Application of Highly Homogeneous Monomeric Streptavidin Tetramer in Immunochemiluminescence Platform

Abstract

The present invention provides an application of a highly homogeneous monovalent streptavidin tetramer in the field of detection. Specifically, the present invention provides a detection system, and the detection system includes a multi-component complex having a structure shown in Formula I or Formula II, (A-B)-(C-D m ) n (Formula I) Each component in the formula is as described in the specification. The detection system provided by the present invention can improve the stability of the detection reagent and ensure the sensitivity and accuracy of the detection.

Description

Technical Field

[0001] The present invention relates to applications in the field of in vitro detection technology and other detection fields, and particularly relates to an application of a highly homogeneous monomeric streptavidin tetramer in improving the correlation, stability and repeatability in the field of immuno-chemiluminescence platform detection. Background Art

[0002] Streptavidin (hereinafter referred to as SA) is an extracellular secretion of Streptomyces avidinii, a protein with similar biological characteristics to avidin. Its molecular weight and ability to bind biotin are similar to those of avidin in egg white, with an isoelectric point of 6.0 and much lower non-specific binding than avidin; SA is a tetrameric protein with a size of 66KDa. One molecule of streptavidin can specifically bind to four molecules of biotin with extremely strong affinity, and the dissociation constant of the streptavidin-biotin complex is in the order of 10mol / L.

[0003] In the current in vitro diagnostic industry (IVD) and biological research, the tetramer of streptavidin is mainly applied to enzyme-linked immunosorbent assay and combined with biotin for biological research. When developing an immuno-chemiluminescence platform detection kit in the in vitro diagnostic industry, chemiluminescent markers are used.

[0004] However, in current biological research and diagnostic applications, tetravalent streptavidin still has some significant defects. First, in the components of the immuno-chemiluminescence kits developed in the market, one of the components is an antibody directly labeled with a chemiluminescent reagent as a component of the kit. When studying the functionality of the kit later, the correlation performance of some projects of the kit fails to meet the recognized standards in the market and the standards developed using the same kit, and this poor performance directly hinders the development of the kit; second, the application of tetravalent streptavidin in the chemiluminescence platform and the application of direct antibody labeling with acridinium ester often suffer from poor repeatability performance. Third, the developers of the kit try to label the antibody with biotin and label the tetravalent streptavidin with a chemiluminescent reagent, and then the mixture of the two is combined to form a component of the kit. When this kit is tested and stability tested later, the signal is unstable and decreases continuously in the short term. Later research found that there is a drawback in this labeling method. The mixture of biotin-labeled antibody and chemiluminescent reagent-labeled tetravalent streptavidin is prone to aggregation to form aggregates and then precipitate within a certain period of time. This instability also affects the research and development of the kit. The kits developed using the above methods still have unsatisfactory effects, poor stability performance of some projects, poor correlation performance of some projects, and poor repeatability performance.

[0005] Therefore, there is an urgent need in the art to develop an improved method for in vitro diagnostic detection or biological research using streptavidin, which can significantly improve the stability performance, correlation performance, and repeatability performance of the detection system. SUMMARY OF THE INVENTION

[0006] The object of the present invention is to provide an improved method for in vitro diagnostic detection or biological research using streptavidin, which can significantly improve the stability performance, correlation performance, and repeatability performance of the detection system.

[0007] In the first aspect of the present invention, a detection system is provided, and the detection system includes:

[0008] (a) A multi-component complex having a structure shown in Formula I,

[0009] (A - B)-(C - D m ) n (Formula I)

[0010] Each element in the formula includes:

[0011] A is a second binding protein that can specifically bind to a target analyte;

[0012] B is biotin;

[0013] C is a monovalent streptavidin tetramer;

[0014] D is a chemiluminescent label;

[0015] “-” is a bond or a linking group;

[0016] m is 0.9 - 6, preferably 0.9 - 2;

[0017] n is 0.8 - 4, preferably 1 - 2;

[0018] (b) A first binding protein crosslinked to a solid-phase carrier Z0, and the first binding protein can specifically bind to a target analyte;

[0019] (c) Optionally, a target analyte;

[0020] Wherein, the first binding protein and the second binding protein can respectively bind to the target analyte, and there is no competition between them.

[0021] In another preferred embodiment, the chemiluminescent label is selected from the group consisting of acridinium esters.

[0022] In the second aspect of the present invention, a detection system is provided, and the detection system includes:

[0023] (a) A multi-component complex having a structure as shown in Formula II,

[0024] (A-B)(C-Z0) p (Formula II)

[0025] Each component in the formula includes:

[0026] A is a first binding protein that can specifically bind to a target analyte;

[0027] B is biotin;

[0028] C is a monomeric streptavidin tetramer;

[0029] Z0 is a solid-phase carrier;

[0030] “-” is a bond or a linking group;

[0031] And, p is from 1 to 200, preferably from 1 to 10, more preferably from 2 to 4;

[0032] p is the molar ratio of antibody-biotin to magnetic beads of monomeric SA molecules

[0033] (b) A second binding protein crosslinked to a chemiluminescent label D and capable of specifically binding to a target analyte;

[0034] (c) Optionally, a target analyte;

[0035] Wherein, the first binding protein and the second binding protein can respectively bind to the target analyte, and there is no competition between them.

[0036] In another preferred embodiment, the chemiluminescent label is acridinium ester.

[0037] In another preferred embodiment, the material of the solid-phase carrier is selected from the group consisting of: metal, glass, colloid, plastic, or a combination thereof.

[0038] In another preferred embodiment, the material of the solid-phase carrier includes: homopolymer, copolymer, or a combination thereof.

[0039] In another preferred embodiment, the material of the solid-phase carrier is selected from the group consisting of: polystyrene, polyethylene, polypropylene, or a combination thereof.

[0040] In another preferred embodiment, the material of the solid-phase carrier is selected from the group consisting of: microspheres, microtiter plates, strips, test tubes, or a combination thereof.

[0041] In another preferred embodiment, the Z0 is a microsphere (bead), particle, or magnetic bead.

[0042] In another preferred example, in the monovalent streptavidin tetramer, it contains wild-type streptavidin monomers and mutant streptavidin monomers.

[0043] In another preferred example, the mutant streptavidin monomer does not have the function of binding biotin.

[0044] In another preferred example, in the monovalent streptavidin tetramer, the molar ratio of the wild-type streptavidin monomer to the mutant streptavidin monomer is 1:3.

[0045] In another preferred example, the monovalent streptavidin tetramer may include a tag for protein purification.

[0046] In another preferred example, the sequence of the tag is located at the N-terminus, C-terminus or middle of the wild-type streptavidin monomer and / or the mutant streptavidin monomer.

[0047] In another preferred example, the tag includes: His tag, GST tag, Trx tag, MBP tag, HA tag, c-Myc tag, Flag tag, or a combination thereof.

[0048] In another preferred example, the second binding protein is selected from the group consisting of: antigen, antibody, ligand, receptor, or a combination thereof.

[0049] In another preferred example, the concentration of the multi-component complex is 0.01 to 1 mg / ml, preferably 0.02 to 0.8 mg / ml, more preferably 0.02 to 0.5 mg / ml; and / or

[0050] In the detection system, the concentration of Z0 varies greatly due to different detection platforms, and is 1×10 4 to 5.0×10 8 per mL, preferably 1×10 7 to 5×10 8 per mL, more preferably 5×10 7 to 1×10 8 per mL.

[0051] In another preferred example, the first binding protein is selected from the group consisting of: antigen, antibody, ligand, receptor, or a combination thereof.

[0052] In another preferred example, the target analyte includes: antigen, antibody, ligand, receptor, small molecule, or a combination thereof.

