Fusion proteins, complexes, reagent combinations, products and uses for detecting the amount of abeta 1-42

Abstract

The present application relates to the biomedical technology field, specifically relates to a kind of fusion protein, complex, reagent combination, product for detecting A beta 1-42 content and application.The fusion protein provided by the present application includes detection antibody, avidin tag and cyan fluorescent protein;The detection antibody can specifically bind to the antigen A beta 1-42 to be measured, and the detection antibody includes Fc region;The avidin tag and cyan fluorescent protein are sequentially connected to the carboxyl end of the Fc region.The fusion protein provided by the present application is low in cost, and batch difference is stable and controllable, simple to operate, high in sensitivity for detecting A beta 1-42 in serum, accurate in detection result, and short in reaction time.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a fusion protein, complex, reagent combination, product for detecting Aβ1-42 content, and its application. Background Technology

[0002] Alzheimer's disease (AD) is a chronic, progressive neurodegenerative disease. The main clinical symptoms include cognitive impairment (such as memory decline, language impairment, and executive function impairment), behavioral abnormalities (such as hallucinations and aggressive behavior), and a decline in daily living abilities. It is one of the most common causes of cognitive impairment in old age. The pathological mechanism involves the formation of neurofibrillary tangles due to abnormal phosphorylation of tau protein within neurons and plaques (commonly known as senile plaques) in the brain caused by the deposition of β-amyloid-beta-protein (Aβ). Aβ is composed of two polypeptide fragments: one consisting of 40 amino acids and the other of 42 amino acids, formed by the continuous hydrolysis of β-amyloid precursor protein (APP) by β-protease and γ-protease. The former, Aβ1-40, is present in the brain of all individuals; the latter, Aβ1-42, is mainly found in the brains of AD patients. Aβ circulates in the blood, cerebrospinal fluid, and cerebrospinal fluid, mostly bound to chaperone protein molecules, with a small amount existing in a free state. In human cerebrospinal fluid and blood, the levels of Aβ1-40 are 10 times and 1.5 times higher than those of Aβ1-42, respectively. Recent studies have shown that the long peptide chain of Aβ1-42 is highly unstable and readily polymerizes to form oligomers and insoluble protofibrils and fibrils. These insoluble Aβ fibers deposit in brain tissue, forming senile plaques, and increased Aβ1-42 expression is closely related to the pathogenesis of Alzheimer's disease (AD).

[0003] Clinically, common methods for measuring Aβ protein in the brain include cerebrospinal fluid analysis and positron emission tomography (PET). However, cerebrospinal fluid testing is invasive and has low patient acceptance, while the high cost of PET examinations limits its widespread application. Therefore, studying Aβ protein through peripheral body fluids has attracted considerable attention. Aβ monomers can be cleared from the brain into the bloodstream through various pathways, such as the blood-brain barrier clearance system and the cerebrospinal fluid absorption and clearance circulatory system. However, due to the very low concentrations of Aβ1-40 and Aβ1-42 in plasma, the varying sensitivities of different detection techniques lead to significant discrepancies in results. Currently, the median Aβ1-42 level in normal human plasma is approximately 73.4-82.7 pg / mL, while the median level in AD patients is approximately 56.5-70.9 pg / mL. Therefore, there is an urgent need for highly sensitive reagent kits for clinical diagnosis.

[0004] Currently available testing products, such as colloidal gold immunochromatography, suffer from significant batch-to-batch variability and low sensitivity. Due to their physical adsorption method, antigens / antibodies easily detach from the gold particle surface, leading to unstable labeling. They can only provide qualitative or semi-quantitative results. Enzyme-linked immunosorbent assays (ELISA) are complex, time-consuming, require specialized personnel, and are susceptible to numerous influencing factors, easily resulting in false positives and false negatives. While the Simoa-based single-molecule immunoassay system from CaiKe Biotechnology boasts a sensitivity of up to 0.75 pg / mL, it requires a specialized AXL / SXL single-molecule immunoassay analyzer, making it expensive and hindering widespread adoption. Similarly, while liquid-phase chip assays offer high sensitivity (0.5-5 pg / mL) and can detect multiple indicators, they are time-consuming and costly, limiting their application in general hospitals outside of tertiary hospitals. Therefore, developing a chemiluminescent assay (such as a test kit) for detecting serum Aβ1-42 levels—with high sensitivity, accurate results, short reaction time, simple operation, low cost, and minimal batch-to-batch variability—is essential. Summary of the Invention

