An antibody detection kit for HER2 biological agents and a preparation method thereof

Abstract

The present invention discloses an antibody detection kit for HER2 biological agents and a preparation method thereof, which relates to the technical field of antibody detection. The kit includes an antibody capture device and an antibody detection device. The antibody capture device includes a solid-phase carrier coated with HER2 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, heavy-chain variable regions or complementary determining regions of HER2 biological agents. The detection kit provided by the present invention has the advantages of high capture efficiency, high accuracy and high sensitivity, can effectively avoid the occurrence of false positives and false negatives, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody detection, and more particularly, to a kit for detecting antibodies against HER2 biological agents and a preparation method thereof. Background Art

[0002] HER2 (human epidermal growth factor receptor 2) is a member of the epidermal growth factor receptor family with tyrosine kinase activity. The polymerization of the receptor leads to the phosphorylation of receptor tyrosine residues and initiates multiple signaling pathways resulting in cell proliferation and tumorigenesis. HER2 gene amplification or overexpression occurs in approximately 15% - 30% of breast cancers and 10% - 30% of gastric cancers. The overexpression of the HER2 gene is not only closely related to the occurrence and development of tumors but also an important indicator of clinical prognosis.

[0003] Monoclonal antibody drugs designed targeting HER2 include Trastuzumab and Pertuzumab, which are approved by the FDA for the treatment of diseases such as breast cancer, metastatic breast cancer, or metastatic gastric cancer.

[0004] Although clinical practice has proven that such biological agents have good efficacy in the treatment of the above diseases, not all patients respond effectively. Some patients are ineffective from the beginning (primary ineffectiveness), and more patients initially show effectiveness but become resistant and ineffective after multiple uses (secondary ineffectiveness). Further research has found that secondary ineffectiveness is related to the production of antibodies against biological agent drugs in the body. Therefore, detecting antibodies against biological agents in the blood is an important companion diagnosis.

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

[0006] The purpose of the present invention is to provide a kit for detecting antibodies against HER2 biological agents and a preparation method thereof to solve the above technical problems.

[0007] Existing methods for detecting antibodies against HER2 have problems of false positives and false negatives. Through research by the inventors, it is found that the root cause of the false negative problem is that after capturing antibodies against HER2 in the sample by the double - antigen sandwich method, there is a problem with the binding efficiency to the subsequent detection antibody element, resulting in a positive sample that cannot be detected, leading to false negatives.

[0008] The root cause of the false positive problem is that it is interfered by a large number of irrelevant antibodies in the sample, resulting in detection distortion such as false positives.

[0009] The present invention is implemented as follows:

[0010] The present invention provides an antibody detection kit for a HER2 biological agent, which comprises an antibody capture device and an antibody detection device. 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 label; and a HER2 biological agent is coated on the solid-phase carrier, and a functional fragment without a label 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 HER2 biological agent.

[0011] The inventors found that: when using the HER2 biological agent as a capture antigen, due to the large spatial contact surface, it is easier to capture the anti-antibody in the sample, and has the advantage of high capture efficiency. And by means of the double-antigen sandwich method to realize the detection of the anti-antibody, a large number of irrelevant antibody interferences can be avoided to a great extent, the detection accuracy can be improved, and the occurrence of false positives can be avoided.

[0012] The antigen-antibody complex formed after capture itself has a large steric hindrance. If the molecular weight of the antibody detection device is large (such as a biological agent), it is more difficult to bind to the other Fab of the anti-antibody in the complex. The inventors found that the variable region, heavy-chain variable region or complementary determining region of the HER2 biological agent with a small molecular weight has the advantage of small steric hindrance and is more easily bound by the anti-antibody Fab. Using it as the antibody detection device can improve the binding efficiency of the antigen-antibody complex and the detection antibody element, improve the detection sensitivity, and avoid the occurrence of false negatives. To achieve the purpose of companion diagnosis of the HER2 biological agent drug.

