Application of a highly homogeneous monovalent streptavidin tetramer

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) a multi-component complex having a structure shown in Formula I, (A-B)-(C-D m ) n (Formula I) Each component in the formula includes: A is a second binding protein that can specifically bind to a target analyte; B is the biotin; C is a monovalent streptavidin tetramer; D is a fluorescent label; "-" is a bond or a linking group; and, m is a real number between 1 and 6, and n is a real number between 2 and 8; (b) a first binding protein crosslinked to a solid-phase carrier Z0, and the first binding protein can specifically bind to the target analyte; (c) an optional target analyte; the first binding protein and the second binding protein can respectively bind to the target analyte, and there is no competition between the two. 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 the field of biotechnology, and in particular to an application of a high-homogeneity monovalent streptavidin tetramer for improving stability in the detection field. Background Art

[0002] Streptavidin (SA) is an exosome of Streptomyces avidinii, a protein with similar biological properties to avidin. Its molecular weight and ability to bind biotin are similar to avidin in egg white, with an isoelectric point of 6.0 and a much lower nonspecific binding than avidin. SA is a tetrameric protein with a size of 66KDa. One molecule of streptavidin can bind to four molecules of biotin with high specificity, and the affinity between the two is extremely strong. The dissociation constant of the streptavidin-biotin complex is at the order of 10 mol / L.

[0003] In the current in vitro diagnostic industry (IVD) and biological research, the tetramer of streptavidin is mainly used in enzyme-linked immunosorbent assays and combined with biotin for biological research. When developing immunoassay kits in the in vitro diagnostic industry, markers are used.

[0004] However, in current biological research and diagnostic applications, tetravalent streptavidin still has some significant defects. First, among the components of the kit developed on the market, one of the components is an antibody-labeled biotin, which is combined with a mixture of tetravalent streptavidin-labeled fluorescent markers to form a kit component. During the later testing and stability testing of the kit composed of this method, the signal has been unstable and will decrease in the short term. Later studies found that this labeling method has a disadvantage. When the antibody-labeled biotin is combined with the mixture of tetravalent streptavidin-labeled fluorescent markers, it will easily aggregate to form large aggregates, and then precipitation will occur in a short time. This instability hinders the development of the kit. Second, when developing the kit, people use antibodies to directly label fluorescent markers to replace the above-mentioned methods. After testing the signal and stability experiments, it was found that although this method can reduce the generation of precipitation through adjustment, it is still easy to produce precipitation and instability still exists.

[0005] The effect of the kit developed using the above method is still not ideal, and precipitation is still easily formed.

[0006] Therefore, there is an urgent need in the art to develop an improved method for performing in vitro diagnostic testing or biological research using streptavidin that can significantly improve the stability of the detection system. Summary of the invention

[0007] The purpose of the present invention is to provide an improved method for in vitro diagnostic testing or biological research using streptavidin, which can significantly improve the stability of the detection system.

[0008] In a first aspect of the present invention, a detection system is provided, the detection system comprising:

[0009] (a) a multi-component complex having a structure as shown in Formula I,

[0010] (AB)-(CD m ) n (Formula I)

[0011] The components in the formula include:

[0012] A is a second binding protein, which can specifically bind to the target analyte;

[0013] B is biotin;

[0014] C is the monovalent streptavidin tetramer;

[0015] D is a fluorescent marker;

[0016] “-” is a bond or a connecting group;

[0017] And, m is a real number between 1 and 6;

[0018] And, n is a real number between 2 and 8;

[0019] (b) a first binding protein, wherein the first binding protein is cross-linked to the solid phase carrier Z0, and the first binding protein can specifically bind to the target analyte;

[0020] (c) optional target analyte;

[0021] The first binding protein and the second binding protein can bind to the target analyte respectively, and are not competitive with each other.

[0022] In another preferred embodiment, the fluorescent marker is phycoerythrin rPE.

[0023] In another preferred embodiment, the monovalent streptavidin tetramer comprises a streptavidin wild-type monomer and a streptavidin mutant monomer.

[0024] In another preferred embodiment, the streptavidin mutant monomer does not have the function of binding to biotin.

[0025] In another preferred embodiment, the amino acid sequence of the wild-type streptavidin monomer is shown in SEQ ID NO:1.

[0026] In another preferred embodiment, the streptavidin mutant monomer includes the following mutations based on the streptavidin wild-type monomer: N12A, S16D and S34A.

[0027] In another preferred embodiment, the amino acid sequence of the streptavidin mutant monomer is shown in SEQ ID NO:2.

[0028] In another preferred embodiment, in the monovalent streptavidin tetramer, the molar ratio of the streptavidin wild-type monomer to the streptavidin mutant monomer is 1:3.

[0029] In another preferred embodiment, the monovalent streptavidin tetramer may include a tag for protein purification.

[0030] In another preferred embodiment, the sequence of the tag is located at the N-terminus, C-terminus or the middle of the streptavidin wild-type monomer and / or the streptavidin mutant monomer.

[0031] In another preferred embodiment, the tag includes: His tag, GST tag, Trx tag, MBP tag, HA tag, c-Myc tag, Flag tag, or a combination thereof.

[0032] In another preferred embodiment, the second binding protein is selected from the following group: antigen, antibody, ligand, receptor or a combination thereof.

[0033] In another preferred embodiment, m is a positive integer between 1 and 6.

[0034] In another preferred embodiment, m is 1, 2, 3, 4, 5 or 6.

[0035] In another preferred embodiment, m is 2, 3 or 4.

[0036] In another preferred embodiment, n is a positive integer between 3 and 8.

[0037] In another preferred embodiment, n is 3, 4, 5, 6, 7 or 8.

[0038] In another preferred embodiment, n is 3 or 4.

[0039] In another preferred embodiment, the concentration of the multi-complex is 0.01 to 0.4 mg / ml, preferably 0.015 to 0.25 mg / ml, and more preferably 0.03 to 0.07 mg / ml.

[0040] In another preferred embodiment, the first binding protein is selected from the following group: antigen, antibody, ligand, receptor or a combination thereof.

[0041] In another preferred embodiment, the solid phase carrier material is selected from the following group: metal, glass, colloid, plastic or a combination thereof.

[0042] In another preferred embodiment, the solid phase carrier material includes: a homopolymer, a copolymer, or a combination thereof.

[0043] In another preferred embodiment, the solid phase carrier material is selected from the following group: polystyrene, polyethylene, polypropylene, or a combination thereof.

[0044] In another preferred embodiment, the solid phase carrier material is selected from the following group: microspheres, microplates, strips, test tubes, or a combination thereof.

[0045] In another preferred embodiment, the Z0 is a microsphere (bead), a particle (particle) or a magnetic bead.

[0046] In another preferred embodiment, in the detection system, the concentration of Z0 has a large deviation due to different detection platforms, which is 1×10 4 to 1.0×10 8 / mL, preferably 1×10 4 to 1.5×10 7 / mL, more preferably 2×10 4 to 1.5×10 7 Pieces / mL.

[0047] In another preferred embodiment, the target analyte includes: an antigen, an antibody, a ligand, a receptor, a small molecule, or a combination thereof.

[0048] In a second aspect of the present invention, there is provided a use of the detection system according to the first aspect of the present invention, for detecting whether a sample contains a target analyte.

[0049] In another preferred embodiment, the sample is an in vitro sample or an in vitro sample.

[0050] In another preferred embodiment, the sample is derived from whole blood, preferably serum.

[0051] In another preferred embodiment, when the sample contains the target analyte, the fluorescent signal emitted by the fluorescent marker can be detected from the solid phase carrier separated from the detection system.

[0052] In another preferred embodiment, when the sample does not contain the target analyte, the fluorescent signal emitted by the fluorescent marker cannot be detected from the solid phase carrier separated from the detection system.