[0005] The purpose of this invention is to provide a fusion protein, a complex, a reagent combination, a product for detecting Aβ1-42 content, and its application. The fusion protein provided by this invention is low in cost, has stable and controllable batch-to-batch differences, is simple to operate, and has high sensitivity, accurate detection results, and short reaction time for detecting Aβ1-42 in serum.

[0006] The first aspect of the present invention provides a fusion protein comprising a detection antibody, an avidin tag, and a cyan fluorescent protein; The detection antibody can specifically bind to the antigen Aβ1-42 to be tested, and the detection antibody includes an Fc region; The avidin tag and cyan fluorescent protein are sequentially linked to the carboxyl terminus of the Fc region.

[0007] In some embodiments, the avidin tag includes at least one of Avi-tag and BSP-tag; In some embodiments, the avidin tag is inserted in either a forward or reverse direction. In some embodiments, the number of avidin tags is 1 to 4, and optionally 1 to 2.

[0008] In some embodiments, the cyan fluorescent protein includes mTurquoise2.

[0009] In some embodiments, the detection antibody includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes CDRH1 to CDRH3 as shown in SEQ ID NO: 1 to 3 respectively; the light chain variable region includes CDRL1 to CDRL3; the amino acid sequence of CDRL1 is shown in SEQ ID NO: 4, the amino acid sequence of CDRL2 is TAS, and the amino acid sequence of CDRL3 is shown in SEQ ID NO: 5. In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 6; In some embodiments, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 7; In some embodiments, the detection antibody is a monoclonal antibody; In some embodiments, the antibody further includes a light chain constant region selected from kappa or lambda isotypes; In some embodiments, the antibody further includes a heavy chain constant region selected from IgG1, IgG2, IgG3, and IgG4 subtypes.

[0010] The fusion protein of this invention is produced by directionally inserting one or more avidin tags (such as Avi-Tag) and cyan fluorescent proteins (such as mTurquoise2) into the Fc terminus of an antibody using genetic engineering recombination technology. This allows for direct and mild biotinylation modification within the cell. The expressed fusion protein does not affect the binding site at the Fab terminus of the antibody, solving the problem of uncontrollable batch-to-batch variability caused by random coupling of arbitrary functional groups in traditional biotin labeling. Simultaneously, it reduces the high background value caused by the amplification cascade effect of the biotin-streptavidin-acridone ester complex, i.e., reducing background noise and the probability of false positives, thereby significantly improving the positive signal-to-noise ratio and enhancing reagent sensitivity and accuracy. Furthermore, the generation of the fusion protein as a gene expression product is subject to strict biological regulatory network constraints. This regulation exhibits high robustness and consistency, resulting in smaller batch-to-batch variability in the prepared fusion protein.

[0011] In some embodiments, the fusion protein is an expression product of gene recombination.

[0012] In some embodiments, the fusion protein is obtained by expressing a fusion protein encoding gene in a host cell; A second aspect of the present invention provides a complex comprising the above-described fusion protein and biotin; wherein the biotin is attached to the avidin tag.

[0013] A third aspect of the present invention provides a reagent combination comprising: Reagent R1 comprises a solid support and a capture antibody coupled to the surface of the solid support; Reagent R2 comprises streptavidin (SA) and an acridine derivative coupled to said streptavidin; Reagent R3 includes the above-mentioned fusion protein or the above-mentioned complex.

[0014] In some embodiments, the solid support in reagent R1 comprises magnetic microparticles; In some embodiments, the surface of the magnetic microspheres has active groups; in some embodiments, the active groups include carboxyl groups. In some embodiments, the capture antibody specifically binds to the test antigen Aβ1-42, and the detection antibody, the test antigen Aβ1-42, and the capture antibody can form a double antibody sandwich structure.

