Compound protein as well as preparation method and application thereof

Abstract

The invention discloses a compound protein as well as a preparation method and application thereof, and relates to the field of biology. The invention develops a polyphosphorylated tau compound protein which comprises streptavidin, and an antigen A-biotin A and an antigen B-biotin B which are connected with the streptavidin, so that the problem that the difference between existing calibrators and natural epitopes causes overlarge batch difference of reagents is solved, the calibration performance is improved in a breakthrough manner, and the accuracy of calibration is improved. The kit can be adapted to various detection platforms such as chemiluminescence and enzyme-linked immunosorbent assay, is high in universality and has important clinical value and industrial significance.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biology, in particular to a complex protein and a preparation method and application thereof. BACKGROUND

[0002] Alzheimer's Disease (AD), commonly known as senile dementia, is a central nervous system degenerative disease that mainly occurs in old age. Its typical clinical symptoms include memory loss, emotional changes, cognitive decline, difficulty speaking, difficulty writing, and difficulty walking, which seriously affect the quality of life of patients.

[0003] Currently, the diagnosis of Alzheimer's disease is mainly based on various cognitive tests, routine laboratory tests and structural imaging (MRI or CT scan). To improve the accuracy of diagnosis, amyloid positron emission tomography (PET) can be used as a confirmation standard for amyloid pathology, but this detection method still has limitations such as high cost and exposure to radiation.

[0004] Compared with traditional neuroimaging examination and clinical evaluation, cerebrospinal fluid and blood detection have significant advantages in the diagnosis of Alzheimer's disease (AD). They can capture core pathological changes such as Aβ deposition and Tau protein phosphorylation 10-20 years before the onset of symptoms, enabling ultra-early diagnosis. As one of the "gold standards" for diagnosis, cerebrospinal fluid detection has an accuracy comparable to amyloid PET (up to 90% consistency), but is more cost-effective, easier to obtain and non-radiative. Blood detection, with its non-invasive, low-cost and high-convenience features, breaks through the limitations of expensive PET examination and invasive cerebrospinal fluid puncture, providing the possibility for large-scale community screening and long-term disease monitoring, and greatly improving the accessibility of early diagnosis.

[0005] In the clinical diagnosis of neurodegenerative diseases such as Alzheimer's disease, the phosphorylation level of Tau protein is one of the core biomarkers, and the accurate detection of polyphosphorylated Tau protein is crucial for early diagnosis and disease progression assessment. Currently, commercial recombinant phosphorylated Tau antigens (such as p-Tau181 and p-Tau217 single-site antigens) have significant limitations when used as calibrators in immunoassays. The detection results of different matrix samples (serum / cerebrospinal fluid) show inconsistent bias, and the difference between the calibrator and the natural antigen epitope causes batch-to-batch variation (bias of 30%-60%), which seriously affects the accuracy and reliability of the detection results and becomes a key pain point restricting the development of the in vitro diagnostic industry.

[0006] In view of this, the present application is proposed. SUMMARY

[0007] The present application aims to provide a complex protein and a preparation method and application thereof.

[0008] The present application is implemented as follows: In a first aspect, the present application provides a complex protein, comprising: streptavidin and antigen A-biotin A and antigen B-biotin B connected to the streptavidin; the antigen A and the antigen B are phosphorylated Tau protein antigens with different phosphorylation sites.

[0009] In a second aspect, the present application provides a preparation method of the complex protein as described in the preceding embodiments, comprising: mixing and reacting the antigen A-biotin A, the antigen B-biotin B and the streptavidin in the complex protein.

[0010] In a third aspect, the present application provides a composition, comprising: the complex protein as described in the preceding embodiments or prepared by the preparation method as described in the preceding embodiments.

[0011] In a fourth aspect, the present application provides a kit, comprising: the complex protein as described in the preceding embodiments or prepared by the preparation method as described in the preceding embodiments or the composition as described in any of the preceding embodiments.

[0012] In a fifth aspect, the present application provides application of the complex protein as described in the preceding embodiments or prepared by the preparation method as described in the preceding embodiments or the composition as described in the preceding embodiments in preparation of a kit for detecting Tau protein.

[0013] In a sixth aspect, the present application provides application of the complex protein as described in the preceding embodiments or prepared by the preparation method as described in the preceding embodiments or the composition as described in the preceding embodiments or the kit as described in the preceding embodiments in detecting Tau protein for non-disease diagnosis or treatment purposes.