[0053] In the third aspect of the present invention, a kit is provided, comprising: a container and raw material reagents located in the container for forming the detection system as described in the first aspect of the present invention, wherein the raw material reagents do not include the target analyte in the sample to be tested.

[0054] In another preferred embodiment, the kit comprises:

[0055] (a) a first container and a second binding protein A in the detection system according to the first aspect of the present invention located in the first container;

[0056] (b) a second container and biotin B in the detection system according to the first aspect of the present invention located in the second container;

[0057] (c) a third container and the monovalent streptavidin tetramer C in the detection system according to the first aspect of the present invention located in the third container;

[0058] (d) a fourth container and a fluorescent marker D in the detection system according to the first aspect of the present invention located in the fourth container;

[0059] (e) a fifth container and the first binding protein in the detection system according to the first aspect of the present invention located in the fifth container;

[0060] (f) a sixth container and a solid phase carrier in the detection system according to the first aspect of the present invention located in the sixth container.

[0061] In another preferred embodiment, the first container, the second container, the third container and the fourth container may be the same or different containers.

[0062] In another preferred example, the fifth container and the sixth container may be the same or different containers.

[0063] In another preferred embodiment, 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.

[0064] In another preferred embodiment, the kit further comprises:

[0065] (g) a calibrator in and located within a seventh container;

[0066] (h) an eighth container and a sample diluent located in the eighth container;

[0067] (i) a ninth container and a wash solution in the ninth container; and / or

[0068] (j) a tenth container and a buffer solution for a reaction system located in the tenth container.

[0069] In a fourth aspect of the present invention, a method for detecting whether a target analyte is present in a sample is provided, the method comprising the following steps:

[0070] (I) providing a detection system as described in the first aspect of the present invention, wherein the detection system does not contain a target analyte, mixing the detection system not containing the target analyte with a sample or a dilution thereof to form a mixed solution, and reacting the mixture;

[0071] (II) separating the solid phase carrier from the mixed solution obtained in step (I); and

[0072] (III) Detecting whether the solid phase carrier carries the fluorescent signal of the fluorescent marker in the detection system described in the first aspect of the present invention.

[0073] In another preferred embodiment, in step (I), the sub-steps may be included:

[0074] (i) cross-linking the first binding protein in the detection system according to the first aspect of the present invention with the solid phase carrier to form a system i containing the first binding protein cross-linked to the solid phase carrier;

[0075] (ii) mixing the system i obtained in step (i) with the sample or its dilution to form a system ii;

[0076] (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 mixed solution described in step (I).

[0077] In another preferred embodiment, step (II) further includes a step of washing the separated solid phase carrier, and this step can be repeated 1-5 times, preferably 3 times.

[0078] In another preferred embodiment, in step (III), the detection includes fluorescence detection.

[0079] In another preferred embodiment, the fluorescence detection method is performed using an enzyme reader or a fluorescence spectrophotometer.

[0080] In another preferred embodiment, the detection is performed in vitro or ex vivo.

[0081] In another preferred embodiment, the detection is non-diagnostic and non-therapeutic.

[0082] 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 embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 The electrophoresis diagram of the tetramer of highly homogeneous monovalent streptavidin prepared and purified in the present invention is shown.

[0084] Numbers 1-4 in the figure represent boiled monovalent SA, divalent SA, trivalent SA and tetravalent SA, respectively; numbers 5-8 in the figure represent unboiled inactive SA tetramers, monovalent SA tetramers, divalent SA tetramers and tetravalent SA tetramers.

[0085] Figure 2 A schematic diagram showing labeling of phycoerythrin (rPE) with monovalent streptavidin at 1:1, 1:2, 1:3 and 1:4 in one example of the present invention.

[0086] Among them, ①-④ are schematic diagrams of monovalent SA-labeled rPE molecular ratios of 1:1, 1:2, 1:3 and 1:4, respectively.

[0087] Figure 3 Schematic diagram showing the commercially available tetravalent streptavidin labeled phycoerythrin (rPE) with a molecular ratio of 1:1

[0088] Figure 4 Shown is an example of an antibody labeled with biotin and Figure 2 Schematic diagram of mixing different molecular ratios of mixtures in which the molecular ratios of monovalent streptavidin-labeled phycoerythrin (rPE) are 1:1 and 1:3 respectively.

[0089] in, Figure 4 ① and ② respectively represent: when the molecular ratio monovalent SA:rPE=1:1, the mixed schematic diagram of the molecular ratio Ab-Biotin:SA-rPE=1:3 and 1:4; Figure 4 ③ and ④ respectively represent: when the molecular ratio of monovalent SA:rPE=1:3, the mixing schematic diagrams of the molecular ratio of Ab-Biotin:SA-rPE=1:3 and 1:4.

[0090] Figure 5 The biotin-labeled antibody is shown Figure 3 Schematic diagram of a mixture of commercially available tetravalent streptavidin-labeled phycoerythrin (rPE) with a molecular ratio of 1:1 and a mixture with a molecular ratio of 1:0.86.

[0091] Figure 6 Shown is a schematic diagram of antibody direct labeling of phycoerythrin (rPE).

[0092] Figure 7 Shown is a stability comparison picture when the present invention is used for immunoassay of carcinoembryonic antigen project.

[0093] in, Figure 7 A and Figure 7 In B, control group 1 is a mixture of commercially available tetravalent streptavidin-labeled phycoerythrin and antibody-biotin, control group 2 is a mixture of antibody-directly labeled phycoerythrin and antibody-biotin. , the experimental group is a mixture of monovalent streptavidin-labeled phycoerythrin and antibody-biotin.

[0094] Figure 8 Shown is a graph showing the change trend of the fluorescence signal data corresponding to the control group 1 in the conventional stability test in Example 2.

[0095] in, Figure 8 A is the corresponding data change trend chart of calibration products CAL1-CAL6 and quality control products CON1-CON2. Figure 8 B is the data change trend chart of the quality control products CON1-CON2 and samples S1-S4. DETAILED DESCRIPTION

[0096] The present inventors have conducted extensive and in-depth research and, after extensive screening, have for the first time developed a method for detecting target analytes using highly homogeneous monovalent streptavidin tetramers.

[0097] The inventors have discovered for the first time that by utilizing the binding properties of monovalent streptavidin, adjusting the ratio of its binding to fluorescent markers, and adjusting the ratio of monovalent streptavidin-fluorescent markers to antibody-biotin binding, the size of the mixture of streptavidin-labeled fluorescent markers and antibody-labeled biotin can be effectively controlled, the precipitation phenomenon caused by aggregation can be reduced, and the problem of unstable fluorescence signal measurement can be solved. Specifically, the inventors have discovered that by preparing monovalent streptavidin and then mixing it with phycoerythrin at a certain ratio of 1:1 to 1:6, the antibody-biotin complex formed therewith binds at a ratio of 1:3 to 1:8 to form a multi-complex. This multi-complex is used as one of the components in the sandwich flow fluorescence immunoassay kit, namely the secondary antibody, which can completely solve the problem of poor stability caused by the development of the kit.

[0098] The experimental results show that by mixing monovalent streptavidin with fluorescent markers and applying them to immunoassays, the amount of fluorescent protein bound to it can be effectively controlled, and the amount of the monovalent streptavidin-labeled fluorescent protein mixture and the antibody-biotin mixture can be strictly controlled, which can greatly reduce the possibility of aggregation, delay or reduce precipitation, improve the efficiency of raw material use, and avoid waste of raw materials. At the same time, it can effectively increase the stability of the streptavidin-fluorescent protein aggregate and the stability of the antibody-biotin binding mixture, improve the stability of the reagents, and ensure the effectiveness of the test results.

[0099] On this basis, the present invention has been completed.

[0100] the term

[0101] As used herein, the terms "tetravalent streptavidin" and "tetravalent SA" are used interchangeably, and the four monomeric proteins constituting the streptavidin tetramer are all active, that is, one molecule of tetravalent streptavidin can bind to four molecules of biotin with high specificity. The present invention uses commercially available tetravalent streptavidin.

