CN114264814A

CN114264814A - Antibody detection kit for PD-1 biological agent and preparation method thereof

Info

Publication number: CN114264814A
Application number: CN202111641580.9A
Authority: CN
Inventors: 王莹; 陈菲; 霍如松
Original assignee: Suzhou Herui Biotechnology Co ltd
Current assignee: Suzhou Herui Biotechnology Co ltd
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2022-04-01

Abstract

The invention discloses an antibody detection kit for a PD-1 biological agent and a preparation method thereof, and relates to the technical field of antibody detection. The kit comprises an antibody capture device and an antibody detection device, wherein the antibody capture device comprises a solid phase carrier coated with a PD-1 biological agent, and the antibody detection device is a cellulose membrane for fixing a functional fragment or a conjugate coupled with the functional fragment; the functional fragment is a variable region, a heavy chain variable region or a complementarity determining region of the PD-1 biological agent. The detection kit provided by the invention has the advantages of high capture efficiency, high accuracy and high sensitivity, can effectively avoid the occurrence of false positive and false negative, and has good application prospect.

Description

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

Technical Field

The invention relates to the technical field of antibody detection, in particular to an antibody detection kit of a PD-1 biological agent and a preparation method thereof.

Background

PD-1 (programmed death receptor 1), an important immunosuppressive molecule, is an immunoglobulin superfamily, a membrane protein of 288 amino acid residues. The immunoregulation taking PD-1 as a target point has important significance for resisting tumor, infection, autoimmune disease, organ transplantation survival and the like.

Monoclonal antibody drugs designed to target PD-1 include convilumab (Sintilimab), Nivolumab (Nivolumab), tirelinzumab (tiselizumab), pamrolizumab (Pembrolizumab), carprilizumab (Camrelizumab), avizumab (Avelumab), atelizumab (Atezolizumab), and teriprizumab (tropilizab) that are approved by the FDA for the treatment of metastatic melanoma, advanced (metastatic) non-small cell lung cancer, recurrent or refractory classical hodgkin lymphoma and locally advanced or metastatic urothelial cancer, advanced non-squamous non-small cell lung cancer, and the like.

Although clinical practice has shown that such biological agents are effective in treating the above-mentioned diseases, not all patients are effective, some are initially ineffective (primary ineffective), and more patients appear to be initially effective, but develop resistance after multiple uses and are ineffective (secondary ineffective). Further development has found that secondary effectors are involved in the in vivo production of antibodies against the biologic, and therefore, detection of antibodies against the biologic in the blood is an important companion diagnostic.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide an antibody detection kit for a PD-1 biological agent and a preparation method thereof so as to solve the technical problems.

The existing anti-PD-1 antibody detection method has the problems of false positive and false negative. The inventor researches to find that the problem of false negative is rooted in that: the double antigen sandwich method has the problem of binding efficiency with a subsequent detection antibody element after capturing an anti-PD-1 antibody in a sample, so that the sample is positive and cannot be detected, and false negative is caused.

The problem of false positives stems from: the interference of a large amount of irrelevant antibodies in a sample causes detection distortion such as false positive and the like.

The invention is realized by the following steps:

the invention provides an antibody detection kit of 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 the solid phase carrier is coated with PD-1 biological agent, and the cellulose membrane is fixed with functional fragments without markers; the functional fragment is a variable region, a heavy chain variable region or a complementarity determining region of the PD-1 biological agent.

The inventor finds that: the PD-1 biological agent is used as the capture antigen, and the anti-antibody in the sample is easier to capture due to the large space contact surface, so that the capture antigen has the advantage of high capture efficiency. The detection of the anti-antibody is realized by means of a double-antigen sandwich method, so that a large amount of irrelevant antibody interference can be avoided to a great extent, the detection accuracy is improved, and the occurrence of false positive is avoided.

The antigen-antibody complex formed after capture has a large steric hindrance itself, and if the molecular weight of the antibody detection device is large (such as a biological agent), it is difficult to bind to another Fab of the anti-antibody in the complex. The inventor finds that the variable region, the heavy chain variable region or the complementarity determining region of the PD-1 biological agent with small molecular weight has the advantages of small steric hindrance and easy combination by an anti-antibody Fab, and the variable region, the heavy chain variable region or the complementarity determining region can be used as an antibody detection device to improve the combination efficiency of an antigen-antibody compound and a detection antibody element, improve the detection sensitivity and avoid the occurrence of false negative. The purpose of concomitant diagnosis of PD-1 biological agent medicines is achieved.

In an alternative embodiment, the functional fragment will generally have the same binding specificity as the antibody from which it is derived. The detection antigens for the above variable region (Fv) were: fv (variable region) in which the variable regions of the heavy chain and the variable regions of the light chain are linked by a linker peptide or a disulfide bond to maintain the stability of the Fv. The complementarity determining regions include CDR1, CDR2 and CDR3 in the heavy chain complementarity determining region and CDR1, CDR2 and CDR3 in the light chain complementarity determining region. It will be appreciated by those skilled in the art, based on the teachings herein, that 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 cleave disulfide bonds. Based on the disclosure of the structure of the intact antibody, the above-described functional fragments are readily available to those skilled in the art.

Functional fragments of the above antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art (e.g., recombinant expression by means of gene recombination) or by synthesis, for example, by an automated peptide synthesizer such as those sold by Applied BioSystems and the like.

In a preferred embodiment of the invention, the PD-1 biological agent is selected from monoclonal antibody drugs of PD-1.

