Antibody detection kit for integrin biological preparations and preparation method thereof

Abstract

The present invention discloses an antibody detection kit for integrin biological preparations and a preparation method thereof, and relates to the technical field of anti-integrin antibody detection. The kit 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 an integrin biological preparation is coated on the solid phase carrier, and a functional fragment without a marker is fixed on the cellulose membrane; the functional fragment is a variable region, a heavy chain variable region or a complementary determining region of the integrin biological preparation. The kit overcomes the defects of false positive and false negative at the same time. It has the advantages of high capture efficiency and high detection sensitivity.

Description

Technical Field

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

[0002] Integrin is a type of cell membrane surface receptor, a heterodimeric transmembrane glycoprotein composed of α subunit and β subunit. It is the main factor mediating the interaction between cells and extracellular matrix and plays an important role in immune regulation. Integrin is involved in a variety of physiological and pathological processes such as inflammation, thrombosis, malignant tumor growth, infiltration and metastasis.

[0003] Currently, monoclonal antibody drugs or biological agents designed to target integrins, including monoclonal antibodies against α4β7 integrin (Vedolizumab, humanized monoclonal antibody) and monoclonal antibodies against integrin α4 (Natalizumab), have been approved by the FDA for the treatment of ulcerative colitis, Crohn's disease, multiple sclerosis, etc.

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

[0005] The detection method of anti-integrin biological agent antibodies is mainly carried out through immunological reactions, including indirect methods or double antigen sandwich methods. For detection schemes with washing steps such as ELISA and tube chemiluminescence, the indirect method is an efficient and effective detection method. However, for detection schemes such as chromatography detection that lack washing steps, the indirect method is interfered by a large amount of irrelevant antibodies in the sample, resulting in false positives and other detection distortions. Although the double antigen sandwich method can effectively avoid the interference of irrelevant antibodies in the sample, the false negatives are more serious in the published double antigen sandwich method technical scheme.

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

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

[0008] The present invention is achieved in that:

[0009] The present invention provides an antibody detection kit for integrin biological preparations, 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 integrin biological preparation is coated on the solid phase carrier, and the functional fragment without a marker is fixed on the cellulose membrane; the functional fragment is a variable region, a heavy chain variable region or a complementary determining region of the integrin biological preparation.

[0010] The inventor provides an antibody detection kit for integrin biological preparations, which overcomes the defects of false positive and false negative at the same time. In view of the defect of false positive, the inventor uses integrin biological preparations as antibody capture devices. Since integrin biological preparations have a large spatial contact surface, they are easier to capture anti-antibodies in the sample and have the advantage of high capture efficiency.

[0011] To address the defect of false negatives, the inventors fixed the Fv (variable region), Hv (heavy chain variable region) or CDRs (complementarity determining regions) of integrin biologics on the detection device, and reduced the molecular weight of the coating on the detection device, thereby reducing the spatial steric hindrance of the coating, making it easier for the fixed substance on the detection device to bind to the Fab fragment of the anti-antibody, so that even lower concentrations of anti-antibodies in the sample can achieve specific binding to the fixed substance on the detection device, thereby improving the sensitivity of the detection and achieving the purpose of companion diagnosis of integrin biologics.

[0012] The principle of anti-antibody detection in the above kit is: using integrin biological preparations as capture antigens, combining with antibodies against anti-integrin biological preparations in the sample, and then using the variable region, heavy chain variable region or complementary determining region of the labeled integrin monoclonal antibody or the variable region, heavy chain variable region or complementary determining region of the integrin monoclonal antibody fixed on the membrane to qualitatively or quantitatively detect the anti-antibody.

[0013] The method of using the above kit for anti-antibody detection is: the sample to be tested acts on the antibody capture device to form an antigen-antibody complex, and then the antigen-antibody complex acts on the antibody detection device to form an antigen-antibody-antigen complex. Quantitative or qualitative detection is performed using a known color development method.

[0014] In an optional embodiment, the above functional fragments generally have the same binding specificity as the antibody from which they are derived. Fv (variable region) is composed of a heavy chain variable region and a light chain variable region connected by a connecting peptide or a disulfide bond to maintain the stability of Fv. CDRs (complementarity determining regions) include CDR1, CDR2 and CDR3 parts in the heavy chain variable region and the light chain variable region. It is easy for those skilled in the art to understand based on the contents described in the present invention that the functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, those skilled in the art can easily obtain the above functional fragments.

[0015] Including but not limited to any one of HV, F(ab')2, Fab', Fab, Fv, CDRs and scFv selected from the above-mentioned integrin biological agents.

[0016] The functional fragments of the above antibodies can also be synthesized by recombinant genetic techniques known to those skilled in the art (such as recombinant expression by gene recombination) or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems.

[0017] In a preferred embodiment of the present invention, the integrin biological agent is selected from integrin monoclonal antibody drugs.

[0018] In an optional embodiment, the monoclonal antibody drug for integrin is a monoclonal antibody drug that specifically recognizes α1β1, α2β1, α4β1, α5β1, αLβ2, αMβ2, αⅡbβ3, αvβ3, α6β4, α4β7, α4, and α6 integrins.

[0019] As long as the monoclonal antibody drug can specifically recognize the above integrins, it is within the protection scope of the present invention. In an optional embodiment, the α4 monoclonal antibody drug is selected from Natalizumab; the α4β7 monoclonal antibody drug is selected from Vedolizumab.

[0020] In a preferred embodiment of the present invention, the solid phase carrier is selected from fluorescent microspheres, latex microspheres, resin microspheres, magnetic microspheres, colloidal gold particles, quantum dots, microporous plates or microporous membranes. The colloidal gold is a colloidal solution obtained by reducing tetrachloroauric acid with a reducing agent. In other embodiments, the colloidal gold may also be other colloidal substances, such as colloidal carbon, colloidal silver or colloidal selenium.

[0021] In an optional embodiment, the quantum dots are core-shell quantum dots, such as quantum dots formed by ZnS / CdSe or ZnS / CdTe. As needed, the quantum dots can be adjusted to quantum dots formed by a single compound, such as cadmium selenide (CdSe), zinc sulfide (ZnS), cadmium telluride (CdTe), cadmium sulfide (CdS), zinc selenide (ZnSe), indium phosphide (InP) or indium arsenide (InAs), or a nanocrystal or semiconductor nanocrystal composed of a layer of ZnS or CdS wrapped on a CdSe core.