[0102] As used herein, the term "marker" refers to a class of substances that can bind to antigens, antibodies, and some proteins, and can be used in immunoassays, clinical diagnosis, and bioengineering techniques such as fluorescence microscopy and flow cytometry. The main commonly used markers are radionuclides, fluorescent substances, enzyme markers, chemiluminescent agents, quantum dots, and colloidal gold. Commonly used fluorescent substances include organic compound fluorescein, rare earth ion chelates, and fluorescent substrates, and organic compound fluorescein includes fluorescein isothiocyanate, tetraethyl rhodamine, tetramethyl rhodamine isothiocyanate, and phycoerythrin, etc. In one embodiment of the present invention, commercially available phycoerythrin, referred to as rPE, is used.

[0103] As used herein, 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 bound to substances such as biotin and markers using conventional labeling methods. They are often used in the fields of immunoassays. The present invention uses commercially available antibodies.

[0104] As used herein, the terms "biotin" and "biotin" are used interchangeably and refer to small molecules that can bind to streptavidin with high specificity and can be used to label antibodies and in the fields of immunoassays. The present invention uses commercially available biotin.

[0105] As used herein, the terms "antibody-labeled fluorescent marker" and "Ab-rPE" are used interchangeably and refer to a mixture formed by direct labeling of phycoerythrin with an antibody using conventional labeling methods. It is commonly used in immunoassays. In the present invention, it is used as a secondary antibody.

[0106] As used herein, the terms "antibody-labeled biotin" and "Ab-Biotin" are used interchangeably and refer to a conventional labeling method in which an antibody is directly labeled with biotin to form a mixture, and then combined with a mixture of streptavidin and phycoerythrin. It is often used in immunoassays.

[0107] As used herein, the terms "streptavidin-labeled fluorescent marker" and "SA-rPE" are used interchangeably and refer to a conventional labeling method, in which streptavidin is labeled with phycoerythrin to form a mixture, which is then combined with a mixture of antibody and biotin (this mixture is used as a secondary antibody in the present invention). It is often used in immunoassays.

[0108] Monovalent Streptavidin (Monovalent SA)

[0109] In the existing technology, people have developed a monovalent streptavidin (abbreviated as monovalent SA). In the monovalent SA, only one monomer of the tetrameric protein constituting streptavidin is active, and the other three monomers are inactive, that is, one molecule of monovalent streptavidin can bind to one molecule of biotin with high specificity.

[0110] In an embodiment of the present invention, the amino acid sequence of the wild-type streptavidin monomer is shown in SEQ ID NO:1.

[0111] MAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS(SEQ ID NO:1)

[0112] In an embodiment of the present invention, the amino acid sequence of the streptavidin mutant monomer is shown in SEQ ID NO:2.

[0113] MAEAGITGTWYAQLGDTFIVTAGADGALTGTYEAAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS(SEQ ID NO:2)

[0114] Currently, monovalent streptavidin is used in neuroglial testing, studying cell surface protein transport, etc. In addition, some reports mentioned that mutant streptavidin or mutant streptavidin subunits can be bound 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, magnetic resonance imaging, PET probes and neutron capture therapy with boron 10 labeling can also be bound to mutant streptavidin or mutant streptavidin subunits.

[0115] The monovalent streptavidin used in the present invention is prepared by referring to the method provided in the patent "Monovalent Streptavidin Complex" of US Patent Application US20070099248, the entire content of which is incorporated herein by reference.

[0116] In the present invention, the mixing range of different ratios of monovalent streptavidin and phycoerythrin refers to the molecular ratio of SA to rPE, preferably SA:rPE=1:1 to 1:6, and the preferred molecular ratio is 1:3.

[0117] The theoretical ratio range of monovalent streptavidin-phycoerythrin and antibody-biotin binding refers to the molecular ratio of the two, Ab-Biotin:SA-rPE=1:1 to 1:n. In order to ensure the practicality of the test signal and take into account the saving of raw materials, the ratio range of the experiment of the present invention is Ab-Biotin:SA-rPE=1:2 to 1:8, and the preferred molecular ratio is 1:4.

[0118] In order to facilitate understanding of the present invention, the inventor provides the following basic principles. However, it should be understood that the protection scope of the present invention is not limited to the basic principles of the present invention.

[0119] (1) Preparation of highly homogeneous monovalent streptavidin tetramers

[0120] 1.1 The wild type and active deletion mutant of streptavidin were cloned into the ECROI and XHOI restriction sites of the pET21a vector to obtain two recombinant plasmids;

[0121] 1.2 The recombinant plasmid was transformed into E. coli competent cells BL21 (DE3). When the cells were cultured to OD600 = 0.7, the inducer IPTG was added to a final concentration of 1 mM for induction. After induction at 37°C for 5 hours, the cells were collected and stored at -20°C.

[0122] 1.3 The frozen cells were resuspended in lysis buffer at a ratio of 5 ml / g of cell weight, and then ultrasonically disrupted in an ice bath. After centrifugation, the precipitate was resuspended in washing buffer, stirred and washed at 16°C for 30 minutes, centrifuged and the supernatant was discarded. Repeat the washing process until the purity of the inclusion bodies reaches more than 90%.

[0123] 1.4 The wild-type and mutant streptavidin inclusion bodies dissolved in urea were fully mixed at a mass ratio of 1:3, and the inclusion bodies were renatured using the rapid dilution renaturation method. The renatured sample was stirred at 4°C for more than 4 hours, and the precipitate was discarded by centrifugation;

[0124] 1.5 The obtained streptavidin tetramer was resuspended with Ni-A buffer, the precipitate was discarded by centrifugation, and the obtained supernatant was affinity purified with NTA medium and eluted using an imidazole concentration gradient.

[0125] The C-terminus of the wild-type streptavidin is a recombinant protein with a His8-tag label.

[0126] The streptavidin activity-deficient mutant does not contain a His8-tag.

[0127] The lysis buffer and the washing buffer in steps 1.3 and 1.4) are both made of 10 mM PBS PH8.0.

[0128] The dissolution buffer described in step 1.4) is made of 10 mM PBS PH8.0, 8 M Urea.

[0129] The rapid dilution refolding method for refolding the inclusion bodies is specifically as follows: adding the dissolved inclusion bodies dropwise into a refolding buffer (10 mM PBS pH 8.0) at 4°C and stirring sufficiently, and controlling the volume of the refolding buffer to ensure that the concentration of urea during the refolding process is not higher than 0.5 M;

[0130] The Ni-A buffer described in step 1.5) is made of 10mM PBS pH 8.0, 5% glycerol buffer; the gradient imidazole concentration refers to 20mM imidazole, 70mM imidazole, 100mM imidazole, 125mM imidazole and 250mM imidazole solution

[0131] The beneficial effects of the present invention are as follows: the present invention provides a streptavidin compatible with wild-type and mutants, and the wild-type and mutant streptavidin inclusion bodies are mixed in a mass ratio of 1:3, and multiple active streptavidin tetramers are obtained by renaturation, and the streptavidin monovalent tetramer obtained by multi-step separation and purification has high uniformity, no recombinant tags, and can tolerate multiple factors while maintaining the stability of the monovalent tetramer.