[0015] In some embodiments, the acridine derivative in reagent R2 includes at least one of acridine ester, acridine acid, acridine amide, and acridine sulfonamide; In some embodiments, the acridine derivative includes at least one selected from the group consisting of: NSP-SA-NHS, NSP-DMAE-NHS, ME-DMAE-NHS, NSP-DMAE-HEG-Glu-NHS, NSP-SA-ADH, and DMAE-NHS.

[0016] A fourth aspect of the present invention provides a product for detecting the content of Aβ1-42, the product comprising the above-described fusion protein, the above-described complex, or the above-described reagent combination; In some embodiments, the product includes at least one of the following: reagents, reagent kits, and instrument platforms.

[0017] The fifth aspect of the present invention provides the use of the above-described fusion protein, the above-described complex, the above-described reagent combination, or the above-described product in the preparation of a product for diagnosing Alzheimer's disease.

[0018] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: The fusion protein provided by this invention is low in cost, has stable and controllable batch-to-batch differences, and is simple to operate. It has high detection sensitivity, accurate detection results, and short detection reaction time for detecting Aβ1-42 in serum.

[0019] The fusion protein provided by this invention fuses an avidin tag and a cyan fluorescent protein gene sequence with the Fc terminus of an antibody using genetic engineering recombination technology. One or more Avi-Tag and mTurquoise2 fluorescent proteins are inserted into the Fc terminus of the antibody, and the biotinylation modification is carried out directly and mildly in the cell. The resulting novel biotin-antibody multifunctional fusion protein complex does not affect the binding site at the Fab terminus of the antibody, thus solving the problem of uncontrollable batch-to-batch differences in random conjugation of arbitrary functional groups during traditional biotin labeling.

[0020] The reagent combination provided by this invention, used for chemiluminescent immunoassay, can reduce the high background value caused by the amplification cascade effect of the biotin and streptavidin-acridone ester complex, i.e., reduce background noise and false positive probability, thereby significantly improving the positive signal-to-noise ratio and thus enhancing reagent sensitivity and accuracy. Furthermore, the generation of fusion proteins as gene expression products is subject to strict biological regulatory networks. This regulation is highly robust and consistent, ensuring the uniformity and large-scale production of fusion protein products, which is beneficial for controlling batch-to-batch variation of fusion proteins, thus resulting in smaller batch-to-batch variation in the combined reagents.

[0021] The detection method of the kit prepared by the fusion protein-biotin complex provided by this invention is simple, easy to operate, and less time-consuming. The batch-to-batch variation of the reagent is stable and controllable, which can significantly improve the signal-to-noise ratio and sensitivity of the detection reagent. Attached Figure Description

[0022] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. In the drawings: Figure 1 A schematic diagram of the structural simulation of the fusion protein-biotin complex prepared in Example 1 of this invention and the control detection antibody prepared in Comparative Example 1; Figure 2 SDS-PAGE results of the fusion protein-biotin complex in Example 1 of this invention; Figure 3 Purity identification of the fusion protein-biotin complex in Example 1 of this invention; Figure 4 The curve of sample concentration versus luminescence value in Example 2 of reagent combination performance testing; Figure 5 A schematic diagram illustrating the binding principle of each component in the formulation combination of the present invention in Example 2; Figure 6 A schematic diagram illustrating the binding principle of each component in the formulation combination of Comparative Example 1 of this invention. Detailed Implementation

[0023] Exemplary embodiments of this disclosure will now be described in more detail. It should be understood that this disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of this disclosure to those skilled in the art.

[0024] Aβ1-42 peptide, mainly found in the brains of Alzheimer's disease patients, circulates in the blood, cerebrospinal fluid, and cerebrospinal fluid. Studies have shown that increased expression levels of Aβ1-42 are closely related to the course of AD. However, due to the extremely low concentration of Aβ1-42 in plasma and the varying sensitivity of detection techniques, there are significant differences in the results of peripheral fluid tests for Alzheimer's disease.

