solution to terminate the color development reaction. Detect the OD value at a wavelength of 450nm and compare it with the OD value of the standard or reference to determine the content of anti-antibodies in the sample to be tested.

[0184] Example 3

[0185] This embodiment provides an atezolizumab antibody chemiluminescence assay kit and a preparation method.

[0186] The anti-atezolizumab antibody chemiluminescent detection kit includes: biotinylated atezolizumab, anti-atezolizumab antibody calibrator, anti-atezolizumab antibody quality control, acridinium ester-labeled atezolizumab heavy chain variable region (Hv), magnetic particle reagent, excitation solution and cleaning solution.

[0187] The sequence of the atezolizumab heavy chain variable region is as follows:

[0188] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA.

[0189] The preparation method is as follows:

[0190] 1) Biotinylation of Atezolizumab

[0191] Take 1 mg of atezolizumab and place it in a 5 mL centrifuge tube. Add 50 μL of 10 mg / mL activated biotin (Sulfo-NHS-LC-Bintin). React at room temperature for 1 hour. Take out the reaction solution and dialyze it against 0.02M PBS overnight. Take out the biotinylated atezolizumab antibody, add 0.03% Proclin300 for preservation, and store at 4 degrees for later use.

[0192] 2) Acridinium ester labeling of atezolizumab heavy chain variable region (Hv)

[0193] The heavy chain variable region (Hv) of atezolizumab was synthesized by an amino acid synthesis method. It was verified that the synthetic polypeptide coated on an ELISA plate could react with anti-atezolizumab antibodies.

[0194] Take 2 mg of atezolizumab heavy chain variable region (Hv), add activated acridinium ester at a molar ratio of antibody: acridinium ester = 1:10~50, and react at room temperature for 30 minutes. After dialyzing the reaction solution with 0.05M CB solution, take it out, add an equal volume of glycerol, and store it at -20℃ for later use.

[0195] 3) Detection steps

[0196] This embodiment uses acridinium ester chemiluminescent immunoassay technology combined with biotin-avidin magnetic particle separation technology to achieve anti-antibody detection. The detection principle is as follows Figure 3 : The sample (diluted 20 times) is mixed with streptavidin magnetic particles (1) and biotinylated atezolizumab (2) to obtain a streptavidin magnetic particle-biotinylated atezolizumab-sample complex (3). After washing, acridinium ester-labeled atezolizumab heavy chain variable region (Hv) is added for reaction to form a streptavidin magnetic particle-biotinylated atezolizumab-sample-acridinium ester-labeled atezolizumab heavy chain variable region (Hv) complex. A luminescent excitation solution is added to measure the luminescent intensity, and compared with the value of the standard or reference product to determine the content of the anti-antibody in the sample to be tested.

[0197] Experimental Example 1

[0198] This experimental example performs quantitative detection on the kit of Example 1.

[0199] 1.1 Preparation of standard curve

[0200] 1.1.1 Obtain a set of anti-sintilimab antibody calibrators. The specific values ​​are shown in Table 1.

[0201] Table 1

[0202]

[0203] 1.1.2 Detection Method

[0204] Take 90 μL of the calibrator and add it directly into the sample window in the chromatography strip; after 10 minutes, use a time-resolved fluorescence quantitative analyzer to quantitatively detect the signal value.

[0205] Each calibrator was tested twice and the average T / C value was taken. The specific results are shown in Table 2.

[0206] Table 2

[0207]

[0208] 1.1.3 Preparation of standard curve

[0209] According to the above test results, with the concentration value as the X-axis and the T / C value as the Y-axis, a linear regression is performed to obtain the linear equation: y = 0.0026x + 0.0553, R² = 0.9979, the curve is shown in Figure 4 .