[0132] (2) Types of streptavidin (monovalent and commercially available tetravalent) labeled phycoerythrin (rPE)

[0133] 2.1 Commercially available tetravalent streptavidin labeled phycoerythrin at a molecular ratio of 1:1

[0134] The commercially available tetravalent streptavidin was first activated with the reducing agent DTT and then purified and dialyzed, and then cross-linked with the cross-linking agent SPDP; phycoerythrin (rPE) was purified by a hydrophobic column (HIC) and then cross-linked with the cross-linking agent SMCC. When labeling the tetravalent streptavidin protein, phycoerythrin with a molecular weight of 1 was added for labeling, and the resulting labeled product was recorded as A1. See the labeling mixture Figure 3 Schematic diagram;

[0135] 2.2 Monovalent streptavidin labeled phycoerythrin at molecular ratios of 1:1, 1:2, 1:3, 1:4, 1:5 and 1:6

[0136] Monovalent streptavidin was labeled in the same way as tetravalent streptavidin. When labeling monovalent streptavidin protein, phycoerythrin with 1, 2, 3, 4, 5 and 6 times molecular weight was added for labeling. The obtained labeled products were recorded as A'1, A'2, A'3, A'4, A'5 and A'6. Some of the labeled products were mixed. Figure 2 Schematic diagram;

[0137] (3) Types of products labeled with streptavidin and phycoerythrin mixed with a secondary antibody labeled with biotin

[0138] 3.1 Mixing of labeled product A1 and secondary antibody-biotin

[0139] The commercially available tetravalent streptavidin with a molecular weight of 0.86 times and the product labeled with phycoerythrin were added to the second antibody labeled with biotin and mixed. At this time, one molecule of streptavidin can bind to four biotins. The combined complexes are respectively recorded as B1-1, which is the second antibody in the experiment. Figure 5 Schematic diagram;

[0140] 3.2 Labeled products A'1, A'2, A'3, A'4, A'5 and A'6 are mixed with the second antibody-biotin

[0141] The products labeled with different molecular ratios of monovalent streptavidin and phycoerythrin were added to the second antibody labeled with biotin at 3 and 4 times the molecular weight, respectively, and mixed. At this time, because streptavidin is monovalent, it can only bind to one biotin. The combined complexes are recorded as B'1-1, B'1-2, B'2-1, B'2-2, B'3-1, B'3-2, B'4-1, B'4-2, B'5-1, B'5-2, B'6-1 and B'6-2, which are all the second antibodies in the experiment. Some of them are mixed as shown in Figure 4 Schematic diagram;

[0142] (4) A mixture of phycoerythrin-streptavidin-biotin-secondary antibody is used to improve the stability of immunoassay signals in in vitro diagnostics

[0143] 4.1 Phycoerythrin-labeled streptavidin

[0144] Take phycoerythrin of different mass ratios (stored in 60% (NH4)2SO4 solution) and label streptavidin (tetravalent and monovalent at the same time), protect the reaction from light, dialyze to remove the reducing agent DTT, and store for later use.

[0145] 4.2 Biotinylated Antibodies

[0146] Take a certain amount of purified and dialyzed antibodies that specifically recognize antigen (Ag), add dimethyl sulfoxide (DMSO) solution of biotin, react in the dark, dialyze to remove unreacted biotin, and store for later use.

[0147] 4.3 Phycoerythrin-labeled mycoavidin binds to biotin-labeled antibodies

[0148] Take phycoerythrin-streptavidin (monovalent and tetravalent) of different molecular ratios and add them to the second antibody solution labeled with biotin for binding. The resulting product is phycoerythrin-streptavidin (monovalent and tetravalent)-biotin-antibody, which is the secondary antibody and is recorded as solution B.

[0149] 4.4 Capture Antibody Coated Microspheres

[0150] The capture antibody against a certain antigen (Ag) is covalently cross-linked with carboxylated polystyrene microspheres (Beads) to obtain a coupling mixture of Beads and capture antibody, which is Ab-Beads, that is, the primary antibody, recorded as C solution.

[0151] 4.5 Preparation of antigen calibrator solution

[0152] Prepare a standard solution of a certain concentration using a standard antigen (Ag).

[0153] 4.6 In vitro diagnostic immune response and signal detection

[0154] The test was conducted according to the Tesmi F3999+Luminex multifunctional flow cytometer, and the corresponding A solution, calibration substance, B solution and C solution were manually tested in turn according to the conventional 15+15min mode on the machine. There were control group and experimental group, respectively. The control group had two types of commercially available tetravalent streptavidin-labeled phycoerythrin (rPE) and antibody-labeled biotin mixture and antibody-directly labeled phycoerythrin (rPE) mixture; the experimental group was a sample of a homemade monovalent streptavidin-labeled phycoerythrin (rPE) and antibody-labeled biotin mixture.

[0155] 4.7 In vitro diagnostic immunoassay signal stability

[0156] The experimental operation process is the same as described in 4.6. The stability test is divided into short-term conventional stability test and short-term accelerated stability test:

[0157] (1) Short-term routine stability test: ① Control group 1 and 2: The storage temperature of the commercially available tetravalent streptavidin-labeled phycoerythrin (rPE) and antibody-labeled biotin mixture and the antibody-directly labeled phycoerythrin (rPE) mixture was 2-8°C; ② Experimental group: The storage temperature of the homemade monovalent streptavidin-labeled phycoerythrin (rPE) and antibody-labeled biotin mixture was also 2-8°C; ③ The stability test time for control group 1 and 2 and the experimental group was 1 day, 4 days, 7 days, 14 days, 38 days and 68 days, respectively.

[0158] (2) Short-term accelerated stability test: ① Control group 1 and 2: The commercially available tetravalent streptavidin-labeled phycoerythrin (rPE) and antibody-labeled biotin mixture and the antibody-directly labeled phycoerythrin (rPE) mixture were stored at room temperature; ② Experimental group: The homemade monovalent streptavidin-labeled phycoerythrin (rPE) and antibody-labeled biotin mixture was also stored at room temperature; ③ The stability test time for control group 1 and 2 and the experimental group was 1 day, 4 days, 7 days and 14 days, respectively.

[0159] (The stability of labeling by the invention is evaluated by the percentage of signal change in each test)

[0160] The main advantages of the present invention include:

[0161] 1) Through monovalent SA, it can highly specifically bind to a molecule of biotin. During the polymerization / labeling process, the size of the complex after the mixture of phycoerythrin-monovalent streptavidin and antibody-biotin is controlled to avoid the formation of huge polymers, thereby forming precipitation, etc., improving the stability of intermediates and then the stability of detection reagents, ensuring the stability and accuracy of the detection.

[0162] 2) Monovalent SA can highly specifically bind to a molecule of biotin, without reducing the detection sensitivity, specificity and other performances, and without precipitation formation, ensuring stability, which greatly reduces the amount of antibodies and marker phycoerythrin, saving raw material costs. At the same time, stable intermediates and reagents can further extend the use time of reagents, and to a certain extent, reduce the cost and time waste caused by reconfiguration due to instability.

[0163] 3) Save a lot of time and cost for setting up kits and immunoassays, and improve research and development level and efficiency. By using the present invention, the time and labor cost of repeated measurements to ensure the stability of the tetravalent-streptavidin-phycoerythrin and antibody-biotin complexes and the direct labeling mode antibody-phycoerythrin complexes can be omitted, and the efficiency is higher.

[0164] 4) The monovalent SA of the present invention can be applied not only to the flow platform in immunoassay, but also to the chemiluminescence platform and other platforms, and has a wide range of applications.

[0165] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0166] Example 1: Commercially available tetravalent streptavidin and homemade monovalent streptavidin were used for CEA immunoassay detection

[0167] 1. Preparation of monovalent streptavidin

[0168] 1.1 Raw materials

[0169] pET21a vector;

[0170] E. coli competent cells BL21 (DE3);

[0171] Inducer IPTG;

[0172] 10 mM PBS solution, 10 mM PBS + 5% glycerol solution, and 10 mM PBS + 8 M Urea solution;

[0173] 20 mM imidazole, 70 mM imidazole, 100 mM imidazole, 125 mM imidazole and 250 mM imidazole solutions;

[0174] SDS-PAGE (15%) electrophoresis material;

[0175] Ultrasonic cell disruptor (SCIENTZ-II17);

[0176] Hitachi high-speed refrigerated centrifuge (CR21G).