[0025] Currently available testing products, such as colloidal gold immunochromatography, suffer from significant batch-to-batch variability and low sensitivity. Due to their physical adsorption method, antigens / antibodies easily detach from the gold particle surface, leading to unstable labeling. They can only provide qualitative or semi-quantitative results. Enzyme-linked immunosorbent assays (ELISA) are complex, time-consuming, require specialized personnel, and are susceptible to numerous influencing factors, resulting in false positives and false negatives. While the Simoa-based single-molecule immunoassay system from CaiKe Biotechnology boasts a sensitivity of 0.75 pg / mL, it requires a specialized AXL / SXL single-molecule immunoassay analyzer, making it expensive and hindering widespread adoption. Similarly, while liquid-phase chip assays offer sensitivities of 0.5-5 pg / mL and can detect multiple indicators, they are time-consuming and costly, limiting their application in general hospitals outside of tertiary hospitals. Therefore, developing a chemiluminescent assay (such as a test kit) for detecting serum Aβ1-42 levels that offers high sensitivity, accurate results, short reaction time, simple operation, low cost, and minimal batch-to-batch variability is essential.

[0026] The biotin-avidin system (BAS) is a novel biological reaction amplification system applied in immunology and experiencing rapid development. Avidin and biotin have an extremely strong affinity, with an affinity constant (Ka) of 10⁻⁶. 15M, once bound to biotin, avidin is unaffected by pH, temperature, organic solvents, and denaturing agents (dissociation only occurs when using denaturing agents and at temperatures above 90°C). Due to its high affinity for biotin and avidin and its multi-stage amplification effect, it can be organically combined with immunolabeling techniques such as fluorescein, enzymes, and isotopes, further improving the specificity and sensitivity of various immunoassays. Biotin can bind with high affinity to avidin and streptavindin (SA), with an affinity millions of times higher than that of antigen-antibody binding, making it a useful label for protein detection and purification. Simultaneously, one SA can bind to four biotin molecules, facilitating cascade signal amplification during detection. However, existing biotin labeling involves random labeling of amino, carbonyl, thiol, and carboxylic acid groups on proteins, which can lead to uncontrollable batch-to-batch variations due to binding efficiency issues. If the antigen binding site is occupied by biotin, it may prevent the double-antibody sandwich from binding to the analyte, resulting in missed detections. Biotin protein ligase (BPL) catalyzes the biotinylation of lysine (K) at position 10 of the avidin tag (Avi-Tag), covalently linking biotin to the target protein in vivo. Therefore, Avi-tag technology can utilize BPL-catalyzed reactions to label biotin, achieving targeted and quantitative labeling and effectively controlling batch-to-batch variations.

[0027] Green fluorescent protein (GFP) is a protein composed of approximately 238 amino acids that can be excited by light ranging from blue to ultraviolet, emitting green fluorescence. In cell biology and molecular biology, the GFP gene is commonly used as a reporter gene. Through genetic engineering, the GFP gene can be transferred into the genomes of different species and continuously expressed in offspring. Cyan fluorescent protein mTurquoise2 (K206A mutant) is a variant of GFP, with an excitation wavelength of 433 nm and an emission wavelength of 475 nm. Its excitation wavelength falls within the luminescence range of acridine ester, achieving a luminescence efficiency of up to 93%, significantly higher than GFP, and exhibiting stronger photostability than the commonly used FITC luciferin. When conjugated with an antibody and then with acridine ester to form a complex, it can absorb photons released by the electronically excited N-methylacridone state of acridine ester in alkaline H2O2 solution. This luminescence significantly enhances luminescence efficiency and stably increases the positive signal. The NSP-SA-NHS acridine sulfonamide selected in this invention has an emission wavelength of approximately 430 nm. Its key feature is the attachment of a sulfonamide group (-SO2NHR) at the C-9 position of the acridine ring. This group enhances water solubility and hydrolytic stability, while simultaneously reducing photosensitivity through a conjugation effect, resulting in greater stability. Furthermore, NSP-SA-NHS acridine sulfonamide can be conjugated to biomolecules (such as streptavidin, antibodies, and antigens) via the NHS active ester. Its combination with the cyan fluorescent protein mTurquoise2 effectively controls background levels.