[0014] The present application has the following beneficial effects: The present application constructs a multi-phosphorylated tau complex protein by using specific biotin combination, solves the problem of excessive batch difference of reagents caused by the difference between existing calibrators and natural antigen epitopes, and breakthroughly improves the calibration performance. The present application can be adapted to various detection platforms such as chemiluminescence and enzyme-linked immunoassay, has strong universality, and has important clinical value and industry significance. DETAILED DESCRIPTION

[0015] In order to make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. The specific conditions not mentioned in the embodiments are carried out according to conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be obtained by market purchase.

[0016] The inventors discovered that commercially available recombinant phosphorylated Tau antigens cannot mimic the polyphosphorylation co-conformation of natural Tau proteins (such as the coexistence of multiple sites like p181 / p217), leading to inconsistent test results for different matrix samples (serum / cerebrospinal fluid). Differences between calibrators and natural antigen epitopes cause batch-to-batch variations (30%-60%), severely impacting the accuracy and reliability of test results and becoming a key bottleneck restricting the development of the in vitro diagnostics industry. The polyphosphorylated tau complex protein developed in this application can solve the problem of excessive batch-to-batch variations caused by differences between existing calibrators and natural antigen epitopes, possessing significant clinical value and industry implications.

[0017] On one hand, embodiments of the present invention provide a composite protein comprising: streptavidin and antigen A-Biotin A and antigen B-Biotin B linked to the streptavidin; wherein antigen A and antigen B are phosphorylated Tau protein antigens with different phosphorylation sites.

[0018] In an optional embodiment, biotin A and biotin B are the same or different activated biotin.

[0019] In an optional embodiment, the activated biotin includes at least one of NHS-PEG12-Biotin, Sulfo-NHS-LC-LC-Biotin, Sulfo-NHS-LC-Biotin, and NHS-PEG4-Biotin.

[0020] In an optional embodiment, biotin A is NHS-PEG12-Biotin, and biotin B is Sulfo-NHS-LC-LC-Biotin.

[0021] In an optional embodiment, biotin A is Sulfo-NHS-LC-LC-Biotin, and biotin B is Sulfo-NHS-LC-Biotin.

[0022] In an optional embodiment, biotin A is Sulfo-NHS-LC-Biotin, and biotin B is NHS-PEG4-Biotin.

[0023] In an optional embodiment, biotin A is NHS-PEG12-Biotin, and biotin B is Sulfo-NHS-LC-Biotin.

[0024] In an optional embodiment, antigen A and antigen B are monophosphorylated Tau protein antigens with different phosphorylation sites; antigen A is recombinant p-Tau181 and antigen B is p-Tau217; or antigen B is recombinant p-Tau181 and antigen A is p-Tau217.

[0025] On the other hand, embodiments of the present invention provide a method for preparing a composite protein as described in any of the foregoing embodiments, comprising: mixing and reacting antigen A-biotin A, antigen B-biotin B and streptavidin in the composite protein.

[0026] In an optional embodiment, antigen A-Biotin A and antigen B-Biotin B are mixed in a molar ratio of antigen A:antigen B of (1~3):(1~3); the molar ratio can be any one or any two of 1:1, 2:1, 3:1, 1:2, 1:3 and 2:3.

[0027] In an optional embodiment, antigen A-Biotin A and antigen B-Biotin B are mixed at a molar ratio of antigen A:antigen B of 1:1.

[0028] In an optional embodiment, the streptavidin is mixed at a molar ratio of streptavidin to total antigen of 1:(4~8), wherein the total antigen is the sum of antigen A and antigen B. This molar ratio can be any one of 1:4, 1:5, 1:6, 1:7, and 1:8, or a range between any two of these ratios.

[0029] In an optional embodiment, the conditions for the mixing reaction include: reacting at room temperature in the dark for 5 to 30 minutes.

[0030] Room temperature generally refers to 18~25℃, with 20~25℃ being ideal; this temperature can be any one or any two of 18, 19, 20, 21, 22, 23, 24 and 25℃; this time can be any one or any two of 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30 minutes.

[0031] In an optional embodiment, the preparation method further includes the preparation of a biotinylated antigen, wherein the biotinylated antigen is selected from the preparation of antigen A-Biotin A or antigen B-Biotin B.