[0022] In an alternative embodiment, the integrin biological preparation coated on the solid carrier is modified with avidin or biotin.

[0023] In an optional embodiment, the fluorescent microspheres are selected from time-resolved fluorescent microspheres, and the magnetic microspheres are selected from magnetic beads. In an optional embodiment, the time-resolved fluorescent microspheres are selected from a combination of a time-resolved fluorescent substance and latex or nanoparticles, and the time-resolved fluorescent substance is one of a lanthanide element, a combination of a lanthanide element and latex, and a chelate of a lanthanide element; 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 integrin biological agent. The coated full length has a large spatial contact surface, which makes it easier to capture anti-antibodies in the sample and has the advantage of high capture efficiency.

[0025] A detectable marker refers to a substance that has properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which the corresponding target can be qualitatively or quantitatively detected. In a preferred embodiment of the present invention, the detectable marker is selected from fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent agents, or nanoparticle markers.

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

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

[0028] In an alternative embodiment, the radioactive isotope includes but is 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 Such as 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y. 88 Y. 121 Sn, 161 Tb, 166 Ho, 105Rh, 177 Lu, 172 Lu or 18 F.

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

[0030] In an alternative embodiment, the nanoparticle markers include but are not limited to nanoparticles or colloids.

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

[0032] In an alternative embodiment, the nanoparticles include but are not limited to organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles or rare earth complex nanoparticles.

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

[0034] In an alternative embodiment, the cellulose membrane includes but is not limited to a cellulose acetate membrane or a nitrocellulose membrane.

[0035] In a preferred embodiment of the present invention, the variable region of the above-mentioned vedolizumab is connected by a heavy chain variable region and a light chain variable region through a connecting peptide or a disulfide bond. The heavy chain variable region of vedolizumab is shown in SEQ ID NO.1:

[0036] QVQLVQSGAEVKKPGASVKVSCKGSGYTFTSYWMHWVRQAPGQAIEWIGEIDPSESNTNYNQKTKGRVTLTVDISASTAYMELSSLRSEDTAVYYCARGGKDGWDYAIDYWGQGTLVTVSS.

[0037] The light chain variable region of vedolizumab is shown in SEQ ID NO.2:

[0038] DVVMTQSPLSLPVTPGEPASISCRSSQSLAKSYGNTYLSWYLQKPGQSPQLLIYGISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGTHQPYTFGQGTKVEIKRTV.

[0039] The complementary determining regions of Vidol beads include CDR-H1, CDR–H2, CDR–H3, CDR-L1, CDR–L2, and CDR–L3 connected by disulfide bonds or linker peptides:

[0040] CDR-H1: SYWMH; CDR-H2: EIDPSESNTNYNQKTKG; CDR-H3: GGKDGWDYAIDY;

[0041] CDR-L1: RSSQSLAKSYGNTYLS; CDR-L2: GISNRFS; CDR-L3: LQGTHQPYT.

[0042] The variable region of natalizumab includes a heavy chain variable region and a light chain variable region connected by a disulfide bond or a linker peptide.

[0043] The heavy chain variable region is shown in SEQ ID NO.3:

[0044] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQRLEWMGRIDPANGYTKYDPKFQGRVTITADTSASTAYMELSSLRSEDEAVYYCAREGYYGNYGVYAMDYWGQGTLVTVSS.

[0045] The light chain variable region of natalizumab is shown in SEQ ID NO.4:

[0046] DIQMTQSPSSSLSASVGDRVTITCKTSQDINKYMAWYQQTPGKAPRLLIHYTSALQPGIPSRFSGSGSGRDYTFTISSLQPEDIATYYCLQYDNLWTFGQGTKVEIKRTV.

[0047] The complementary determining regions of natalizumab include CDR-H1, CDR–H2, CDR–H3, CDR-L1, CDR–L2, and CDR–L3, which are connected by disulfide bonds or linkers:

[0048] CDR-H1: DTYIH; CDR-H2: RIDPANGYTKYDPKFQG; CDR-H3: EGYYGNYGVYAMDY;

[0049] CDR-L1: KTSQDINKYMA; CDR-L2: YTSALQP; CDR-L3: LQYDNLWT.

[0050] It should be noted that, in other embodiments, the amino acid sequence of the heavy chain variable region of the integrin biological agent provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the above-mentioned corresponding variable region sequence, heavy chain variable region or complementary determining region.

[0051] The present invention also provides a method for preparing an antibody detection kit for integrin biological preparations, which comprises: fixing the integrin biological preparations on a solid phase carrier as an antibody capture device, combining or fixing functional fragments without markers on a cellulose membrane or marking the functional fragments with markers as an antibody detection device.

[0052] In a preferred embodiment of the present invention, when the solid phase carrier is selected from magnetic microspheres, the preparation method includes first modifying streptavidin on the magnetic microspheres.

[0053] In a preferred embodiment of the present invention, the antibody detection kit for integrin biological preparations is a quantitative detection kit or a qualitative detection kit.

[0054] In a preferred embodiment of the present invention, the antibody detection kit for integrin biological preparations is a time-resolved 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.

[0055] The present invention has the following beneficial effects:

[0056] The present invention provides an antibody detection kit for integrin biological preparations, which overcomes the defects of false positive and false negative at the same time. In view of the defect of false positive, the inventor uses integrin biological preparations as antibody capture devices. Since integrin biological preparations have a large spatial contact surface, they are more likely to capture anti-antibodies in the sample and have the advantage of high capture efficiency.

