An antibody detection kit for CD biological agents and its preparation method

Abstract

The present invention discloses an antibody detection kit for CD biological agents and a preparation method thereof, which relates to the technical field of immunoassay. The kit includes an antibody capture device and an antibody detection device. The antibody capture device includes a solid-phase carrier coated with CD biological agents, and the antibody detection device is a cellulose membrane immobilized with functional fragments or a conjugate conjugated with functional fragments; the functional fragments are variable regions or heavy chain variable regions or heavy chain hypervariable regions of CD biological agents. The present invention overcomes the false positive and false negative defects in the prior art. By coating CD biological agents on the capture device, the occurrence of false positives is avoided. The inventor sets that the detection antigen with a small molecular weight, such as the variable region, heavy chain variable region or complementarity-determining region of CD biological agents, has the advantages of small steric hindrance and easy binding to the Fab of anti-antibody, making the detection sensitivity higher and achieving the purpose of companion diagnosis of CD biological agent drugs. Thus, the phenomenon of false negatives is avoided.

Description

Technical Field

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

[0002] Human leukocyte differentiation antigens (CD molecules) are cell surface molecules that appear or disappear at different stages during the normal differentiation of leukocytes. Most of them are proteins or glycoproteins. Among them, lymphocyte differentiation antigens play important regulatory roles in cell activation, signal transduction, differentiation and activation of hematopoietic cells and blood formed elements. Abnormal expression of them can lead to the occurrence and development of immune diseases, hematological malignancies such as leukemia, non-Hodgkin lymphoma and other diseases.

[0003] There are many kinds of monoclonal antibody drugs or biological agents designed with CD antigens as targets, including monoclonal antibodies targeting CD20 (Rituximab, Ofatumumab, Tositumomab, Obinutuzumab), monoclonal antibodies targeting CD3 (Muromonab-CD3), monoclonal antibodies targeting CD52 (Alemtuzumab), monoclonal antibodies targeting CD38 (Daratumumab), and monoclonal antibodies targeting CD-11α (Efalizumab) which are approved by the FDA for the treatment of non-Hodgkin lymphoma, chronic lymphocytic leukemia, rheumatoid arthritis, heart and kidney transplant rejection, relapsing-remitting multiple sclerosis, multiple myeloma, psoriasis and so on.

[0004] Although clinical practice has proved that such biological agents have good curative effects in the treatment of the above diseases, not all patients are effective. Some patients are ineffective from the beginning (primary ineffectiveness), and more patients are effective at the beginning, but become drug-resistant and ineffective after multiple uses (secondary ineffectiveness). Further research has found that secondary ineffectiveness is related to the production of antibodies against biological agents in the body. Therefore, detecting antibodies against biological agents in the blood is an important companion diagnosis.

[0005] The detection methods of antibodies against CD antigen biological agents mainly proceed through immunological reactions, including the indirect method or the double antigen sandwich method. In Comparative Document Patent 201180042537.9, the Fab of a therapeutic monoclonal antibody is used to coat a solid-phase carrier to capture the immune complex formed by the target antibody, and the content of the immune complex is detected by a labeling method. This method belongs to the indirect method. In Comparative Document Patent 201180060851.X, a therapeutic antibody is used to capture the anti-antibody in the sample, and molecular exclusion is used to distinguish specific anti-antibodies. The principle is to capture with a full-length monoclonal antibody and then detect by high-performance liquid chromatography, which belongs to the indirect method plus the liquid phase method. In detection schemes with washing steps such as ELISA and tube chemiluminescence, the indirect method is an efficient and effective detection method. However, in detection schemes like chromatography that lack a washing step, the indirect method is interfered by a large number of irrelevant antibodies in the sample, resulting in detection distortion such as false positives. The double antigen sandwich method can effectively avoid the interference of irrelevant antibodies in the sample. For example, in the determination of anti-drug antibodies disclosed in Patent CN200780002957.8, a double antigen bridging immunoassay is mentioned, that is, a mixture of drug antibodies is used as the capture antigen and a mixture of drug antibodies is used as the detection antigen. In Comparative Document Patent 201510788470.3, biotinylated TNFα monoclonal antibody is used as the capture antigen, and AP-labeled TNFα monoclonal antibody is used as the detection antibody. Its principle is essentially to form a double antigen sandwich with two full-length monoclonal antibodies. However, in the disclosed double antigen sandwich method technical solution, if the binding efficiency of the immune complex formed after capture and the detection antigen is not fully considered, false negative results will occur.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] The purpose of the present invention is to provide an antibody detection kit for CD biological agents and a preparation method to solve the above technical problems.

[0008] The inventors creatively discovered that in the double antigen sandwich method technical solution, the root cause of false negative results is that the binding efficiency of the immune complex formed after capture and the detection antigen is not fully considered, so that even if the target antibody in the positive sample binds to the capture antigen to form an immune complex, it cannot bind to the detection antigen with high sensitivity, resulting in misjudgment of the detection result for positive samples (causing false negatives) during subsequent detection.

[0009] The present invention is implemented as follows:

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

[0011] The present invention overcomes the false positive and false negative defects in the prior art. Using the CD biological agent as the capture antigen, due to the large spatial contact surface, it is easier to capture the antibody against the CD biological agent (i.e., anti-antibody) in the sample, and has the advantage of high capture efficiency. By coating the CD biological agent on the capture device, the occurrence of false positives is avoided.

[0012] The antigen-antibody complex formed after capture itself has a large steric hindrance. If the biological agent is coated on the antibody detection device, since the molecular weight of the biological agent (i.e., the detection antigen) is large, it is difficult for the biological agent coated on the detection device to bind to the other Fab of the antibody against the CD biological agent in the complex formed after capture. Therefore, the inventor sets that the detection antigen with a small molecular weight, such as the variable region, heavy chain variable region or complementary determining region of the biological agent, has the advantages of small steric hindrance and easy binding to the Fab of the anti-antibody, making the detection sensitivity higher and achieving the purpose of companion diagnosis of the CD biological agent drug. This also avoids the misjudgment of the subsequent sample detection results and the appearance of false negative phenomena.

[0013] In an optional embodiment, the above-mentioned functional fragment usually has the same binding specificity as its source antibody. Fv (variable region) is composed of the heavy chain variable region and the light chain variable region connected by a linker peptide or a disulfide bond to maintain the stability of Fv. CDRs (complementary determining regions) include CDR1, CDR2 and CDR3 parts in the heavy chain variable region and the light chain variable region. Those skilled in the art can easily understand from the content recorded in the present invention that the above-mentioned functional fragment of the antibody can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemically reducing and cleaving disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, those skilled in the art can easily obtain the above-mentioned functional fragment.

[0014] Including but not limited to any one selected from HV, F(ab’)2, Fab’, Fab, Fv, CDRs and scFv of the above-mentioned CD biological agent.

[0015] Functional fragments of the above antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art (such as recombinant expression by genetic recombination methods) or by synthesis using, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems.

[0016] In a preferred embodiment of the application of the present invention, the above CD biological agent is selected from monoclonal antibody drugs against CD.

[0017] In an alternative embodiment, the monoclonal antibody drugs against CD are selected from Rituximab, Ofatumumab, Tositumomab, Obinutuzumab, Muromonab-CD3, Alemtuzumab, Daratumumab or Efalizumab.

[0018] In a preferred embodiment of the application of the present invention, the above solid-phase carrier is selected from fluorescent microspheres, latex microspheres, resin microspheres, magnetic microspheres, colloidal gold particles, quantum dots, microtiter plates or microporous membranes. The above colloidal gold is a colloidal solution obtained by reducing chloroauric acid with a reducing agent. In other embodiments, the above colloidal gold can also be other colloidal substances, such as colloidal carbon, colloidal silver or colloidal selenium.