[0032] The preparation of the biotinylated antigen includes: mixing the antigen and biotin at a molar ratio of 1:(10~30), adding buffer solution to the mixture to bring the antigen concentration to 0.5~2 mg / mL, and reacting at 25℃~37℃ for 1~2 h. The molar ratio can be any one or any two of 1:10, 1:12, 1:14, 1:16, 1:18, 1:20, 1:22, 1:24, 1:26, 1:28, and 1:30; the concentration can be any one or any two of 0.5, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, and 2 mg / mL; the temperature can be any one or any two of 25, 26, 28, 30, 32, 34, 36, and 37℃; and the time can be any one or any two of 1, 1.2, 1.4, 1.6, 1.8, and 2 h.

[0033] In an optional embodiment, the pH of the buffer solution is 7 to 8, specifically any one or any two of 7, 7.2, 7.4, 7.6, 7.8 and 8.

[0034] In an optional embodiment, after the mixing reaction, the preparation method further includes: blocking and / or purification.

[0035] In an optional embodiment, the blocking includes: adding a blocking agent to block the biotin binding site on the linker avidin.

[0036] In an optional embodiment, the sealing is achieved by adding free biotin and incubating at room temperature for at least 10 minutes; In an optional embodiment, the molar ratio of the free biotin to the streptomycin is 1 to 3:1, and can be 2:1.

[0037] In an optional embodiment, the purification includes: purification by molecular sieve chromatography, with the eluent being PBS buffer containing 0.02% (w / v) sodium azide, a flow rate of 0.5 mL / min, collection of the main peak component, and obtaining a composite antigen with a purity ≥90%.

[0038] On the other hand, embodiments of the present invention provide a composition comprising: the composite protein described in any of the foregoing embodiments or the composite protein prepared by the preparation method described in any of the foregoing embodiments.

[0039] In an optional embodiment, the composition is a calibrator.

[0040] In an optional embodiment, the calibrator further includes a diluent.

[0041] In an optional embodiment, the diluent comprises: a Tris-HCl buffer containing 5%–15% fetal bovine serum and 0.05%–2% BSA, with a pH of 7.6–8.0; the volume fraction of fetal bovine serum can be any one or any two of 5%, 6%, 8%, 10%, 12%, 14%, and 15%; the mass-volume fraction of BSA can be any one or any two of 0.05%, 0.06%, 0.08%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.5%, and 2%; and the pH can be any one or any two of 7.6, 7.7, 7.8, 7.9, and 8.0.

[0042] In an optional embodiment, the concentration of the complex protein can be 1~1000 pg / mL.

[0043] On the other hand, embodiments of the present invention provide a kit comprising: the composite protein described in any of the foregoing embodiments, the composite protein prepared by the preparation method described in any of the foregoing embodiments, or the composition described in any of the foregoing embodiments.

[0044] On the other hand, embodiments of the present invention provide the use of the composite protein as described in any of the foregoing embodiments, or the composite protein prepared by the preparation method described in any of the foregoing embodiments, or the composition described in any of the foregoing embodiments, in the preparation of a kit for Tau protein detection.

[0045] Furthermore, embodiments of the present invention provide the application of the composite protein as described in any of the foregoing embodiments, or the composite protein prepared by the preparation method described in any of the foregoing embodiments, or the composition described in any of the foregoing embodiments, or the kit described in any of the foregoing embodiments, in the detection of Tau protein for non-disease diagnostic or therapeutic purposes.

[0046] There are many scenarios for detecting Tau protein for non-disease diagnostic or therapeutic purposes, such as drug screening (rapidly screening lead compounds from a vast library of compounds that can inhibit abnormal Tau phosphorylation, aggregation, or promote its clearance), antibody and therapeutic molecular evaluation, and the development of new detection methods (using recombinant Tau protein or brain homogenate samples of known concentrations as standards and quality controls to validate the sensitivity, specificity, and repeatability of new methods).