[0057] To address the defect of false negatives, the inventors fixed the heavy chain variable region of the integrin biologic on the detection device, and reduced the molecular weight of the coating on the detection device, thereby reducing the spatial steric hindrance of the coating, making it easier for the fixed substance on the detection device to bind to the Fab fragment of the anti-antibody, so that even lower concentrations of anti-antibodies in the sample can achieve specific binding to the fixed substance on the detection device, thereby improving the sensitivity of the detection and achieving the purpose of companion diagnosis of integrin biologics. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0059] Figure 1 It is a device diagram of the anti-vedolizumab antibody time-resolved fluorescent immunochromatographic test paper of the present invention;

[0060] Figure 2 It is a device diagram of the anti-natalizumab antibody colloidal gold immunochromatographic test paper of the present invention;

[0061] Figure 3 It is a device diagram of the anti-vedolizumab antibody quantum dot fluorescent immunochromatographic test paper of the present invention;

[0062] Figure 4 This is a detection principle diagram of the anti-natalizumab antibody enzyme-linked immunosorbent assay kit of the present invention;

[0063] Figure 5 This is a detection principle diagram of the anti-vedolizumab antibody chemiluminescence assay kit of the present invention;

[0064] Figure 6 is a calibration curve regression graph of the anti-vedolizumab antibody time-resolved fluorescent immunochromatography kit of the present invention;

[0065] Figure 7 It is a regression graph of the calibration curve of the anti-natalizumab antibody colloidal gold immunochromatography kit of the present invention;

[0066] Figure 8 is a regression graph of the calibration curve of the anti-vedolizumab antibody quantum dot fluorescent immunochromatography kit of the present invention;

[0067] Fig. 9 is a calibration curve regression diagram of the anti-natalizumab antibody enzyme-linked immunosorbent assay kit of the present invention;

[0068] Fig.10It is a regression graph of the calibration curve of the anti-vedolizumab antibody chemiluminescence assay kit of the present invention.

[0069] Reference numerals: 1 - sample pad; 2 - binding pad; 3 - nitrocellulose membrane; 4 - detection line; 5 - quality control line; 6 - water-absorbing membrane; 7 - back substrate. DETAILED DESCRIPTION

[0070] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0071] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0072] Example 1

[0073] This embodiment provides an anti-vedolizumab time-resolved fluorescent immunochromatography quantitative detection kit and a preparation method thereof.

[0074] Anti-Vediobead Antibody Immunochromatographic Test Strips Figure 1 As shown, the test paper is provided with a nitrocellulose membrane 3, a sample pad 1, a conjugation pad 2 and a water-absorbing membrane 6 on a back substrate 7, the sample pad 1 is stacked on one end of the conjugation pad 2, and the other end of the conjugation pad 2 and the water-absorbing membrane 6 are respectively stacked on both ends of the nitrocellulose membrane 3; the conjugation pad 2 is coated with vedolizumab labeled with time-resolved fluorescent microspheres and goat anti-chicken IgY antibody labeled with time-resolved fluorescent microspheres; the nitrocellulose membrane 3 is provided with a detection line 4 (T line) and a quality control line 5 (C line), the detection line 4 (T line) is coated with the heavy chain variable region (Hv) of vedolizumab, and the quality control line 5 (C line) is coated with chicken IgY antibody.

[0075] The heavy chain variable region of vedolizumab is as follows:

[0076] QVQLVQSGAEVKKPGASVKVSCKGSGYTFTSYWMHWVRQAPGQAIEWIGEIDPSESNTNYNQKTKGRVTLTVDISASTAYMELSSLRSEDTAVYYCARGGKDGWDYAIDYWGQGTLVTVSS.

[0077] The preparation and detection methods of the kit are as follows:

[0078] 1) Time-resolved fluorescent microsphere labeling.

[0079] Take 500 μL of time-resolved fluorescent microspheres (300 nm) (1% stock solution), centrifuge at 13000 rpm, 4°C for 10 min, and discard the supernatant. Add 10 mg / ml EDC, react for 15 min; centrifuge, discard the supernatant, add 1000 μL of borate buffer (20 mM, pH 8.0), and mix by ultrasound for 10 seconds; add 100 μg of labeled antibody (vedolizumab or goat anti-chicken IgY) (labeling concentration 100 μg / mL), react at room temperature for 2 h; centrifuge, discard the supernatant, add blocking solution, react for 1 h; centrifuge, discard the supernatant, add 1000 μL of borate buffer, mix by ultrasound, label well, and store at 4°C for use.

[0080] 2) Treatment of sample pad and conjugate pad

[0081] The polyester membrane was pretreated by immersing it in a buffer solution containing a surfactant (formula: 20 mM pH 8.0 BB, containing 2% BSA, 1% casein, 2% Tween-20, 1% S9 and 5% trehalose), and dried at 37° C. overnight to prepare a binding pad; the prepared binding pad was taken, and vedolizumab labeled with time-resolved fluorescent microspheres and goat anti-chicken IgY antibody were mixed using a gold-labeled HM3035 gold spray film instrument, and then sprayed onto the pretreated binding pad with a width of 1 cm at 4 μL / cm, and dried at 37° C. for 3 hours to prepare a specific binding pad.

[0082] The glass cellulose membrane was pre-blocked by soaking in a buffer solution containing a surfactant (formula: 100 mM pH 7.4 PB, containing 2% BSA, 0.5% S9 and 5% sucrose), dried at 37° C. overnight, and cut into 30*1.5 cm to prepare a sample pad.

[0083] 3) Coating of nitrocellulose membrane (NC membrane)

[0084] The heavy chain variable region of Vedolizumab was synthesized by an amino acid synthesis method, and it was verified that the synthetic polypeptide could react with anti-Vedolizumab antibodies.

[0085] The NC membrane was attached to the designated position of the backing substrate, and the heavy chain variable region (Hv) of vedolizumab was diluted to 1.5 mg / mL with 50 mM pH 7.4 phosphate buffer for preparing T lines; the chicken IgY antibody was diluted to 0.5 mg / mL with 50 mM pH 7.4 phosphate buffer for preparing C lines; the two diluted antibodies were evenly streaked onto the NC membrane using a gold standard HM3035 gold spray film streaker at a streaking volume of 1 μL / cm to prepare T lines and C lines; the streaked NC membrane was placed in a 37°C drying oven to dry overnight.

[0086] 4) Assembly

[0087] The conjugate pad of step 2) is stacked on one end of the nitrocellulose membrane obtained in step 3), and the absorbent membrane is fixedly stacked on the other end of the nitrocellulose membrane. Finally, the sample pad obtained in step 2) is stacked on the other end of the conjugate pad, and the sample pad is cut into strips of 4 mm width using a film cutter and loaded into the chromatography strip shell to obtain the finished product.

[0088] 5) Detection

[0089] Dilute the serum 20 times, take 90 μL and add it to the sample well. After reacting for 10 minutes, place it in the fluorescence reader for detection. Compare it with the value of the standard or reference product to determine the content of anti-antibodies in the sample to be tested.