[0019] In an alternative embodiment, the above quantum dots are core-shell quantum dots, such as quantum dots formed by ZnS / CdSe or ZnS / CdTe. According to needs, the above 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 coated on a CdSe core, etc.

[0020] In an alternative embodiment, the CD biological agent coated on the solid-phase carrier is modified with avidin or biotin. It should be noted that in an alternative embodiment, avidin or biotin can also be optionally not modified on the biological agent according to needs.

[0021] In an alternative embodiment, the fluorescent microspheres are selected from time-resolved fluorescent microspheres, and the magnetic microspheres are selected from magnetic beads.

[0022] In an alternative embodiment, the solid phase carrier is coated with the full length of the CD biological agent. Using the full length of the CD biological agent as the capture antigen, due to the large spatial contact surface, it is easier to capture the anti-CD biological agent antibody (i.e., anti-antibody) in the sample, and has the advantage of high capture efficiency. By coating the full length of the CD biological agent on the capture device, the occurrence of false positives can be better avoided.

[0023] In an alternative embodiment, the time-resolved fluorescence microspheres are selected from the conjugates of time-resolved fluorescent substances and latex or nanoparticles, and the time-resolved fluorescent substances are one of lanthanide elements, conjugates of lanthanide elements and latex, and chelates of lanthanide elements; the lanthanide element can be any one of europium, terbium, samarium or dysprosium.

[0024] The solid phase carrier is coated with the full length of the CD biological agent. Coating the full length has a large spatial contact surface and is easier to capture the anti-antibody in the sample, and has the advantage of high capture efficiency.

[0025] The detectable marker refers to a class of substances with characteristics such as luminescence, color development, radioactivity, etc. that can be directly observed by the naked eye or detected or detected by an instrument. Through this characteristic, qualitative or quantitative detection of the corresponding target can be achieved.

[0026] In a preferred embodiment of the application of the present invention, the above-mentioned detectable markers include, but are not limited to, fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents or nanoparticle-based markers.

[0027] In an alternative embodiment, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (such as, but not limited to, fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (such as, but not limited to, red rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (such as, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (such as, 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 (such as, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0028] In an alternative embodiment, the enzymes that catalyze substrate chromogenesis include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0029] In an alternative embodiment, the radioisotopes include, but are not limited to 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 Ru, 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.

[0030] In an alternative embodiment, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, rhodamine and its derivatives, or peroxyoxalate and its derivatives.

[0031] In an alternative embodiment, the nanoparticle-based markers include, but are not limited to, nanoparticles and colloids.

[0032] In an alternative embodiment, the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latexes; in an alternative embodiment, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, colloidal carbon, or colloidal selenium.

[0033] In an alternative embodiment, the nanoparticles include, but are not limited to, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, or rare earth complex nanoparticles.

[0034] In a preferred embodiment of the application of the present invention, a quality control antibody is also immobilized on the cellulose membrane as a quality control T line, and the functional fragment and the quality control antibody are arranged at intervals.

[0035] In an alternative embodiment, the cellulose membrane includes, but is not limited to, a cellulose acetate membrane or a nitrocellulose membrane.

[0036] In a preferred embodiment of the application of the present invention, the variable region of rituximab includes a heavy chain variable region and a light chain variable region connected by a disulfide bond or a linker peptide. The heavy chain variable region and the light chain variable region of rituximab are shown in SEQ ID NO.1-2 respectively; the complementarity-determining regions of rituximab include CDR-H1, CDR2–H2, CDR3–H3, CDR-L1, CDR2–L2, and CDR3–L3 connected by a disulfide bond or a linker peptide;

[0037] CDR-H1: SYNMH; CDR-H2: AIYPGNGDTSYNQKFKG; CDR-H3: STYYGGDWYFNV; CDR-L1: RASSSVSYIH; CDR-L2: ATSNLAS; CDR-L3: QQWTSNPPT.

[0038] The variable region of ofatumumab includes a heavy chain variable region and a light chain variable region connected by a disulfide bond or a linker peptide. The heavy chain variable region and the light chain variable region of ofatumumab are shown in SEQ ID NO.3-4 respectively;

[0039] The complementarity-determining regions of ofatumumab include CDR-H1, CDR2-H2, CDR3-H3, CDR-L1, CDR2-L2, and CDR3-L3 connected by a disulfide bond or a linker peptide:

[0040] CDR-H1: DYAMH; CDR-H2: TISWNSGSIGYADSVKG; CDR-H3: DIQYGNYYYGMDV; CDR-L1: RASQSVSSYLA; CDR-L2: DASNRAT; CDR-L3: QQRSNWPIT.

[0041] The variable regions of obinutuzumab include a heavy-chain variable region and a light-chain variable region linked by a disulfide bond or a linker peptide. The heavy-chain variable region and the light-chain variable region of obinutuzumab are shown in SEQ ID NO.5-6 respectively; the complementarity-determining regions of obinutuzumab include CDR-H1, CDR2-H2, CDR3-H3, CDR-L1, CDR2-L2 and CDR3-L3 linked by a disulfide bond or a linker peptide:

[0042] CDR-H1: YSWIN; CDR-H2: RIFPGDGDTDYNGKFKG; CDR-H3: NVFDGYWLVY; CDR-L1: RSSKSLLHSNGITYLY; CDR-L2: QMSNLVS; CDR-L3: AQNLELPYT.

[0043] The variable regions of muromonab-CD3 include a heavy-chain variable region and a light-chain variable region linked by a disulfide bond or a linker peptide. The heavy-chain variable region and the light-chain variable region of muromonab-CD3 are shown in SEQ ID NO.7-8 respectively; the complementarity-determining regions of muromonab-CD3 include CDR-H1, CDR2-H2, CDR3-H3, CDR-L1, CDR2-L2 and CDR3-L3 linked by a disulfide bond or a linker peptide:

[0044] CDR-H1: RYTMH; CDR-H2: YINPSRGYTNYNQKFKD; CDR-H3: YYDDHYCLDY; CDR-L1: SASSSVSYMN; CDR-L2: DTSKLAS; CDR-L3: QQWSSNPFT.

[0045] The variable regions of alemtuzumab include a heavy-chain variable region and a light-chain variable region linked by a disulfide bond or a linker peptide. The heavy-chain variable region and the light-chain variable region of alemtuzumab are shown in SEQ ID NO.9-10 respectively; the complementarity-determining regions of alemtuzumab include CDR-H1, CDR2-H2, CDR3-H3, CDR-L1, CDR2-L2 and CDR3-L3 linked by a disulfide bond or a linker peptide:

[0046] CDR-H1: DFYMN; CDR-H2: FIRDKAKGYTTEYNPSVKG; CDR-H3: EGHTAAPFDY; CDR-L1: KASQNIDKYLN; CDR-L2: NTNNLQT; CDR-L3: LQHISRPRT.