[0053] In another preferred embodiment, when the multi-component complex has the structure shown in formula (I), the concentration of the multi-component complex is 0.1-1 mg / mL, preferably 0.2-0.8 mg / mL, more preferably 0.2-0.5 mg / mL; the concentration of the multi-component complex in claim 2 is 3.5×10 8- 5.6×10 8 per mL, preferably 4.0×10 8- 5.6×10 8 per mL, more preferably 4.8×10 8- 5.5×10 8 per mL

[0054] In another preferred embodiment, the target analyte is selected from the group consisting of: carbohydrate antigen 199 (CA199), neuron-specific enolase (NSE), carbohydrate antigen 724 (CA724).

[0055] In a third aspect of the present invention, there is provided a use of the detection system as described in the first or second aspect of the present invention for detecting whether a sample contains a target analyte.

[0056] In another preferred embodiment, the sample is an ex vivo sample or an in vitro sample.

[0057] In another preferred embodiment, the sample is derived from whole blood, preferably serum.

[0058] In another preferred embodiment, when the sample contains the target analyte, the light signal emitted by the chemiluminescent label can be detected from the solid-phase carrier separated from the detection system.

[0059] In another preferred embodiment, when the sample does not contain the target analyte, the light signal emitted by the chemiluminescent label cannot be detected from the solid-phase carrier separated from the detection system.

[0060] In a fourth aspect of the present invention, there is provided a kit, characterized in that the kit includes: a container and raw material reagents located in the container for forming the detection system as described in claim 1, wherein the raw material reagents do not include the target analyte in the sample to be tested.

[0061] In another preferred embodiment, the kit includes:

[0062] (a) a first container and the second binding protein A in the detection system as described in the first aspect of the present invention located in the first container;

[0063] (b) a second container and biotin B in the detection system as described in the first aspect of the present invention located in the second container;

[0064] (c) A third container and the monomeric streptavidin tetramer C in the detection system as described in the first aspect of the present invention located in the third container;

[0065] (d) A fourth container and the chemiluminescent label D in the detection system as described in the first aspect of the present invention located in the fourth container;

[0066] (e) A fifth container and the first binding protein in the detection system as described in the first aspect of the present invention located in the fifth container;

[0067] (f) A sixth container and the solid-phase carrier in the detection system as described in the first aspect of the present invention located in the sixth container.

[0068] In another preferred example, the first container, the second container, the third container, and the fourth container may be the same or different containers.

[0069] In another preferred example, the fifth container and the sixth container may be the same or different containers.

[0070] In another preferred example, the first container, the second container, the third container, the fourth container, the fifth container, and the sixth container may be the same or different containers.

[0071] In another preferred example, the kit further includes:

[0072] (g) A calibrator in a seventh container and located in the seventh container;

[0073] (h) An eighth container and a sample diluent located in the eighth container;

[0074] (i) A ninth container and a washing solution located in the ninth container; and / or

[0075] (j) A tenth container and a buffer for the reaction system located in the tenth container.

[0076] In another preferred example, the kit includes:

[0077] (a) A first container, and the first binding protein A in the detection system as described in the second aspect of the present invention located in the first container;

[0078] (b) A second container, and the biotin B in the detection system as described in the second aspect of the present invention located in the second container;

[0079] (c) A third container, and a solid-phase carrier Z0 located in the third container, the surface of the Z0 being modified with monomeric streptavidin tetramer C;

[0080] (d) A fourth container, and a chemiluminescent label D in the detection system as described in the second aspect of the present invention located in the fourth container;

[0081] (e) A fifth container and a second binding protein in the detection system as described in the second aspect of the present invention located in the fifth container.

[0082] In another preferred example, the kit is used for analyzing target analytes selected from the group consisting of carbohydrate antigen 199 (CA199), neuron-specific enolase (NSE), and carbohydrate antigen 724 (CA724).

[0083] In another preferred example, the first binding protein and the second binding protein are each independently selected from the group consisting of a CA19-9 labeled antibody and CA199.

[0084] The fifth aspect of the present invention provides a method for detecting the presence of a target analyte in a sample, the method comprising the following steps:

[0085] (I) Providing the detection system as described in the first or second aspect of the present invention, which does not contain the target analyte, mixing the detection system without the target analyte with the sample or its dilution to form a mixture, and allowing it to react;

[0086] (II) Separating the solid-phase carrier from the mixture obtained in step (I); and

[0087] (III) Detecting whether the solid-phase carrier carries a luminescence signal from the chemiluminescent label in the detection system according to claim 1.

[0088] In another preferred example, in step (I), it may include sub-steps:

[0089] (i) Crosslinking the first binding protein in the detection system as described in the first aspect of the present invention with the solid-phase carrier to form a system i containing the first binding protein crosslinked to the solid-phase carrier;

[0090] (ii) Mixing the system i obtained in step (i) with the sample or its dilution to form a system ii;

[0091] (iii) Providing a multi-component complex in the detection system as described in the first aspect of the present invention and mixing it with the system ii obtained in step (ii) to form the mixture described in step (I).

[0092] In another preferred example, in step (I), it may include sub-steps:

[0093] (i) Mixing the multi-component complex in the detection system as described in the second aspect of the present invention with the sample or its dilution to form a system i;

[0094] (ii) Provide the second binding protein in the detection system as described in the first aspect of the present invention, and mix it with the system i obtained in step (i) to form the mixed solution described in step (I).

[0095] In another preferred embodiment, in step (II), it further includes the step of washing the separated solid-phase carrier, and this step can be repeated 1-5 times, preferably 3 times.

[0096] In another preferred embodiment, in step (III), the detection includes chemiluminescence detection method.

[0097] In another preferred embodiment, the chemiluminescence detection method is performed using a chemiluminescence detector.

[0098] In another preferred embodiment, the detection is carried out in vitro or ex vivo.

[0099] In another preferred embodiment, the detection is non-diagnostic and non-therapeutic.

[0100] The present invention is applied to immunoassay, with broad generality and a wide application scope.

[0101] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Brief Description of the Drawings

[0102] Figure 1 Shows the electrophoresis pattern after the preparation and purification of the tetramer of highly homogeneous monovalent streptavidin in the present invention. The numbers 1-4 in the figure respectively represent the monovalent SA, divalent SA, trivalent SA, and tetravalent SA after boiling; the numbers 5-8 in the figure represent the inactive SA tetramer, monovalent SA tetramer, divalent SA tetramer, and tetravalent SA tetramer without boiling.

[0103] Figure 2 Shows the test data of the first hospital sample in Jiangsu, Zhejiang and Shanghai regarding the correlation performance of the chemiluminescence platform CA199 project in Example 1; among them, Figure 2 A is the correlation analysis diagram of the antibody directly labeled with acridinium ester for testing the first hospital sample, Figure 2 B is the correlation analysis diagram of the mixture of tetravalent SA labeled with acridinium ester and antibody biotin for testing the first hospital sample, Figure 2 C is the correlation analysis diagram of the mixture of monovalent SA labeled with acridinium ester and antibody biotin for testing the first hospital sample.

[0104] Figure 3Shows the test data of samples from the second hospital in Jiangsu, Zhejiang and Shanghai regarding the test correlation performance of the CA199 project on the chemiluminescence platform; among them, Figure 3 Analysis chart of the correlation of the second hospital samples tested with A antibody directly labeled acridinium ester; Figure 3 B is the analysis chart of the correlation of the second hospital samples tested with the mixture of tetravalent SA labeled acridinium ester and antibody biotin; Figure 3 C is the analysis chart of the correlation of the second hospital samples tested with the mixture of monovalent SA labeled acridinium ester and antibody biotin.

[0105] Figure 4 Shows the test data of samples from the third hospital in Jiangsu, Zhejiang and Shanghai regarding the test correlation performance of the CA199 project on the chemiluminescence platform; among them, Figure 4 A is the analysis chart of the correlation of the third hospital samples tested with antibody directly labeled acridinium ester; Figure 4 B is the analysis chart of the correlation of the third hospital samples tested with the mixture of tetravalent SA labeled acridinium ester and antibody biotin; Figure 4 C is the analysis chart of the correlation of the third hospital samples tested with the mixture of monovalent SA labeled acridinium ester and antibody biotin.