[0090] In this embodiment, the time-resolved fluorescent substance latex microspheres are europium-containing latex microspheres, that is, the time-resolved fluorescent substance is a combination of lanthanide element europium and latex. In other embodiments, the time-resolved fluorescent substance can also be adjusted to one of lanthanide elements, a combination of lanthanide elements and latex, and a chelate of lanthanide elements as needed; the lanthanide element can be one of europium, terbium, samarium or dysprosium.

[0091] Example 2

[0092] This embodiment provides an anti-natalizumab colloidal gold immunochromatography assay kit and a preparation method thereof.

[0093] Anti-natalizumab immunochromatographic test strip reference Figure 2 As shown, the test paper is provided with a nitrocellulose membrane 3, a sample pad 1, a conjugation pad 2 and a water-absorbing membrane 6 on a back substrate 7, the sample pad 1 is stacked on one end of the conjugation pad 2, and the other end of the conjugation pad 2 and the water-absorbing membrane 6 are respectively stacked on both ends of the nitrocellulose membrane 3; the conjugation pad 2 is coated with colloidal gold-labeled biotinylated natalizumab and goat anti-chicken IgY antibody 2; the sample pad 1 contains streptavidin; the nitrocellulose membrane 3 is provided with a detection line 4 (T line) and a quality control line 5 (C line), the detection line 4 (T line) is coated with the variable region (Fv) of natalizumab, and the quality control line 5 (C line) is coated with chicken IgY antibody.

[0094] The variable region of natalizumab includes a heavy chain variable region and a light chain variable region connected by a linker peptide.

[0095] The heavy chain variable region of natalizumab is as follows:

[0096] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQRLEWMGRIDPANGYTKYDPKFQGRVTITADTSASTAYMELSSLRSEDEAVYYCAREGYYGNYGVYAMDYWGQGTLVTVSS.

[0097] The light chain variable region of natalizumab is as follows:

[0098] DIQMTQSPSSSLSASVGDRVTITCKTSQDINKYMAWYQQTPGKAPRLLIHYTSALQPGIPSRFSGSGSGRDYTFTISSLQPEDIATYYCLQYDNLWTFGQGTKVEIKRTV.

[0099] The connecting peptide is selected from GGGGSGGGGSGGGGS.

[0100] 1) Preparation of colloidal gold

[0101] Wash the round-bottom flask and glass reagent bottle used to prepare colloidal gold, soak them in a secondary strong acid solution, wash them thoroughly, and dry them. Take a clean round-bottom flask, add 100mL of ultrapure water, add 1mL of 1% chloroauric acid, and boil. Quickly add 2mL of 1% sodium citrate, wait until the color turns purple-red, continue the reaction for 10 minutes, and stop heating. After cooling, add ultrapure water to 100mL and set aside at 4 degrees.

[0102] 2) Biotinylation of labeled antibodies

[0103] Take 1 mg of natalizumab and place it in a 5 mL centrifuge tube, add 60 μL of 10 mg / mL activated biotin (Sulfo-NHS-LC-Bintin), add 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 natalizumab antibody, add 0.03% Proclin300 for preservation, and store at 4 degrees for later use.

[0104] 3) Colloidal gold labeling

[0105] Take 10mL of the prepared colloidal gold, add 300μL of 0.2M potassium carbonate and mix well, then add 150μg of biotinylated natalizumab or goat anti-chicken IgY and react at room temperature for 30min, add 1% BSA to block for 30min. Centrifuge at 12000rpm at 4℃ for 30min, take the precipitate and dissolve it in 1mL of resuspension solution (formula: 20mM pH 8.0BB, which contains 2% BSA, 2% Tween-20 and 5% sucrose), and store at 4℃ for later use.

[0106] 3) Treatment of sample pad and conjugate pad

[0107] Using a gold standard HM3035 gold spraying film scribing instrument, the biotinylated natal beads labeled with colloidal gold and goat anti-chicken IgY antibody were mixed, sprayed onto a glass fiber pad with a width of 1 cm*30 cm at 3 μL / cm, and dried at 37°C for 3 hours to prepare a colloidal gold binding pad.

[0108] The glass cellulose membrane was pre-blocked by soaking in a buffer containing streptavidin (formula: 100 mM pH 7.4 PB, containing 1% streptavidin, 1% BSA, 0.5% casein, 0.5% S9 and 2% sucrose), dried at 37°C overnight, and cut into 30*1.5 cm to prepare a sample pad.

[0109] 4) Coating of nitrocellulose membrane (NC membrane)

[0110] The variable region of natalizumab was synthesized by an amino acid synthesis method, and it was verified that the synthetic polypeptide could react with anti-natalizumab antibodies.

[0111] The NC membrane was attached to the designated position of the backing substrate, and the variable region (Fv) of natal beads was diluted to 1.5 mg / mL with 50 mM pH 7.4 phosphate buffer for preparing T lines; the chicken IgY antibody was diluted to 0.5 mg / mL with 50 mM pH 7.4 phosphate buffer for preparing C lines; the two diluted antibodies were evenly streaked onto the NC membrane using a gold standard HM3035 gold spray film streaker at a streaking volume of 1 μL / cm to prepare T lines and C lines; the streaked NC membrane was placed in a 37°C drying oven to dry overnight.

[0112] 5) Assembly

[0113] The conjugate pad of step 2) is stacked on one end of the nitrocellulose membrane obtained in step 3), and the absorbent membrane is fixedly stacked on the other end of the nitrocellulose membrane. Finally, the sample pad obtained in step 2) is stacked on the other end of the conjugate pad, and the sample pad is cut into strips of 4 mm width using a film cutter and loaded into the chromatography strip shell to obtain the finished product.

[0114] 6) Detection

[0115] Dilute the serum 20 times, take 90 μL and add it to the sample well. After reacting for 10 minutes, place it in the colloidal gold reader for detection. Compare it with the value of the standard or reference product to determine the content of anti-antibodies in the sample to be tested.

[0116] In this embodiment, colloidal gold is a colloidal solution obtained by reducing tetrachloroauric acid with a reducing agent; in other embodiments, the marker can be adjusted to one of colloidal carbon and colloidal silver as needed.

[0117] Example 3

[0118] This embodiment provides an anti-vedolizumab antibody quantum dot fluorescence immunochromatography assay kit and a preparation method thereof.