[0047] The variable regions of Ofatumumab include a heavy chain variable region and a light chain variable region linked by a disulfide bond or a linker peptide. The heavy chain variable region and the light chain variable region of Ofatumumab are shown in SEQ ID NO.11-12 respectively; the complementarity determining regions of Ofatumumab include CDR-H1, CDR2-H2, CDR3-H3, CDR-L1, CDR2-L2 and CDR3-L3 linked by a disulfide bond or a linker peptide:

[0048] CDR-H1: GHWMN; CDR-H2: MIHPSDSETRYNQKFKD; CDR-H3: GIYFYGTTYFDY; CDR-L1: RASKTISKYLA; CDR-L2: SGSTLQS; CDR-L3: QQHNEYPLT

[0049] The variable regions of Tositumomab include a heavy chain variable region and a light chain variable region linked by a disulfide bond or a linker peptide. The heavy chain variable region and the light chain variable region of Tositumomab are shown in SEQ ID NO.13-14 respectively; the complementarity determining regions of Tositumomab include CDR-H1, CDR2-H2, CDR3-H3, CDR-L1, CDR2-L2 and CDR3-L3 linked by a disulfide bond or a linker peptide:

[0050] CDR-H1: SYNMH; CDR-H2: AIYPGNGDTSYNQKFKG; CDR-H3: VVYYSNSYWYFDV; CDR-L1: RASSSVSYMH; CDR-L2: APSNLAS; CDR-L3: QQWSFNPPT

[0051] The variable regions of Daratumumab include a heavy chain variable region and a light chain variable region linked by a disulfide bond or a linker peptide. The heavy chain variable region and the light chain variable region of Daratumumab are shown in SEQ ID NO.15-16 respectively; the complementarity determining regions of Daratumumab include CDR-H1, CDR2-H2, CDR3-H3, CDR-L1, CDR2-L2 and CDR3-L3 linked by a disulfide bond or a linker peptide:

[0052] CDR-H1: SFAMS; CDR-H2: AISGSGGGTYYADSVKG; CDR-H3: DKILWFGEPVFDY; CDR-L1: RASQSVSSYLA; CDR-L2: DASNRAT; CDR-L3: QQRSNWPPT

[0053] It should be noted that in other embodiments, the amino acid sequences of the variable region, heavy chain variable region or complementary determining region of the CD 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 corresponding variable region, heavy chain variable region or complementary determining region.

[0054] The present invention also provides a method for preparing an antibody detection kit for a CD biological agent, which includes: immobilizing the CD biological agent on a solid-phase carrier as an antibody capture device, and binding or immobilizing a functional fragment on a cellulose membrane or labeling the functional fragment with a labeling agent as an antibody detection device.

[0055] In a preferred embodiment of the application of the present invention, when the solid-phase carrier is selected from a microplate, before immobilizing the CD biological agent on the microplate, it further includes coating streptavidin on the microplate, and then incubating the CD biological agent modified with biotin with the microplate coated with streptavidin.

[0056] In a preferred embodiment of the application of the present invention, the antibody detection kit for the CD biological agent is a quantitative detection kit or a qualitative detection kit.

[0057] In a preferred embodiment of the application of the present invention, the antibody detection kit for the CD biological agent is a time-resolved fluorescence immunochromatographic detection kit, a colloidal gold immunochromatographic detection kit, a quantum dot fluorescence immunochromatographic detection kit, an enzyme-linked immunosorbent assay kit or a chemiluminescence detection kit.

[0058] The present invention has the following beneficial effects:

[0059] The present invention overcomes the false positive and false negative defects in the prior art. Using the CD biological agent as a capture antigen, due to the large spatial contact surface, it is easier to capture the antibody against the CD biological agent (i.e., anti-antibody) in the sample, and has the advantage of high capture efficiency. By coating the CD biological agent on the capture device, the occurrence of false positives is avoided.

[0060] The antigen-antibody complex formed after capture itself has a large steric hindrance. If a biological agent is coated on the antibody detection device, since the molecular weight of the biological agent (i.e., the detected antigen) is large, it is difficult for the biological agent coated on the detection device to bind to the other Fab of the antibody against the CD biological agent in the complex formed after capture. Therefore, the inventor sets that the detection antigen with a small molecular weight, such as the variable region, heavy chain variable region or complementary determining region of the CD biological agent, has the advantages of small steric hindrance and easy binding to the Fab of the anti-antibody, making the detection sensitivity higher and achieving the purpose of companion diagnosis of the CD biological agent drug. This also avoids subsequent misjudgment of the sample test results and the occurrence of false negative phenomena. Brief Description of the Drawings

[0061] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0062] Figure 1 It is a device diagram of the time-resolved fluorescence immunochromatographic test strip for the anti-rituximab antibody of the present invention;

[0063] Figure 2 It is a device diagram of the colloidal gold immunochromatographic test strip for the anti-muromonab-CD3 antibody of the present invention;

[0064] Figure 3 It is a device diagram of the quantum dot fluorescence immunochromatographic test strip for the anti-alemtuzumab antibody of the present invention;

[0065] Figure 4 It is a detection principle diagram of the enzyme-linked immunosorbent assay kit for the anti-daratumumab antibody of the present invention;

[0066] Figure 5 It is a detection principle diagram of the chemiluminescence assay kit for the anti-efalizumab antibody of the present invention;

[0067] Figure 6 It is a calibration curve regression diagram of the time-resolved fluorescence immunochromatographic kit for the anti-rituximab antibody of the present invention;

[0068] Figure 7 It is a calibration curve regression diagram of the colloidal gold immunochromatographic kit for the anti-muromonab-CD3 antibody of the present invention;

[0069] Figure 8 It is a calibration curve regression diagram of the quantum dot fluorescence immunochromatographic kit for the anti-alemtuzumab antibody of the present invention;

[0070] Figure 9It is the calibration curve regression graph of the anti-daratumumab antibody ELISA kit of the present invention;

[0071] Figure 10 It is the calibration curve regression graph of the anti-efalizumab antibody chemiluminescence assay kit of the present invention

[0072] Reference numerals: 1 - sample pad; 2 - conjugate pad; 3 - nitrocellulose membrane; 4 - test line; 5 - control line; 6 - absorbent membrane; 7 - backing base plate. Detailed implementation manners

[0073] To make the objectives, 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. For those not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are followed. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0074] The working principle of the present invention is as follows: 1) Fix the solid-phase carrier with the CD biological agent to capture the anti-antibody in the sample to be detected. The CD biological agent has a large molecular spatial action surface and can efficiently capture the anti-antibody in the sample to form an antigen-antibody complex; 2) Use the variable region, heavy chain variable region or complementary determining region of the CD biological agent as the detection antigen. Since the molecule is small, 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, and this complex is quantitatively or qualitatively detected by well-known colorimetric methods.

[0075] The features and performance of the present invention will be further described in detail below in conjunction with the embodiments.

[0076] Embodiment 1

[0077] This embodiment provides a preparation method for an anti-rituximab antibody time-resolved fluorescence immunochromatographic quantitative detection kit.

[0078] This embodiment provides an anti-rituximab antibody immunochromatographic test strip, as Figure 1 shown. The test strip is provided with a nitrocellulose membrane 3, a sample pad 1, a conjugate pad 2 and an absorbent membrane 6 on a backing base plate 7. The sample pad 1 is laminated at one end of the conjugate pad 2, and the other end of the conjugate pad 2 and the absorbent membrane 6 are respectively laminated at both ends of the nitrocellulose membrane 3; the conjugate pad 2 is coated with time-resolved fluorescence microsphere-labeled anti-rituximab and time-resolved fluorescence microsphere-labeled goat anti-chicken IgY antibody 2; the nitrocellulose membrane 3 is provided with a test line 4 (T line) and a control line 5 (C line). The test line 4 (T line) is coated with the heavy chain variable region (Hv) of anti-rituximab, and the control line 5 (C line) is coated with chicken IgY antibody.

[0079] The heavy chain variable region of rituximab is as follows:

[0080] qvqlqqpgaelvkpgasvkmsckasgytftsynmhwvkqt pgrglewigaiypgngdtsynqkfkgkatltadkssstaymqlssltsedsavyycarstyyggdwyfnvwgagttvtvsa

[0081] The preparation method of the immunochromatographic test strip is as follows:

[0082] 1) Labeling of time-resolved fluorescence microspheres

[0083] Take 500 μL (300 nm) (1% stock solution) of time-resolved fluorescence microspheres, centrifuge at 13000 rpm for 10 min at 4 °C, and discard the supernatant. Add 10 mg / ml of EDC and react for 15 min; centrifuge and discard the supernatant. Add 1000 μL of borate buffer (20 mM, pH 8.0), and mix well by ultrasonic for 10 s; add 100 μg of labeled antibody (rituximab or goat anti-chicken IgY) (labeling concentration 100 μg / mL), and react at room temperature for 2 h; centrifuge and discard the supernatant. Add the blocking solution and react for 1 h; centrifuge and discard the supernatant. Add 1000 μL of borate buffer, mix well by ultrasonic, label it, and store it at 4 °C for later use.