[0047] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0048] I. Some of the experimental materials used in the examples Tau antigen monophosphorylation: recombinant p-Tau181 (molecular weight 55kD, purity 98%), recombinant p-Tau217 (molecular weight 58kD, purity 97%), catalog numbers: P00003CA, P00003CE, purchased from Iboson; Activated biotin: NHS-PEG12-Biotin, Sulfo-NHS-LC-LC-Biotin, Sulfo-NHS-LC-Biotin, NHS-PEG4-Biotin, purchased from Thermo Fisher Scientific. Streptavidin: purity ≥99%, molecular weight 65kD, catalog number: 434302, purchased from Sigma-Aldrich; Clinical plasma samples (from 30 Alzheimer's patients and 30 healthy individuals), and immunoassay reagents (self-prepared pTau181 antigen test reagents, 3 different batches, referred to as reagent 1, reagent 2, and reagent 3).

[0049] II. Implementation Examples and Experimental Parameter Design 1. Reaction parameters for biotinylated antigen: antigen (1 mg / mL, 0.1 M PBS pH 7.2), biotin (10 mM), biotin to antigen molar ratio (20:1), reaction temperature (25℃), reaction time (2 h), removal of unreacted biotin, and collection of biotinylated antigen.

[0050] 2. Streptavidin bridging: Streptavidin (SA) (1 mg / mL), 0.1 M PBS pH 7.2. Biotinylated antigen 1 and biotinylated antigen 2, prepared separately in step 1, were mixed at a molar ratio of 1:1. PBS was added to a total volume of 1 mL. SA was added (at a streptavidin:total antigen molar ratio of 1:4), and the mixture was reacted at room temperature in the dark for 15 minutes.

[0051] 3. Termination of reaction: Add free biotin (molar ratio of free biotin to SA = 2:1) and react at room temperature for 10 minutes (to block unbound biotin sites and avoid non-specific binding).

[0052] 4. Purification: Unbound free biotin and biotinylated antigen are removed by molecular sieve chromatography to obtain a complex antigen (biotinylated antigen 1-streptavidin-biotinylated antigen 2 complex) with a purity ≥85%.

[0053] 5. Examples and biotin selection are as follows.

[0054] Table 1 Examples and Biotin Selection

[0055] Experimental Example 1: Epitope Exposure Rate The antigen epitope binding ability was detected using a competitive ELISA method with known specific antibodies (anti-p-Tau181 and / or p-Tau217 antibodies), and the epitope exposure rate (the percentage of antigen bound to the antibody out of the total antigen) was calculated.

[0056] In this study, control 1 was detected using an anti-p-Tau181 specific antibody, control 2 was detected using a p-Tau217 specific antibody, and the remaining examples were detected using anti-p-Tau181 and p-Tau217 specific antibodies, respectively. The exposure rate was the average of the epitope exposure rates obtained from the two antibody tests.

[0057] The results are shown in Table 2.

[0058] Table 2 Results

[0059] As shown in Table 2, the epitope exposure rate of Example 1 was significantly higher than that of other groups, while the epitope exposure rates of Examples 4-6 were significantly lower than those of other groups, and the differences among the remaining groups were not significant.

[0060] Experimental Example 2: Immunological Detection and Signal Recording Each polyphosphorylated antigen complex obtained from conjugation was diluted with diluent to create five gradient concentration complex antigen solutions. The luminescence values ​​of each gradient complex antigen solution and 30 clinical samples were detected using three different batches of pTau181 antigen detection reagents (reagent 1, reagent 2, and reagent 3). Each concentration / sample was tested three times, and the average value was taken as the final luminescence value. The luminescence value deviation between the clinical samples and the complex antigen solutions was calculated. A deviation within ±15% was considered acceptable for the antigen. Further statistical analysis of the test results was performed, calculating the difference between the deviation of the complex antigen solution and the clinical sample. A difference within ±15% was considered acceptable for further evaluation.

[0061] The results are as follows.

[0062] Table 3 Clinical Sample Testing and Deviation

[0063] Table 4. Comparison of commercial p-tau181 antigen detection and deviation.

[0064] Table 5. Detection and bias of commercial p-tau217 antigen in control 2.