[0119] Anti-vedolizumab antibody immunochromatographic test strip reference Figure 3As shown, the test paper is provided with a nitrocellulose membrane 3, a sample pad 1, a conjugation pad 2 and a water-absorbing membrane 6 on a back substrate 7, the sample pad 1 is stacked on one end of the conjugation pad 2, and the other end of the conjugation pad 2 and the water-absorbing membrane 6 are respectively stacked on both ends of the nitrocellulose membrane 3; the conjugation pad 2 is coated with quantum dot-labeled vedolizumab and goat anti-chicken IgY antibody; the nitrocellulose membrane 3 is provided with a detection line 4 (T line) and a quality control line 5 (C line), the detection line 4 (T line) is coated with the heavy chain variable region (Hv) of vedolizumab, and the quality control line 5 (C line) is coated with chicken IgY antibody.

[0120] The heavy chain variable region (Hv) of vedolizumab is the same as that shown in Example 1.

[0121] 1) Quantum dot labeling

[0122] Take 100 μL of quantum dots, centrifuge at 13000 rpm, 4°C for 10 min, discard the supernatant, add 1000 μL of MES buffer, mix by ultrasound, weigh 20 mg EDC and 20 mg NHS and slowly add them to the microsphere mixture, mix by ultrasound in time, and react for 15 minutes; centrifuge, discard the supernatant, add 1000 μL of borate buffer (20 mM, pH 8.0), mix by ultrasound; add 100 μg of labeled antibody (vedolizumab or goat anti-chicken IgY) (labeling concentration 100 μg / mL), react at room temperature for 2 h; centrifuge, discard the supernatant, add 1000 μL of blocking solution, mix by ultrasound with ice water, and react at room temperature for 1 h; centrifuge, discard the supernatant, add 1000 μL of borate buffer (20 mM PH8.0), mix by ultrasound for 10-30 seconds, label, and store at 4°C for use.

[0123] 2) Treatment of sample pad and conjugate pad

[0124] The polyester membrane was pre-blocked by immersing it in a buffer solution containing a surfactant (formula: 20 mM pH 8.0 BB, containing 2% BSA, 2% Tween-20, 1% S9 and 5% trehalose), and dried at 37° C. overnight to prepare a binding pad; the prepared binding pad was taken, and vedolizumab labeled with quantum dots and goat anti-chicken IgY antibody were mixed using a Gold Standard HM3035 gold spray film instrument, and then sprayed onto a pre-treated binding pad with a width of 1 cm at 4 μL / cm, and dried at 37° C. for at least 3 hours to prepare a specific binding pad.

[0125] The glass cellulose membrane was pre-blocked by soaking in a buffer solution containing a surfactant (formula: 100 mM pH 7.4 PB, containing 1% BSA, 0.5% casein, 0.1% Tween-20, 0.5% S9 and 2% sucrose), dried at 37°C overnight, and cut into 30*1.5 cm pieces to prepare a sample pad.

[0126] 3) Coating of nitrocellulose membrane (NC membrane)

[0127] The heavy chain variable region of vedolizumab was synthesized by an amino acid synthesis method. It was verified that the synthetic polypeptide coated on an ELISA plate could react with anti-vedolizumab antibodies.

[0128] The NC membrane was attached to the designated position of the backing substrate, and the heavy chain variable region (Hv) of vedolizumab was diluted to 1.5 mg / mL with 50 mM pH 7.4 phosphate buffer for preparing T lines; the chicken IgY antibody was diluted to 0.5 mg / mL with 50 mM pH 7.4 phosphate buffer for preparing C lines; the two diluted antibodies were evenly streaked onto the NC membrane using a gold standard HM3035 gold spray film streaker at a streaking volume of 1 μL / cm to prepare T lines and C lines; the streaked NC membrane was placed in a 37°C drying oven to dry overnight.

[0129] 4) Assembly

[0130] The conjugate pad of step 2) is stacked on one end of the nitrocellulose membrane obtained in step 3), and the absorbent membrane is fixedly stacked on the other end of the nitrocellulose membrane. Finally, the sample pad obtained in step 2) is stacked on the other end of the conjugate pad, and the sample pad is cut into strips of 4 mm width using a film cutter and loaded into the chromatography strip shell to obtain the finished product.

[0131] 5) Detection

[0132] Dilute the serum 20 times, take 70 μL and add it to the sample well. After reacting for 10 minutes, place it in the fluorescence reader for detection. Compare it with the value of the standard or reference product to determine the content of anti-antibodies in the sample to be tested.

[0133] In this embodiment, the quantum dots are core-shell quantum dots, quantum dots formed by ZnS / CdSe or ZnS / CdTe. In other embodiments, the quantum dots can be adjusted to quantum dots formed by a single compound as needed, such as cadmium selenide (CdSe), zinc sulfide (ZnS), cadmium telluride (CdTe) / cadmium sulfide (CdS), etc.

[0134] Example 4

[0135] This embodiment provides an anti-natalizumab antibody enzyme-linked immunosorbent assay kit and a preparation method thereof.

[0136] The invention discloses an enzyme-linked immunosorbent assay kit for anti-natalizumab, comprising an ELISA plate, biotinylated natalizumab, an anti-natalizumab calibrator, an anti-natalizumab quality control product, an enzyme conjugate, a cleaning solution, and a stop solution. The enzyme conjugate is a natalizumab complementary determining region (CDRs) labeled with horseradish peroxidase (HRP). The ELISA plate is coated with streptavidin.

[0137] The complementary determining regions of natalizumab include CDR-H1, CDR–H2, CDR–H3, CDR-L1, CDR–L2, and CDR–L3, which are connected by disulfide bonds:

[0138] CDR-H1: DTYIH; CDR-H2: RIDPANGYTKYDPKFQG; CDR-H3: EGYYGNYGVYAMDY;

[0139] CDR-L1: KTSQDINKYMA; CDR-L2: YTSALQP; CDR-L3: LQYDNLWT.

[0140] Preparation process reference Figure 4 As shown, the preparation method and detection method are as follows:

[0141] 1) Biotinylation of natalizumab

[0142] Take 1 mg of natalizumab and place it in a 5 mL centrifuge tube, add 60 μL of 10 mg / mL activated biotin (Sulfo-NHS-LC-Bintin), add 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 natalizumab full-length antibody, add 0.03% Proclin300 for preservation, and store at 4 degrees for use.