[0013] In an optional embodiment, the above-mentioned functional fragment generally has the same binding specificity as the antibody from which it is derived. The detection antigen of the above-mentioned variable region (Fv) is: Fv (variable region) in which the heavy-chain variable region and the light-chain variable region are connected by a linker peptide or a disulfide bond to maintain the stability of the Fv. The complementary determining region includes CDR1, CDR2 and CDR3 in the heavy-chain complementary determining region and CDR1, CDR2 and CDR3 in the light-chain complementary determining region. Those skilled in the art can easily understand according to 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 chemical reduction to cleave 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] The above-mentioned functional fragment of the antibody 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 automatic peptide synthesizer, such as an automatic peptide synthesizer sold by Applied BioSystems, etc.

[0015] In a preferred embodiment of the application of the present invention, the above-mentioned HER2 biological agent is selected from monoclonal antibody drugs of HER2.

[0016] In an alternative embodiment, the anti-HER2 monoclonal antibody drugs include, but are not limited to, Trastuzumab and Pertuzumab.

[0017] In an alternative embodiment, the 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-mentioned colloidal gold is a colloidal solution obtained by reducing chloroauric acid with a reducing agent. In other embodiments, the above-mentioned colloidal gold may also be other colloidal substances, such as colloidal carbon, colloidal silver or colloidal selenium.

[0018] In an alternative embodiment, the above-mentioned quantum dots are core-shell quantum dots, such as quantum dots formed by ZnS / CdSe or ZnS / CdTe. In addition, in other embodiments, according to needs, the above-mentioned 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.

[0019] In an alternative embodiment, the anti-HER2 biological agent coated on the solid-phase carrier is modified with avidin or biotin. Modifying avidin or biotin is beneficial to further improve the amplification of the detection signal and the sensitivity of the detection. It should be noted that in an alternative embodiment, avidin or biotin can also be optionally not modified on the anti-HER2 biological agent according to needs.

[0020] In an alternative embodiment, the full length of the anti-HER2 biological agent is coated on the solid-phase carrier. Using the full length of the anti-HER2 biological agent as the capture antigen, due to the large spatial contact surface, it is easier to capture the antibody (i.e., anti-antibody) against the anti-HER2 biological agent in the sample, and has the advantage of high capture efficiency. By coating the full length of the anti-HER2 biological agent on the capture device, the occurrence of false positives can be better avoided.

[0021] In an alternative embodiment, the fluorescent microspheres are selected from time-resolved fluorescent microspheres or fluorescent microspheres containing fluorescein, and the magnetic microspheres are selected from magnetic beads. In an alternative embodiment, the fluorescence excitation wavelength of the above-mentioned fluorescent microspheres containing fluorescein is 300 - 520 nm, the emission wavelength is 340 - 630 nm, and the microsphere diameter ranges from 100 nm to 1000 nm.

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

[0023] The time-resolved fluorescent substance is one of lanthanide elements, the combination 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 detectable label 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 instruments. Through these characteristics, qualitative or quantitative detection of the corresponding target can be achieved.

[0025] In an optional embodiment, the detectable labels include, but are not limited to, fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents or nanoparticle-based labels.

[0026] In an optional 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, Cy7, 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, 633, 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 (PerCP), etc.).

[0027] In an optional embodiment, the enzymes that catalyze substrate color development include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase and 6-phosphogluconate dehydrogenase.

[0028] In an optional embodiment, the radioisotopes are 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 and 18 F;

[0029] In an alternative embodiment, the chemiluminescent reagent includes, but is 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.

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

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

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

[0033] In an alternative embodiment, a quality control antibody is further immobilized on the cellulose membrane as a quality control T line, and the functional fragment and the quality control antibody are separately arranged at intervals;

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

[0035] In an alternative embodiment,

[0036] The variable region (Fv) comprises 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 trastuzumab are shown in SEQ ID NO.1-2 respectively:

[0037] SEQ ID NO.1:

[0038] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS;

[0039] SEQ ID NO.2:

[0040] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ HYTTPPTFGQGTKVEIKRTV。

[0041] The complementarity-determining regions of trastuzumab include CDR1-VH1, CDR2 –VH2, CDR3–VH3, CDR1-VL1, CDR2 –VL2, and CDR3–VL3 that are linked by disulfide bonds or linker peptides:

[0042] CDR1-VH1: DTYIH;

[0043] CDR2-VH2: RIYPTNGYTRYADSVKG;

[0044] CDR3-VH3: WGGDGFYAMDY;

[0045] CDR1-VL1: RASQDVNTAVA;

[0046] CDR2-VL2: SASFLYS;

[0047] CDR3-VL3: QQHYTTPPT.