[0210] 1.2 Precision test:

[0211] 1.2.1 Take 10 prepared chromatography strips and prepare a concentration (50 ng / mL) of anti-sintilimab antibody working calibrator;

[0212] 1.2.2 Take 90 μL of calibrator and add it directly into the sample well of the reagent strip;

[0213] 1.2.3 After the calibrator was chromatographed for 10 minutes, the time-resolved fluorescence quantitative analyzer was used for detection. The results of 10 reagent strips were measured according to the above linear equation. The results are shown in Table 3, indicating that the anti-sintilimab antibody time-resolved fluorescence immunochromatography assay kit of this method has good precision.

[0214] Table 3

[0215]

[0216] 1.3 Compared with other methods:

[0217] Method 2 is to label the microspheres with sintilimab and coat the NC membrane with sintilimab; method 3 is to label the microspheres with sintilimab fab and coat the NC membrane with sintilimab fab. The three methods have the same conditions except for the labeling antibody and coating antibody. The comparison of the three results is shown in Table 4.

[0218] Table 4

[0219]

[0220] Experimental Example 2

[0221] This experimental example performs quantitative detection on the kit of Example 2.

[0222] 2.1 Detection Method

[0223] 2.1.1 Obtain a set of anti-nivolumab antibody calibrators. See Table 5 for specific values.

[0224] Table 5

[0225]

[0226] 2.1.2 Testing steps

[0227] 1) Add 100 μL of calibrators and controls to each well of the microplate, incubate at 37°C for 2 hours, and wash the plate 4 times.

[0228] 2) Add HRP-labeled nivolumab complementarity determining region, incubate for 1 hour, and wash the plate 4 times.

[0229] 3) Add substrate TMB to the microplate, incubate for 15 minutes, add stop solution to terminate the color reaction, and detect the OD value at a wavelength of 450nm to determine the performance of the kit. See Table 6 for specific results.

[0230] Table 6

[0231]

[0232] 2.1.3 Standard curve

[0233] According to the above test results, the OD value is the X-axis and the concentration value is the Y-axis. The curve equation is: y = 0.0047x + 0.1247, R² = 0.997, the curve is shown in Figure 5 .

[0234] 2.2 Precision test:

[0235] According to the OD value of the precision quality control product tested, the result value was calculated using the curve equation. The results are shown in Table 7, indicating that the anti-nivolumab antibody enzyme-linked immunosorbent assay kit of this method has good precision.

[0236] Table 7

[0237]

[0238] 2.3 Compared with other methods:

[0239] Method 2 is to label HRP with nivolumab and coat the microplate with biotinylated nivolumab; and method 3 is to label HRP with nivolumab fab and coat the microplate with nivolumab fab. The three methods have the same conditions except for the labeling antibody and coating antibody. The comparison of the three results is shown in Table 8.

[0240] Table 8

[0241]

[0242] Experimental Example 3

[0243] This experimental example performs quantitative detection on the kit of Example 3.

[0244] 3.1 Detection method

[0245] 3.1.1 Obtain a set of anti-atezolizumab antibody calibrators. See Table 9 for specific values.

[0246] Table 9

[0247]

[0248] 2.1.2 Testing steps

[0249] 1) Take 20 μL of calibrator, 20 μL of streptavidin magnetic particles, and 50 μL of biotinylated atezolizumab and add them to the microwells, incubate at 37 degrees for 15 minutes, and wash three times.

[0250] 2) Add acridinium ester-labeled atezolizumab heavy chain variable region (Hv), incubate at 37°C for 15 min, and wash three times.

[0251] 3) Add luminescence excitation liquid and measure the luminescence intensity. See Table 10 for specific results.

[0252] Table 10

[0253]

[0254] 3.1.3 Standard curve

[0255] According to the above test results, with the concentration value as the X-axis and the luminescence value as the Y-axis, a linear regression is performed to obtain the linear equation: y = 1042.1x + 15675, R² = 0.9982, the curve is shown in Figure 6 .