[0143] 2) HRP labeled complementarity determining regions (CDRs) of natalizumab

[0144] The complementary determining region of natalizumab was synthesized by an amino acid synthesis method, and it was verified that the synthetic polypeptide could react with anti-natalizumab antibodies.

[0145] Weigh 5mg HRP into a 5mL centrifuge tube, add 1mL pure water to dissolve, add 1mL NaIO4 solution (10mg / mL, prepared immediately before use), and react at 4 degrees in the dark for 30min. Add 0.02mL ethylene glycol and react at room temperature for 15min. Take out the above solution after dialyzing. Add 1mg certol bead heavy chain variable region (HCDR) to the above activated solution, react at room temperature in the dark for 2 hours, add 0.2mL NaBH4 solution (5mg / mL, prepared immediately before use), mix well, and dialyze overnight with 0.05M CB. Take out the labeled enzyme-labeled antibody, add an equal volume of glycerol, and store at -20℃ for later use.

[0146] 3) Coating ELISA plate

[0147] Dilute streptavidin to 1 μg / mL in CB buffer, coat the microplate with 100 μL per well and incubate at 37°C for 2 hours. Aspirate the coating solution, add 200 μL per well of blocking buffer containing 2% BSA, incubate at 37°C for 2 hours, tap off the blocking solution, and dry at 37°C for later use.

[0148] 4) Coating with biotinylated natalizumab

[0149] Dilute biotinylated natalizumab to 1 μg / mL with neutral phosphate buffer, add 100 μL to each well, and incubate at 37°C for 2 hours. Wash the plate 3 times, add 200 μL of blocking buffer containing 2% BSA to each well, and incubate at 37°C for 2 hours. Remove the blocking solution and dry at 37°C for later use.

[0150] 5) Dilute the serum sample to be tested 100 times, add 100 μL to each well of the microplate, incubate at 37 degrees for 2 hours, and wash the plate 4 times.

[0151] 6) Add HRP-labeled natalizumab complementary determining regions (CDRs), incubate for 1 hour, and wash the plate 4 times.

[0152] 7) Add TMB, the substrate of HRP enzyme, to the microplate and incubate for 15 minutes. Add stop solution HCL solution to terminate the color development reaction. Detect the OD value at a wavelength of 450nm and compare it with the OD value of the standard or reference to determine the content of anti-antibodies in the sample to be tested.

[0153] Example 5

[0154] This embodiment provides an anti-vedolizumab antibody chemiluminescence assay kit and a preparation method thereof.

[0155] The anti-vedolizumab antibody chemiluminescent detection kit includes biotinylated vedolizumab, anti-vedolizumab antibody calibrator, anti-vedolizumab antibody quality control, acridinium ester-labeled vedolizumab heavy chain variable region, magnetic particle reagent, excitation solution and cleaning solution.

[0156] The variable region of the heavy chain of VEDO is the same as that in Example 1.

[0157] The preparation and testing steps are as follows:

[0158] 1) Biotinylation of Vedolizumab

[0159] Take 1 mg of vedolizumab and place it in a 5 mL centrifuge tube, add 50 μL of 10 mg / mL activated biotin (Sulfo-NHS-LC-Bintin), add 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 vedolizumab full-length antibody, add 0.03% Proclin 300 for preservation, and store at 4 degrees for later use.

[0160] 2) Acridinium ester labeling of the heavy chain variable region of Vidol

[0161] The heavy chain variable region of vedolizumab was synthesized by an amino acid synthesis method, and it was verified that the synthetic polypeptide could react with anti-vedolizumab antibodies.

[0162] Take 2 mg of the heavy chain variable region of vedol beads, add activated acridinium ester at a molar ratio of antibody: acridinium ester = 1:10-50, and react at room temperature for 30 minutes. After dialyzing the reaction solution with 0.05M CB solution, take it out, add an equal volume of glycerol, and store it at -20°C for later use.

[0163] 3) Detection steps

[0164] This method uses acridinium ester chemiluminescent immunoassay technology combined with biotin-avidin magnetic particle separation technology to achieve anti-antibody detection. Figure 5 :The sample (diluted 20 times) is mixed with streptavidin magnetic particles (1) and biotinylated vedolizumab (2) to obtain a streptavidin magnetic bead-biotinylated vedolizumab-sample complex (3), and after washing, acridinium ester-labeled vedolizumab heavy chain variable region is added for reaction to form a streptavidin magnetic bead-biotinylated vedolizumab-sample-acridinium ester-labeled vedolizumab heavy chain variable region complex. A luminescent excitation solution is added to measure the luminescent intensity, and compared with the value of a standard or reference product to determine the content of the anti-antibody in the sample to be tested.

[0165] Experimental Example 1

[0166] This experimental example performs quantitative testing on the kit provided in Example 1 to verify its performance.

[0167] 1.1 Preparation of standard curve

[0168] 1.1.1 Obtain a set of anti-vedol antibody calibrators. See Table 1 for specific values.

[0169] Table 1

[0170]

[0171] 1.1.2 Detection Method

[0172] Take 90 μL of the calibrator and add it directly into the sample window in the chromatography strip; after 10 minutes, use a time-resolved fluorescence quantitative analyzer to quantitatively detect the signal value.

[0173] Each calibrator was tested twice and the average T / C value was taken. The specific results are shown in Table 2.

[0174] Table 2

[0175]

[0176]

[0177] 1.1.3 Preparation of standard curve

[0178] According to the above test results, the concentration value is the X-axis and the T / C value is the Y-axis. Linear regression is performed to obtain the linear equation: y = 0.0095x + 0.2589, R 2 =0.9928, curve Figure 6 .

[0179] 1.2 Precision test:

[0180] 1.2.1 Take 10 prepared chromatography strips and prepare a concentration (100 ng / mL) of anti-vidol bead antibody working calibrator;

[0181] 1.2.2 Take 90 μL of calibrator and add it directly into the sample well of the reagent strip;

[0182] 1.2.3 After the calibration sample was chromatographed for 10 minutes, the time-resolved fluorescence quantitative analyzer was used for detection. The results of 10 reagent strips were measured according to the above linear equation. The results are shown in Table 3, indicating that the anti-vidol bead antibody time-resolved fluorescence immunochromatography assay kit of this method has good precision.