In an alternative embodiment, the mab drug of PD-1 includes, but is not limited to, tudinizumab (Sintilimab), Nivolumab (Nivolumab), tirezuzumab (tiselizumab), parbolizumab (Pembrolizumab), carpriluzumab (Camrelizumab), avilamumab (Avelumab), atelizumab (Atezolizumab), or teriprizumab (toriplalimab).

In an alternative embodiment, the solid support is selected from fluorescent microspheres, latex microspheres, resin microspheres, magnetic microspheres, colloidal gold particles, quantum dots, microwell plates, or microporous membranes. The colloidal gold is a colloidal solution obtained by reducing tetrachloroauric acid by a reducing agent. In other embodiments, the colloidal gold may be other colloidal substances, such as colloidal carbon, colloidal silver, or colloidal selenium.

In an alternative embodiment, the quantum dots are core-shell quantum dots, such as ZnS/CdSe or ZnS/CdTe quantum dots. In addition, in other embodiments, the quantum dots may be adjusted to quantum dots formed by a single compound, such as one of 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 formed by wrapping a layer of ZnS or CdS on a CdSe core, according to needs.

In an alternative embodiment, the PD-1 biological agent coated on the solid phase carrier is modified with avidin or biotin, which is beneficial to further improve the amplification of the detection signal and improve the detection sensitivity. It should be noted that in an alternative embodiment, it is also possible to select unmodified avidin or biotin on the PD-1 biological agent as desired.

In an alternative embodiment, the solid support is coated with the full length of the PD-1 substance preparation. The whole length of the PD-1 biological agent is used as the capture antigen, and the antibody (namely the anti-antibody) of the anti-PD-1 biological agent in the sample is easier to capture due to the large space contact surface, so that the method has the advantage of high capture efficiency. By coating the PD-1 biological agent over the full length of the capture device, the occurrence of false positives can be better avoided.

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

Fluorescent microspheres containing fluorescein include, but are not limited to: blue fluorescent microspheres, green fluorescent microspheres, red fluorescent microspheres or green and red dry fluorescent microspheres.

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

Detectable labels are substances having properties, such as luminescence, color development, radioactivity, etc., which can be observed directly by the naked eye or detected by an instrument, by which qualitative or quantitative detection of the respective target substance can be achieved.

In an alternative embodiment, the detectable label includes, but is not limited to, a fluorescent dye, an enzyme that catalyzes the development of a substrate, a radioisotope, a chemiluminescent reagent, or a nanoparticle-based label.

In an alternative embodiment, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (e.g., including, but not limited to, Fluorescein Isothiocyanate (FITC) hydroxyphoton (FAM), tetrachloro-fluorescein (TET), etc. or analogs thereof), rhodamine-based dyes and derivatives thereof (e.g., including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine b (tritc), etc. or analogs thereof), Cy-series dyes and derivatives thereof (e.g., including, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc. or analogs thereof), Alexa-series dyes and derivatives thereof (e.g., including, but not limited to, Alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or analogs thereof), and protein-based dyes and derivatives thereof (e.g., including, but not limited to, Phycoerythrin (PE), Phycocyanin (PC), phycocyanin, phycoerythrin, Phycocyanin (PC), phycocyanin, and derivatives thereof), and derivatives thereof, Allophycocyanin (APC), polymethacrylic flavin-chlorophyll protein (precP), etc.).

In an alternative embodiment, enzymes that catalyze the development of a substrate include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxyenzyme.

In an alternative embodiment, the radioisotope is selected from the group consisting of²¹²Bi、¹³¹I、¹¹¹In、⁹⁰Y、¹⁸⁶Re、²¹¹At、¹²⁵I、¹⁸⁸Re、¹⁵³Sm、²¹³Bi、³²P、⁹⁴mTc、⁹⁹mTc、²⁰³Pb、⁶⁷Ga、⁶⁸Ga、⁴³Sc、⁴⁷Sc、¹¹⁰mIn、⁹⁷Ru、⁶²Cu、⁶⁴Cu、⁶⁷Cu、⁶⁸Cu、⁸⁶Y、⁸⁸Y、¹²¹Sn、¹⁶¹Tb、¹⁶⁶Ho、¹⁰⁵Rh、¹⁷⁷Lu、¹⁷²Lu and¹⁸F；

in an alternative embodiment, the chemiluminescent reagent comprises, without limitation, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridyl ruthenium and its derivatives, acridinium ester and its derivatives, dioxirane and its derivatives, lotucine and its derivatives, or peroxyoxalate and its derivatives.

In an alternative embodiment, the nanoparticle-based label is selected from the group consisting of nanoparticles and colloids.

In an alternative embodiment, the colloid is selected from the group consisting of colloidal metals, disperse dyes, dye-labeled microspheres, and latexes; in an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, colloidal carbon, or colloidal selenium.

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

In an alternative embodiment, a quality control antibody is further fixed on the cellulose membrane as a quality control T line, and the functional fragment and the quality control antibody are respectively arranged at intervals;

in an alternative embodiment, the cellulose membrane is selected from a cellulose acetate membrane or a cellulose nitrate membrane.