[0048] The heavy chain variable region and light chain variable region of pertuzumab are shown in SEQ ID NO.3-4 respectively:

[0049] SEQ ID NO.3:

[0050] EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS。

[0051] SEQ ID NO.4:

[0052] DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTV。

[0053] The complementarity-determining regions of pertuzumab include CDR1-VH1, CDR2-VH2, CDR3-VH3, CDR1-VL1, CDR2-VL2, and CDR3-VL3 linked by disulfide bonds or linker peptides:

[0054] CDR1-VH1: DYTMD;

[0055] CDR2-VH2: DVNPNSGGSIYNQRFKG;

[0056] CDR3-VH3: NLGPSFYFDY;

[0057] CDR1-VL1: KASQDVSIGVA;

[0058] CDR2-VL2: SASYRYT;

[0059] CDR3-VL3: QQYYIYPYT.

[0060] It should be noted that in other embodiments, the amino acid sequences of the variable region, heavy chain variable region, or complementarity-determining region of the HER2 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 complementarity-determining region.

[0061] In an alternative embodiment, the antibody detection kit for the HER2 biological agent is a quantitative detection kit or a qualitative detection kit.

[0062] In an alternative embodiment, the antibody detection kit for the HER2 biological agent is a 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;

[0063] In an alternative embodiment, the fluorescence immunochromatographic detection kit is a time-resolved fluorescence immunochromatographic detection kit.

[0064] The present invention also provides a method for preparing an antibody detection kit for HER2 biological agents, which includes: immobilizing the full-length HER2 biological agent on a solid-phase carrier as an antibody capture device, and binding or immobilizing a functional fragment without a label on a cellulose membrane or labeling the functional fragment with a label as an antibody detection device.

[0065] In an alternative embodiment, when the solid-phase carrier is selected from a microplate, before immobilizing the full-length HER2 biological agent on the microplate, it further includes coating streptavidin on the microplate, and then incubating the full-length HER2 biological agent modified with biotin with the microplate coated with streptavidin.

[0066] The present invention has the following beneficial effects:

[0067] The present invention uses the HER2 biological agent as a capture antigen. Due to the large spatial contact surface, it is easier to capture the anti-antibody in the sample, and has the advantage of high capture efficiency. By using the double-antigen sandwich method to detect the anti-antibody, a large number of irrelevant antibody interferences can be avoided to a great extent, the detection accuracy can be improved, and the occurrence of false positives can be avoided.

[0068] The antigen-antibody complex formed after capture itself has a large steric hindrance. The inventors found that the variable region, heavy-chain variable region or complementary determining region of the HER2 biological agent with a small molecular weight has the advantage of small steric hindrance and is more easily bound by the anti-antibody Fab. Using it as an antibody detection device can improve the binding efficiency between the antigen-antibody complex and the detection antibody element, improve the detection sensitivity, and avoid the occurrence of false negatives. To achieve the purpose of companion diagnosis of HER2 biological agent drugs. Description of the Drawings

[0069] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the embodiments will be briefly introduced below. 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, other related drawings can be obtained based on these drawings without creative efforts.

[0070] Figure 1 is the device diagram of the time-resolved fluorescence immunochromatographic test strip for anti-trastuzumab antibody of the present invention;

[0071] Figure 2 is the detection principle diagram of the enzyme-linked immunosorbent assay kit for anti-pertuzumab antibody of the present invention;

[0072] Figure 3 is the detection principle diagram of the chemiluminescence assay kit for anti-trastuzumab antibody of the present invention;

[0073] Figure 4It is the calibration curve regression graph of the time-resolved fluorescence immunochromatography kit for anti-trastuzumab antibody of the present invention;

[0074] Figure 5 It is the calibration curve regression graph of the enzyme-linked immunosorbent assay kit for anti-patritumab antibody of the present invention;

[0075] Figure 6 It is the calibration curve regression graph of the chemiluminescence assay kit for anti-trastuzumab antibody of the present invention;

[0076] Reference numerals: 1 - sample pad; 2 - conjugate pad; 3 - nitrocellulose membrane; 4 - test line; 5 - quality control line; 6 - absorbent membrane; 7 - backing bottom plate. Detailed implementation manners

[0077] 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. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can all be obtained as conventional products through commercial purchase.