[0084] 2) Treatment of the sample pad and the conjugate pad

[0085] The polyester membrane is pre-blocked by soaking in a buffer containing a surfactant (formula: 20 mM pH 8.0 BB, containing 2% BSA, 0.5% casein, 1% Tween-20, 0.5% S9, and 2% trehalose), dried at 37 °C for 4 h to obtain the conjugate pad; take the prepared conjugate pad, use a gold-labeled HM3035 gold-spraying and membrane-stripping instrument, mix the rituximab labeled with time-resolved fluorescence microspheres and the goat anti-chicken IgY antibody, and spray it onto the pre-treated conjugate pad with a width of 1 cm at 5 μL / cm, and dry it at 37 °C for 3 h to obtain the specific conjugate pad.

[0086] The glass fiber membrane is pre-blocked by soaking in a buffer containing a surfactant (formula: 100 mM pH 7.4 PB, containing 1% BSA, 1% S9, and 2% trehalose), dried overnight at 37 °C, and cut into 30 * 1.5 cm to obtain the sample pad.

[0087] 3) Coating of the nitrocellulose membrane (NC membrane)

[0088] The heavy chain variable region of anti-rituximab is synthesized by the amino acid synthesis method. It has been verified that coating the enzyme-linked immunosorbent assay (ELISA) plate with this synthetic polypeptide can react with the anti-rituximab antibody.

[0089] Attach the NC membrane to the designated position on the backing bottom plate. Dilute the heavy chain variable region (Hv) of rituximab to 1.5 mg / mL with 50 mM phosphate buffer solution at pH 7.4 for preparing the T line; dilute chicken IgY antibody to 0.5 mg / mL with 50 mM phosphate buffer solution at pH 7.4 for preparing the C line; according to the liquid application amount of 1 μL / cm, use a gold-labeled HM3035 spraying and membrane scribing instrument to evenly scribe the above two diluted antibodies onto the NC membrane to prepare the T line and the C line; place the scribed NC membrane in a drying oven at 37 °C and dry for 2 h.

[0090] 4) Assembly

[0091] Stack and press the conjugate pad obtained in step 2) at one end of the nitrocellulose membrane obtained in step 3), fix and stack the absorbent membrane at the other end of the nitrocellulose membrane, and finally stack and press the sample pad obtained in step 2) at the other end of the conjugate pad. Cut it with a membrane cutting instrument at a width of 4 mm per strip and load it into the chromatographic strip housing to obtain the finished product.

[0092] 5) Detection

[0093] Dilute the serum 20 times, take 60 μL and add it to the sample loading hole. After reacting for 10 min, place it in a fluorescence reader for detection. Compare with the values of the standard product or reference product to judge the content of anti-antibody in the sample to be detected.

[0094] In this example, the time-resolved fluorescent substance latex microspheres are europium-containing latex microspheres, that is, the time-resolved fluorescent substance is a conjugate of the lanthanide element europium and latex. In other embodiments, as needed, the time-resolved fluorescent substance can be adjusted to be one of a lanthanide element, a conjugate of a lanthanide element and latex, and a chelate of a lanthanide element; the lanthanide element can be one of europium, terbium, samarium or dysprosium.

[0095] Example 2

[0096] This example provides a muromonab-CD3 antibody colloidal gold immunochromatographic assay kit and its preparation method.

[0097] The anti-muromonab-CD3 immunochromatographic test strip provided in this example is as Figure 2 shown. The test strip is provided with a nitrocellulose membrane 3, a sample pad 1, a conjugate pad 2 and an absorbent membrane 6 on a backing bottom plate 7. The sample pad 1 is stacked and pressed at one end of the conjugate pad 2, and the other end of the conjugate pad 2 and the absorbent membrane 6 are respectively stacked and pressed at both ends of the nitrocellulose membrane 3; the conjugate pad 2 is coated with colloidal gold-labeled biotinylated muromonab-CD3 and goat anti-chicken IgY antibody 2; the sample pad 1 contains streptavidin; the nitrocellulose membrane 3 is provided with a test line 4 (T line) and a quality control line 5 (C line). The test line 4 (T line) is coated with the variable region (Fv) of muromonab-CD3, and the quality control line 5 (C line) is coated with chicken IgY antibody.

[0098] Its variable region (Fv) contains a heavy chain variable region and a light chain variable region linked by a disulfide bond, and the heavy chain variable region sequence is as follows:

[0099] QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS

[0100] The light chain variable region is as follows:

[0101] QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRAD

[0102] The preparation method is as follows:

[0103] 1) Preparation of colloidal gold

[0104] Wash the round-bottom flask and glass reagent bottle used for preparing colloidal gold. After soaking in a strong acid cleaning solution, thoroughly wash and dry. Take a clean round-bottom flask, add 100 mL of ultrapure water to it, add 1 mL of 1% chloroauric acid, and boil. Quickly add 2.2 mL of 1% sodium citrate to it. After the color turns purple-red, continue the reaction for 10 min and stop heating. After cooling, add ultrapure water to 100 mL and store at 4 °C for later use.

[0105] 2) Biotinylation of the labeled antibody

[0106] Take 1 mg of muromonab-CD3 and place it in a 5 mL centrifuge tube. Add 60 μL of 10 mg / mL activated biotin (Sulfo-NHS-LC-Bintin), and make up with 0.02 M PBS to a volume of 1 mL. React at room temperature for 1 hour. Take out the reaction solution and dialyze it against 0.02 M PBS overnight. Take out the biotinylated muromonab-CD3, add 0.03% Proclin300 for anti-corrosion, and store at 4 °C for later use.

[0107] 3) Labeling of colloidal gold

[0108] Take 10 mL of the prepared colloidal gold, add 300 μL of 0.2 M potassium carbonate, mix well, then add 150 μg of biotinylated muromonab-CD3 or goat anti-chicken IgY and react at room temperature for 30 min. Add 1% BSA for blocking for 30 min. Centrifuge at 12,000 rpm at 4 °C for 30 min, take the precipitate, dissolve it in 1 mL of resuspension solution (formula: 20 mM PB at pH 8.0, containing 2% BSA, 0.5% Tween-20 and 5% sucrose), and keep it at 4 °C for later use.

[0109] 3) Treatment of sample pad and conjugate pad

[0110] Using a gold-labeled HM3035 gold-spraying and membrane-stripping instrument, after mixing the biotinylated muromonab-CD3 full length and goat anti-chicken IgY antibody labeled with colloidal gold, spray it onto a glass fiber pad with a width of 1 cm * 30 cm at a rate of 3 μL / cm, and dry it at 37 °C for 3 hours to prepare a colloidal gold conjugate pad.

[0111] Soak the glass cellulose membrane in a buffer containing streptavidin (formula: 100 mM PB at pH 7.4, containing 1% streptavidin, 1% BSA, 0.5% casein, 0.5% S9 and 2% sucrose) for pre-blocking, then dry it overnight at 37 °C, and cut it into 30 * 1.5 cm to prepare a sample pad.

[0112] 4) Coating of nitrocellulose membrane (NC membrane)

[0113] Synthesize approximately 200 amino acids of the variable region of muromonab-CD3 by amino acid synthesis method. It has been verified that the synthetic polypeptide can react with anti-muromonab-CD3 antibody.