In an alternative embodiment, the variable region (Fv) comprises a heavy chain variable region and a light chain variable region linked by a disulfide bond or a linking peptide. The heavy chain variable region and the light chain variable region of the Xindilizumab are respectively shown as SEQ ID NO. 1-2:

SEQ ID NO.1：

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGLIIPMFDTAGYAQKFQGRVAITVDESTSTAYMELSSLRSEDTAVYYCARAEHSSTGTFDYWGQGTLVTVSS。

SEQ ID NO.2：

DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLISAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANHLPFTFGGGTKVEIKRTV。

the complementarity determining regions of the sillimumab include CDR1-VH1, CDR 2-VH 2, CDR 3-VH 3, CDR1-VL1, CDR 2-VL 2, and CDR 3-VL 3 connected by disulfide bonds or connecting peptides:

CDR1-H1：SYAIS；

CDR2-H2：LIIPMFDTAGYAQKFQG；

CDR3-H3：AEHSSTGTFDY；

CDR1-VL1：RASQGISSWLA；

CDR2-VL2：AASSLQS；

CDR3-VL3：QQANHLPFT。

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

SEQ ID NO.3：

QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS

SEQ ID NO.4：

STGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTV。

the complementarity determining regions of nivolumab include CDR1-VH1, CDR 2-VH 2, CDR 3-VH 3, CDR1-VL1, CDR 2-VL 2 and CDR 3-VL 3 connected by disulfide bonds or connecting peptides:

CDR1-VH1：NSGMH；

CDR2-VH2：VIWYDGSKRYYADSVKG；

CDR3-VH3：NDDY；

CDR1-VL1：RASQSVSSYLA；

CDR2-VL2：DASNRAT；

CDR3-VL3：QQSSNWPRT。

the heavy chain variable region and the light chain variable region of the tirezumab are respectively shown as SEQ ID NO. 5-6:

SEQ ID NO.5：

QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVHWIRQPPGKGLEWIGVIYADGSTNYNPSLKSRVTISKDTSKNQVSLKLSSVTAADTAVYYCARAYGNYWYIDVWGQGTTVTVSS。

SEQ ID NO.6：

DIVMTQSPDSLAVSLGERATINCKSSESVSNDVAWYQQKPGQPPKLLINYAFHRFTGVPDRFSGSGYGTDFTLTISSLQAEDVAVYYCHQAYSSPYTFGQGTKLEIKRTV。

the complementarity determining regions of tirizumab include CDR1-VH1, CDR 2-VH 2, CDR 3-VH 3, CD1R-VL1, CDR 2-VL 2, and CDR 3-VL 3 connected by disulfide bonds or connecting peptides:

CDR1-VH1：SYGVH；

CDR2-VH2：VIYADGSTNYNPSLKS；

CDR3-VH3：AYGNYWYIDV；

CDR1-VL1：KSSESVSNDVA；

CDR2-VL2：YAFHRFT；

CDR3-VL3：HQAYSSPYT。

the heavy chain variable region and the light chain variable region of the Pabollizumab are respectively shown as SEQ ID NO. 7-8:

SEQ ID NO.7：

QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS。

SEQ ID NO.8：

EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTV。

the complementarity determining regions of pabollizumab include CDR1-VH1, CDR 2-VH 2, CDR 3-VH 3, CDR1-VL1, CDR 2-VL 2, and CDR 3-VL 3 connected by disulfide bonds or connecting peptides:

CDR1-VH1：NYYMY；

CDR2-VH2：GINPSNGGTNFNEKFKN；

CDR3-VH3：RDYRFDMGFDY；

CDR1-VL1：RASKGVSTSGYSYLH；

CDR2-VL2：LASYLES；

CDR3-VL3：QHSRDLPLT。

the heavy chain variable region and the light chain variable region of the Abamelumab are respectively shown as SEQ ID NO. 9-10:

SEQ ID NO.9：

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSS。

SEQ ID NO.10：

QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQP。

the complementarity determining regions of avilamumab include CDR1-VH1, CDR 2-VH 2, CDR 3-VH 3, CDR1-VL1, CDR 2-VL 2 and CDR 3-VL 3 connected by disulfide bonds or connecting peptides:

CDR1-VH1：SYIMM；

CDR2-VH2：SIYPSGGITFYADTVKG；

CDR3-VH3：IKLGTVTTVDY；

CDR1-VL1：TGTSSDVGGYNYVS；

CDR2-VL2：DVSNRPS；

CDR3-VL3：SSYTSSSTRV。

the heavy chain variable region and the light chain variable region of the atezumab are respectively shown as SEQ ID NO. 11-12:

SEQ ID NO.11：

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA。

SEQ ID NO.12：

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVA。

the complementarity determining regions of attrituzumab include CDR1-VH1, CDR 2-VH 2, CDR 3-VH 3, CDR1-VL1, CDR 2-VL 2, and CDR 3-VL 3 connected by disulfide bonds or connecting peptides:

CDR1-VH1：DSWIH；

CDR2-VH2：WISPYGGSTYYADSVKG；

CDR3-VH3：RHWPGGFDY；

CDR1-VL1：RASQDVSTAVA；

CDR2-VL2：SASFLYS；

CDR3-VL3：QQYLYHPAT。

the heavy chain variable region and the light chain variable region of the Tereprinizumab are respectively shown as SEQ ID NO. 13-14:

SEQ ID NO.13：

QGQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPIHGLEWIGVIESETGGTAYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREGITTVATTYYWYFDVWGQGTTVTVSS。

SEQ ID NO.14：

DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIKRTV。

the complementarity determining regions of Teraprimab include CDR1-VH1, CDR 2-VH 2, CDR 3-VH 3, CDR1-VL1, CDR 2-VL 2, and CDR 3-VL 3, which are linked by disulfide bonds or linking peptides:

CDR1-VH1：DYEMH；

CDR2-VH2：VIESETGGTAYNQKFKG；

CDR3-VH3：EGITTVATTYYWYFDV；

CDR1-VL1：RSSQSIVHSNGNTYLE；

CDR2-VL2：KVSNRFS；

CDR3-VL3：FQGSHVPLT。

in other embodiments, the amino acid sequence of the variable region, heavy chain variable region, or complementarity determining region of a PD-1 biological agent provided by the invention can 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 to the corresponding variable region, heavy chain variable region, or complementarity determining region.