[0106] Figure 5 Shows the test data of samples from the fourth hospital in Jiangsu, Zhejiang and Shanghai regarding the test correlation performance of the CA199 project on the chemiluminescence platform; among them, Figure 5 A is the analysis chart of the correlation of the fourth hospital samples tested with antibody directly labeled acridinium ester; Figure 5 B is the analysis chart of the correlation of the fourth hospital samples tested with the mixture of tetravalent SA labeled acridinium ester and antibody biotin; Figure 5 B is the analysis chart of the correlation of the fourth hospital samples tested with the mixture of monovalent SA labeled acridinium ester and antibody biotin.

[0107] Figure 6 Shows the test data analysis charts of samples from four hospitals; Figure 6 A is the correlation data analysis chart of the production intermediate (Ab - AE); Figure 6 B is the correlation data analysis chart of tetravalent Ab - Biotin - SA - AE; Figure 6 C is the correlation data analysis chart of monovalent Ab - Biotin - M - SA - AE.

[0108] Figure 7 Shows the signal data change trend charts during the stability test of the application of monovalent streptavidin on the CA199 project of the chemiluminescence platform and during the stability test on the NSE project of the chemiluminescence platform. Among them, Figure 7 A is the test data change trend chart of antibody direct labeling acridinium ester; Figure 7Figure showing the trend of test data for the mixture of tetravalent streptavidin-labeled acridinium ester and antibody biotin; Figure 7 Figure showing the trend of test data for the mixture of monovalent streptavidin-labeled acridinium ester and antibody biotin.

[0109] Figure 8 Figure showing the trend of signal data during the stability test of monovalent streptavidin applied to the CA199 project on the chemiluminescence platform and the stability test on the NSE project of chemiluminescence. Among them, Figure 8 Figure A shows the trend of test data for direct-labeled acridinium ester of antibody; Figure 8 Figure B shows the trend of test data for the mixture of tetravalent streptavidin-labeled acridinium ester and antibody biotin; Figure 8 Figure C shows the trend of test data for the mixture of monovalent streptavidin-labeled acridinium ester and antibody biotin.

[0110] Figure 9 Figure showing the data analysis of the correlation performance of monovalent streptavidin-coated magnetic beads applied to the CA724 project test on the chemiluminescence platform. Figure 9 Figure A represents the data analysis of the correlation of tetravalent Ab - Biotin - SA - Beads; Figure 9 Figure B represents the data analysis of the correlation of monovalent Ab - Biotin - M - SA - Beads. Detailed implementation method

[0111] Through extensive and in - depth research and a large number of screenings, the inventor has developed for the first time a method for detecting target analytes on the chemiluminescence platform using highly homogeneous monovalent streptavidin tetramers.

[0112] The inventor has discovered for the first time that this patent utilizes the binding characteristics of monovalent streptavidin. Monovalent streptavidin can react with chemiluminescent markers and then specifically bind to biotin - labeled antibodies, forming a component of the chemiluminescent kit. It can effectively control the size of the mixture of tetravalent streptavidin - labeled chemiluminescent markers and antibody - labeled biotin, reduce the precipitation phenomenon caused by aggregation, and solve the problem of unstable measured values. Moreover, the method of mixing monovalent streptavidin - labeled acridinium ester and antibody biotin can solve the problem of poor correlation performance in some chemiluminescence projects, and can effectively improve the repeatability performance of the project without affecting the performance of other project kits.

[0113] The experimental results show that when monovalent streptavidin is mixed with chemiluminescent markers and applied to chemiluminescent immunoassay, it can effectively control the dosage of the chemiluminescent markers bound thereto, strictly control the dosage of the mixture of monovalent streptavidin-labeled luminescent markers and antibody-biotin mixture, greatly reduce the possibility of aggregation, delay or reduce precipitation, improve the utilization efficiency of raw materials, avoid waste of raw materials, and at the same time can effectively increase the stability of streptavidin and fluorescent protein aggregates and the stability of the antibody-biotin binding mixture, improve the reagent stability, and ensure the effectiveness of the test results; moreover, it can effectively improve the correlation performance of some project products, and at the same time has great superiority in improving the repeatability performance of project products.

[0114] Term

[0115] In this article, tetravalent streptavidin, abbreviated as SA, is a tetrameric protein that makes up streptavidin and all of its components are active, that is, one molecule of tetravalent streptavidin can highly specifically bind four molecules of biotin. The commercially available tetravalent streptavidin is used in this patent.

[0116] Monovalent streptavidin (monovalent-SA) is abbreviated as M-SA in this article. When only one of the tetrameric proteins that make up streptavidin is active and the other three are inactive, one molecule of monovalent streptavidin is formed. M-SA can highly specifically bind one molecule of biotin. Currently, monovalent streptavidin is applied in aspects such as neuroglin testing and studying cell surface protein transport. The monovalent streptavidin used in the present invention is prepared by referring to the method provided in the patent of US Patent Application US20070099248.

[0117] The term "chemiluminescent marker" refers to a class of substances that can bind to antigens, antibodies, and some proteins and can be used for immunoassays in immunoassay techniques. Commonly used chemiluminescent markers include luminol, isoluminol and its derivatives, adamantane-1,2-dioxetane and its derivatives, acridinium esters, and acridinamide compounds. In a preferred embodiment of the present invention, commercially available acridinium ester (abbreviated as AE) is used as the chemiluminescent marker. In the present invention, the commonly used types are acridinium esters and acridinamide compounds. A particularly preferred acridinium ester is NSP-DMAE-NHS, and the structure is as follows:

[0118]

[0119] Generally, the ester group of the acridinium ester binds to the amino group on streptavidin, thereby forming a covalent bond.

[0120] The terms "antibody" and "Ab" are used interchangeably and refer to a class of immunoglobulins secreted by plasma cells that can specifically bind to antigens and can be conjugated with substances such as biotin and labels using conventional labeling methods for use in fields such as immunoassays. In this patent, a preferred class of antibodies are self-made antibodies.

[0121] In this text, the terms "biotin" and "Biotin" are used interchangeably and refer to small molecule substances that can bind highly specifically to streptavidin and can be used to label antibodies for use in fields such as immunoassays. The biotin used in this patent is commercially available.

[0122] In this text, the terms "antibody-labeled chemiluminescent label" and "Ab-AE" are used interchangeably and refer to a mixture formed by directly labeling an antibody with acridinium ester using conventional labeling methods for use in immunoassays and is referred to as the secondary antibody in the experiments of this patent.

[0123] In this text, the terms "antibody-labeled biotin" and "Ab-Biotin" are used interchangeably and refer to a mixture formed by directly labeling an antibody with biotin using conventional labeling methods, which then binds to a mixture of streptavidin and acridinium ester for use in immunoassays.

[0124] In this text, the term "streptavidin" refers to monomeric and tetrameric streptavidin.

[0125] As used herein, the term "streptavidin-labeled chemiluminescent label" refers to a mixture formed by labeling streptavidin with acridinium ester using conventional labeling methods. Among them, the labeling of tetrameric streptavidin with acridinium ester is abbreviated as SA-AE; the labeling of monomeric streptavidin with acridinium ester, the English name is monovalent-SA-AE, abbreviated as M-SA-AE, which then binds to a mixture of antibody and biotin (this mixture is referred to as the secondary antibody in this text) for use in immunoassays.