[0065] Table 6. Antigen Detection and Deviation in Example 1

[0066] Table 7 Antigen Detection and Deviation in Example 2

[0067] Table 8. Antigen Detection and Deviation in Example 3

[0068] Table 9. Antigen Detection and Deviation in Example 4

[0069] Table 10. Antigen Detection and Deviation in Example 5

[0070] Table 11 Antigen Detection and Deviation in Example 6

[0071] Table 12 Antigen Detection and Deviation in Example 7

[0072] As shown in Tables 3-12, the deviations in testing multiple clinical samples using different batches of pTau181 antigen detection reagents were all within ±15%. However, the deviations in testing commercially available p-tau181 antigen or p-tau217 antigen solutions exceeded the acceptable deviation values. Testing with different concentrations of composite antigen solutions showed that Examples 1-3 and 7 met the requirements, while Examples 4-6 did not.

[0073] Further analysis of the discrepancies between the various composite antigen solutions and clinical samples yielded the following statistical results.

[0074] Table 13: Deviations between various embodiments and clinical samples

[0075] As shown in Table 13, the commercially available p-tau181 antigen or p-tau217 antigen Examples 4-6 did not meet the requirements, with a difference exceeding ±15%, while Examples 1-3 and 7 met the requirements.

[0076] Experimental Example 3 The antigens in Examples 1, 2, 3, and 7 were validated and evaluated. Standard curves were prepared using the antigen complex solutions from Examples 1, 2, 3, and 7, respectively. After calibrating different batches of reagents, the average luminescence value of the 30 clinical samples tested above was substituted into the standard curves of the corresponding batches of reagents obtained from the corresponding antigen complex solutions to obtain the concentration values ​​of the corresponding samples. The mean concentration deviation (MAD) of the samples was calculated. The concentration deviations of the same antigen complex solution obtained from the standard curves of three batches of reagents were compared, and the average value was calculated. An average deviation within 15% is acceptable. (The standard curves were fitted with four parameters, with the coordinate type X-log(Y), where X represents concentration and Y represents luminescence value.) The sample concentration deviations were calculated, and the results are as follows.

[0077] Table 14 shows the deviation results of different batches of reagents calibrated with antigen complex solutions in each example.

[0078] The results in Table 14 show that the reagent calibration results in Example 1 are the best.

[0079] Test Example 4 The antigens of Examples 1, 2, and 7 were subjected to stability testing: the qualified antigens were stored at 2-8℃ for 7 days and at 37℃ for 7 days, respectively. The deviation of the luminescence value under the two storage conditions was calculated, and the deviation was within ±10%, which is considered acceptable. The stability test results are as follows.

[0080] Table 15 Stability Results

[0081] Results and Analysis: The length, flexibility, and water solubility of the biotin spacer arm significantly affect the calibration results. Example 1 (NHS-PEG12-Biotin + SulfoNHS-LC-LC-Biotin / SA) effectively avoids steric hindrance between large proteins by combining the extremely long spacer arm of PEG12 (approximately 50 Å) with the long spacer arm of the dual LC (approximately 30 Å). Simultaneously, the water solubility of the Sulfo-NHS group protects the antigen conformation and phosphorylation sites, ultimately achieving a batch-to-batch concentration deviation of only 4.16%, with stability performance far superior to other combinations. Other combinations, due to their single spacer arm length or insufficient flexibility, result in low epitope exposure rates, weak coupling, and other issues, leading to batch-to-batch differences significantly exceeding the acceptable range and poor stability.

[0082] Experimental Example 5 Under the conditions of the biotin combination in Example 1 (NHS-PEG12-Biotin+SulfoNHS-LC-LC-Biotin / SA), the reaction system concentration (antigen concentration), the molar ratio of antigen to biotin, the reaction temperature of antigen to biotin, and the reaction time of antigen to biotin were adjusted and compared with those in Example 1. The remaining reaction parameters and steps were kept consistent with those in Example 1.

[0083] The evaluation results for the comparative examples are as follows.

[0084] Table 16 Experimental Setup and Results

[0085] Results Analysis and Conclusions Under different experimental conditions, the performance of Comparative Examples 4-6 was lower than that of Example 1, but overall met the requirements.

[0086] Effect of antigen concentration: Comparative Example 1 (0.5 mg / mL) had a low concentration, resulting in insufficient biotinylation reaction efficiency and a significantly lower epitope exposure rate than Example 1; Comparative Example 2 (2 mg / mL) had a high concentration, resulting in increased steric hindrance between antigen molecules, decreased biotin conjugation uniformity, and an epitope exposure rate and calibration effect that were still inferior to Example 1.