[0078] The features and performance of the present invention will be further described in detail below in conjunction with the embodiments.

[0079] The working principle of the kit provided by the present invention is as follows: 1) Use a solid-phase carrier fixed with a HER2 biological agent to capture the anti-antibody in the sample to be tested. Since the molecular spatial interaction surface of the HER2 biological agent is large, it 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 HER2 biological agent as the detection antigen. Since the molecular weight 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.

[0080] Example 1

[0081] This example provides a time-resolved fluorescence immunochromatographic quantitative detection kit for anti-trastuzumab antibody and its preparation method.

[0082] Refer to Figure 1As shown, on the backing substrate 7, there are a nitrocellulose membrane 3, a sample pad 1, a conjugate pad 2, and an absorbent membrane 6. 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 trastuzumab labeled with time-resolved fluorescence microspheres and goat anti-chicken IgY antibody 2 labeled with time-resolved fluorescence microspheres. 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 trastuzumab, and the quality control line 5 (C line) is coated with chicken IgY antibody.

[0083] The variable region (Fv) of trastuzumab contains the trastuzumab heavy chain variable region and the trastuzumab light chain variable region connected by a disulfide bond. The sequence of the trastuzumab heavy chain variable region is as follows: SEQ ID NO.1:

[0084] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS;

[0085] The sequence of the trastuzumab light chain variable region is as follows: SEQ ID NO.2:

[0086] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ HYTTPPTFGQGTKVEIKRTV.

[0087] The preparation method is as follows:

[0088] 1) Labeling of time-resolved fluorescence microspheres

[0089] Take 500 μL (300 nm) of time-resolved fluorescence microspheres (1% stock solution), 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 seconds. Add 100 μg of labeled antibody (ustekinumab 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.

[0090] 2) Treatment of the sample pad and the conjugate pad

[0091] The polyester film is pre-blocked by soaking it in a buffer solution containing a surfactant (formulation: 20 mM pH 8.0 BB, containing 2% BSA, 0.5% S9, and 2% trehalose), dried overnight at 37 °C, and a conjugate pad is prepared; the prepared conjugate pad is taken, and a gold-labeled HM3035 gold spraying and membrane scribing instrument is used to mix trastuzumab labeled with time-resolved fluorescent microspheres and goat anti-chicken IgY antibody, and then sprayed onto the pre-treated conjugate pad with a width of 1 cm at 3 μL / cm, and dried at 37 °C for 3 h to obtain a specific conjugate pad.

[0092] The glass cellulose membrane is pre-blocked by soaking it in a buffer solution containing a surfactant (formulation: 100 mM pH 7.4 PB, containing 2% BSA, 0.5% S9), dried overnight at 37 °C, and cut into 30 * 1.5 cm to obtain a sample pad.

[0093] 3) Coating of nitrocellulose membrane (NC membrane)

[0094] The variable region of trastuzumab is synthesized by the amino acid synthesis method. It has been verified that coating an enzyme-linked immunosorbent assay plate with this synthetic polypeptide can react with anti-trastuzumab antibody.

[0095] The NC membrane is attached to the designated position of the backing bottom plate. The heavy chain variable region of trastuzumab is diluted to 1.5 mg / mL with 50 mM pH 7.4 phosphate buffer for preparing the T line; the chicken IgY antibody is diluted to 0.5 mg / mL with 50 mM pH 7.4 phosphate buffer for preparing the C line; according to the liquid scribing volume of 1 μL / cm, the above two diluted antibodies are evenly scribed onto the NC membrane through a gold-labeled HM3035 gold spraying and membrane scribing instrument to prepare the T line and the C line; the scribed NC membrane is placed in a drying oven at 37 °C and dried overnight.

[0096] 4) Assembly

[0097] The conjugate pad obtained in step 2) is laminated at one end of the nitrocellulose membrane obtained in step 3), and the absorbent membrane is fixed and laminated at the other end of the nitrocellulose membrane. Finally, the sample pad obtained in step 2) is laminated at the other end of the conjugate pad, cut with a membrane cutting instrument at a width of 4 mm per strip, and loaded into a chromatographic strip housing to obtain the finished product.