[0126] Magnetic beads, also known as magnetic microspheres (Beads), are mainly composed of materials such as cellulose, gelatin, polystyrene, polyacrylic acid (ester) and its copolymers, polyamides, and polyaniline, etc. Their surfaces can be endowed with functional groups (such as -OH, -COOH, -CHO, -NH 2 etc.) through methods such as copolymerization and surface modification. The magnetic beads used in this patent are those with -COOH functional groups. The magnetic beads can be in various diameter ranges. In a preferred embodiment, the diameter of the magnetic beads used is 1.5 um - 3 um.

[0127] The term "streptavidin-coated magnetic beads" refers to magnetic microspheres coated with streptavidin by a conventional coating method (wherein tetravalent streptavidin-coated magnetic beads are abbreviated as SA-Beads; monovalent streptavidin-coated magnetic beads are abbreviated as M-SA-Beads), and then combined with a mixture of antibody and biotin (this mixture is referred to as the primary antibody in this article), and is applied to immunoassay.

[0128] Monovalent streptavidin (monovalent SA)

[0129] In the existing technology, a kind of monovalent streptavidin (abbreviated as monovalent SA) has been developed. In monovalent SA, only one monomer of the tetrameric protein that makes up streptavidin has activity, and the other three monomers are activity-deficient bodies, that is, one molecule of monovalent streptavidin can specifically bind one molecule of biotin with high specificity.

[0130] In an embodiment of the present invention, the amino acid sequence of the wild-type monomer of streptavidin is shown in SEQ ID NO:1.

[0131] MAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPA TDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAW KSTLVGHDTFTKVKPSAAS(SEQ ID NO:1)

[0132] In an embodiment of the present invention, the amino acid sequence of the mutant monomer of streptavidin is shown in SEQ ID NO:2.

[0133] MAEAGITGTWYAQLGDTFIVTAGADGALTGTYEAAVGNAESRYVLTGRYDSAP ATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANA WKSTLVGHDTFTKVKPSAAS(SEQ ID NO:2)

[0134] Currently, monovalent streptavidin is applied to glial fibrillary acidic protein testing, studying cell surface protein trafficking, etc. In addition, some reports mention that mutant streptavidin or mutant streptavidin subunits can bind to labels, such as radioactive labels, fluorescent labels, luminescent labels, chromophore labels, and substances and enzymes that produce detectable substrates, such as horseradish peroxidase, luciferase or alkaline phosphatase. Moreover, boron-10 labeling for magnetic resonance imaging, PET probes and neutron capture therapy can also bind to mutant streptavidin or mutant streptavidin subunits.

[0135] The monomeric streptavidin used in the present invention is prepared by referring to the method provided in the patent "Monomeric Streptavidin Complex" of US Patent Application US20070099248.

[0136] In the present invention, the mixing range of monomeric streptavidin and acridinium ester in different proportions refers to the molecular ratio of S-SA to AE. Preferably, M-SA:AE = 1:0.9 to 1:6, and the preferred molecular ratio is 1:2.

[0137] The theoretical proportion range of the binding of monomeric streptavidin acridinium ester to antibody-biotin refers to the molecular ratio of the two, Ab-Biotin: (M-SA-AE) = 1:0.8 to 1:n. To ensure the practicality of the test signal and considering the saving of raw material usage, the proportion range in the experiments of the present invention is Ab-Biotin: (M-SA-AE) = 1:0.8 to 1:4, and the preferred molecular ratio is 1:2.

[0138] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0139] General method

[0140] (1) Preparation of highly homogeneous monomeric streptavidin tetramer

[0141] 1.1 Clone the wild-type streptavidin and the active-site deleted mutant into the ECROI and XHOI restriction enzyme sites in the pET21a vector to obtain two recombinant plasmids;

[0142] 1.2 Transform the recombinant plasmids into E. coli competent cells BL21(DE3). When the cell culture reaches OD600 = 0.7, add the inducer IPTG to a final concentration of 1 mM for induction. After inducing at 37 °C for 5 hours, collect the cells and store them at -20 °C;

[0143] 1.3 Resuspend the frozen cells in the lysis buffer at a ratio of buffer: cell weight of 5 ml / g and perform ultrasonic disruption in an ice bath. After centrifugation, take the precipitate and resuspend it in the washing buffer, stir and wash at 16 °C for 30 min, centrifuge and discard the supernatant, and repeat the washing until the purity of the inclusion body reaches more than 90%.

[0144] 1.4 Thoroughly mix the wild-type and mutant streptavidin inclusion bodies dissolved in urea at a mass ratio of 1:3, and perform renaturation of the inclusion bodies using the rapid dilution renaturation method. The renatured sample is stirred at 4 °C for more than 4 hours, and the precipitate is discarded by centrifugation;

[0145] 1.5 Resuspend the obtained streptavidin tetramer with Ni-A buffer, centrifuge to discard the precipitate, and purify the obtained supernatant by affinity purification using NTA medium, and elute it using an imidazole concentration gradient.

[0146] The streptavidin wild type, the streptavidin activity-deficient mutant, and various buffers are the same as those described in the cited patent.

[0147] (2) Process of labeling antibody Ab with acridinium ester (AE)

[0148] The labeling antibody directly labels acridinium ester (AE). The reaction concentration of acridinium ester is fixed and unified this time. The labeling antibody and acridinium ester (AE) are added in a volume ratio of 10:1 and then purified by an ion column. The obtained product is the secondary antibody in the experiment.

[0149] (3) Process of labeling streptavidin (monovalent and commercially available tetravalent) with acridinium ester (AE)

[0150] Acridinium ester (AE) directly labels commercially available tetravalent streptavidin and self-made monovalent streptavidin respectively, and then passes through an ion column for purification. Acridinium ester (AE) is added to commercially available tetravalent streptavidin and self-made monovalent streptavidin in a volume ratio of 10:1. The obtained labeled products are denoted as A1 and A2.

[0151] (4) Process of mixing the products labeled with streptavidin (tetravalent and monovalent) and acridinium ester with the biotin-labeled secondary antibody

[0152] 4.1 Mixing labeled product A1 with secondary antibody-biotin

[0153] Add 0.5 - 1 times the molecular weight of commercially available tetravalent streptavidin and the product labeled with acridinium ester to the biotin-labeled secondary antibody for mixing. At this time, one molecule of streptavidin can bind four biotins. The combined complexes are denoted as B1-1 respectively, which are the secondary antibodies in the experiment.

[0154] 4.2 Mixing labeled product A2 with secondary antibody-biotin

[0155] Add 1 - 2 times the molecular weight of monovalent streptavidin and the product labeled with acridinium ester to the biotin-labeled secondary antibody for mixing. At this time, because streptavidin is monovalent, it can only bind one biotin. The combined complexes are denoted as B2-1 or B2-2 respectively, which are both the secondary antibodies in the experiment.

[0156] (5) The mixture of acridinium ester-monovalent streptavidin-biotin-secondary antibody is used to improve the performance of some items in chemiluminescence platform immunoassay for in vitro diagnosis

[0157] 5.1 Labeling streptavidin with acridinium ester

[0158] Prepare acridinium ester solutions with different molar ratios (dissolved in dimethyl sulfoxide (DMSO)) to label streptavidin (both tetravalent and monovalent) simultaneously, perform light shielding treatment, then purify through an ion column to remove excess acridinium ester, and store for later use.

[0159] 5.2 Biotin-labeled antibody

[0160] Take a certain mass of antibody that specifically recognizes antigen (Ag) after dialysis purification, add a dimethyl sulfoxide (DMSO) solution of biotin, perform a light shielding reaction, dialyze to remove unreacted biotin, and store for later use.

[0161] 5.3 Binding of acridinium ester-labeled streptavidin to biotin-labeled antibody

[0162] Take acridinium ester-streptavidin with different molar ratios (monovalent and tetravalent), add it to the solution of the second antibody labeled with biotin for binding, and the resulting product is acridinium ester-streptavidin (monovalent and tetravalent)-biotin-antibody, which is the secondary antibody and denoted as Solution B.