In an alternative embodiment, the antibody detection kit for the PD-1 biological agent is a quantitative detection kit or a qualitative detection kit.

In an optional embodiment, the antibody detection kit for the PD-1 biological agent is a fluorescence immunochromatography detection kit, a colloidal gold immunochromatography detection kit, a quantum dot fluorescence immunochromatography detection kit, an enzyme-linked immunosorbent assay kit, or a chemiluminescence detection kit;

in an alternative embodiment, the fluorescence immunochromatographic assay kit is a time-resolved fluorescence immunochromatographic assay kit.

The invention also provides a preparation method of the antibody detection kit for the PD-1 biological agent, which comprises the following steps: the PD-1 biological agent is fixed on a solid phase carrier in full length to be used as an antibody capture device, and the functional fragment without the label is combined or fixed on a cellulose membrane or the functional fragment is labeled by the label to be used as an antibody detection device.

In an alternative embodiment, when the solid support is selected from a microplate, streptavidin is coated on the microplate before the PD-1 biological agent is immobilized in the whole length of the microplate, and then the biotin-modified PD-1 biological agent is incubated in the whole length of the microplate coated with streptavidin.

The invention has the following beneficial effects:

the PD-1 biological agent is used as the capture antigen, and the anti-antibody in the sample can be captured more easily due to the large space contact surface, so that the method has the advantage of high capture efficiency. The detection of the anti-antibody is realized by means of a double-antigen sandwich method, so that a large amount of irrelevant antibody interference can be avoided to a great extent, the detection accuracy is improved, and the occurrence of false positive is avoided.

The antigen-antibody complex formed after capture has larger steric hindrance, and the inventor finds that the variable region, the heavy chain variable region or the complementarity determining region of the PD-1 biological agent with small molecular weight has the advantages of small steric hindrance and easy combination by the anti-antibody Fab, and the antigen-antibody complex can be used as an antibody detection device to improve the combination efficiency of the antigen-antibody complex and a detection antibody element, improve the detection sensitivity and avoid the occurrence of false negative. The purpose of concomitant diagnosis of PD-1 biological agent medicines is achieved.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a diagram of the apparatus of the anti-Sedum aibizumab antibody time-resolved fluorescence immunochromatographic test paper of the present invention;

FIG. 2 is a schematic diagram of the detection of the ELISA kit for detecting anti-Nwaruzumab antibody according to the present invention;

FIG. 3 is a schematic diagram of the detection of the anti-astuzumab antibody chemiluminescence assay kit of the present invention;

FIG. 4 is a regression plot of the calibration curve of the anti-Sedum aibizumab antibody time-resolved fluorescence immunochromatographic kit of the present invention;

FIG. 5 is a regression graph of the calibration curve of the ELISA kit for anti-Nwaruzumab antibody according to the present invention;

FIG. 6 is a regression plot of the calibration curve of the anti-astuzumab antibody chemiluminescence assay kit of the present invention;

reference numerals: 1-sample pad; 2-a conjugate pad; 3-nitrocellulose membrane; 4-detection line; 5-quality control line; 6-water absorbing film; 7-backing the base plate.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

The kit provided by the invention has the following working principle that 1) the immobilized solid phase carrier of the PD-1 biological agent is used for capturing the anti-antibody in a sample to be detected, and the molecular space action area of the PD-1 biological agent is large, so that the anti-antibody in the sample can be captured efficiently to form an antigen-antibody compound; 2) the variable region, the heavy chain variable region or the complementarity determining region of the PD-1 biological agent is used as a detection antigen, and due to the small molecular weight, the steric hindrance can be effectively overcome, the detection antigen is combined with the other Fab of the antibody in the antigen-antibody complex to form an antigen-antibody-antigen complex, and the antigen-antibody-antigen complex is quantitatively or qualitatively detected by a well-known color development method.

Example 1

The embodiment provides an anti-Cedilizumab antibody time-resolved fluorescence immunochromatography quantitative detection kit and a preparation method thereof.

Referring to fig. 1, the test paper is provided with a nitrocellulose membrane 3, a sample pad 1, a combination pad 2 and a water absorption membrane 6 on a backing substrate 7, wherein the sample pad 1 is laminated on one end of the combination pad 2, and the other end of the combination pad 2 and the water absorption membrane 6 are respectively laminated on two ends of the nitrocellulose membrane 3; the combination pad 2 is coated with a Xindilizumab labeled by a time-resolved fluorescent microsphere and a goat anti-chicken IgY antibody 2 labeled by the time-resolved fluorescent microsphere; 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 a variable region Fv of the Xindilizumab, and the quality control line 5(C line) is coated with a chicken IgY antibody.