[0114] Attach the NC membrane to the designated position on the backing bottom plate. Dilute the variable region (Fv) of muromonab-CD3 to 1.5 mg / mL with 50 mM phosphate buffer at pH 7.4 for preparing the T line; dilute the chicken IgY antibody to 0.5 mg / mL with 50 mM phosphate buffer at pH 7.4 for preparing the C line; according to the liquid application amount of 1 μL / cm, use a gold-labeled HM3035 gold-spraying and membrane-stripping instrument to evenly apply the above two diluted antibodies onto the NC membrane to prepare the T line and C line; place the membrane-striped NC membrane in a drying oven at 37 °C and dry it overnight.

[0115] 5) Assembly

[0116] Stack the conjugate pad obtained in step 2) on one end of the nitrocellulose membrane obtained in step 3), fix and stack the absorbent membrane on the other end of the nitrocellulose membrane, and finally stack the sample pad obtained in step 2) on the other end of the conjugate pad. Cut it with a membrane cutter at a width of 4 mm per strip and load it into the chromatographic strip housing to obtain the finished product.

[0117] 6) Detection

[0118] Dilute the serum 20-fold, take 80 μL and add it to the sample well. After reacting for 10 min, place it in a colloidal gold reader for detection. Compare with the values of the standard product or reference product to judge the content of anti-antibody in the sample to be tested.

[0119] In this example, the colloidal gold is a colloidal solution obtained by reducing chloroauric acid with a reducing agent; according to needs, the label can be adjusted to any one of colloidal carbon, colloidal silver, and colloidal selenium.

[0120] Example 3

[0121] This example provides a preparation method of a quantum dot fluorescence immunochromatographic assay kit for anti-alemtuzumab.

[0122] The anti-alemtuzumab immunochromatographic test strip is as Figure 3 shown. The test strip is provided with a nitrocellulose membrane 3, a sample pad 1, a conjugate pad 2, and an absorbent membrane 6 on a backing substrate 7. The sample pad 1 is laminated at one end of the conjugate pad 2, and the other end of the conjugate pad 2 and the absorbent membrane 6 are respectively laminated at both ends of the nitrocellulose membrane 3; the conjugate pad 2 is coated with quantum dot-labeled alemtuzumab and goat anti-chicken IgY antibody; the nitrocellulose membrane 3 is provided with a test line 4 (T line) and a control line 5 (C line). The test line 4 (T line) is coated with the complementarity-determining regions (CDRs) of alemtuzumab, and the control line 5 (C line) is coated with chicken IgY antibody.

[0123] The complementarity-determining regions (CDRs) of alemtuzumab include heavy chain complementarity-determining regions and light chain complementarity-determining regions. Among them, in the heavy chain complementarity-determining regions: CDR-H1: DFYMN; CDR-H2: FIRDKAKGYTTEYNPSVKG; CDR-H3: EGHTAAPFDY; in the light chain complementarity-determining regions: CDR-L1: KASQNIDKYLN; CDR-L2: NTNNLQT; CDR-L3: LQHISRPRT. The complementarity-determining regions are connected by a linker peptide, and the linker peptide is selected from GGGGSGGGGSGGGGS.

[0124] The preparation method is as follows:

[0125] 1) Labeling of quantum dots

[0126] Take 100 μL of commercially available quantum dots, centrifuge at 13,000 rpm at 4 °C for 10 min, discard the supernatant after centrifugation, add 1000 μL of MES buffer, mix well by ultrasound, weigh 20 mg of EDC and 20 mg of NHS, slowly add them to the microsphere mixture, mix well by ultrasound in time, and react for 15 minutes; centrifuge, discard the supernatant, add borate buffer (20 mM, pH 8.0), mix well by ultrasound; add 100 μg of labeled antibody (alemtuzumab or goat anti-chicken IgY) (labeling concentration 100 μg / mL), react at room temperature for 2 h; centrifuge, discard the supernatant, add blocking solution, mix well by ultrasound in ice water, and react at room temperature for 1 h; centrifuge, discard the supernatant, add 1000 μL of borate buffer (20 mM, pH 8.0), mix well by ultrasound, make labels, and store at 4 °C for later use.

[0127] 2) Treatment of sample pad and conjugate pad

[0128] The polyester membrane is pre-blocked by soaking in a buffer containing surfactant (formula: 20 mM pH 8.0 BB, containing 2% BSA, 1% Tween-20, 1% S9, and 3% trehalose), dried overnight at 37 °C to prepare the conjugate pad; take the prepared conjugate pad, use a gold-labeled HM3035 spraying and scribing instrument, mix the alemtuzumab labeled with quantum dots and goat anti-chicken IgY antibodies, and spray them onto the pre-treated conjugate pad with a width of 1 cm at 4 μL / cm, dry at 37 °C, and the drying time is 3 hours to prepare the specific conjugate pad.

[0129] The glass fiber membrane is pre-blocked by soaking in a buffer containing surfactant (formula: 100 mM pH 7.4 PB, containing 1% BSA, 0.5% casein, 0.1% Tween-20, 1% S9, and 5% sucrose), dried overnight at 37 °C, and cut into 30 * 1.5 cm to prepare the sample pad.

[0130] 3) Coating of nitrocellulose membrane (NC membrane)

[0131] The complementary determining region of alemtuzumab is synthesized by amino acid synthesis method. After verification, coating the enzyme-linked immunosorbent assay plate with this synthetic polypeptide can react with the anti-alemtuzumab antibody.

[0132] Attach the NC membrane to the designated position on the backing plate, dilute the complementary determining region of alemtuzumab to 1.5 mg / mL with 50 mM phosphate buffer at pH 7.4 for preparing the T line; dilute the chicken IgY antibody to 0.5 mg / mL with 50 mM phosphate buffer at pH 7.4 for preparing the C line; according to the liquid scribing volume of 1 μL / cm, evenly scribe the above two diluted antibodies onto the NC membrane to prepare the T line and C line through a gold-labeled HM3035 spraying and scribing instrument; place the scribed NC membrane in a drying oven at 37 °C and dry overnight.

[0133] 4) Assembly

[0134] Press the combination pad obtained in step 2) onto one end of the nitrocellulose membrane obtained in step 3), fix and press the water-absorbing membrane onto the other end of the nitrocellulose membrane, and finally press the sample pad obtained in step 2) onto the other end of the combination pad. Cut it with a film cutter at a width of 4 mm per strip and load it into the chromatographic strip housing to obtain the finished product.

[0135] 5) Detection

[0136] Dilute the serum 20 times, take 80 μL and add it to the sample well. After reacting for 10 min, place it in a fluorescence reader for detection. Compare with the values of the standard product or reference product to judge the content of anti-antibody in the sample to be tested.

[0137] In this example, the quantum dots are core-shell quantum dots, quantum dots formed by ZnS / CdSe or ZnS / CdTe. According to needs, the quantum dots can be adjusted to quantum dots formed by a single compound, such as any one of cadmium selenide (CdSe), zinc sulfide (ZnS), cadmium telluride (CdTe) / cadmium sulfide (CdS), etc.

[0138] Example 4

[0139] This example provides a preparation method of an anti-daratumumab enzyme-linked immunosorbent assay kit.

[0140] The anti-daratumumab enzyme-linked immunosorbent assay kit includes an enzyme-labeled plate, biotinylated daratumumab, an anti-daratumumab antibody calibrator, an anti-daratumumab antibody quality control product, an enzyme conjugate, a washing solution, and a termination solution. The enzyme conjugate is the heavy chain variable region (Hv) of daratumumab labeled with horseradish peroxidase (HRP). The enzyme-labeled plate is coated with streptavidin.