[0163] 5.4 Coating of capture antibody on microspheres

[0164] Covalently crosslink the capture antibody against a certain antigen (Ag) with carboxylated polystyrene microspheres (Beads) to obtain a coupling mixture of Beads and the capture antibody, which is Ab-Beads and denoted as the primary antibody, Solution C.

[0165] 5.5 Preparation of antigen calibrator solution

[0166] Prepare a standard solution with a certain concentration using the standard of antigen (Ag).

[0167] 5.6 In vitro diagnostic immune reaction and signal detection

[0168] Perform tests according to the Abbott i2000-121 chemiluminescence fully automatic instrument. Automatically test the corresponding calibrator (sample), Solution B, and Solution C in sequence according to the conventional 18 + 4 min mode on this machine. There are a control group and an experimental group. In the control group, there are two types: a commercially available mixture of tetravalent streptavidin-labeled acridinium ester (AE) and antibody-labeled biotin, and a mixture of antibody directly labeled with acridinium ester (AE); the experimental group is an example of a self-made mixture of monovalent streptavidin-labeled acridinium ester (AE) and antibody-labeled biotin.

[0169] 5.7 Raw materials and instruments in Examples 1, 2, and 3

[0170] Monovalent streptavidin (Lot No. 20191211, Concentration: 1.3 mg / mL); Carbohydrate antigen CA19-9 antibody (Lot No. 20191218, Concentration: 2.89 mg / mL); Carbohydrate antigen CA724 antibody (Lot No. 20190405, Concentration: 3.12 mg / mL); The company's carbohydrate antigen CA9-9 calibrator (20A001), carbohydrate antigen CA724 antigen calibrator (20A002), neuron-specific enolase NSE calibrator (20A001), etc. are self-made raw materials.

[0171] Tetravalent streptavidin (Roche, Specification: 100 mg / mL), neuron-specific enolase NSE antibody, biotin (Thermo, Specification: 50 mg), commercially available acridinium ester (yellow powder), ion column packing materials, etc. are commercially available raw materials.

[0172] Reagents such as reagent GLY, DMSO reagent, self-made PBS (10 mmol / L) solution, 0.5 mol / L boric acid buffer, 10 mMol / L boric acid buffer, and 10 mMol / L boric acid buffer containing 0.5 mol / L NaCl are commercially available and self-prepared solutions.

[0173] Ultrasonic cell disruptor (SCIENTZ-II17), Hitachi high-speed refrigerated centrifuge (CR21G), Abbott i2000-121 chemiluminescence automatic analyzer, etc. are the main instruments used in this patent.

[0174] Example 1

[0175] Self-made monovalent streptavidin was used in the immunoassay detection of carbohydrate antigen CA19-9

[0176] 1. Monovalent streptavidin

[0177] 1.1 Raw materials

[0178] pET21a vector;

[0179] E.coli competent cell BL21(DE3);

[0180] Inducer IPTG;

[0181] 10 mM PBS solution, 10 mM PBS + 5% glycerol solution, and 10 mM PBS + 8M Urea solution;

[0182] 20 mM imidazole, 70 mM imidazole, 100 mM imidazole, 125 mM imidazole, and 250 mM imidazole solutions;

[0183] SDS-PAGE (15%) electrophoresis materials.

[0184] 1.2 Operating Steps

[0185] 01. Conduct experimental operations according to the basic principle of preparing the above-mentioned monovalent streptavidin;

[0186] 02. See Figure 1 in the two schematic diagrams: Use SDS-PAGE electrophoresis to identify the streptavidin tetramer obtained after elution (boiled and unboiled). Take two sets of samples from the separated elution peaks respectively. One set is heated and boiled, and the other set is not heated and boiled. Both use SDS-PAGE (15%) to analyze the results. Because the streptavidin tetramer is relatively stable and remains in the tetramer form during SDS-PAGE without heating, as shown in the pictures corresponding to the numbers 5, 6, 7, and 8 in Figure 1 ; while heating at 100°C for 5 minutes can destroy the streptavidin tetramer and form monomers, as shown in the pictures corresponding to the numbers 1, 2, 3, and 4 in Figure 1 . Therefore, comprehensive analysis of the results of the two sets of SDS-PAGE can reveal the composition of each elution peak. Finally, it is confirmed that the elution peak contains a monovalent streptavidin tetramer (W1M3) composed of 1 wild-type monomer (w) and 3 mutant monomers (M) with high purity at an imidazole concentration of 70 mM;

[0187] 2. The monovalent streptavidin is used in chemiluminescent immunoassay detection

[0188] Here, taking the detection of carbohydrate antigen CA19-9 by Abbott i2000-121 fully automatic chemiluminescent instrument as an example, it is illustrated that this patent can improve the effectiveness of the correlation performance of this project.

[0189] According to the elaboration of the basic principle, the self-made monovalent streptavidin is used in immunoassay. Under the condition of ensuring the normal other characteristics of the project, it should be able to achieve the signal of the commercially available tetravalent streptavidin and should also be consistent with or higher than the signal of the conventional antibody direct label. Moreover, the correlation performance should be significantly improved mainly in some projects.

[0190] 2.1 Test the correlation of this project by directly labeling the antibody corresponding to carbohydrate antigen CA19-9 with acridinium ester and its labeled biotin and the mixture of (tetravalent / monovalent) streptavidin-acridinium ester

[0191] Experimental scheme: Set three groups of tests. Group A: Direct labeling method, that is, the antibody is directly labeled with acridinium ester (AE); Group B: Indirect labeling method 1, that is, tetravalent streptavidin is labeled with acridinium ester (AE); Group C: Indirect labeling method 2, that is, the self-made monovalent streptavidin is labeled with acridinium ester (AE); Then Group B and Group C are respectively mixed with antibody biotin as a component in the test. The molecular ratio of antibody biotin to Group B mixture is 1:0.86; the molecular ratio of antibody biotin to Group C mixture is 1:2.

[0192] For the correlation performance test, first, each group separately tested the carbohydrate antigen CA19-9 calibrators CAL1 - CAL6 (with concentrations of 0 U / mL, 30 U / mL, 100 U / mL, 250 U / mL, 600 U / mL, and 1200 U / mL respectively), showing the signals at the same concentration for each group; then, a total of 144 clinical samples from four hospitals in Jiangsu, Zhejiang, and Shanghai were tested. The correlation coefficients of each hospital were statistically analyzed in three groups, and the total correlation coefficient of the four hospitals was statistically analyzed in three groups.

[0193] 2.1.1 Raw materials

[0194] The raw materials for the carbohydrate antigen CA199 project described in 5.7 above

[0195] 2.1.2 Operations

[0196] 01. Take appropriate amounts of commercially available tetravalent streptavidin and self-made monovalent streptavidin, and dilute them to a concentration of 2 mg / mL with 0.5 mol / L boric acid buffer solution;

[0197] 02. Take an appropriate amount of acridinium ester (AE), dissolve it with DMSO solution, and then react with (tetravalent / monovalent) streptavidin at a mass ratio of acridinium ester:streptavidin = 1:12.5 - 1:150 (preferably 1:25 - 1:100). The reaction conditions are 2℃ - 8℃, and the reaction is terminated with GLY. After the reaction, it is purified by passing through an ion column to remove the unreacted acridinium ester;

[0198] 03. Take an appropriate amount of the carbohydrate antigen CA19-9 labeled antibody (concentration: 2.0 mg / mL) and 0.92 mg of biotin, and prepare them to a concentration of 0.5 mg / mL with DMSO, and react them at a certain mass ratio (m / m). The reaction conditions are 2 - 8℃; the reaction is terminated with GLY;

[0199] 04. Take a part of the reacted CA199-Biotin and SA-AE and mix them at a conventional molecular ratio (CA199-Biotin:SA-AE = 1:0.86). Take another part of the reacted CA199-Biotin and M-SA-AE and mix them at a molecular ratio of 1:2 (CA199-Biotin:M-SA-AE = 1:2), and then dilute and store them with the chemiluminescence conjugate diluent;