The variable region (Fv) of the sediizumab comprises a sediizumab heavy chain variable region and a sediizumab light chain variable region linked by a disulfide bond, wherein the sediizumab heavy chain variable region sequence is as follows:

the sequence of the variable region of the Xindilizumab light chain is as follows:

the preparation method comprises the following steps:

1) time-resolved labeling of fluorescent microspheres

500. mu.L (300 nm) of time-resolved fluorescent microspheres (1% stock solution) are centrifuged at 13000rpm at 4 ℃ for 10min, and the supernatant is discarded. Adding 10mg/ml EDC, and reacting for 15 min; centrifuging, removing supernatant, adding 1000 μ L borate buffer (20 mM, pH8.0), and mixing by ultrasonic for 10 s; adding 100 mu g of labeled antibody (Usneuzumab or goat anti-chicken IgY) (labeled concentration is 100 mu g/mL), and reacting for 2h at room temperature; centrifuging, removing supernatant, adding confining liquid, and reacting for 1 h; centrifuging, removing supernatant, adding 1000. mu.L borate buffer solution, ultrasonically mixing, labeling, and storing at 4 deg.C.

2) Sample pad, conjugate pad treatment

The polyester film was pre-blocked by soaking in a buffer solution containing a surfactant (formulation: 20mM pH8.0 BB, containing 2% BSA, 0.5% S9 and 2% trehalose), and dried overnight at 37 ℃ to prepare a conjugate pad; and (3) mixing the prepared binding pad with a gold-labeled HM3035 gold spraying and membrane scratching instrument, spraying 3 mu L/cm of the mixture of the Xindilizumab labeled with the time-resolved fluorescent microspheres and the goat anti-chicken IgY antibody onto the binding pad with the width of 1cm, drying at 37 ℃, and drying for 3 hours to prepare the specific binding pad.

The glass cellulose membrane was pre-blocked by soaking in a buffer containing a surfactant (formulation: 100mM pH7.4 PB containing 2% BSA, 0.5% S9), dried overnight at 37 ℃ and cut into 30X 1.5cm pieces to prepare sample pads.

3) Coating of nitrocellulose membranes (NC membranes)

The amino acid synthesis method synthesizes the variable region of the Xindilizumab, and the synthesized polypeptide is proved to be coated on an enzyme label plate and can react with an anti-Xindilizumab antibody.

Attaching an NC membrane to a designated position of a backing substrate, diluting the heavy chain variable region of the Cedilizumab to 1.5mg/mL with 50mM phosphate buffer solution of pH7.4 for preparing a T-line; diluting chicken IgY antibody to 0.5mg/mL by 50mM phosphate buffer solution with pH7.4 for preparing C line; uniformly scratching the two diluted antibodies on an NC film by a gold mark HM3035 gold spraying and film scratching instrument according to the scratching amount of 1 mu L/cm to prepare a T line and a C line; the scribed NC films were placed in a 37 ℃ dry box to dry overnight.

4) Assembly

Laminating the combination pad obtained in the step 2) on one end of the nitrocellulose membrane obtained in the step 3), fixing and laminating a water absorption membrane on the other end of the nitrocellulose membrane, finally laminating the sample pad obtained in the step 2) on the other end of the combination pad, cutting the sample pad according to the width of 4mm of each pad by using a membrane cutting instrument, and filling the sample pad into a chromatography strip shell to obtain a finished product.

5) Detection of

Diluting the serum by 10 times, adding 90 mu L of the diluted serum into a sample adding hole, reacting for 10min, and detecting by a fluorescence reader. And comparing the value with the value of a standard substance or a reference substance, and judging the content of the anti-antibody in the sample to be detected.

In this embodiment, the time-resolved fluorescent substance latex microspheres are europium-containing latex microspheres, i.e., the time-resolved fluorescent substance is a combination of europium lanthanide and latex; the time-resolved fluorescent substance can be adjusted to be one of lanthanide, a combination of lanthanide and latex, and a lanthanide chelate compound as required; the lanthanide may be one of europium, terbium, samarium or dysprosium.

Example 2

The embodiment provides a preparation method of an enzyme-linked immunoassay kit for an anti-nivolumab antibody. The detection principle diagram is shown in fig. 2.

The kit comprises an ELISA plate, biotinylation nivolumab, an anti-nivolumab antibody calibrator, an anti-nivolumab antibody quality control product, an enzyme conjugate, a cleaning solution and a stop solution. The enzyme conjugate is horseradish peroxidase (HRP) labeled nivolumab Complementarity Determining Regions (CDRs). The microplate is coated with streptavidin.

The Complementarity Determining Regions (CDRs) of nivolumab include CDR1-VH1, CDR 2-VH 2, CDR 3-VH 3, CDR-VL1, CDR 2-VL 2 and CDR 3-VL 3 connected by a connecting peptide:

CDR1-VH1：NSGMH；

CDR2-VH2：VIWYDGSKRYYADSVKG；

CDR3-VH3：NDDY；

CDR1-VL1：RASQSVSSYLA；

CDR2-VL2：DASNRAT；

CDR3-VL3：QQSSNWPRT。

the linker peptide is selected from GGGGSGGGGSGGS.

The preparation method comprises the following steps:

1) biotinylation of Nwaruzumab

1mg of nivolumab was put into a 5 mL centrifuge tube, 50. mu.L of 10mg/mL activated biotin (Sulfo-NHS-LC-Bintin) was added thereto, the mixture was reacted at room temperature for 1 hour, and the reaction mixture was taken out and dialyzed with 0.02M PBS overnight. The biotinylated nivolumab was removed and preserved by adding 0.03% Proclin300 for 4 degrees.

2) HRP-labeled nivolumab Complementarity Determining Regions (CDRs)

The amino acid synthesis method synthesizes the Complementary Determining Regions (CDRs) of the monoclonal antibody of the nivolumab, and the synthetic polypeptide can react with the antibody of the monoclonal antibody of the nivolumab after verification.