[0087] The effect of the biotin-antigen molar ratio: In Comparative Example 3 (1:10), the insufficient biotin ratio led to incomplete biotinylation, resulting in suboptimal epitope exposure rate (78.2%) and calibration batch-to-batch difference (18.3%). Although Comparative Example 4 (1:30) increased the epitope exposure rate (81.5%) with excessive biotin, the residual free biotin interfered with the specificity of the bridging reaction, and the calibration batch-to-batch difference (8.1%) was still higher than that of Example 1. In contrast, the 1:20 molar ratio in Example 1 achieved a precise balance between "sufficient biotin conjugation" and "control of residual free biotin," ultimately achieving optimal epitope exposure rate (86.4%) and calibration batch-to-batch difference (3.95%) across all dimensions.

[0088] Effect of reaction temperature: Although the antiepitope exposure rate of Comparative Example 5 (37°C) was close to that of Example 1, high temperature easily accelerated antigen conformational changes, and the final calibration batch-to-batch difference was still higher than that of Example 1 at 25°C, proving that 25°C is a milder and more efficient reaction temperature.

[0089] Effect of reaction time: In Comparative Example 6 (1h), due to insufficient reaction time, biotin did not bind completely to the antigen, resulting in a significant decrease in epitope exposure rate and calibration effect. In contrast, the 2h reaction time in Example 1 ensured that the coupling reaction was fully completed.

[0090] Through the multi-dimensional comparison of the above comparative examples, it can be fully demonstrated that the reaction parameters of Example 1 (antigen concentration 1 mg / mL, biotin-antigen molar ratio 1:10, reaction at 25°C for 2 h) are the optimal combination that can simultaneously meet the requirements of high epitope exposure rate and ultra-low batch-to-batch variation, and the technical effect is significantly better than other parameter combinations.

[0091] Experimental Example 6 Under the conditions of the biotin combination in Example 1 (NHS-PEG12-Biotin+Sulfo NHS-LC-LC-Biotin / SA), the ratio of biotin antigen to SA and the reaction time were adjusted and compared with Example 1, while the remaining reaction parameters and steps remained consistent with the example.

[0092] The evaluation results for the comparative examples are as follows.

[0093] Table 17 Results

[0094] Results Analysis and Conclusions As can be seen from the comparison of Comparative Examples 7-10, the molar ratio of SA to biotin antigen directly determines the sufficiency and specificity of the bridging reaction: When the molar ratios were 1:1, 1:2 and 1:6, the epitope exposure rate was less than 76% and the inter-batch concentration deviation exceeded 16.8%, indicating that when the SA ratio was insufficient, the biotinylated antigen could not be fully bridged, resulting in an incomplete complex antigen structure and severe epitope masking. When the molar ratio was increased to 1:8, the epitope exposure rate increased significantly to 82.3%, and the calibration batch-to-batch deviation decreased to 11.5%, but it was still higher than the 4.16% of Example 1, indicating that although excessive SA can improve bridging efficiency, it may introduce interference due to non-specific binding. In Example 1, when a molar ratio of 1:4 was used, the epitope exposure rate and calibration effect reached the optimal level (86.4% and 4.16%, respectively), proving that this ratio is a precise balance point between "bridging adequacy" and "response specificity," which exceeds the conventional understanding of those skilled in the art that "the higher the molar ratio, the better the effect."

[0095] Focusing on comparative examples 8 and 11-13 (with a fixed molar ratio of 1:4), it can be seen that the reaction time has a non-linear effect on the bridging effect, first increasing and then decreasing. The reaction time was too short (5 min, Comparative Example 11), the epitope exposure rate was only 79.8%, and the calibration deviation was 12.3%, because the bridging reaction was not fully completed; When the reaction time was too long (20 min, 30 min, Comparative Examples 12-13), the epitope exposure rate was slightly increased (81.2%-80.2%), but the calibration deviation increased to 15.3%-18.4%, presumably due to antigen conformation destruction or SA aggregation caused by the excessively long reaction time. The 15-minute reaction time in Example 1 achieved the optimal balance between "reaction adequacy" and "antigen stability," with significantly better epitope exposure rate and calibration deviation than other time groups.

[0096] This experiment, through multiple comparisons, clearly demonstrates that only when the molar ratio of SA to biotin antigen is 1:4 and the reaction time is 15 min (parameters of Example 1) can the optimal combination of epitope exposure rate (86.4%) and calibration batch-to-batch concentration deviation (4.16%) be achieved.