[0200] 05. Test operation:

[0201] According to the requirements of the correlation performance test, the directly labeled acridinium ester intermediate product CA199-AE of group A, the labeled CA19-9-Biotin-SA-AE of group B, and the CA19-9-Biotin-M-SA-AE and magnetic microspheres of group C were diluted to the corresponding working concentrations with chemiluminescence luminescence conjugate diluent and chemiluminescence solid conjugate diluent respectively;

[0202] 06. According to the above test operations, the results are shown in the following table:

[0203] Table 1: Calibration luminescence signal values of calibrators CAL1-CAL6

[0204]

[0205]

[0206] Table 2: Sample data of the correlation performance test of directly labeled acridinium ester of antibody and (tetravalent and monovalent) streptavidin-labeled acridinium ester and antibody-biotin mixture

[0207]

[0208]

[0209]

[0210]

[0211]

[0212] Table 3: Statistical table of correlation coefficients corresponding to each hospital for the correlation performance study of directly labeled acridinium ester of antibody and (tetravalent and monovalent) streptavidin-labeled acridinium ester and antibody-biotin mixture

[0213]

[0214] It can be seen from Table 1 that by comparing group A and group B, for the test of CA199 item with the monovalent streptavidin-labeled acridinium ester and antibody-biotin mixture (Ab-Biotin-M-SA-AE) in group C, there is a better growth ratio of luminescence signal among calibrators with different concentrations, that is, the concentration differentiation is obvious, and it is better than the common group A and group B.

[0215] Through Table 2, Table 3 and Figures 2 to 6Data analysis shows that: for testing the correlation performance of the CA199 project, the correlation coefficients of each hospital in Group A are between 0.88 and 0.94; those in Group B are between 0.92 and 0.97; and those in Group C are between 0.96 and 0.99. Comparing Group A and Group B, the correlation coefficient of Group C is closer to 1, indicating better correlation performance. (In the linear range, the correlation coefficient r value should be infinitely close to 1, which is the standard requirement for the development of test kits.) This experimental result also confirms the content of the present invention, that is, the application of monomeric streptavidin in the chemiluminescence platform can improve the correlation performance of the project.

[0216] Example 2

[0217] The self-made monomeric streptavidin was used for immunoassay detection of carbohydrate antigen CA199 project and neuron-specific enolase project.

[0218] Here, taking the detection of carbohydrate antigen CA199 and neuron-specific enolase NSE by Abbott i2000-121 chemiluminescence automatic analyzer as an example, it is illustrated that the method of the present invention can improve the repeatability performance of the project.

[0219] According to the elaboration of the basic principle, the self-made monomeric streptavidin is used for immunoassay. Under the condition of ensuring the normal other characteristics of the project, it should be able to achieve the signal of commercially available tetravalent streptavidin and should also be the same as or higher than the signal directly labeled with conventional antibodies, and mainly should be able to improve the repeatability performance in the project.

[0220] 1. The antibodies corresponding to carbohydrate antigen CA199 and the antibodies corresponding to neuron-specific enolase are directly labeled with acridinium ester respectively, and the mixture of its labeled biotin and (tetravalent / monomeric) streptavidin-acridinium ester is used to test the repeatability of this project. The experimental scheme is the same as that in Example 1.

[0221] For the repeatability performance test, first, each group tests the carbohydrate antigen CA19-9 calibrators CAL1-CAL6 (concentrations are 0 U / mL, 30 U / mL, 100 U / mL, 250 U / mL, 600 U / mL, and 1200 U / mL respectively) and neuron-specific enolase NSE calibrators CAL1-CAL6 (concentrations are 0 ng / mL, 5 ng / mL, 25 ng / mL, 100 ng / mL, 200 ng / mL, and 370 ng / mL respectively), showing the signals of each item in each group at the same concentration; then test the self-made multi-tumor marker quality control product of our company (batch number: 19A002), and perform 10 replicate hole tests on the low-value quality control product and the high-value quality control product respectively, and calculate the coefficient of variation (CV) (for the two projects in this example, the coefficient of variation should not be greater than 10%, and the lower the CV value, the better the repeatability performance).

[0222] 1.1 Operation

[0223] The general experimental operations of the three test groups were the same as those in Example 1. According to the requirements of the repeatability test, the acridinium ester intermediates CA199-AE and NSE-AE directly labeled with antibodies in Group A of the project, CA19-9-Biotin-SA-AE and NSE-Biotin-SA-AE labeled in Group B of the project, and CA19-9-Biotin-M-SA-AE, NSE-Biotin-M-SA-AE in Group C of the project, as well as the corresponding CA199-magnetic microspheres and NSE-magnetic microspheres were diluted to the corresponding working concentrations with chemiluminescence luminescence conjugate diluent and chemiluminescence solid-phase conjugate diluent respectively;

[0224] According to the above test operations, the results are shown in the following table:

[0225] Table 4: Calibration luminescence signal values of the corresponding calibration products CAL1-CAL6 for the CA199 project

[0226]

[0227] Table 5: Data analysis table for the repeatability test of low-value and high-value quality control products corresponding to the CA199 project

[0228]

[0229] Table 6: Calibration luminescence signal values of the corresponding calibration products CAL1-CAL6 for the NSE project

[0230]

[0231]

[0232] Table 7: Data analysis table for the repeatability test of low-value and high-value quality control products corresponding to the NSE project

[0233]

[0234]

[0235] Table 8: Statistical summary table of repeatability test data for the CA199 project and the NSE project

[0236]

[0237]

[0238] It can be seen from Table 4 and Table 6 that: compared with Group A and Group B, Group C has a better growth ratio of luminescence signals among calibration products with different concentrations, that is, the concentration differentiation is obvious, and it is better than the common Group A and Group B.

[0239] Analysis of the data in Tables 5, 7, and 8 shows that for the repeatability performance test of the CA199 project, the CV values of the low-value and high-value quality control products in Group A are 4.83% and 4.40% respectively; the CV values in Group B are 6.78% and 6.87% respectively; the CV values in Group C are 3.44% and 2.17% respectively. The coefficients of variation of the three groups are all lower than 10%. However, when comparing Group A and Group B, the coefficient of variation of Group C is lower. For the repeatability performance test of the NSE project, the CV values of the low-value and high-value quality control products in Group A are 8.62% and 7.85% respectively; the CV values in Group B are 10.85% and 7.89% respectively; the CV values in Group C are 2.13% and 3.22% respectively. When comparing Group A and Group B, the coefficient of variation of Group C is the lowest. Considering these two projects together, it can be seen that the repeatability performance of Group C is better. The experimental results confirm that using the monovalent streptavidin of the present invention for detection on a chemiluminescence platform can significantly improve the repeatability performance of the project.

[0240] Example 3

[0241] The self-made monovalent streptavidin was used for immunoassay detection of the carbohydrate antigen CA199 project and the neuron-specific enolase project

[0242] Here, taking the detection of carbohydrate antigen CA199 and neuron-specific enolase NSE by the Abbott i2000-121 fully automated chemiluminescence instrument as an example, the effectiveness of this patent in improving the stability performance of the project is illustrated.

[0243] According to the elaboration of the basic principle, the self-made monovalent streptavidin is used for immunoassay. Under the condition of ensuring the normal other characteristics of the project, it should be able to achieve the signal of the commercially available tetravalent streptavidin and should also be the same as or higher than the signal of the conventional antibody direct label. Moreover, its stability performance is theoretically superior.