Weighing 5mg HRP, placing in a 5 mL centrifuge tube, adding 1mL pure water for dissolving, adding 1mL NaIO₄The solution (10 mg/mL, ready to use) was reacted for 30min at 4 ℃ in the dark. 0.02mL of ethylene glycol was added and the reaction was carried out at room temperature for 15 min. Dialyzing the solution and taking out. 1mg of the Complementary Determining Regions (CDRs) of the nivolumab was added to the activated solution, and the mixture was reacted for 2 hours at room temperature with exclusion of light, and 0.2mL of NaBH4 solution (5 mg/mL, prepared as is) was added thereto, mixed well, and dialyzed overnight against 0.05M CB. And (4) taking out the marked enzyme-labeled antibody, adding glycerol with the same volume, and storing at-20 ℃ for later use.

3) Coated enzyme label plate

Streptavidin was diluted to 1. mu.g/mL in CB buffer, coated in 100. mu.L wells in microtiter plates, for 2h at 37 ℃. The coating solution was aspirated off, incubated with 200. mu.L of blocking buffer containing 2% BSA per well for 2 hours at 37 ℃, the blocking solution was tapped off, and dried at 37 ℃ for future use.

4) Coating biotinylation nivolumab

Dilute biotinylated nivolumab to 1. mu.g/mL with neutral phosphate buffer, 100. mu.L per well, 2h at 37 ℃. The plate was washed 3 times, incubated with 200. mu.L of blocking buffer containing 2% BSA per well for 2 hours at 37 ℃, blotted off and dried at 37 ℃ for use.

5) Diluting a serum sample to be detected by 100 times, adding 100 mu L of the serum sample into each hole of the sample, incubating at 37 ℃ for 2 hours, and washing the plate for 4 times.

6) Adding HRP-labeled nivolumab Complementarity Determining Regions (CDRs), incubating for 1 hr, washing the plate 4 times

7) Adding a substrate TMB of HRP enzyme into the microplate, incubating for 15 minutes, adding a termination solution HCL solution to terminate the color reaction, detecting the OD value by using the wavelength of 450nm, and comparing with the OD value of a standard substance or a reference substance to judge the content of the anti-antibody in the sample to be detected.

Example 3

The embodiment provides an astuzumab antibody chemiluminescence assay kit and a preparation method thereof.

The chemiluminescence detection kit for the anti-attrituzumab antibody comprises: biotinylation alemtuzumab, an anti-alemtuzumab antibody calibrator, an anti-alemtuzumab antibody quality control product, an acridinium ester labeled alemtuzumab single-antibody heavy chain variable region (Hv), a magnetic particle reagent, an excitation liquid and a cleaning liquid.

The sequence of the variable region of the Abelizhu single-antibody heavy chain is as follows:

the preparation method comprises the following steps:

1) biotinylation of Abiralizumab

1mg of atuzumab was put into a 5 mL centrifuge tube, 50. mu.L of 10mg/mL activated biotin (Sulfo-NHS-LC-Bintin) was added thereto, the mixture was reacted at room temperature for 1 hour, and the reaction mixture was taken out and dialyzed with 0.02M PBS overnight. The biotinylated alemtuzumab antibody was removed and preserved by adding 0.03% Proclin300 for 4 degrees.

2) Acridinium ester labeled altemant bead single-antibody heavy chain variable region (Hv)

The amino acid synthesis method synthesizes an attrituximab heavy chain variable region (Hv), and the synthetic polypeptide is proved to be capable of reacting with an anti-attrituximab antibody to coat an enzyme label plate.

Taking 2mg of attritor bead single-antibody heavy chain variable region (Hv), and according to the antibody: and (3) adding the activated acridinium ester according to the molar ratio of 1: 10-50, and reacting for 30min at room temperature. Dialyzing the reaction solution with 0.05M CB solution, taking out, adding glycerol with the same volume, and storing at-20 ℃ for later use.

3) Detection step

The present example utilizes acridinium ester chemiluminescence immunoassay technology-coupled biotin-avidin magnetic particle separation technology to achieve anti-antibody detection. The detection principle is as shown in FIG. 3: and mixing the sample (diluted by 20 times), the streptavidin magnetic particles (1) and the biotinylated altlizumab (2) to obtain a streptavidin magnetic bead-biotinylated altlizumab-sample complex (3), washing, adding an acridinium ester labeled altlizumab single-antibody heavy chain variable region (Hv) for reaction to form the streptavidin magnetic bead-biotinylated altlizumab-sample-acridinium ester labeled altlizumab single-antibody heavy chain variable region (Hv) complex. Adding luminous excitation liquid, measuring luminous intensity, comparing with the value of standard or reference, and judging the content of the anti-antibody in the sample to be detected.

Experimental example 1

This example quantitatively detects the kit of example 1.

1.1 Standard Curve preparation

1.1.1A set of anti-Cedilizumab antibody calibrators is taken, and specific values are shown in Table 1.

TABLE 1

1.1.2 detection method

Taking 90 mu L of a calibrator, and directly adding the calibrator into a sample window in the chromatographic strip; after 10 minutes, the signal values were quantitatively determined with a time-resolved fluorescence quantitative analyzer.

Each calibrator was tested 2 times and the T/C values averaged. The specific results are shown in Table 2.

TABLE 2

1.1.3 Standard Curve preparation

According to the detection result, taking the concentration value as an X axis and the T/C value as a Y axis, performing linear regression to obtain a linear equation: y =0.0026x + 0.0553, R = 0.9979, the curve is shown in fig. 4.