[0177] 1.2 Operation steps

[0178] 01. Perform the experiment according to the basic principle of monovalent streptavidin preparation mentioned above;

[0179] 02.See Figure 1Two schematic diagrams in the figure: SDS-PAGE electrophoresis is used to identify the streptavidin tetramer obtained after elution (boiling and non-boiling). Two groups of samples are taken from each elution peak, one group is heated and boiled, and the other group is not heated and boiled. Both groups are analyzed by SDS-PAGE (15%). Because streptavidin tetramers are relatively stable, they remain in the form of tetramers during the SDS-PAGE process when not heated. Figure 1 The numbers 5, 6, 7 and 8 in the figure correspond to the pictures; heating at 100℃ for 5 min can destroy the tetramer of streptavidin and form monomers, such as Figure 1 The pictures corresponding to the numbers 1, 2, 3 and 4 in the figure. Therefore, the comprehensive analysis of the results of the two sets of SDS-PAGE can reveal the composition of each elution peak, and finally confirm that the elution peak contains a monovalent streptavidin tetramer (W1M3) composed of one wild-type monomer (w) and three mutant monomers (M) with high purity when the imidazole concentration is 70mM;

[0180] 2. Commercially available tetravalent streptavidin and monovalent streptavidin were used for immunoassay detection

[0181] Here, the detection of carcinoembryonic antigen using Tesmi F3999+Luminex multifunctional flow cytometer is taken as an example to illustrate the effectiveness of this patent in detecting signals and improving stability.

[0182] According to the explanation of the basic principles, the signal of commercially available tetravalent streptavidin used in immunoassay should theoretically be consistent with or higher than the signal of conventional antibody direct labeling; the signal of homemade monovalent streptavidin used in immunoassay should be able to reach the signal of commercially available tetravalent streptavidin and should also be consistent with or higher than the signal of conventional antibody direct labeling.

[0183] 2.1 CEA-related antibodies directly labeled with phycoerythrin to detect CEA (see Figure 6 )

[0184] 2.1.1 Operation

[0185] 01. Redissolve CAL1-CAL6 in the calibrator of the alpha-fetoprotein / carcinoembryonic antigen quantitative detection kit (lot number 151101) sold by our company;

[0186] 02. Labeled CEA-rPE (concentration: 0.439 mg / mL) and magnetic microspheres (concentration: 1.5*10 7 / mL) were diluted with PBS-TBN diluent (lot number: 180501) to the corresponding working concentrations of 0.8ug / mL and 1.0*10 5 Pieces / mL;

[0187] 03.According to the instructions of the AFP / CEA quantitative detection kit, obtain the fluorescence signals of the six parallel samples of calibrators CAL1-CAL6. The results are shown in the table below:

[0188] Table 1: Fluorescence signal values ​​of calibrators CAL1-CAL6

[0189]

[0190] 2.2 Detection of carcinoembryonic antigen by conventional antibody-biotin and tetravalent streptavidin-phycoerythrin

[0191] 2.2.1 Raw materials

[0192] Commercially available tetravalent streptavidin (SA);

[0193] CEA antibodies purchased by the company;

[0194] Biotin (commercially available);

[0195] Phycoerythrin (20 mg / mL, rPE, commercially available);

[0196] Hydrophobic column (HIC column) Anion column (DEAE column) Packing

[0197] Commercially available cross-linking agents SPDP (concentration 3 mg / 320 ul) and SMCC (2 mg / mL), activator DTT (50 mmol / L), reagent GLY (10%) and DMSO, and homemade PBS (10 mmol / L) solution.

[0198] 2.2.2 Operation

[0199] 01. Take an appropriate amount of commercially available tetravalent streptavidin and prepare it with 10mM PBS solution to a concentration of 2mg / mL;

[0200] 02. Activate the tetravalent streptavidin with SPDP crosslinker and DTT activator so that it can bind to the activated phycoerythrin (rPE);

[0201] 03. Take an appropriate amount of phycoerythrin (rPE), purify it through a hydrophobic column (HIC), activate it with SMCC reagent, and then purify it through an anion column DEAE;

[0202] 04. Mix the activated streptavidin and phycoerythrin in a conventional ratio (i.e., molecular ratio SA:rPE = 1:1) at 2°C-8°C overnight;

[0203] 05. Take an appropriate amount of carcinoembryonic antigen CEA labeled antibody (concentration: 2.0 mg / mL) and 1.42 mg of biotin, prepare them with DMSO to a concentration of 1.5 mg / mL, and react them at a certain mass ratio (m / m) at 2℃-8℃, 2H; terminate with 10% GLY;

[0204] 06. The reacted CEA-Biotin and SA-rPE were mixed at a conventional molecular ratio (CEA-Biotin:SA-rPE=1:0.86) (the concentration of the mixture was 0.4761 mg / ml);

[0205] 07. Test operation: Redissolve CAL1-CAL6 in the calibrator of the alpha-fetoprotein / carcinoembryonic antigen quantitative detection kit (batch number 151101) sold by our company; 7 / mL) were diluted with PBS-TBN diluent to the corresponding working concentrations of 0.8ug / mL and 1.0*10 5 Pieces / mL;

[0206] 08.According to the instructions of the AFP / CEA quantitative detection kit, the fluorescence signals of the six parallel samples of calibrators CAL1-CAL6 were obtained. The results are shown in the following table:

[0207] Table 2: Fluorescence signal values ​​of calibrators CAL1-CAL6

[0208]

[0209]

[0210] 2.3 Detection of carcinoembryonic antigen by antibody-biotin and monovalent streptavidin-phycoerythrin

[0211] 2.3.1 Raw materials

[0212] Homemade monovalent streptavidin

[0213] The other reagents used are the same as those for tetravalent streptavidin above.

[0214] 2.3.2 Operation

[0215] 01. Take an appropriate amount of homemade monovalent streptavidin and prepare it with 10mmol / L PBS solution to a concentration of 1.39mg / mL;

[0216] The operation steps of 02 and 03 are the same as those in 2.2.2 above.

[0217] 04. According to the above basic principle: Mix the activated monovalent streptavidin and phycoerythrin in one of the ratios (molecular ratio SA: rPE = 1:3), at 2°C-8°C, overnight;

[0218] 05. The operation steps are the same as those in 2.2.2 above;

[0219] 06. According to the above basic principle: the reacted CEA-Biotin and SA-rPE are mixed in a molecular ratio. That is, when the molecular ratio SA:rPE=1:3, the mixed molecular ratio is (CEA-Biotin:SA-rPE=1:4), and the concentration of this mixture is 0.0445mg / ml;

[0220] The operation steps of 07 and 08 are the same as those in 2.2.2 above. The fluorescence signals of the six parallel samples of calibrators CAL1-CAL6 are obtained. The results are shown in the table below:

[0221] Table 3: Fluorescence signal values ​​of calibrators CAL1-CAL6 when the molecular ratio SA:rPE=1:3

[0222]

[0223]

[0224] Through the above three immune signal test experiments 2.1, 2.2 and 2.3, it can be seen that when the homemade monovalent streptavidin is used for immunodetection of carcinoembryonic antigen, when SA:rPE=1:3 and at the molecular ratio CEA-Biotin:SA-rPE=1:4, the test fluorescence signal of the monovalent streptavidin-labeled phycoerythrin is higher than the fluorescence signal of the antibody directly labeled phycoerythrin; in addition, compared with the conventional commercially available tetravalent streptavidin-labeled phycoerythrin for immunodetection of carcinoembryonic antigen (that is, when SA:rPE=1:1, CEA-Biotin:SA-rPE=1:0.86), the test fluorescence signal is not much different, and the signals are almost the same.