[0141] The sequence of the heavy chain variable region (Hv) of daratumumab is as follows:

[0142] EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSNVRQA PGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS

[0143] The preparation method and the detection method refer to Figure 4 as shown, and the specific preparation is as follows:

[0144] 1) Biotinylation of daratumumab

[0145] Take 1 mg of daratumumab and place it in a 5 mL centrifuge tube. Add 60 μL of 10 mg / mL activated biotin (Sulfo-NHS-LC-Biotin), and supplement with 0.02 M PBS to a volume of 1 mL. React at room temperature for 1 hour. Take out the reaction solution and dialyze it against 0.02 M PBS overnight. Take out the biotinylated cetuximab antibody, add 0.03% Proclin300 for preservation, and store it at 4 °C for later use.

[0146] 2) HRP-labeled variable heavy chain (Hv) of daratumumab

[0147] Synthesize the variable heavy chain of daratumumab by amino acid synthesis method. It has been verified that this synthetic polypeptide can react with anti-daratumumab antibody.

[0148] Weigh 5 mg of HRP and place it in a 5 mL centrifuge tube. Add 1 mL of pure water to dissolve it, then add 1 mL of NaIO4 solution (10 mg / mL, freshly prepared before use), and react at 4 °C in the dark for 30 min. Add 0.02 mL of ethylene glycol and react at room temperature for 15 min. Take out the above solution after dialysis. Add 1 mg of the variable heavy chain of cetuximab to the above-activated solution, react at room temperature in the dark for 2 h, add 0.2 mL of NaBH4 solution (5 mg / mL, freshly prepared before use), mix well, and dialyze against 0.05 M CB overnight. Take out the labeled enzyme conjugate, add an equal volume of glycerol, and store it at -20 °C for later use.

[0149] 3) Coating the enzyme-labeled plate

[0150] Dilute streptavidin to 1 μg / mL with CB buffer, coat 100 μL per well on the microplate, and incubate at 37 °C for 2 h. Aspirate the coating solution, add 200 μL of blocking buffer containing 2% BSA per well, incubate at 37 °C for 2 hours, pat off the blocking solution, and dry it at 37 °C for later use.

[0151] 4) Coating biotinylated daratumumab

[0152] Dilute biotinylated daratumumab to 1 μg / mL with neutral phosphate buffer, add 100 μL per well, and incubate at 37 °C for 2 h. Wash the plate 3 times, add 200 μL of blocking buffer containing 2% BSA per well, incubate at 37 °C for 2 hours, pat off the blocking solution, and dry it at 37 °C for later use.

[0153] 5) Dilute the serum sample to be tested 100 times, add 100 μL per well to the microplate, incubate at 37 °C for 2 hours, and wash the plate 4 times.

[0154] 6) Add HRP-labeled variable heavy chain of daratumumab, incubate for 1 h, and wash the plate 4 times.

[0155] 7) Add the substrate TMB of HRP enzyme to the microplate, incubate for 15 min, add the stop solution to terminate the reaction, detect the OD value at a wavelength of 450 nm, and compare it with the OD value of the standard or reference product to judge the content of the anti-antibody in the sample to be tested.

[0156] Example 5

[0157] This example provides a preparation method for a chemiluminescence assay kit for anti-efalizumab antibody.

[0158] The chemiluminescence detection kit for anti-efalizumab antibody includes biotinylated efalizumab, anti-efalizumab antibody calibrator, anti-efalizumab antibody quality control product, acridinium ester-labeled efalizumab heavy chain variable region (Hv), magnetic particle reagent, excitation solution and washing solution. The sequence of the efalizumab heavy chain variable region is as follows:

[0159] evqlvesggglvqpggslrlscaasgysftghwmnwvrqapgkglewvgmihpsdsetrynqkfkdrftisvdkskntlylqmnslraedtavyycargiyfygttyfdywgqgtlvtvss

[0160] The preparation and detection steps are as follows:

[0161] 1) Biotinylation of efalizumab

[0162] Take 1 mg of efalizumab and place it in a 5 mL centrifuge tube. Add 50 μL of 10 mg / mL activated biotin (Sulfo-NHS-LC-Bintin), supplement with 0.02 M PBS to a volume of 1 mL, react at room temperature for 1 hour, take out the reaction solution, and dialyze it with 0.02 M PBS overnight. Take out the biotinylated efalizumab, add 0.03% Proclin300 for preservation, and store it at 4 °C for later use.

[0163] 2) Acridinium ester labeling of the efalizumab heavy chain variable region (Hv)

[0164] Synthesize the heavy chain variable region of efalizumab by amino acid synthesis method. It has been verified that the synthesized polypeptide can react with anti-efalizumab antibody.

[0165] Take 2 mg of the efalizumab heavy chain variable region, add the activated acridinium ester according to the molar ratio of antibody: acridinium ester = 1:10 - 50, and react at room temperature for 30 min. Take out the reaction solution after dialysis with 0.05 M CB solution, add an equal volume of glycerol, and store it at -20 °C for later use.

[0166] 3) Detection steps

[0167] This method uses acridinium ester chemiluminescence immunoassay technology - combined with biotin-avidin magnetic particle separation technology to achieve the detection of anti-monoclonal antibodies. The detection principle is as follows Figure 5 : Mix the sample (diluted 20 times), streptavidin magnetic particles (1), and biotinylated efalizumab (2) to obtain a streptavidin magnetic bead-biotinylated efalizumab-sample complex (3). After washing, add acridinium ester-labeled efalizumab Hv for reaction to form a streptavidin magnetic bead-biotinylated efalizumab-sample-acridinium ester-labeled efalizumab Hv complex. Add the luminescence excitation solution, measure the luminescence intensity, compare it with the values of the standard product or reference product, and judge the content of the anti-antibody in the sample to be tested.

[0168] Experimental Example 1

[0169] This experimental example performs a quantitative test on the kit provided in Example 1 to test its performance.

[0170] 1.1 Standard curve preparation

[0171] 1.1.1 Take a set of anti-rituximab antibody calibration products, and the specific values are shown in Table 1.

[0172] Table 1

[0173]

[0174] 1.1.2 Detection method

[0175] Take 80 μL of the calibration product and directly add it to the sample window of the chromatography strip; after 10 minutes, quantitatively detect the signal value with a time-resolved fluorescence quantitative analyzer.

[0176] Each calibration product is detected 2 times, and the average value of the T / C value is taken. The specific results are shown in Table 2.

[0177] Table 2

[0178]

[0179] 1.1.3 Standard curve preparation

[0180] According to the above detection results, with the T / C value as the X-axis and the concentration value as the Y-axis, perform linear regression to obtain the linear equation: y = 0.0123x + 0.1292, R² = 0.9975. The curve is shown in Figure 6 .

[0181] 1.2 Precision test:

[0182] 1.2.1 Take 10 prepared chromatography strips and configure an anti-rituximab antibody working calibration product with a concentration of (50 ng / mL);

[0183] 1.2.2 Take 80 μL of the calibrator and directly add it to the sample well of the test strip;

[0184] 1.2.3 After the calibrator has chromatographed for 10 minutes, use a time-resolved fluorescence quantitative analyzer for detection. According to the above linear equation, the results of 10 test strips are measured. The results are shown in Table 3, indicating that the precision of the time-resolved fluorescence immunochromatographic assay kit for anti-rituximab antibody is good.

[0185] Table 3

[0186]

[0187] 1.3 Compared with other methods:

[0188] Using rituximab-labeled microspheres and rituximab-coated NC membrane as Method 2; using rituximab fab-labeled microspheres and rituximab fab-coated NC membrane as Method 3. For the three methods, except for the different labeled antibodies and coated antibodies, other conditions are the same. The three results are compared as shown in Table 4.