[0183] Table 3

[0184]

[0185] 1.3 Compared with other methods:

[0186] Method 2 is to label the microspheres with vedolizumab and coat the NC membrane with vedolizumab; Method 3 is to label the microspheres with vedolizumab and coat the NC membrane with vedolizumab. The three methods have the same conditions except for the labeling antibody and the coating antibody. The comparison of the three results is shown in Table 4.

[0187] Table 4

[0188] This embodiment Method 2 Method 3 Detection limit <5ng / ml <10ng / ml <20ng / ml Detection range 5-500ng / ml 10-400ng / ml 20-500ng / ml Linear R2 0.9928 0.9883 0.9876 Precision 4.51％ 15.90％ 18.82％ Non-specific background interference Low high Low

[0189] Experimental Example 2

[0190] This experimental example performs quantitative testing on the kit provided in Example 2 to verify its performance.

[0191] 2.1 Preparation of standard curve

[0192] 2.1.1 Obtain a set of anti-natalizumab antibody calibrators. See Table 5 for specific values.

[0193] Table 5

[0194]

[0195] 2.1.2 Detection methods

[0196] Take 90 μL of the calibrator and add it directly to the sample window in the chromatography strip; after 10 minutes, use a colloidal gold reader to detect the signal value. Each calibrator is tested twice and the average T / C value is taken. The specific results are shown in Table 6.

[0197] Table 6

[0198]

[0199] 2.1.3 Preparation of standard curve

[0200] According to the above test results, the concentration value is the X-axis and the T / C value is the Y-axis. Linear regression is performed to obtain the linear equation: y = 0.004x + 0.1157, R 2 =0.9951, curve Figure 7 .

[0201] 2.2 Precision test:

[0202] 2.2.1 Take 10 prepared chromatography strips and prepare a concentration (50 ng / mL) of anti-natalizumab antibody working calibrator;

[0203] 2.2.2 Take 90 μL of calibrator and add it directly into the sample well of the reagent strip;

[0204] 2.2.3 After the calibration sample was chromatographed for 10 minutes, the colloidal gold reader was used for detection. The results of 10 reagent strips were measured according to the above linear equation. The results are shown in Table 7, indicating that the anti-natalizumab antibody colloidal gold immunochromatography assay kit of this method has good precision.

[0205] Table 7

[0206]

[0207] 2.3 Compared with other methods:

[0208] Taking this example as a control, colloidal gold was labeled with natalizumab and NC membrane was coated with natalizumab as method 2; colloidal gold was labeled with natalizumab fab and NC membrane was coated with natalizumab fab as method 3. The three methods were the same except for the labeling antibody and the coating antibody, and the results were compared in Table 8.

[0209] Table 8

[0210] This embodiment Method 2 Method 3 Detection limit <5ng / ml <10ng / ml <20ng / ml Detection range 5-500ng / ml 10-500ng / ml 20-400ng / ml Linear R2 0.9951 0.9901 0.9874 Precision 5.99％ 16.42％ 18.39％ Non-specific background interference Low high Low

[0211] Experimental Example 3

[0212] This experimental example performs quantitative testing on the kit provided in Example 3 to verify its performance.

[0213] 3.1 Preparation of standard curve

[0214] 3.1.1 Obtain a set of anti-vedolizumab antibody calibrators. See Table 9 for specific values.

[0215] Table 9

[0216]

[0217] 3.1.2 Detection methods

[0218] Take 70 μL of the calibrator and add it directly to the sample window in the chromatography strip; after 10 minutes, use a fluorescence quantitative analyzer to detect the signal value. Each calibrator is tested twice and the average T / C value is taken. The specific results are shown in Table 10.

[0219] Table 10

[0220]

[0221]

[0222] 3.1.3 Preparation of standard curve

[0223] According to the above test results, the curve fitting is performed with the T / C value as the X axis and the concentration value as the Y axis, and the curve equation is obtained: y = 45.082x 2 -14.298x-5.829, R 2 =0.9983, see the curve Figure 8 .

[0224] 3.2 Precision test:

[0225] 3.2.1 Take 10 prepared chromatography strips and prepare a concentration (50 ng / mL) of anti-vedolizumab antibody working calibrator;

[0226] 3.2.2 Take 70 μL of calibrator and add it directly into the sample well of the reagent strip;

[0227] 3.2.3 After the calibration sample was chromatographed for 10 minutes, the fluorescence quantitative analyzer was used for detection. The results of 10 reagent strips were measured according to the above equation. The results are shown in Table 11, indicating that the anti-vedolizumab antibody quantum dot fluorescence immunochromatography assay kit of this method has good precision.

[0228] Table 11

[0229]

[0230]

[0231] 3.3 Compared with other methods:

[0232] Method 2 is to label quantum dots with vedolizumab and coat NC membrane with vedolizumab; Method 3 is to label quantum dots with vedolizumab fab and coat NC membrane with vedolizumab fab. The three methods have the same conditions except for the labeling antibody and coating antibody. The comparison of the three results is shown in Table 12.

[0233] Table 12

[0234]

[0235] Experimental Example 4

[0236] In this experimental example, the kit provided in Example 4 was quantitatively tested to verify its performance.

[0237] 4.1 Detection Method

[0238] 4.1.1 Obtain a set of anti-natalizumab antibody calibrators (customized). See Table 13 for specific values.

[0239] Table 13

[0240]

[0241] 4.1.2 Detection steps

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

[0243] 2) Add HRP-labeled natalizumab complementarity determining region, incubate for 1 hour, and wash the plate 4 times.

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

[0245] Table 14

[0246]

[0247] Raw Data{Wavelength:450.0}

[0248] 1 2 3 4 5 A 0.072 0.069 0.494 0.489 0.052 B 0.126 0.121 0.472 0.467 0.031 C 0.256 0.247 0.463 0.468 D 0.587 0.592 0.476 0.472 E 0.832 0.827 0.046 0.048 F 1.446 1.408 0.032 0.059 G 2.489 2.441 0.037 0.04 H 0.473 0.482 0.028 0.038

[0249] 4.1.3 Standard curve

[0250] According to the above test results, the OD value is the X-axis and the concentration value is the Y-axis, and the curve equation is obtained: y = 54.77x2 + 70.01x-6.2404, R2 =0.9991, see the curve Fig. 9 .

[0251] 4.2 Precision test:

[0252] According to the OD value of the precision quality control product tested, the result value was calculated using the curve equation. The results are shown in Table 15, indicating that the anti-natalizumab antibody enzyme-linked immunosorbent assay kit of this method has good precision.