[0225] Example 2: Stability experiment of a mixture ratio of monovalent streptavidin and phycoerythrin and a mixture ratio of monovalent streptavidin and phycoerythrin and antibody biotin for immunoassay

[0226] Here, the use of Tesmi F3999+Luminex multifunctional flow cytometer to detect carcinoembryonic antigen is used as an example to illustrate the effectiveness of this patent in improving stability.

[0227] According to the explanation of the basic principles and the embodiment of the schematic diagram, the application of the monovalent streptavidin of this patent should not only have a test signal that can reach or exceed the signal of the commercially available tetravalent streptavidin and the antibody directly labeled with phycoerythrin, but also have better and higher stability than the commercially available tetravalent streptavidin and the currently used antibody directly labeled with the luminescent substance when used for immunoassay.

[0228] 1. Short-term conventional stability test and accelerated stability test experiment

[0229] 1.1 Raw materials

[0230] Our company sells the “Alpha-fetoprotein / Carcinoembryonic Antigen Quantitative Detection Kit Calibrator” (Batch No. 151101);

[0231] Carcinoembryonic antigen quality control (con-1, con-2, batch number: 180501);

[0232] 4 sera related to carcinoembryonic antigen (S1, S2, S3, and S4);

[0233] Antibody (CEA) coated magnetic microspheres (concentration: 1.5*10 7 / mL, batch number 180401);

[0234] Control group 1: Secondary antibody labeled by our production department (0.439 mg / mL, CEA-rPE, batch number 180701-1) Control group 2: Commercially available tetravalent streptavidin SA:rPE = 1:1, while the molecular ratio is CEA-Biotin:SA-rPE = 1:0.86 (concentration: 0.4761 mg / ml)

[0235] Experimental group: According to the above basic principle: select homemade monovalent streptavidin SA: rPE = 1:3, and the molecular ratio is

[0236] CEA-Biotin:SA-rPE=1:4 (concentration: 0.0445 mg / ml)

[0237] The reagents in the control group and experimental group were stored at 2-8°C and room temperature, and 0.02% of the preservative NaN3 was added;

[0238] A solution (batch number: 180301) and D solution (sheath fluid) for tumor markers on the Tesmi F3999+Luminex multifunctional flow cytometer detection platform;

[0239] PBS-TBN diluent (lot number: 180501).

[0240] 1.2 Operation

[0241] 01. Redissolve CAL1-CAL6 in the calibrator of the AFP / CEA quantitative detection kit; at the same time, place the quality control and sample serum at room temperature for use.

[0242] 02. Dilute the labeled CEA-rPE, homemade CEA-Biotin:SA-rPE and magnetic microspheres with PBS-TBN diluent to the corresponding working concentrations of 0.8ug / mL and 1.0*10 5 Pieces / mL;

[0243] 03.According to the instructions of the AFP / CEA quantitative detection kit, obtain the fluorescence signals of the six parallel samples of calibrators CAL1-CAL6

[0244] 04. The stability test results are shown in Table 1, Table 2, Table 3 and Table 4, and the stability appearance pictures are shown in Figure 7 A and Figure 7 B

[0245] According to the basic principles of the above stability test, the calibrators used in the stability test are always from the same batch, with the same concentration and validity period, and the concentration and batch of the paired antibodies (antibodies coated with magnetic microspheres) used during this period are always consistent; the working concentration and batch used in the control group 1, control group 2 and experimental group during the stability test are also consistent; in addition, the quality control products and samples during the stability test are always from the same batch, the same sample and the same concentration, of which there are 2 quality control products (the batch is always the same, one batch is randomly selected), and there are 4 test samples (4 samples are randomly selected from high, medium and low concentrations respectively)

[0246] Table 1: Fluorescence signal values ​​of calibrators CAL1-CAL6 during routine short-term stability testing (stored at 2-8°C)

[0247]

[0248]

[0249]

[0250] Table 2: Fluorescence signal values ​​of calibrators CAL1-CAL6 during short-term accelerated stability test (room temperature storage)

[0251]

[0252]

[0253] Combined with Table 1 and Table 2 and Appendix Figure 8It can be seen that the change trends of the experimental data of each group are consistent. In the test, the CAL1-CAL6 signals, quality control product signals and sample signals of the control group and the experimental group all change synchronously, and the decline ratio and fluctuation trend are almost the same. Due to the large amount of test data, the CAL4 signal in the calibrator in the control group and the experimental group and the S3 in the sample are selected as representatives to perform statistical test signal changes. The summary is shown in Tables 3 and 4.

[0254] Table 3: Short-term conventional stability detection signals (taking CAL4 and S3 in the test as examples) and phenomenon summary table

[0255]

[0256]

[0257] Table 4: Short-term acceleration stability detection signal (taking CAL4 and S3 in the test as an example) and phenomenon summary table

[0258]

[0259] It can be seen from Tables 3 and 4 that the fluorescence signals of the initial control group 2 and the experimental group are similar, both higher than those of the control group 1.

[0260] From the conventional stability test, control group 1 became turbid on the 4th day and precipitated on the 14th day. At this time, the fluorescence signal value represented by calibrator CAL4 and sample S3 had dropped by nearly half. Control group 2 also had precipitation on the 14th day, and the fluorescence signal also dropped by nearly half. In the experimental group, the signal in the conventional stability test hardly dropped before 38 days. After 38 days, the signal dropped by about 10%, but the sample was still transparent.

[0261] In the accelerated stability test, the fluorescence signals of the control group 1 represented by the calibrator CAL4 and sample S3 decreased by 76.5% and 80.1% on the 14th day, and precipitation was produced. The corresponding fluorescence signals of the control group 2 decreased by 82.6% and 83.3%, and precipitation was also produced. The experimental group only decreased by 0.8%, which effectively avoided the formation of precipitation and ensured the stability of the fluorescence signal.

[0262] Example 3: Application of other ratios of monovalent streptavidin and phycoerythrin in carcinoembryonic antigen immunoassay

[0263] Here, the detection of carcinoembryonic antigen using the same instrument as in Example 2 is used as an example to illustrate that the scope of this patent is relatively large.

[0264] According to the explanation of the basic principle, the monovalent streptavidin of the present invention is mixed with phycoerythrin in different proportions within a certain range, and then the mixtures in these proportions are mixed with antibody biotin in a certain proportion for experiment.

[0265] Other ratios of monovalent streptavidin and phycoerythrin were used for the stability test of carcinoembryonic antigen. According to the experimental design of Example 2, since the test data was too large, we chose to test it under the condition of conventional storage at 2-8°C based on its stability test time and data rules. Only the corresponding calibrators (CAL1-CAL6) were selected as the objects of the stability test, and the test time was selected on the 1st day and the 50th day as representatives; the batch number of the calibrator used during the stability test was always the same.

[0266] 1. Experimental study on signal and stability of monovalent streptavidin and phycoerythrin (i.e., SA:rPE = 1:1 and 1:6) using carcinoembryonic antigen

[0267] 1.1 Raw materials

[0268] The calibrators and antibody-coated microspheres used were the same as those in Example 2;

[0269] Secondary antibody control group 1 labeled by our production department: CEA-rPE (concentration: 0.439 mg / mL)

[0270] Control group 2: Commercially available tetravalent streptavidin SA:rPE = 1:1, while the molecular ratio is CEA-Biotin:SA-rPE = 1:0.86 (concentration: 0.4761 mg / ml)

[0271] Experimental group 1: Self-made monovalent streptavidin SA: rPE = 1:1, and the molecular ratio is

[0272] ①CEA-Biotin: SA-rPE=1:4 (C=0.1020mg / ml),

[0273] Experimental group 2: Self-made monovalent streptavidin SA: rPE = 1:6, and the molecular ratio is

[0274] ②CEA-Biotin: SA-rPE=1:4 (C=0.0242mg / ml),

[0275] The samples of the control group and the experimental group were stored at 2-8°C and 0.02% of the preservative NaN3 was added.