[0244] 1. The antibodies corresponding to the carbohydrate antigen CA199 and the antibodies corresponding to the neuron-specific enolase are directly labeled with acridinium ester, and the mixture of its labeled biotin and (tetravalent / monovalent) streptavidin-acridinium ester is used to test the stability of this project

[0245] Experimental scheme: Similar to the correlation performance test in Example 1, three groups of tests are set up, namely Group A, Group B, and Group C; then they are all mixed with antibody biotin in corresponding proportions as a component in the test.

[0246] Conventional stability performance test, using our company's commercially available carbohydrate antigen CA19-9 calibrators CAL1-CAL6 (concentrations are 0U / mL, 30U / mL, 100U / mL, 250U / mL, 600U / mL and 1200U / mL) and neuron-specific enolase NSE calibrators CAL1-CAL6 (concentrations are 0ng / mL, 5ng / mL, 25ng / mL, 100 ng / mL, 200ng / mL and 370ng / mL), showing the signal of each item and group at the same concentration and our company's self-made multi-tumor marker material control (batch number: 19A002), the stability test time is 1 day, 7 days, 14 days, 30 days and 60 days. During the test, the calibrators and quality controls were always stored at the same batch number, concentration and temperature.

[0247] 1.1 Operation

[0248] 01. The general experimental operation of the three test groups is the same as that of Example 1

[0249] 02 According to the requirements of the stability performance test, the antibody direct-labeled acridinium ester intermediates CA199-AE and NSE-AE of group A, the labeled CA19-9-Biotin-SA-AE and NSE-Biotin-SA-AE of group B, and the CA19-9-Biotin-M-SA-AE and NSE-Biotin-M-SA-AE of group C and the corresponding CA199-magnetic microspheres and NSE-magnetic microspheres were diluted to the corresponding working concentrations with chemiluminescent conjugate diluent and chemiluminescent solid conjugate diluent respectively; 06. According to the above test operation, the calibration luminescent signals of the six parallel samples of the calibrators CAL1-CAL6 corresponding to each stability test and the duplicate well test data of the corresponding quality control products were obtained. The results are shown in the following table:

[0250] Table 9: CA199 project corresponding stability test data

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258] Table 10: Stability test data corresponding to the NSE project

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265] As can be seen from Table 9 and Table 10 above: For the chemiluminescence CA199 project and the chemiluminescence NSE project, when comparing Group A, Group B, and Group C among different concentration calibrators, the luminescence signal has a good growth ratio, that is, the concentration discrimination is obvious, and it is better than the common Group A and Group B.

[0266] Combined with the Figure 7 and Figure 8 of the attached drawings, it can be known that the change trends of the experimental data of each group of each project are basically the same. During the test, the CAL1-CAL6 signals and the quality control product signals of the three groups change synchronously, and the decreasing ratio and the fluctuation trend are almost the same. Due to the large amount of test data, the CAL2 signal and the high-value quality control product in the calibrators of the three groups in each project are selected as representatives to statistically test the signal change situation. Selecting the CAL2 value is on the one hand to show the signal change in the low-concentration area, and on the other hand to distinguish it from the serum quality control product to make the experimental test more comprehensive; selecting the high-value quality control product is on the one hand to represent the measured value change at high concentration, and on the other hand because the serum quality control product is close to the serum sample and is distinguished from the calibrator measured value to make the experimental test representative. The summary is shown in Table 11 and Table 12.

[0267] Table 11: Summary table of stability test data of biotinylated streptavidin monomer applied in the chemiluminescence CA199 project

[0268]

[0269]

[0270] Table 12: Summary table of stability test data of biotinylated streptavidin monomer applied in the chemiluminescence NSE project

[0271]

[0272] It can be seen from Table 11 that for the chemiluminescent CA199 project, the percentage change of the stability test of Group A fluctuated between 0.5% and 5%, which is a normal level fluctuation; the percentage change of the stability test of Group B decreased by 9% to 41%, that is, the signal was very unstable; the percentage change of the stability test of Group C fluctuated between 0.09% and 3%, which is a normal test signal level fluctuation.

[0273] It can be seen from Table 12 that for the chemiluminescent NSE project, the percentage change of the stability test of Group A fluctuated between 0.5% and 5%, which is a normal level fluctuation; the percentage change of the stability test of Group B decreased by 9% to 44%, that is, the signal was very unstable; the percentage change of the stability test of Group C fluctuated between 0.2% and 2%, which is a normal test signal level fluctuation.

[0274] Combining the results of Example 1 and Example 2, the antibody directly labeled with acridinium ester in Group A and the monovalent streptavidin labeled with acridinium ester in Group C and the antibody biotin mixed to improve the project relevance performance and repeatability are superior, and the stability test of its application project is also superior. This test result confirms that the monovalent streptavidin in the invention of this patent binds to one molecule of biotin, will not bind to other biotins, will not aggregate into a large mixture, and no precipitation is formed, so it is applied on the chemiluminescence platform for project stability;

[0275] The stability test signal of the mixture of tetravalent streptavidin labeled acridinium ester and antibody biotin in group B gradually decreased. The calibration signal and quality control signal corresponding to the chemiluminescent CA199 project decreased by 40.61% and 37.61% respectively after 60 days; the calibration signal and quality control signal corresponding to the chemiluminescent NSE project decreased by 43.18% and 39.89% respectively in 60 days. The percentage change of the CAL2 calibration test is larger than that of the quality control product, because its concentration is lower, and the fluctuation of each test will be larger. Since the matrix of the high-value quality control product is close to the serum sample, if the corresponding sample is tested, there will be corresponding signal changes. This experimental result shows that tetravalent streptavidin can bind to four molecules of biotin. When labeling acridinium ester and antibody, a large polymer will be formed in a shorter period of 60 days, and precipitation is easily formed, so the signal will decrease by about 40% in a shorter time, confirming the content of the present invention.

[0276] Example 4

[0277] Homemade monovalent streptavidin was used for immunoassay detection of carbohydrate antigen CA724 project on chemiluminescent platform

[0278] Here, the Abbott i2000-121 chemiluminescence fully automatic instrument is used as an example to detect the carbohydrate antigen CA724 project to illustrate that a different application method of this patent can also improve the effectiveness of the project correlation performance.

[0279] According to the elaboration of the basic principle, streptavidin can be used as a label. Therefore, in this patent, commercially available tetravalent streptavidin and self-made monovalent streptavidin are both coated on magnetic beads, and then mixed with antibody biotin. This mixture can be used as the solid-phase conjugate component of the kit. Comparing the experimental principle and experimental scheme of Example 1, the experimental scheme of coating magnetic beads with monovalent streptavidin can also have the same effect on the chemiluminescence platform. For example, it can improve the correlation performance of some items. The self-made monovalent streptavidin is used in immunoassay. Under the condition of ensuring the normal performance of other characteristics of the item, it should be able to reach or be higher than the signal of commercially available tetravalent streptavidin, and theoretically it is also superior in its stability performance.

[0280] 1. Test the correlation performance of this item with the mixture of the coated antibody labeled with biotin corresponding to carbohydrate antigen CA724 and (tetravalent / monovalent) streptavidin-magnetic beads

[0281] 1.1 Raw materials: Magnetic beads (commercially available, product number: D beads, concentration 6*10 8 per mL), EDC reagent (Thermo, specification: 5 g), and NHS reagent (Thermo, specification: 5 g)

[0282] 0.1 mol / L Mes buffer and self-made PBS-TBN solution (10 mmol / L).

[0283] Samples from clinical hospitals

[0284] Other raw materials are the same as those in 2.1.1 of Example 1

[0285] 1.2 Operations

[0286] 01. Take appropriate amounts of commercially available tetravalent streptavidin and self-made monovalent streptavidin, and dissolve them in 10 mM PBS solution to a concentration of 1 mg / mL each.