1.2 precision test:

1.2.1 preparing 10 prepared chromatographic strips, and preparing an anti-Truelizumab antibody working calibration product with the concentration (50 ng/mL);

1.2.2 directly adding 90 mu L of calibrator into a sample adding hole of the reagent strip;

1.2.3 after the calibrator is chromatographed for 10min, a time-resolved fluorescence quantitative analyzer is used for detecting, the results of 10 reagent strips are measured according to the linear equation, the results are shown in table 3, and the precision of the anti-Tradix monoclonal antibody time-resolved fluorescence immunochromatography assay kit is good.

TABLE 3

1.3 compared to other methods:

labeling the microspheres with the Xindilizumab, and coating an NC membrane with the Xindilizumab as a method 2; labeling the microspheres with the Cedilizumab fab, and coating the NC membrane with the Cedilizumab fab as method 3. The conditions were the same for the three methods except that the labeled antibody and the coated antibody were different, and the results were shown in Table 4.

TABLE 4

Experimental example 2

This example quantitatively detects the kit of example 2.

2.1 detection method

2.1.1 taking a set of anti-Natuzumab antibody calibrator, the specific values are shown in Table 5

TABLE 5

2.1.2 detection procedure

1) Adding 100 microliter of calibrator and quality control material into each well, incubating at 37 deg.C for 2 hr, and washing for 4 times.

2) Adding HRP-labeled nivolumab complementarity determining region, incubating for 1h, and washing the plate for 4 times.

3) Adding a substrate TMB into the microporous plate, incubating for 15min, adding a stop solution to stop the color reaction, detecting the OD value by using the wavelength of 450nm, and judging the performance of the kit. The specific results are shown in Table 6.

TABLE 6

2.1.3 Standard Curve

According to the detection result, curve fitting is carried out by taking the OD value as an X axis and the concentration value as a Y axis to obtain a curve equation: y = 0.0047x + 0.1247, R = 0.997, the curve is shown in fig. 5.

2.2 precision test:

according to the OD value of the detected precision quality control product, the result value is calculated by a curve equation, and the result is shown in table 7, which shows that the anti-nivolumab antibody enzyme-linked immunoassay kit has good precision.

TABLE 7

2.3 compared to other methods:

labeling HRP with nivolumab, coating the microporous plate with biotinylation nivolumab as method 2; labeling HRP with nivolumab fab, and coating the microplate with nivolumab fab as method 3. The conditions were the same for the three methods except that the labeled antibody and the coated antibody were different, and the results were shown in Table 8.

TABLE 8

Experimental example 3

This example quantitatively detects the kit of example 3.

3.1 detection method

3.1.1 taking a set of anti-attrituzumab antibody calibrator, the specific values are shown in Table 9

TABLE 9

2.1.2 detection procedure

1) Respectively taking 20 mu L of calibrator, 20 mu L of streptavidin magnetic particles and 50 mu L of biotinylation attrituximab, adding the calibrators, the streptavidin magnetic particles and the biotinylation attrituximab into the micropores, mixing, incubating at 37 ℃ for 15min, and cleaning for 3 times.

2) Adding acridinium ester labeled altemant bead single-antibody heavy chain variable region (Hv), incubating at 37 deg.C for 15min, and washing 3 times.

3) Adding luminous excitation liquid, and measuring luminous intensity. The specific results are shown in Table 10.

Watch 10

3.1.3 Standard Curve

According to the detection result, taking the concentration value as an X axis and the luminous value as a Y axis, performing linear regression to obtain a linear equation: y =1042.1x + 15675, R = 0.9982, the curve is shown in fig. 6.

3.2 precision test:

adding 20 μ L of precision quality control product, 20 μ L of streptavidin magnetic particle, and 50 μ L of biotinylation attrituximab into the micropore, mixing, incubating at 37 deg.C for 15min, and cleaning for 3 times; adding acridinium ester labeled altemant bead single-antibody heavy chain variable region (Hv), incubating at 37 ℃ for 15min, and washing for 3 times; adding luminous excitation liquid, and measuring luminous intensity. And calculating a result value according to the curve equation. The anti-alemtuzumab antibody chemiluminescence immunoassay kit has high precision. The specific results are shown in Table 11.

TABLE 11

3.3 compared to other methods:

labeling acridinium ester with astuzumab, labeling biotin with astuzumab as method 2; labeling acridinium ester with atuzumab fab, labeling biotin with atuzumab fab as method 3. The three methods were performed under the same conditions except that the acridinium ester-labeled antibody and the biotin-labeled antibody were different, and the results were shown in Table 12.

TABLE 12

In conclusion, the detection kit provided by the invention has the advantages of high capture efficiency, high accuracy and high sensitivity, and can effectively avoid the occurrence of false positive and false negative. Has good application prospect.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

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Claims

1. The antibody detection kit of the PD-1 biological agent is characterized by comprising 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 the functional fragment without the label is fixed on the cellulose membrane; the functional fragment is a variable region, a heavy chain variable region or a complementarity determining region of the PD-1 biological agent.

2. The antibody detection kit for the PD-1 biological agent according to claim 1, wherein the PD-1 biological agent is selected from the group consisting of monoclonal antibody drugs of PD-1;

preferably, the monoclonal antibody drug of PD-1 is selected from the group consisting of certolizumab (Sintilimab), Nivolumab (Nivolumab), tirelinzumab (tiselizumab), pamrolizumab (Pembrolizumab), carprilizumab (Camrelizumab), avizumab (Avelumab), atezumab (Atezolizumab), and teriprizumab (tropilimumab).