[0276] 1.2 Operation

[0277] 01. Test operation: The specific experimental operation steps are the same as those in Example 2;

[0278] 02.According to the instructions of the AFP / CEA quantitative detection kit, obtain the fluorescence signals of the six parallel samples of calibrators CAL1-CAL6.

[0279] The monovalent streptavidin mixed with phycoerythrin (ie, SA:rPE = 1:1 and 1:6) was used for the test data of carcinoembryonic antigen. The results are shown in Table 5.

[0280] Table 5: Fluorescence signal values ​​of monovalent streptavidin mixed with phycoerythrin (ie, SA:rPE = 1:1 and 1:6)

[0281]

[0282] As can be seen from Table 5, the data change trend is similar to the data change in Example 2, so the signal value change of the calibrator CAL5 is selected as a representative for summary in the above two ratio tests.

[0283] Table 6: Summary of test data of monovalent streptavidin mixed with phycoerythrin (ie, SA:rPE = 1:1 and 1:6) (represented by calibrator CAL5)

[0284]

[0285] From the data in Tables 5 and 6, it can be seen that at two random ratios within the mixing ratio range of monovalent streptavidin and phycoerythrin (i.e., SA:rPE = 1:1 and 1:6), the test fluorescence signals of control group 2, experimental group 1 and experimental group 2 are all higher than that of control group 1.

[0286] On the first day, the signals of control group 1, control group 2, experimental group 1 and experimental group 2 did not decrease, and the samples were all transparent. After 50 days, the fluorescence signals of control group 1 and control group 2 decreased by 80.5% and 84.5% respectively, and the samples were almost completely precipitated; the fluorescence signals of experimental group 1 and experimental group 2 only decreased by 3.7%, which largely controlled the aggregation of samples and the generation of precipitation, and effectively maintained the stability of the test fluorescence signal. It is then shown that monovalent streptavidin and phycoerythrin are applicable in the range of mixing ratio SA:rPE=1:1-1:6.

[0287] Example 4: Mixtures of monovalent streptavidin and phycoerythrin mixed with antibody biotin in different proportions for use in carcinoembryonic antigen immunoassay detection

[0288] Here, the detection of carcinoembryonic antigen using the same instrument as in Example 2 is used as an example to illustrate that the scope of this patent is relatively large.

[0289] According to the explanation of the basic principle, the monovalent streptavidin of this patent is randomly mixed with phycoerythrin in a certain ratio within a certain range, and then mixed with antibody biotin in different ratios for experiment. This experiment randomly selects two ratios for experiment.

[0290] The design of this experiment is the same as that of Example 2, so the test was performed under the condition of conventional storage at 2-8°C. Only the corresponding calibrators (CAL1-CAL6) were selected as the objects of the stability test, and the test time was also selected on the 1st day and the 50th day as representatives; the batch number of the calibrators used during the stability test was always the same.

[0291] 1. The mixture of monovalent streptavidin and phycoerythrin was mixed with antibody biotin in different ratios (i.e., when SA:rPE=1:3, Ab-Biotin:SA:rPE=1:3 and 1:8) and the signal and stability test of carcinoembryonic antigen was performed

[0292] 1.1 Raw materials

[0293] The calibrators and antibody-coated microspheres used were the same as in Example 2;

[0294] Control group 1: Commercially available tetravalent streptavidin SA:rPE = 1:1, while the molecular ratio is CEA-Biotin:SA-rPE = 1:0.86 (concentration: 0.4761 mg / ml)

[0295] When the homemade monovalent streptavidin SA:rPE=1:3, the molecular ratio is

[0296] Experimental group 1: ①CEA-Biotin:SA-rPE=1:3 (C=0.0581 mg / ml),

[0297] Experimental group 2: ②CEA-Biotin:SA-rPE=1:8 (C=0.0227 mg / ml),

[0298] The samples of the control group and the experimental group were stored at 2-8°C and 0.02% of the preservative NaN3 was added.

[0299] 1.2 Operation

[0300] 01. Test operation: The specific experimental operation steps are the same as those in Example 2;

[0301] 02.According to the instructions of the AFP / CEA quantitative detection kit, obtain the fluorescence signals of the six parallel samples of calibrators CAL1-CAL6.

[0302] The test results of the mixture of monovalent streptavidin and phycoerythrin mixed with antibody biotin in different ratios (i.e., when SA:rPE=1:3, CEA-Biotin:SA:rPE=1:3 and 1:8) are shown in Table 7

[0303] Table 7: Fluorescence signal values ​​of the mixtures at different ratios (i.e., CEA-Biotin:SA:rPE=1:3 and 1:8)

[0304]

[0305]

[0306] It can be seen from Table 7 that the data change trend is similar to that in Example 2, so the signal value change of the calibrator CAL5 can be selected as a representative for the summary of the above two ratio tests.

[0307] Table 8: Summary of test data (represented by calibrator CAL5) of different mixture ratios (i.e., CEA-Biotin:SA:rPE=1:3 and 1:8)

[0308]

[0309] From the data in Tables 7 and 8, it can be seen that when the mixture of monovalent streptavidin and phycoerythrin was mixed with antibody biotin in different ratios (i.e., CEA-Biotin: SA: rPE = 1:3 and 1:8), the fluorescence signal of experimental group 1 was always higher than that of the control group. The fluorescence signal of experimental group 2 was lower than that of the control group at the beginning, and later the signal was higher than that of the control group and remained stable.

[0310] On the first day, the signals of control group 1, experimental group 1 and experimental group 2 did not decrease, and the samples were all transparent. After 50 days, the fluorescence signal of control group 1 decreased by 80.3%, and the samples were almost completely precipitated; the fluorescence signals of experimental group 1 and experimental group 2 decreased by 5.0% and 2.7%, respectively, which largely controlled the aggregation of samples and the generation of precipitation, and effectively maintained the stability of the test fluorescence signal. It is then explained that the mixture ratio is applicable to CEA-Biotin: SA: rPE = 1:3 and 1:8, and this range can be higher.

[0311] The above examples 1, 2, 3 and 4 all describe tumor markers in immunoassays as experimental objects. This experiment shows that the present invention is universal in tumor markers. By analogy, it is also generally applicable to other immune items. That is, the present invention is also applicable to autoimmune items, myocardial markers, liver function tests, ToRCH and respiratory tract infections in immunoassays.

[0312] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. 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 claims attached to this application. Sequence Listing <110> Shanghai TouJing Life Sciences Co., Ltd. Shanghai Toujing Diagnostics Technology Co., Ltd. <120> Application of a highly homogeneous monovalent streptavidin tetramer <130> P2019-0291 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 128 <212> PRT <213> Streptomyces avidinii <400> 1 Met Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser 1 5 10 15 Thr Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr 20 25 30 Glu Ser Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg 35 40 45 Tyr Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp 50 55 60 Thr Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr 65 70 75 80 Trp Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln 85 90 95 Trp Leu Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr 100 105 110 Leu Val Gly His Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser 115 120 125 <210> 2 <211> 128 <212> PRT <213> Artificial Sequence <400> 2 Met Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Ala Gln Leu Gly Asp 1 5 10 15 Thr Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr 20 25 30 Glu Ala Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg 35 40 45 Tyr Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp 50 55 60 Thr Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr 65 70 75 80 Trp Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln 85 90 95 Trp Leu Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr 100 105 110 Leu Val Gly His Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser 115 120 125

Claims

1. A detection system, It is characterized in that The detection system comprises: (a) a multi-component complex having a structure as shown in Formula I, (AB)-(CD m ) n (Formula I) The components in the formula include: A is a second binding protein, which specifically binds to the target analyte; B is biotin; C is a monovalent streptavidin tetramer; D is a fluorescent marker, and the fluorescent marker is phycoerythrin rPE; "-" is a bond or a connecting group; And, m is a real number between 1 and 6; And, n is a real number between 3 and 8; (b) a first binding protein, wherein the first binding protein is cross-linked to the solid phase carrier Z0, and the first binding protein specifically binds to the target analyte; (c) optional target analyte; Wherein, there is no competition between the binding of the first binding protein and the second binding protein to the target analyte respectively; The concentration of the multi-complex is 0.015 to 0.25 mg / ml.