[0098] 5) Detection

[0099] The serum is diluted 10 times, 90 μL is taken and added to the sample well, and after reacting for 10 min, it is placed in a fluorescence reader for detection. By comparing with the values of the standard product or reference product, the content of anti-antibody in the sample to be detected is judged.

[0100] In this embodiment, 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; according to needs, 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.

[0101] Example 2

[0102] This embodiment provides a preparation method for an anti-patuzumab antibody enzyme-linked immunosorbent assay kit. The detection principle diagram is referred to Figure 2 as shown.

[0103] The kit includes an enzyme-labeled plate, biotinylated patuzumab, an anti-patuzumab antibody calibrator, an anti-patuzumab antibody quality control product, an enzyme conjugate, a washing solution, and a termination solution. The enzyme conjugate is patuzumab complementarity-determining regions (CDRs) labeled with horseradish peroxidase (HRP). The enzyme-labeled plate is coated with streptavidin.

[0104] The complementarity-determining regions (CDRs) of patuzumab include CDR1-VH1, CDR2 –VH2, CDR3–VH3, CDR1-VL1, CDR2 –VL2, and CDR3–VL3 connected by a linker peptide:

[0105] CDR1-VH1: DYTMD;

[0106] CDR2-VH2: DVNPNSGGSIYNQRFKG;

[0107] CDR3-VH3: NLGPSFYFDY;

[0108] CDR1-VL1: KASQDVSIGVA;

[0109] CDR2-VL2: SASYRYT;

[0110] CDR3-VL3: QQYYIYPYT.

[0111] The linker peptide is selected from GGGGSGGGGSGGGGS.

[0112] The preparation method is as follows:

[0113] 1) Biotinylation of patuzumab

[0114] Take 1 mg of pertuzumab and place it in a 5 mL centrifuge tube. Add 50 μL of 10 mg / mL activated biotin (Sulfo-NHS-LC-Biotin), react at room temperature for 1 h, take out the reaction solution, and dialyze it overnight with 0.02 M PBS. Take out the biotinylated pertuzumab, add 0.03% Proclin300 for preservation, and store it at 4 °C for later use.

[0115] 2) HRP-labeled complementarity-determining regions (CDRs) of pertuzumab

[0116] Synthesize the complementarity-determining regions (CDRs) of pertuzumab by amino acid synthesis method. It has been verified that the synthetic polypeptide can react with anti-pertuzumab antibody.

[0117] 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), 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 complementarity-determining regions (CDRs) of pertuzumab 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), mix well, and dialyze overnight with 0.05 M CB. Take out the labeled enzyme-linked antibody, add an equal volume of glycerol, and store it at -20 °C for later use.

[0118] 3) Coating the enzyme-linked immunosorbent assay (ELISA) plate

[0119] Dilute streptavidin with CB buffer to 1 μg / mL, 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 h, tap off the blocking solution, and dry it at 37 °C for later use.

[0120] 4) Coating biotinylated pertuzumab

[0121] Dilute biotinylated pertuzumab with neutral phosphate buffer to 1 μg / mL, 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 h, tap off the blocking solution, and dry it at 37 °C for later use.

[0122] 5) Dilute the serum sample to be tested 100 times, add 100 μL per well to the microplate, incubate at 37 °C for 2 h, and wash the plate 4 times.

[0123] 6) Add HRP-labeled complementarity-determining regions (CDRs) of pertuzumab, incubate for 1 h, and wash the plate 4 times

[0124] 7) Add the substrate TMB of HRP enzyme to the microplate, incubate for 15 minutes, add the termination solution HCL solution to terminate the color reaction, detect the OD value at a wavelength of 450 nm, and compare it with the OD value of the standard product or reference product to judge the content of the anti-antibody in the sample to be tested.

[0125] Example 3

[0126] This example provides a chemiluminescent immunoassay kit for trastuzumab antibody and its preparation method.