[0189] Table 4

[0190]

[0191] Experimental Example 2

[0192] This experimental example quantitatively detects the kit provided in Example 2 to test its performance.

[0193] 2.1 Standard curve preparation

[0194] 2.1.1 Take a set of anti-muromonab-CD3 antibody calibrators, and the specific values are shown in Table 5.

[0195] Table 5

[0196]

[0197] 2.1.2 Detection method

[0198] Take 80 μL of the calibrator and directly add it to the sample window of the chromatographic strip; after 10 minutes, use a colloidal gold reader to detect the signal value. Each calibrator is detected 2 times, and the average value of the T / C value is taken. The specific results are shown in Table 6.

[0199] Table 6

[0200]

[0201] 2.1.3 Standard curve preparation

[0202] According to the above detection results, with the T / C value as the X-axis and the concentration value as the Y-axis, a linear regression is performed to obtain the linear equation: y = 0.0049x + 0.1022, R² = 0.993. The curve is shown in Figure 7 .

[0203] 2.2 Precision test:

[0204] 2.2.1 Take 10 prepared chromatographic strips and prepare a working calibration product of anti-muromonab-CD3 antibody with a concentration of 50 ng / mL;

[0205] 2.2.2 Take 80 μL of the calibration product and directly add it to the sample application hole of the reagent strip;

[0206] 2.2.3 After the calibration product has chromatographed for 10 minutes, use a colloidal gold reader for detection. According to the above linear equation, the results of 10 reagent strips are measured. The results are shown in Table 7, indicating that the precision of the anti-muromonab-CD3 antibody colloidal gold immunochromatographic assay kit of this method is good.

[0207] Table 7

[0208]

[0209] 2.3 Compared with other methods:

[0210] Taking this example as a control, using muromonab-CD3 labeled colloidal gold and muromonab-CD3 coated NC membrane as Method 2; using muromonab-CD3fab labeled colloidal gold and muromonab-CD3fab coated NC membrane as Method 3. For the three methods, except for the different labeled antibodies and coated antibodies, other conditions are the same. The results are compared in Table 8.

[0211] Table 8

[0212]

[0213] Experimental Example 3

[0214] This experimental example quantitatively detects the kit provided in Example 3 to test its performance.

[0215] 3.1 Standard curve preparation

[0216] 3.1.1 Take a set of anti-alemtuzumab antibody calibration products, and the specific values are shown in Table 9.

[0217] Table 9

[0218]

[0219] 3.1.2 Detection method

[0220] Take 80 μL of the calibration product and directly add it to the sample window of the chromatographic strip; after 10 minutes, detect the signal value with a fluorescence quantitative analyzer. Each calibration product is detected 2 times, and the average value of the T / C value is taken. The specific results are shown in Table 10.

[0221] Table 10

[0222]

[0223] 3.1.3 Standard curve preparation

[0224] According to the above test results, with the T / C value as the X-axis and the concentration value as the Y-axis, perform curve fitting to obtain the curve equation: y = 39.892x 2 + 29.01x - 8.1918, R² = 0.9981, the curve is shown in Figure 8 .

[0225] 3.2 Precision test:

[0226] 3.2.1 Take 10 prepared chromatographic strips and prepare a working calibration product of anti-alemtuzumab antibody with a concentration of (50 ng / mL);

[0227] 3.2.2 Take 80 μL of the calibration product and directly add it to the sample loading hole of the reagent strip;

[0228] 3.2.3 After the calibration product has chromatographed for 10 min, detect it with a fluorescence quantitative analyzer, and measure the results of 10 reagent strips according to the above equation. The results are shown in Table 11, indicating that the precision of the quantum dot fluorescence immunoassay kit for anti-alemtuzumab antibody of this method is good.

[0229] Table 11

[0230]

[0231] 3.3 Compared with other methods:

[0232] Using alemtuzumab-labeled quantum dots and alemtuzumab-coated NC membrane as Method 2; using alemtuzumab fab-labeled quantum dots and alemtuzumab fab-coated NC membrane as Method 3. For the three methods, except for the different labeled antibodies and coated antibodies, other conditions are the same. The three results are compared as shown in Table 12.

[0233] Table 12

[0234]

[0235] Experimental Example 4

[0236] This experimental example performs quantitative detection on the kit provided in Example 4 to test its performance.

[0237] 4.1 Detection Method

[0238] 4.1.1 Take a set of anti-daratumumab antibody calibration products, and the specific values are shown in Table 13.

[0239] Table 13

[0240]

[0241] 4.1.2 Detection Steps

[0242] 1) Add 100 μL of the calibration product and the quality control product to each well of the microplate, incubate at 37 °C for 2 h, and wash the plate 4 times.

[0243] 2) Add HRP-labeled daratumumab heavy chain variable region, incubate for 1 hour, and wash the plate 4 times

[0244] 3) Add the substrate TMB of HRP enzyme to the microplate, incubate for 15 min, add the stop solution to terminate the reaction, and detect the OD value at a wavelength of 450 nm to judge the performance of the kit. The specific results are shown in Table 14.

[0245] Table 14

[0246]

[0247] 4.1.3 Standard Curve

[0248] According to the above detection results, with the OD value as the X-axis and the concentration value as the Y-axis, perform curve fitting to obtain the curve equation: y = 59.013x 2 + 89.592x - 10.62, R² = 0.9977, and the curve is shown in Figure 9 .

[0249] 4.2 Precision Test:

[0250] According to the OD values of the precision quality control products detected, calculate the result values using the curve equation. The results are shown in Table 15, indicating that the precision of the anti-daratumumab antibody ELISA kit of this method is good.

[0251] Table 15

[0252]

[0253] 4.3 Comparison with Other Methods:

[0254] Using daratumumab labeled with HRP and daratumumab-coated microplate as Method 2; using daratumumab fab labeled with HRP and daratumumab fab-coated microplate as Method 3. For the three methods, except for the different labeled antibodies and coated antibodies, other conditions are the same, and the three results are compared as shown in Table 16.

[0255] Table 16

[0256]

[0257] Experimental Example 5

[0258] This experimental example quantitatively detected the kit provided in Example 5 to test its performance.

[0259] 5.1 Detection method

[0260] 5.1.1 Take a set of anti-efalizumab antibody calibration products, and the specific values are shown in Table 17

[0261] Table 17

[0262]

[0263] 5.1.2 Detection steps

[0264] 1) Respectively take 20 μL of calibration product, 20 μL of streptavidin magnetic particles, and 50 μL of biotinylated efalizumab and add them to the microplate for mixing, incubate at 37 °C for 15 min, and wash 3 times.

[0265] 2) Add acridinium ester-labeled efalizumab heavy chain variable region (Hv), incubate at 37 °C for 15 min, and wash 3 times

[0266] 3) Add the luminescence excitation solution and measure the luminescence intensity. The specific results are shown in Table 18.

[0267] Table 18

[0268]

[0269] 5.1.3 Standard curve

[0270] According to the above detection results, with the logarithm of the concentration value as the X-axis and the logarithm of the luminescence value as the Y-axis, perform linear regression to obtain the linear equation: y = 0.9798x + 2.8353, R² = 0.9932, and the curve is shown in Figure 10 .

[0271] 5.2 Precision test:

[0272] Take 20 μL of precision quality control product, 20 μL of streptavidin magnetic particles, and 50 μL of biotinylated efalizumab and add them to the microplate for mixing, incubate at 37 °C for 15 min, and wash 3 times; add acridinium ester-labeled efalizumab heavy chain variable region (Hv), incubate at 37 °C for 15 min, and wash 3 times; add the luminescence excitation solution and measure the luminescence intensity. According to the above curve equation, calculate the result value. The results are shown in Table 15, indicating that the precision of the anti-efalizumab antibody chemiluminescence immunoassay kit of this method is good. The specific results are shown in Table 19.