2. The detection system according to claim 1, It is characterized in that The monovalent streptavidin tetramer comprises a streptavidin wild-type monomer and a streptavidin mutant monomer.

3. The detection system according to claim 2, It is characterized in that The streptavidin mutant monomer does not have the function of binding to biotin.

4. The detection system according to claim 2, It is characterized in that The amino acid sequence of the wild-type streptavidin monomer is shown in SEQ ID NO:

1.

5. The detection system according to claim 2, It is characterized in that The streptavidin mutant monomer includes the following mutations based on the streptavidin wild-type monomer: N12A, S16D and S34A.

6. The detection system according to claim 2, It is characterized in that The amino acid sequence of the streptavidin mutant monomer is shown in SEQ ID NO:

2.

7. The detection system according to claim 2, It is characterized in that In the monovalent streptavidin tetramer, the molar ratio of the streptavidin wild-type monomer to the streptavidin mutant monomer is 1:

3.

8. The detection system according to claim 2, It is characterized in that The monovalent streptavidin tetramer includes a tag for protein purification.

9. The detection system according to claim 8, It is characterized in that The tag sequence is located at the N-terminus, C-terminus or the middle of the streptavidin wild-type monomer and / or the streptavidin mutant monomer.

10. The detection system according to claim 8, It is characterized in that The tag includes: His tag, GST tag, Trx tag, MBP tag, HA tag, c-Myc tag, Flag tag, or a combination thereof.

11. The detection system according to claim 1, It is characterized in that The second binding protein is selected from the group consisting of an antigen, an antibody, a ligand, a receptor or a combination thereof.

12. The detection system according to claim 1, It is characterized in that The m is a positive integer between 1 and 6.

13. The detection system according to claim 1, It is characterized in that The m is 2, 3 or 4.

14. The detection system according to claim 1, It is characterized in that The n is a positive integer between 3 and 8.

15. The detection system according to claim 1, It is characterized in that The n is 3 or 4.

16. The detection system according to claim 1, It is characterized in that The concentration of the multi-complex is 0.03 to 0.07 mg / ml.

17. The detection system according to claim 1, It is characterized in that The first binding protein is selected from the group consisting of an antigen, an antibody, a ligand, a receptor or a combination thereof.

18. The detection system according to claim 1, It is characterized in that The solid phase carrier material is selected from the following group: metal, glass, colloid, plastic or a combination thereof.

19. The detection system according to claim 1, It is characterized in that The solid phase carrier material includes: homopolymer, copolymer, or a combination thereof.

20. The detection system according to claim 1, It is characterized in that The solid phase carrier material is selected from the following group: polystyrene, polyethylene, polypropylene, or a combination thereof.

21. The detection system according to claim 1, It is characterized in that The solid phase carrier material is selected from the following group: microspheres, microplates, strips, test tubes, or a combination thereof.

22. The detection system according to claim 1, It is characterized in that The Z0 is a microsphere (bead), a particle (particle) or a magnetic bead.

23. The detection system according to claim 1, It is characterized in that In the detection system, the concentration of Z0 is 1×10 4 to 1.0×10 8 Pieces / mL.

24. The detection system according to claim 1, It is characterized in that In the detection system, the concentration of Z0 is 1×10 4 to 1.5×10 7 Pieces / mL.

25. The detection system according to claim 1, It is characterized in that In the detection system, the concentration of Z0 is 2×10 4 to 1.5×10 7 Pieces / mL.

26. The detection system according to claim 1, It is characterized in that The target analyte includes: antigen, antibody, ligand, receptor, small molecule, or a combination thereof.

27. A use of the detection system according to any one of claims 1 to 26 for non-disease detection purposes or non-disease treatment purposes, It is characterized in that Used to detect whether the sample contains the target analyte.

28. The use according to claim 27, It is characterized in that The sample is an ex vivo sample or an in vitro sample.

29. The use according to claim 27, It is characterized in that The sample was derived from whole blood.

30. The use according to claim 27, It is characterized in that The sample is serum.

31. The use according to claim 27, It is characterized in that When the sample contains the target analyte, the fluorescent signal emitted by the fluorescent marker is detected from the solid phase carrier separated from the detection system.

32. The use according to claim 27, It is characterized in that When the sample does not contain the target analyte, the fluorescent signal emitted by the fluorescent marker cannot be detected from the solid phase carrier separated from the detection system.

33. A kit, It is characterized in that The kit comprises: a container and a raw material reagent located in the container for forming a detection system as described in any one of claims 1 to 26, wherein the raw material reagent does not include a target analyte in a sample to be tested.

34. The kit according to claim 33, It is characterized in that The kit comprises: (a) a first container and a second binding protein A in the detection system according to any one of claims 1 to 26 located in the first container; (b) a second container and the biotin B in the detection system according to any one of claims 1 to 26 located in the second container; (c) a third container and the monovalent streptavidin tetramer C in the detection system according to any one of claims 1 to 26 located in the third container; (d) a fourth container and a fluorescent marker D in the detection system according to any one of claims 1 to 26 located in the fourth container; (e) a fifth container and a first binding protein in the detection system according to any one of claims 1 to 26 located in the fifth container; (f) a sixth container and a solid phase carrier in the detection system according to any one of claims 1 to 26 located in the sixth container.

35. The kit according to claim 34, It is characterized in that The first container, the second container, the third container and the fourth container are the same or different containers.

36. The kit according to claim 34, It is characterized in that The fifth container and the sixth container are the same or different containers.

37. The kit according to claim 34, It is characterized in that The first container, the second container, the third container, the fourth container, the fifth container and the sixth container are the same or different containers.

38. The kit of claim 34, It is characterized in that The kit also includes: (g) a calibrator in and located within a seventh container; (h) an eighth container and a sample diluent located in the eighth container; (i) a ninth container and a wash solution in the ninth container; and / or (j) a tenth container and a buffer solution for a reaction system located in the tenth container.

39. A method for detecting the presence of a target analyte in a sample for purposes other than disease detection or disease treatment. It is characterized in that The method comprises the following steps: (I) providing a detection system according to any one of claims 1 to 26, wherein the detection system does not contain a target analyte, mixing the detection system not containing the target analyte with a sample or a dilution thereof to form a mixed solution, and reacting the mixture; (II) separating the solid phase carrier from the mixed solution obtained in step (I); and (III) Detecting whether the solid phase carrier carries a fluorescent signal from the fluorescent marker in the detection system of any one of claims 1 to 26.

40. The method of claim 39, It is characterized in that In step (I), the sub-steps are included: (i) cross-linking the first binding protein in the detection system according to any one of claims 1 to 26 with the solid phase carrier to form a system i containing the first binding protein cross-linked to the solid phase carrier; (ii) mixing the system i obtained in step (i) with the sample or its dilution to form a system ii; (iii) providing a multi-component complex in the detection system according to any one of claims 1 to 26, and mixing it with the system ii obtained in step (ii) to form the mixed solution described in step (I).

41. The method of claim 39, It is characterized in that In step (II), a step of washing the separated solid phase carrier is also included, and this step is repeated 1 to 5 times.

42. The method of claim 39, It is characterized in that In step (II), a step of washing the separated solid phase carrier is also included, and this step is repeated 3 times.

43. The method of claim 39, It is characterized in that In step (III), the detection comprises fluorescence detection.

44. The method of claim 43, It is characterized in that The fluorescence detection method uses an enzyme marker or a fluorescence spectrophotometer for detection.

45. The method of claim 39, It is characterized in that The detection is performed in vitro or ex vivo.

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