[0127] The chemiluminescent immunoassay kit for anti-trastuzumab antibody includes: biotinylated trastuzumab, anti-trastuzumab antibody calibrator, anti-trastuzumab antibody quality control product, acridinium ester-labeled trastuzumab heavy chain variable region (Hv), magnetic particle reagent, excitation solution and washing solution.

[0128] The sequence of the trastuzumab heavy chain variable region is shown in Example 1.

[0129] The preparation method is as follows:

[0130] 1) Biotinylation of trastuzumab

[0131] Take 1 mg of trastuzumab and place it in a 5 mL centrifuge tube. Add 50 μL of 10 mg / mL activated biotin (Sulfo-NHS-LC-Bintin), react at room temperature for 1 h, take out the reaction solution, and dialyze it overnight with 0.02 M PBS. Take out the biotinylated trastuzumab antibody, add 0.03% Proclin300 for preservation, and store it at 4 °C for later use.

[0132] 2) Acridinium ester labeling of trastuzumab heavy chain variable region (Hv)

[0133] Synthesize the trastuzumab heavy chain variable region (Hv) by amino acid synthesis method. It has been verified that the enzyme-labeled plate coated with this synthetic polypeptide can react with the anti-trastuzumab antibody.

[0134] Take 2 mg of trastuzumab heavy chain variable region (Hv), 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.

[0135] 3) Detection steps

[0136] This example uses acridinium ester chemiluminescent immunoassay technology - combined with biotin-avidin magnetic particle separation technology to achieve anti-antibody detection. Its detection principle is as Figure 3:Mix the sample (diluted 20 times), streptavidin magnetic particles (1), and biotinylated trastuzumab (2) to obtain a streptavidin magnetic bead-biotinylated trastuzumab-sample complex (3). After washing, add acridinium ester-labeled heavy chain variable region of trastuzumab (Hv) for reaction to form a streptavidin magnetic bead-biotinylated trastuzumab-sample-acridinium ester-labeled heavy chain variable region of trastuzumab (Hv) complex. Add the luminescence excitation solution, measure the luminescence intensity, compare with the values of the standard product or reference product, and judge the content of the anti-antibody in the sample to be tested.

[0137] Experimental Example 1

[0138] This experimental example performs a quantitative detection on the kit of Example 1.

[0139] 1.1 Standard curve preparation

[0140] 1.1.1 Take a set of anti-trastuzumab antibody calibration products, and the specific values are shown in Table 1.

[0141] Table 1

[0142]

[0143] 1.1.2 Detection method

[0144] Take 90 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.

[0145] 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.

[0146] Table 2

[0147]

[0148] 1.1.3 Standard curve preparation

[0149] According to the above detection results, with the concentration value as the X-axis and the T / C value as the Y-axis, perform linear regression to obtain the linear equation: y = 0.0024x + 0.042, R2 = 0.9987. The curve is shown in Figure 4 .

[0150] 1.2 Precision test:

[0151] 1.2.1 Take 10 prepared chromatography strips and configure a working calibration product of anti-trastuzumab antibody with a concentration of (50 ng / mL);

[0152] 1.2.2 Take 90 μL of the calibration product and directly add it to the sample loading hole of the reagent strip;

[0153] After the chromatography of the calibrator to be calibrated for 10 minutes, it was detected with a time-resolved fluorescence quantitative analyzer. The results of 10 test strips were measured according to the above linear equation. The results are shown in Table 3, indicating that the time-resolved fluorescence immunoassay kit for anti-trastuzumab antibody of this method has good precision.

[0154] Table 3

[0155]

[0156] 1.3 Compared with other methods:

[0157] Using trastuzumab-labeled microspheres and trastuzumab-coated NC membrane as Method 2; using trastuzumab fab-labeled microspheres and trastuzumab 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.

[0158] Table 4

[0159]

[0160] Experimental Example 2

[0161] This experimental example quantitatively detected the kit of Example 2.

[0162] 2.1 Detection method

[0163] 2.1.1 Take a set of anti-patuzumab antibody calibrators, and the specific values are shown in Table 5

[0164] Table 5

[0165]

[0166] 2.1.2 Detection steps

[0167] 1) Add 100 μL of the calibrator and the quality control product to each well of the microplate, incubate at 37 °C for 2 hours, and wash the plate 4 times.