[0273] Table 19

[0274]

[0275] 5.3 Compared with other methods:

[0276] Using efalizumab to label acridinium ester and using efalizumab to label biotin as Method 2; using efalizumab fab to label acridinium ester and using efalizumab fab to label biotin as Method 3. For the three methods, except that the antibodies for labeling acridinium ester and the antibodies for labeling biotin are different, other conditions are the same. The three results are compared as shown in Table 20.

[0277] Table 20

[0278]

[0279] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention. Sequence Listing <110> Suzhou Herui Biotechnology Co., Ltd. <120> Antibody Detection Kit for CD Biological Agent and Preparation Method <160> 16 <170> SIPOSequenceListing 1.0 <210> 1 <211> 121 <212> PRT <213> Artificial Sequence <400> 1 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ala 115 120 <210> 2 <211> 109 <212> PRT <213> Artificial Sequence <400> 2 Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile 20 25 30 His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val 100 105 <210> 3 <211> 122 <212> PRT <213> Artificial Sequence <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 4 <211> 110 <212> PRT <213> Artificial Sequence <400> 4 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 Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val 100 105 110 <210> 5 <211> 119 <212> PRT <213> Artificial Sequence <400> 5 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 Tyr Ala Phe Ser Tyr Ser 20 25 30 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly 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 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 6 <211> 115 <212> PRT <213> Artificial Sequence <400> 6 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val 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 Ala Gln Asn 85 90 95 Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val 115 <210> 7 <211> 119 <212> PRT <213> Artificial Sequence <400> 7 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 8 <211> 109 <212> PRT <213> Artificial Sequence <400> 8 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Arg Ala Asp 100 105 <210> 9 <211> 121 <212> PRT <213> Artificial Sequence <400> 9 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Phe Thr Asp Phe 20 25 30 Tyr Met Asn Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile Arg Asp Lys Ala Lys Gly Tyr Thr Thr Glu Tyr Asn Pro 50 55 60 Ser Val Lys Gly Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln 65 70 75 80 Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly His Thr Ala Ala Pro Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Ser Leu Val Thr Val Ser Ser 115 120 <210> 10 <211> 110 <212> PRT <213> Artificial Sequence <400> 10 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 Lys Ala Ser Gln Asn Ile Asp Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asn Thr Asn Asn Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln His Ile Ser Arg Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 <210> 11 <211> 121 <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 Tyr Ser Phe Thr Gly His 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Phe Thr Ile Ser Val Asp Lys 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 Gly Ile Tyr Phe Tyr Gly Thr Thr Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 12 <211> 110 <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 Lys Thr Ile Ser Lys Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Gly Ser Thr 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 His Asn Glu Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 <210> 13 <211> 122 <212> PRT <213> Artificial Sequence <400> 13 Gln Ala Tyr Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Met His Trp Val Lys Gln Thr Pro Arg Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp 100 105 110 Gly Thr Gly Thr Thr Val Thr Val Ser Gly 115 120 <210> 14 <211> 109 <212> PRT <213> Artificial Sequence <400> 14 Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val 100 105 <210> 15 <211> 122 <212> PRT <213> Artificial Sequence <400> 15 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 Val Ser Gly Phe Thr Phe Asn Ser Phe 20 25 30 Ala Met Ser Asn Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser 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 Phe Cys 85 90 95 Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 16 <211> 110 <212> PRT <213> Artificial Sequence <400> 16 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 Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110

Claims

1. An antibody detection kit for CD biological agents, 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 label; the solid-phase carrier is selected from a microplate or a microporous membrane; the detectable label is selected from a fluorescent dye, an enzyme that catalyzes a substrate to develop color, a radioisotope, a chemiluminescent reagent, or a nanoparticle-based label; Or, the antibody capture device includes a solid-phase carrier, and the antibody detection device is a cellulose membrane fixed with a functional fragment without a label; the solid-phase carrier is selected from fluorescent microspheres, latex microspheres, resin microspheres, magnetic microspheres, colloidal gold particles, or quantum dots; The full length of a CD biological agent is coated on the solid-phase carrier, and the full length of the CD biological agent is the full length of Rituximab, Muromonab-CD3, Alemtuzumab, Daratumumab, or Efalizumab; correspondingly, the functional fragment is the heavy-chain variable region of Rituximab, the variable region of Muromonab-CD3, the complementarity-determining region of Alemtuzumab, the heavy-chain variable region of Daratumumab, or the heavy-chain variable region of Efalizumab; The variable region (Fv) is composed of a heavy-chain variable region and a light-chain variable region connected by a disulfide bond or a linker peptide; the complementarity-determining region includes CDR1-VH1, CDR2 –VH2, CDR3–VH3, CDR-VL1, CDR2 –VL2, and CDR3–VL3 connected by a disulfide bond or a linker peptide.

2. The antibody detection kit for CD biological agents according to claim 1, wherein The CD biological agent coated on the solid-phase carrier is modified with avidin or biotin.

3. The antibody detection kit for CD biological agents according to claim 1, characterized in that, The fluorescent microspheres are selected from time-resolved fluorescent microspheres, and the magnetic microspheres are selected from magnetic beads.

4. The antibody detection kit for CD biological agents according to claim 1 or 2, characterized in that The fluorescent dye is selected from fluorescein-based dyes and their derivatives, rhodamine-based dyes and their derivatives, Cy series dyes and their derivatives, Alexa series dyes and their derivatives, or protein-based dyes and their derivatives; The enzyme that catalyzes a substrate to develop color is selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, or 6-phosphogluconate dehydrogenase; The radioactive isotope 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 Ru, 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 reagent is selected from luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, rosamide and its derivatives, or peroxyoxalate and its derivatives; The nanoparticle-based label is selected from nanoparticles or colloids; The colloid is selected from colloidal metals, disperse dyes, dye-labeled microspheres, or latex; The nanoparticles are selected from organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, or rare earth complex nanoparticles.

5. The antibody detection kit for CD biological agents 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 a cellulose acetate membrane or a nitrocellulose membrane.

6. The antibody detection kit for CD biological agents according to claim 1 or 2, characterized in that, The heavy chain variable region of rituximab is as shown in SEQ ID NO.1; the heavy chain variable region and the light chain variable region of muromonab-CD3 are as shown in SEQ ID NOs. 7-8 respectively; the heavy chain variable region and the light chain variable region of alemtuzumab are as shown in SEQ ID NOs. 9-10 respectively; the heavy chain variable region of efalizumab is as shown in SEQ ID NO.11; the heavy chain variable region of daratumumab is as shown in SEQ ID NO.

15.

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

8. The antibody detection kit for CD biological agents according to claim 1 or 2, characterized in that, The antibody detection kit for the CD biological agent is a time-resolved fluorescence immunochromatographic detection kit, a colloidal gold immunochromatographic detection kit, a quantum dot fluorescence immunochromatographic detection kit, an enzyme-linked immunosorbent assay kit or a chemiluminescence detection kit.

9. A method for preparing an antibody detection kit for a CD biological agent according to any one of claims 1-8, characterized in that, It includes: Fixing the CD biological agent on a solid phase carrier as an antibody capture device, binding or fixing a functional fragment without a label on a cellulose membrane or labeling the functional fragment with a label as an antibody detection device.

10. The preparation method of the antibody detection kit for the CD biological agent according to claim 9, characterized in that, When the solid phase carrier is selected from a microplate, before fixing the CD biological agent on the microplate, it further includes coating streptavidin on the microplate, and then incubating the CD biological agent modified with biotin with the microplate coated with streptavidin.

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