[0256] 3.2 Precision test:

[0257] Take 20μL precision quality control, 20μL streptavidin magnetic particles, and 50μL biotinylated atezolizumab and add them to the microwells, mix, incubate at 37 degrees for 15 minutes, and wash 3 times; add acridinium ester labeled atezolizumab heavy chain variable region (Hv), incubate at 37 degrees for 15 minutes, wash 3 times; add luminescent excitation liquid and measure the luminescence intensity. According to the above curve equation, calculate the result value. The anti-atezolizumab antibody chemiluminescent immunoassay kit of this method has good precision. The specific results are shown in Table 11.

[0258] Table 11

[0259]

[0260] 3.3 Compared with other methods:

[0261] Method 2 is to label acridinium ester with atezolizumab and label biotin with atezolizumab; method 3 is to label acridinium ester with atezolizumab fab and label biotin with atezolizumab fab. The three methods are the same except that the antibodies for labeling acridinium ester and the antibodies for labeling biotin are different. The comparison of the three results is shown in Table 12.

[0262] Table 12

[0263]

[0264] In summary, the detection kit provided by the present invention has the advantages of high capture efficiency, high accuracy and high sensitivity, and can effectively avoid the occurrence of false positives and false negatives. It has good application prospects.

[0265] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. Sequence Listing <110> Suzhou Herui Biotechnology Co., Ltd. <120> Antibody detection kit for PD-1 biological agent and preparation method thereof <160> 14 <170> SIPOSequenceListing 1.0 <210> 1 <211> 120 <212> PRT <213> Artificial Sequence <400> 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Leu Ile Ile Pro Met Phe Asp Thr Ala Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Ala Ile Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Glu His Ser Ser Thr Gly Thr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 110 <212> PRT <213> Artificial Sequence <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn His Leu Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 <210> 3 <211> 113 <212> PRT <213> Artificial Sequence <400> 3 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <210> 4 <211> 113 <212> PRT <213> Artificial Sequence <400> 4 Ser Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu 1 5 10 15 Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val 20 25 30 Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg 35 40 45 Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 65 70 75 80 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn 85 90 95 Trp Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val <210> 5 <211> 118 <212> PRT <213> Artificial Sequence <400> 5 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Tyr Ala Asp Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Tyr Gly Asn Tyr Trp Tyr Ile Asp Val Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 6 <211> 110 <212> PRT <213> Artificial Sequence <400> 6 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Glu Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Asn Tyr Ala Phe His Arg Phe Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys His Gln Ala Tyr Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val 100 105 110 <210> 7 <211> 120 <212> PRT <213> Artificial Sequence <400> 7 Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 8 <211> 114 <212> PRT <213> Artificial Sequence <400> 8 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val <210> 9 <211> 120 <212> PRT <213> Artificial Sequence <400> 9 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 10 <211> 113 <212> PRT <213> Artificial Sequence <400> 10 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln 100 105 110 Pro <210> 11 <211> 118 <212> PRT <213> Artificial Sequence <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 12 <211> 111 <212> PRT <213> Artificial Sequence <400> 12 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 <210> 13 <211> 125 <212> PRT <213> Artificial Sequence <400> 13 Gln Gly Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Ile His Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Glu Ser Glu Thr Gly Gly Thr Ala Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Ile Thr Thr Val Ala Thr Thr Tyr Tyr Trp Tyr Phe 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 14 <211> 115 <212> PRT <213> Artificial Sequence <400> 14 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val 115

Claims

1. A PD-1 biological agent antibody detection kit, characterized in that: It includes an antibody capture device and an antibody detection device; The antibody capture device includes a solid phase carrier, and the antibody detection device is a functional fragment with a detectable marker; the solid phase carrier is selected from a microporous plate or a microporous membrane; the detectable marker is selected from a fluorescent dye, an enzyme that catalyzes the color development of a substrate, a radioactive isotope, a chemiluminescent reagent or a nanoparticle marker; Or, the antibody capture device comprises a solid phase carrier, and the solid phase carrier is selected from fluorescent microspheres, latex microspheres, resin microspheres, magnetic microspheres, colloidal gold particles or quantum dots; the antibody detection device is a cellulose membrane fixed with functional fragments without labels; The solid phase carrier is coated with the full length of the PD-1 biological agent, and the full length of the PD-1 biological agent is the full length of Sintilimab, Nivolumab or Atezolizumab; correspondingly, the functional fragment is the variable region of Sintilimab, the complementary determining region of Nivolumab or the heavy chain variable region of Atezolizumab; The variable region is composed of a heavy chain variable region and a light chain variable region connected by a disulfide bond or a connecting peptide; the complementary determining region includes CDR1-VH1, CDR2-VH2, CDR3-VH3, CDR1-VL1, CDR2-VL2 and CDR3-VL3 connected by a disulfide bond or a connecting peptide.