[0253] Table 15

[0254]

[0255] 4.3 Compared with other methods:

[0256] Method 2 is to label HRP with natalizumab and coat the microplate with natalizumab; and method 3 is to label HRP with natalizumab fab and coat the microplate with natalizumab fab. The three methods have the same conditions except for the labeling antibody and coating antibody. The comparison of the three results is shown in Table 16.

[0257] Table 16

[0258]

[0259] Experimental Example 5

[0260] This experimental example performs quantitative testing on the kit provided in Example 5 to verify its performance.

[0261] 5.1 Detection Method

[0262] 5.1.1 Obtain a set of anti-vedolizumab antibody calibrators. See Table 17 for specific values.

[0263] Table 17

[0264]

[0265]

[0266] 5.1.2 Detection steps

[0267] 1) Take 20 μL of calibrator, 20 μL of streptavidin magnetic particles, and 50 μL of biotinylated vedolizumab and add them to the microwells, mix them, incubate at 37 degrees for 15 minutes, and wash three times.

[0268] 2) Add acridinium ester-labeled vedolizumab heavy chain variable region, incubate at 37 degrees for 15 minutes, and wash three times.

[0269] 3) Add luminescence excitation liquid and measure the luminescence intensity. The specific results are shown in Table 18.

[0270] Table 18

[0271]

[0272] 5.1.3 Standard curve

[0273] According to the above test results, the logarithmic value of the concentration value is used as the X-axis and the logarithmic value of the luminescence value is used as the Y-axis, and a linear regression is performed to obtain the linear equation: y = 0.9339x + 2.8704, R 2 =0.9923, curve Fig.10 .

[0274] 5.2 Precision test:

[0275] Take 20 μL precision quality control, 20 μL streptavidin magnetic particles, 50 μL biotinylated vedolizumab and add them to the microwells, mix, incubate at 37 degrees for 15 minutes, and wash 3 times; add acridinium ester labeled vedolizumab heavy chain variable region, incubate at 37 degrees for 15 minutes, and wash 3 times; 3) add luminescent excitation liquid 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 anti-vedolizumab antibody chemiluminescent immunoassay kit of this method has good precision. The specific results are shown in Table 19.

[0276] Table 19

[0277]

[0278] 5.3 Compared with other methods:

[0279] Method 2 is to label acridinium ester with vedolizumab and label biotin with vedolizumab; method 3 is to label acridinium ester with vedolizumab fab and label biotin with vedolizumab fab. The three methods are the same except that the antibodies for labeling acridinium ester and the antibodies for labeling biotin are different. The comparison of the three results is shown in Table 20.

[0280] Table 20

[0281]

[0282] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. SEQUENCE LISTING <110> Suzhou Herui Biotechnology Co., Ltd. <120> Antibody detection kit for integrin biological preparations and preparation method thereof <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 121 <212> PRT <213> Artificial sequence <400> 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Ala Ile Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Glu Ser Asn Thr Asn Tyr Asn Gln Lys Thr 50 55 60 Lys Gly Arg Val Thr Leu Thr Val Asp Ile Ser Ala 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 Gly Gly Lys Asp Gly Trp Asp Tyr Ala Ile Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 115 <212> PRT <213> Artificial sequence <400> 2 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Ala Lys Ser 20 25 30 Tyr Gly Asn Thr Tyr Leu Ser Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gly Ile Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Gly 85 90 95 Thr His Gln Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val 115 <210> 3 <211> 123 <212> PRT <213> Artificial Sequence <400> 3 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Tyr Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Glu Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Tyr Tyr Gly Asn Tyr Gly Val Tyr Ala Met Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 4 <211> 109 <212> PRT <213> Artificial Sequence <400> 4 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 Thr Ser Gln Asp Ile Asn Lys Tyr 20 25 30 Met Ala Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Arg Leu Leu Ile 35 40 45 His Tyr Thr Ser Ala Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105

Claims

1. An antibody detection kit for integrin biological preparations, characterized in that: It includes an antibody capture device and an antibody detection device; The antibody capture device comprises a solid phase carrier; the antibody detection device is a functional fragment with a detectable marker; the solid phase carrier is selected from a microporous plate or a microporous membrane; the detectable marker is selected from a fluorescent dye, an enzyme that catalyzes the color development of a substrate, a radioactive isotope, a chemiluminescent reagent or a nanoparticle marker; Or, the antibody capture device comprises a solid phase carrier; 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 solid phase carrier is coated with the full length of the integrin biological agent, and the full length of the integrin biological agent is the full length of Vedolizumab or Natalizuma; correspondingly, the functional fragment is the heavy chain variable region of Vedolizumab, or the variable region or complementarity determining region of Natalizuma; The variable region is composed of a heavy chain variable region and a light chain variable region connected by a connecting peptide or a disulfide bond; the complementary determining region includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 connected by a disulfide bond or a connecting peptide.

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

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

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

6. The antibody detection kit for integrin biological preparations according to claim 1 or 2, characterized in that: The heavy chain variable region of the vedolizumab is shown in SEQ ID NO.1; The variable region of natalizumab is composed of a heavy chain variable region and a light chain variable region connected by a connecting peptide or a disulfide bond; the heavy chain variable region and the light chain variable region of natalizumab are shown in SEQ ID NO.3-4 respectively; the complementary determining region of natalizumab includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 connected by a disulfide bond or a connecting peptide: CDR-H1:DTYIH; CDR-H2: RIDPANGYTKYDPKFQG; CDR-H3:EGYYGNYGVYAMDY; CDR-L1:KTSQDINKYMA; CDR-L2: YTSALQP; CDR-L3:LQYDNLWT.

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

8. The antibody detection kit for integrin biological preparations according to claim 1 or 2, characterized in that: The antibody detection kit for the integrin biological preparation is a time-resolved 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.

9. A method for preparing an antibody detection kit for integrin biological preparations according to any one of claims 1 to 8, characterized in that: It includes: The integrin biological preparation is fixed on a solid phase carrier as an antibody capture device, and the functional fragment without a marker is combined or fixed on a cellulose membrane or the functional fragment is labeled with a marker as an antibody detection device.

10. The method for preparing the antibody detection kit for integrin biological preparations according to claim 9, characterized in that: When the solid phase carrier is selected from magnetic microspheres, the preparation method comprises firstly modifying streptavidin on the magnetic microspheres.

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