[0287] 02. Take appropriate amounts of magnetic beads, wash them 3-4 times with 0.1 mol / L Mes buffer, take appropriate amounts of EDC and NHS reagents to activate the magnetic beads for 20-40 min, then wash them 3-4 times with 0.1 mol / L Mes buffer again. Take appropriate amounts of tetravalent streptavidin and monovalent streptavidin and react with the corresponding magnetic beads for 2-3 hours respectively, then wash the mixture 3-4 times with PBS-TBN solution, block it overnight with PBS-TBN solution, and finally store it in the chemiluminescence solid-phase diluent.

[0288] 03. Take an appropriate amount of carbohydrate antigen CA724 coating antibody (concentration: 1.0 mg / mL) and 1.33 mg of biotin, prepare them with DMSO to a concentration of 0.8 mg / mL, and react them at a certain mass ratio (m / m) under the reaction conditions of 2 - 8 °C; terminate the reaction with GLY;

[0289] 04. Take a part of the reacted CA199 - Biotin and SA - Beads (magnetic beads) and mix them in a conventional ratio, take another part of the reacted CA199 - Biotin and M - SA - Beads (magnetic beads) and mix them in a certain ratio, and then dilute and store them with a solid - phase conjugate diluent;

[0290] 05. Test operation: Take CAL1 - CAL6 in the commercially available carbohydrate antigen CA724 calibrator (batch number 20A001) of our company's chemiluminescence platform for testing and the samples from the clinical hospital to be tested for correlation performance; dilute CA19 - 9 - Biotin - SA - Beads, CA19 - 9 - Biotin - M - SA - Beads and the luminescence intermediate reagent to the corresponding working concentrations with a chemiluminescence solid - phase conjugate diluent and a chemiluminescence luminescence conjugate diluent respectively;

[0291] 06. According to the above - mentioned test operation, obtain the luminescence signals of the six parallel samples of the calibrator CAL1 - CAL6 and the correlation performance test data. The results are shown in the following table:

[0292] Table 13: Luminescence signal values of the calibrator CAL1 - CAL6

[0293]

[0294] Table 14: (Tetravalent and monovalent) streptavidin - coated magnetic beads and antibody - biotin mixture research correlation performance test sample data

[0295]

[0296] It can be seen from the data analysis in Table 13 that: compared with the tetravalent streptavidin - coated magnetic beads and antibody - biotin mixture (Ab - Biotin - SA - Beads), the monovalent streptavidin - coated magnetic beads and antibody - biotin mixture (Ab - Biotin - M - SA - Beads) have a better linear growth of the signal in the CA724 test item. Among different - concentration calibrators, the luminescence signals have a better growth ratio, that is, the concentration discrimination is obvious.

[0297] Through Table 14 and Figure 9It can be seen that the correlation coefficient of the CA19-9-Biotin-SA-Beads test is 0.87; the correlation coefficient of the CA19-9-Biotin-M-SA-Beads test is 0.93; the correlation coefficient of the CA19-9-Biotin-M-SA-Beads test is closer to 1, that is, the correlation performance is better. Moreover, it is found that the repeatability of the monovalent streptavidin-coated magnetic beads and antibody-biotin mixture (Ab-Biotin-M-SA-Beads) in testing the CA724 item can reach a CV value between 3% and 5%, and the stability can ensure that the percentage change in the luminescence signal value is within 2% within 30 days (data not shown).

[0298] It can be seen that streptavidin is applied to the chemiluminescence platform by different experimental methods. It has better performance in terms of correlation, repeatability and stability than traditional tetravalent streptavidin.

[0299] All documents mentioned in the present invention are cited herein as references, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. A detection system, characterized in that, the detection system comprises: (a) A multi-component complex having a structure shown in Formula I, (A - B)-(C - D m ) n (Formula I) each component in the formula comprises: A is a second binding protein that can specifically bind to a target analyte; B is biotin; C is a monomeric streptavidin tetramer; D is a chemiluminescent label; "-" is a bond or a linking group; m is 0.9 - 2; n is 1 - 2; (b) A first binding protein crosslinked to a solid-phase carrier Z0 and capable of specifically binding to a target analyte; (c) Optionally, a target analyte; wherein the first binding protein and the second binding protein can respectively bind to the target analyte, and there is no competition between them; the chemiluminescent label is selected from the group consisting of: acridinium ester; and the target analyte is selected from the group consisting of: carbohydrate antigen 199 (CA199), neuron-specific enolase (NSE).

2. A detection system, characterized in that, the detection system comprises: (a) A multi-component complex having a structure shown in Formula II, (A - B)(C - Z0) p (Formula II) each component in the formula comprises: A is a first binding protein that can specifically bind to a target analyte; B is biotin; C is a monomeric streptavidin tetramer; Z0 is a solid-phase carrier; "-" is a bond or a linking group; and p is 2 - 4; (b) A second binding protein crosslinked to a chemiluminescent label D and capable of specifically binding to a target analyte; (c) Optionally, a target analyte; wherein the first binding protein and the second binding protein can respectively bind to the target analyte, and there is no competition between them; the chemiluminescent label is acridinium ester.

3. The detection system according to claim 1 or 2, characterized in that, the concentration of the multi-component complex is 0.01 to 1 mg / ml.

4. The detection system according to claim 1 or 2, characterized in that, the concentration of the multi-component complex is 0.02 to 0.8 mg / ml.

5. The detection system according to claim 1 or 2, characterized in that, the concentration of the multi-component complex is 0.02 to 0.5 mg / ml.

6. The detection system according to claim 1 or 2, characterized in that, In the detection system, the concentration of Z0 is 1×10 4 to 5.0×10 8 cells / mL.

7. The detection system according to claim 1 or 2, characterized in that, In the detection system, the concentration of Z0 is 1×10 7 to 5×10 8 cells / mL.

8. The detection system according to claim 1 or 2, characterized in that, In the detection system, the concentration of Z0 is 5×10 7 to 1×10 8 cells / mL.

9. Use of a detection system according to claim 1 or 2, characterized in that, for detecting whether a target analyte is contained in a sample.

10. A kit, characterized in that, the kit comprises: a container and raw material reagents located in the container for forming the detection system according to claim 1 or 2, wherein the raw material reagents do not include the target analyte in the sample to be tested.

11. The kit according to claim 10, characterized in that, the kit comprises: (a) The first container and the second binding protein A in the detection system as described in claim 1 located in the first container; (b) The second container and biotin B in the detection system as described in claim 1 located in the second container; (c) The third container and the monomeric streptavidin tetramer C in the detection system as described in claim 1 located in the third container; (d) The fourth container and the chemiluminescent label D in the detection system as described in claim 1 located in the fourth container; (e) The fifth container and the first binding protein in the detection system as described in claim 1 located in the fifth container; (f) The sixth container and the solid-phase carrier in the detection system as described in claim 1 located in the sixth container.

12. The kit according to claim 10, wherein, the kit comprises: (a) The first container, and the first binding protein A in the detection system as described in claim 2 located in the first container; (b) The second container, and biotin B in the detection system as described in claim 2 located in the second container; (c) The third container, and the solid-phase carrier Z0 located in the third container, the surface of Z0 being modified with monomeric streptavidin tetramer C; (d) The fourth container, and the chemiluminescent label D in the detection system as described in claim 2 located in the fourth container; (e) The fifth container and the second binding protein in the detection system as described in claim 2 located in the fifth container.

13. The kit according to claim 11 or 12, wherein, the kit is used for analyzing target analytes selected from the group consisting of carbohydrate antigen 199 (CA199), neuron-specific enolase (NSE).

14. A method for detecting the presence of a target analyte in a sample, wherein, the method comprises the following steps: (I) Providing the detection system as described in claim 1 or 2, which does not contain the target analyte, mixing the detection system without the target analyte with the sample or its dilution to form a mixture, and allowing it to react; (II) Separating the solid-phase carrier from the mixture obtained in step (I); and (III) Detecting whether a luminescence signal from the chemiluminescent label in the detection system as described in claim 1 or 2 is carried on the solid-phase carrier.

Images