[0168] 2) Add HRP-labeled complementarity-determining region of patuzumab, incubate for 1 h, and wash the plate 4 times.

[0169] 3) Add the substrate TMB to the microplate, incubate for 15 min, add the stop solution to terminate the color 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 6.

[0170] Table 6

[0171]

[0172] 2.1.3 Standard curve

[0173] According to the above test results, with the OD value on the X-axis and the concentration value on the Y-axis, curve fitting was performed to obtain the curve equation: y = 0.0053x + 0.1398, R² = 0.9961. The curve is shown in Figure 5 .

[0174] 2.2 Precision test:

[0175] Based on the OD values of the precision quality control products detected, the calculated result values were obtained using the curve equation. The results are shown in Table 7, indicating that the precision of the anti-pertuzumab antibody ELISA kit of this method is good.

[0176] Table 7

[0177]

[0178] 2.3 Compared with other methods:

[0179] Using pertuzumab labeled with HRP and biotinylated pertuzumab-coated microtiter plates as Method 2; using pertuzumab fab labeled with HRP and pertuzumab fab-coated microtiter plates 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 in Table 8.

[0180] Table 8

[0181]

[0182] Experimental Example 3

[0183] This experimental example quantitatively detected the kit of Example 3.

[0184] 3.1 Detection method

[0185] 3.1.1 Take a set of anti-trastuzumab antibody calibration products, and the specific values are shown in Table 9

[0186] Table 9

[0187] Number C0 C1 C2 C3 C4 C5 C6 Concentration (ng / mL) 0 5 10 50 100 200 500

[0188] 2.1.2 Detection steps

[0189] 1) Respectively take 20 μL of calibration product, 20 μL of streptavidin magnetic particles, and 50 μL of biotinylated trastuzumab and add them to the microplate for mixing. Incubate at 37 °C for 15 min and wash 3 times.

[0190] 2) Add acridinium ester-labeled trastuzumab heavy chain variable region (Hv), incubate at 37 °C for 15 min, and wash 3 times.

[0191] 3) Add the luminescence excitation solution and measure the luminescence intensity. The specific results are shown in Table 10.

[0192] Table 10

[0193] Number C0 C1 C2 C3 C4 C5 C6 Mean (luminescence value) 1533 12461 43457 95688 143866 253866 671950

[0194] 3.1.3 Standard Curve

[0195] Based on the above test results, with the concentration value on the X-axis and the luminescence value on the Y-axis, linear regression was performed to obtain the linear equation: y = 1304.4x + 13507, R² = 0.9963. The curve is shown in Figure 6 .

[0196] 3.2 Precision Test:

[0197] Take 20 μL of precision quality control product, 20 μL of streptavidin magnetic particles, and 50 μL of biotinylated trastuzumab, add them to the microplate and mix. Incubate at 37 °C for 15 min and wash 3 times; add acridinium ester-labeled trastuzumab 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 chemiluminescent immunoassay kit for anti-trastuzumab antibody of this method has good precision. The specific results are shown in Table 11.

[0198] Table 11

[0199] Number 1 2 3 4 5 6 7 8 9 10 Mean SD CV Concentration ng / mL 48.2 47.5 50.3 49.8 49.7 50.4 51.5 48.7 52.2 50.6 49.9 1.45 2.90%

[0200] 3.3 Compared with Other Methods:

[0201] Using trastuzumab labeled with acridinium ester and trastuzumab labeled with biotin as Method 2; using trastuzumab fab labeled with acridinium ester and trastuzumab fab labeled with biotin as Method 3. For the three methods, except for the different antibodies labeled with acridinium ester and the antibodies labeled with biotin, other conditions are the same. The three results are compared as shown in Table 12.

[0202] Table 12

[0203] This example Method 2 Method 3 Detection limit < 5 ng / ml < 10 ng / ml < 20 ng / ml Detection range 5 - 500 ng / ml 10 - 200 ng / ml 20 - 500 ng / ml Linear R2 0.9963 0.9744 0.9801 Precision 2.90% 15.36% 14.10% Non-specific background interference Low High Low

[0204] In summary, the detection kit provided by the present invention has the advantages of high capture efficiency, high accuracy, and high sensitivity, and can effectively avoid the occurrence of false positives and false negatives. It has good application prospects.