[0028] The first aspect of the present invention provides a fusion protein comprising a detection antibody, an avidin tag, and a cyan fluorescent protein; The detection antibody can specifically bind to the antigen Aβ1-42 to be tested, and the detection antibody includes an Fc region; The avidin tag and cyan fluorescent protein are sequentially linked to the carboxyl terminus of the Fc region.

[0029] In some embodiments, the avidin tag includes at least one of Avi-tag and BSP-tag; In some embodiments, the avidin tag is inserted in either a forward or reverse direction. In some embodiments, the number of avidin tags is 1 to 4, and optionally 1 to 2.

[0030] In some embodiments, the cyan fluorescent protein includes mTurquoise2.

[0031] In some embodiments, the detection antibody includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes CDRH1 to CDRH3 as shown in SEQ ID NO: 1 to 3 respectively; the light chain variable region includes CDRL1 to CDRL3; the amino acid sequence of CDRL1 is shown in SEQ ID NO: 4, the amino acid sequence of CDRL2 is TAS, and the amino acid sequence of CDRL3 is shown in SEQ ID NO: 5. In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 6; In some embodiments, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 7; In some embodiments, the detection antibody is a monoclonal antibody; In some embodiments, the antibody further includes a light chain constant region selected from kappa or lambda isotypes; In some embodiments, the antibody further includes a heavy chain constant region selected from IgG1, IgG2, IgG3, and IgG4 subtypes.

[0032] The fusion protein of this invention is produced by directionally inserting multiple Avi-Tag and mTurquoise2 luciferin proteins into the Fc terminus of an antibody using genetic engineering recombination technology. This allows for direct and mild biotinylation modification within the cell. The expressed fusion protein does not affect the binding site at the Fab terminus of the antibody, solving the problem of uncontrollable batch-to-batch variation in traditional biotinylation with random conjugation of arbitrary functional groups. Simultaneously, it reduces the high background value caused by the amplification cascade effect of the biotin-streptavidin-acridone ester complex, thus lowering the background and false positive probability, significantly improving the positive signal-to-noise ratio, and enhancing reagent sensitivity and accuracy. Furthermore, the generation of the fusion protein as a gene expression product is subject to strict biological regulatory network constraints. This regulation exhibits high robustness and consistency, resulting in smaller batch-to-batch variation in the prepared fusion protein.

[0033] In some embodiments, the fusion protein is an expression product of gene recombination.

[0034] A second aspect of the present invention provides a complex characterized in that it comprises a fusion protein according to any one of claims 1 to 3, and biotin; wherein the biotin is attached to the avidin tag.

[0035] In some embodiments, the fusion protein is obtained by expressing a fusion protein encoding gene in a host cell; In some embodiments, the fusion protein encoding gene includes: a detection antibody encoding gene, an avidin tag encoding gene, and a cyan fluorescent protein encoding gene; In some embodiments, the method for preparing the above-mentioned complex of the present invention includes the following steps: 1) Editing the gene encoding the fusion protein; 2) The fusion protein encoding gene obtained in step 1) is embedded into a vector and then transferred into a host cell for expression to obtain the fusion protein.

[0036] In some implementations, the vector includes plasmids, viruses, bacteriophages, and artificial chromosomes; In some implementations, the host cell includes HEK293F cells.

[0037] Recombinant genetic engineering technology can directionally insert one or more Avi-Tag and mTurquoise2 luciferin proteins into the Fc terminus of antibodies, enabling direct and mild biotinylation modification within cells. The expressed fusion protein does not affect the binding site at the Fab terminus of the antibody, solving the problem of uncontrollable batch-to-batch variation in random conjugation of arbitrary functional groups during traditional biotin labeling. Simultaneously, it can reduce the high background value caused by the amplification cascade effect of the biotin-streptavidin-acridone ester complex, i.e., reducing background noise and false positive probability, thereby significantly improving the positive signal-to-noise ratio and enhancing reagent sensitivity and accuracy.