2. The antibody detection kit for PD-1 biological preparation according to claim 1, characterized in that: The PD-1 biological agent coated on the solid phase carrier is modified with avidin or biotin.

3. The antibody detection kit for PD-1 biological preparation according to claim 1, characterized in that: The fluorescent microspheres are selected from time-resolved fluorescent microspheres or fluorescent microspheres containing fluorescein, and the magnetic microspheres are selected from magnetic beads.

4. The antibody detection kit for PD-1 biological preparation according to claim 1 or 2, characterized in that: The fluorescent dye is selected from fluorescein dyes and their derivatives, rhodamine dyes and their derivatives, Cy series dyes and their derivatives, Alexa series dyes and their derivatives, or protein dyes and their derivatives; The enzyme that catalyzes the color development of the substrate is selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase or 6-phosphoglucose deoxidase; The radioisotope is selected from 212 Bi, 131 I. 111 In, 90 Y. 186 Re, 211 At 125 I. 188 Re, 153 Sm, 213 Bi, 32 P. 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn、 97 Such as 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y. 88 Y. 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu or 18 F; The chemiluminescent agent is selected from luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, or peroxyoxalates and their derivatives; The nanoparticle marker is selected from nanoparticles or colloids; The colloid is selected from colloidal metal, dispersed dye, dye-labeled microspheres or latex; the colloidal metal is selected from colloidal gold or colloidal silver; The nanoparticles are selected from organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles or rare earth complex nanoparticles.

5. The antibody detection kit for PD-1 biological preparation according to claim 1 or 2, characterized in that: A quality control antibody is also fixed on the cellulose membrane as a quality control T line, and the functional fragment and the quality control antibody are arranged at intervals; The cellulose membrane is selected from cellulose acetate membrane or nitrocellulose membrane.

6. The antibody detection kit for PD-1 biological preparation according to claim 1 or 2, characterized in that: The heavy chain variable region and light chain variable region of sintilimab are shown in SEQ ID NO.1-2, respectively, and the heavy chain variable region of atezolizumab is shown in SEQ ID NO.

11.

7. The antibody detection kit for PD-1 biological preparation according to claim 1 or 2, characterized in that: The antibody detection kit for the PD-1 biological agent is a quantitative detection kit or a qualitative detection kit.

8. The antibody detection kit for PD-1 biological preparation according to claim 1 or 2, characterized in that: The antibody detection kit for the PD-1 biological preparation is a fluorescent immunochromatographic detection kit, a colloidal gold immunochromatographic detection kit, an enzyme-linked immunosorbent assay kit or a chemiluminescent detection kit.

9. A method for preparing an antibody detection kit for PD-1 biological agents according to any one of claims 1 to 8, characterized in that: The method comprises: fixing the PD-1 biological agent on a solid phase carrier as an antibody capture device, combining or fixing the functional fragment without a marker on a cellulose membrane or marking the functional fragment with a marker as an antibody detection device.

10. The method for preparing the antibody detection kit for PD-1 biological preparation according to claim 9, characterized in that: When the solid phase carrier is selected from a microplate, before the full length of the PD-1 biological agent is fixed on the microplate, streptavidin is coated on the microplate, and then the PD-1 biological agent modified with biotin is incubated with the microplate coated with streptavidin.

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