[0097] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A complex protein, characterized in that, It includes: Streptavidin and antigen A-Biotin A and antigen B-Biotin B linked to said streptavidin; Antigen A and antigen B are phosphorylated Tau protein antigens with different phosphorylation sites.

2. The composite protein according to claim 1, characterized in that, Biotin A and Biotin B may be the same or different activated biotins; Optionally, the activated biotin is selected from at least one of NHS-PEG12-Biotin, Sulfo-NHS-LC-LC-Biotin, Sulfo-NHS-LC-Biotin, and NHS-PEG4-Biotin; Optionally, biotin A is NHS-PEG12-Biotin, and biotin B is Sulfo-NHS-LC-LC-Biotin; Optionally, biotin A is Sulfo-NHS-LC-LC-Biotin, and biotin B is Sulfo-NHS-LC-Biotin; Optionally, biotin A is Sulfo-NHS-LC-Biotin, and biotin B is NHS-PEG4-Biotin; Optionally, biotin A is NHS-PEG12-Biotin, and biotin B is Sulfo-NHS-LC-Biotin.

3. The composite protein according to claim 1, characterized in that, The antigen A and the antigen B are monophosphorylated Tau protein antigens with different phosphorylation sites; the antigen A is recombinant p-Tau181 and the antigen B is p-Tau217; or the antigen B is recombinant p-Tau181 and the antigen A is p-Tau217.

4. The method for preparing the complex protein according to any one of claims 1 to 3, characterized in that, It includes: The antigen A-Biotin A, antigen B-Biotin B and streptavidin in the complex protein are mixed and reacted. Optionally, antigen A-Biotin A and antigen B-Biotin B are mixed at a molar ratio of antigen A:antigen B of (1~3):(1~3); The streptavidin is mixed at a molar ratio of streptavidin to total antigen of 1:(4~8), wherein the total antigen is the sum of antigen A and antigen B; Optionally, the molar ratio of streptavidin to total biotinylated antigen is 1:4; Optionally, the conditions for the mixing reaction include: reacting at room temperature in the dark for 5 to 30 minutes; Optionally, the conditions for the mixed reaction include: reacting at room temperature in the dark for 14 to 16 minutes.

5. The preparation method according to claim 4, characterized in that, The preparation method further includes the preparation of biotinylated antigen, wherein the biotinylated antigen is selected from the preparation of antigen A-biotin A or antigen B-biotin B; The preparation of the biotinylated antigen includes: mixing the antigen and biotin at a molar ratio of 1:(10~30), adding buffer solution to the mixture until the antigen concentration is 0.5~2mg / mL, and the mixing conditions include reacting at 25℃~37℃ for 1~2h. Optionally, buffer solution is added to the mixture to bring the antigen concentration to 0.8–1.2 mg / mL; Optionally, the molar ratio of the antigen to the biotin is 1:(20~30). Optionally, the mixing conditions include: reacting at 25℃~30℃ for 1.5~2 hours; Optionally, the pH of the buffer solution is 7-8.

6. The preparation method according to claim 4, characterized in that, After the mixing reaction, the preparation method further includes: blocking and / or purification; Optionally, the sealing is achieved by adding free biotin and incubating at room temperature for at least 10 minutes; Optionally, the molar ratio of the free biotin to the streptomycin is 1 to 3:

1.

7. A composition, characterized in that, It includes: the composite protein according to any one of claims 1 to 4 or the composite protein prepared by the preparation method according to any one of claims 4 to 6; Optionally, the composition is a calibrator; Optionally, the calibrator may further include a diluent.

8. A reagent kit, characterized in that, It includes: The composite protein according to any one of claims 1 to 4, or the composite protein prepared by the preparation method according to any one of claims 4 to 6, or the composition according to claim 7.

9. The use of the composite protein according to any one of claims 1 to 4, or the composite protein prepared by the preparation method according to any one of claims 4 to 6, or the composition according to claim 7, in the preparation of a kit for Tau protein detection.

10. The use of the composite protein according to any one of claims 1 to 4, or the composite protein prepared by the preparation method according to any one of claims 4 to 6, or the composition according to claim 7, or the kit according to claim 8, in the detection of Tau protein for non-disease diagnostic or therapeutic purposes.

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