[0205] 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 HER2 Biological Agent and Preparation Method <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 120 <212> PRT <213> Artificial Sequence <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 110 <212> PRT <213> Artificial Sequence <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg 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 Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 <210> 3 <211> 119 <212> PRT <213> Artificial Sequence <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg 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 Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 4 <211> 110 <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 Ala Ser Gln Asp Val Ser Ile Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 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 HER2 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 cellulose membrane or a functional fragment with a detectable label; and the full length of a HER2 biological agent is coated on the solid-phase carrier, and the full length of the HER2 biological agent is the full length of Trastuzumab or Pertuzumab; correspondingly, the functional fragment is the variable region, heavy-chain variable region of Trastuzumab or the complementarity-determining region of Pertuzumab; 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 solid-phase carrier is selected from fluorescent microspheres, latex microspheres, resin microspheres, magnetic microspheres, colloidal gold particles or quantum dots; the antibody detection device is a cellulose membrane fixed with the functional fragment without a label; and the full length of a HER2 biological agent is coated on the solid-phase carrier, and the full length of the HER2 biological agent is the full length of Trastuzumab or Pertuzumab; correspondingly, the functional fragment is the variable region, heavy-chain variable region of Trastuzumab or the complementarity-determining region of Pertuzumab; 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, CDR1-VL1, CDR2–VL2 and CDR3–VL3 connected by a disulfide bond or a linker peptide.

2. The antibody detection kit for HER2 biological agents according to claim 1, characterized in that, The HER2 biological agent coated on the solid-phase carrier is modified with avidin or biotin.

3. The antibody detection kit for HER2 biological agents according to claim 1, wherein The fluorescent microspheres are selected from time-resolved fluorescent microspheres or fluorescent microspheres containing fluorescein, and the magnetic microspheres are selected from magnetic beads.

4. The antibody detection kit for HER2 biological agents according to claim 1 or 2, characterized in that, The fluorescent dyes are 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, and protein-based dyes and their derivatives; The enzymes that catalyze a substrate to develop color are selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 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 and 18 F; The chemiluminescent reagents are selected from luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxane and its derivatives, rosamide and its derivatives, or peroxyoxalate and its derivatives; The nanoparticle-based labels are selected from nanoparticles and colloids; The colloids are selected from colloidal metals, disperse dyes, dye-labeled microspheres and latexes; the colloidal metals are selected from colloidal gold, colloidal silver, colloidal carbon or colloidal selenium; The nanoparticles are selected from organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles or rare earth complex nanoparticles.

5. The antibody detection kit for HER2 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 respectively; The cellulose membrane is selected from cellulose acetate membranes or nitrocellulose membranes.

6. The antibody detection kit for HER2 biological agents according to claim 1 or 2, characterized in that The heavy chain variable region and the light chain variable region of trastuzumab are shown in SEQ ID NO. 1-2 respectively, and the heavy chain variable region and the light chain variable region of pertuzumab are shown in SEQ ID NO. 3-4 respectively; The variable region (Fv) of the trastuzumab and pertuzumab comprises a heavy chain variable region and a light chain variable region linked by a disulfide bond or a linker peptide, and its complementary determining regions include CDR1-VH1, CDR2–VH2, CDR3–VH3, CDR1-VL1, CDR2–VL2 and CDR3–VL3 linked by a disulfide bond or a linker peptide.

7. The antibody detection kit for HER2 biological agents according to claim 1 or 2, characterized in that, The antibody detection kit for the HER2 biological agent is a quantitative detection kit or a qualitative detection kit.

8. The antibody detection kit for HER2 biological agents according to claim 1 or 2, characterized in that The antibody detection kit for the HER2 biological agent is a 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 HER2 biological agent according to any one of claims 1-8, characterized in that, It includes: Fixing the HER2 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 HER2 biological agents according to claim 9, characterized in that, When the solid phase carrier is selected from microtiter plates, before fixing the full length of the HER2 biological agent on the microtiter plate, it further includes coating streptavidin on the microtiter plate, and then incubating the HER2 biological agent modified with biotin with the microtiter plate coated with streptavidin.

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