3. The antibody detection kit for the PD-1 biological agent according to claim 1 or 2, characterized in that the solid phase carrier is selected from fluorescent microspheres, latex microspheres, resin microspheres, magnetic microspheres, colloidal gold particles, quantum dots, microwell plates or microporous membranes;

preferably, the PD-1 biological agent coated on the solid phase carrier is modified with avidin or biotin;

preferably, the fluorescent microspheres are selected from time-resolved fluorescent microspheres or fluorescent microspheres containing fluorescein, and the magnetic microspheres are selected from magnetic beads;

preferably, the solid phase carrier is coated with the full length of the PD-1 biological agent.

4. The antibody detection kit for PD-1 biological agents according to claim 1 or 2, characterized in that said detectable label is selected from fluorescent dyes, enzymes catalyzing the development of a substrate, radioisotopes, chemiluminescent reagents or nanoparticle-based labels;

preferably, the fluorescent dye is selected from fluorescein dyes and derivatives thereof, rhodamine dyes and derivatives thereof, Cy dyes and derivatives thereof, Alexa dyes and derivatives thereof, and protein dyes and derivatives thereof;

preferably, the enzyme catalyzing the color development of the substrate is selected from the group consisting of horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxyenzyme;

preferably, the radioisotope is selected from²¹²Bi、¹³¹I、¹¹¹In、⁹⁰Y、¹⁸⁶Re、²¹¹At、¹²⁵I、¹⁸⁸Re、¹⁵³Sm、²¹³Bi、³²P、⁹⁴mTc、⁹⁹mTc、²⁰³Pb、⁶⁷Ga、⁶⁸Ga、⁴³Sc、⁴⁷Sc、¹¹⁰mIn、⁹⁷Ru、⁶²Cu、⁶⁴Cu、⁶⁷Cu、⁶⁸Cu、⁸⁶Y、⁸⁸Y、¹²¹Sn、¹⁶¹Tb、¹⁶⁶Ho、¹⁰⁵Rh、¹⁷⁷Lu、¹⁷²Lu and¹⁸F；

preferably, the chemiluminescent reagent is selected from luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, lowhine and its derivatives or peroxyoxalate and its derivatives;

preferably, the nanoparticle-based label is selected from the group consisting of nanoparticles and colloids;

preferably, the colloid is selected from the group consisting of colloidal metals, disperse dyes, dye-labeled microspheres, and latexes; preferably, the colloidal metal is selected from colloidal gold, colloidal silver, colloidal carbon or colloidal selenium;

preferably, the nanoparticles are selected from organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles or rare earth complex nanoparticles.

5. The antibody detection kit for the PD-1 biological agent according to claim 1 or 2, characterized in that a quality control antibody is further fixed on the cellulose membrane as a quality control T line, and the functional fragment and the quality control antibody are separately provided at intervals;

preferably, the cellulose membrane is selected from a cellulose acetate membrane or a cellulose nitrate membrane.

6. The antibody detection kit for the PD-1 biological agent according to claim 1 or 2, characterized in that the heavy chain variable region and the light chain variable region of the Cedilizumab are shown in SEQ ID No.1-2, respectively, and the heavy chain variable region and the light chain variable region of the Nawumab are shown in SEQ ID No.3-4, respectively; the heavy chain variable region and the light chain variable region of the tirezumab are respectively shown in SEQ ID NO. 5-6; the heavy chain variable region and the light chain variable region of the Pabollizumab are respectively shown in SEQ ID NO. 7-8; the heavy chain variable region and the light chain variable region of the Abamelumab are respectively shown in SEQ ID NO. 9-10; the heavy chain variable region and the light chain variable region of the atelizumab are respectively shown as SEQ ID NO. 11-12; the heavy chain variable region and the light chain variable region of the Tereprinizumab are respectively shown in SEQ ID NO. 13-14;

preferably, the variable regions (Fv) of said certolizumab, nivolumab, tirezumab, palivizumab, avizumab, atelizumab, terliplizumab comprise heavy and light chain variable regions linked by a disulfide bond or a linking peptide, and the complementarity determining regions thereof comprise CDR1-VH1, CDR 2-VH 2, CDR 3-VH 3, CDR1-VL1, CDR 2-VL 2 and CDR 3-VL 3 linked by a disulfide bond or a linking peptide.

7. The method for preparing the antibody detection kit for the PD-1 biological agent 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 method for preparing the antibody detection kit for the PD-1 biological agent according to claim 1 or 2, characterized in that the antibody detection kit for the PD-1 biological agent is a fluorescence immunochromatography detection kit, a colloidal gold immunochromatography detection kit, a quantum dot fluorescence immunochromatography detection kit, an enzyme-linked immunosorbent assay kit or a chemiluminescence detection kit;

preferably, the fluorescence immunochromatographic assay kit is a time-resolved fluorescence immunochromatographic assay kit.

9. A method of making an antibody detection kit for a PD-1 biological agent as recited in any one of claims 1-8, comprising: PD-1 biological agents are fixed on a solid phase carrier to be used as an antibody capture device, and functional fragments without labels are combined or fixed on a cellulose membrane or the functional fragments are labeled by labels to be used as an antibody detection device.

10. The method for preparing the antibody detection kit for the PD-1 biological agent according to claim 9, wherein when the solid support is selected from a microplate, streptavidin is coated on the microplate before the PD-1 biological agent is immobilized on the microplate in its full length, and then the PD-1 biological agent modified with biotin is incubated with the microplate coated with streptavidin.