[0038] A third aspect of the present invention provides a reagent combination comprising: Reagent R1 comprises a solid support and a capture antibody coupled to the surface of the solid support; Reagent R2 comprises streptavidin (SA) and an acridine derivative coupled to said streptavidin; Reagent R3 includes the above-mentioned fusion protein or the above-mentioned complex.

[0039] In some embodiments, the solid support in reagent R1 comprises magnetic microparticles; In some embodiments, the surface of the magnetic microspheres has active groups; in some embodiments, the active groups include carboxyl groups. In some embodiments, the capture antibody specifically binds to the test antigen Aβ1-42, and the detection antibody, the test antigen Aβ1-42, and the capture antibody can form a double antibody sandwich structure.

[0040] In some embodiments, the acridine derivative in reagent R2 includes at least one of acridine ester, acridine acid, acridine amide, and acridine sulfonamide; In some embodiments, the acridine derivative includes at least one selected from the group consisting of: NSP-SA-NHS, NSP-DMAE-NHS, ME-DMAE-NHS, NSP-DMAE-HEG-Glu-NHS, NSP-SA-ADH, and DMAE-NHS.

[0041] A fourth aspect of the present invention provides a product for detecting the content of Aβ1-42, the product comprising the above-described fusion protein, the above-described complex, or the above-described reagent combination; In some embodiments, the product includes at least one of the following: reagents, reagent kits, and instrument platforms.

[0042] In some embodiments, a kit is provided comprising the above-described fusion protein, the above-described complex, or the above-described reagent combination. In some embodiments, the kit further comprises at least one of HNO3-H2O2 solution, NaOH solution, citrate-sodium citrate buffer, and disodium hydrogen phosphate (12-water)-sodium dihydrogen phosphate buffer.

[0043] The fifth aspect of the present invention provides the use of the above-described fusion protein, the above-described complex, the above-described reagent combination, or the above-described product in the preparation of a product for diagnosing Alzheimer's disease.

[0044] The sixth aspect of this invention provides a method for detecting the content of Aβ1-42, comprising: The sample to be tested is mixed with a solution containing reagent R1 and a solution containing reagent R3 and reacted. The mixture is then magnetically separated. A solution containing reagent R2 is then added for a second reaction. After the second reaction, HNO3-H2O2 solution and NaOH solution are added. The luminescence value is measured using a chemiluminescence immunoassay analyzer. The concentration of Aβ1-42 polypeptide antigen is detected based on the luminescence value. The reagent R1 in the solution containing reagent R1 includes: a solid support and a capture antibody coupled to the surface of the solid support; The reagent R2 in the solution containing reagent R2 includes: streptavidin (SA) and an acridine derivative coupled to the streptavidin; The reagent R3 in the solution containing reagent R3 includes: the above-mentioned fusion protein or the above-mentioned complex.

[0045] In some embodiments, the mass concentration of reagent R1 in the solution containing reagent R1 is 0.1~0.3 mg / mL; The mass concentration of reagent R2 in the solution containing reagent R2 is 0.125~0.5μg / mL; The mass concentration of reagent R3 in the solution containing reagent R3 is 0.2~1.6 μg / mL; The mixing reaction temperature is 36~38℃, and the mixing reaction time is 5~15min; The secondary reaction temperature is 36~38℃, and the mixing reaction time is 3~10min; The volume ratio of the sample to be tested to the solution containing reagent R1 and the solution containing reagent R3 is (8~12):(45~55):(45~55). The volume ratio of the sample to be tested to the solution containing reagent R2 is 10:(40~60). The volume ratio of the sample to be tested to the HNO3-H2O2 solution and the NaOH solution is (8~11):(90~110):(90~110).

[0046] The HNO3-H2O2 solution was selected from the pre-trigger solution (Pre-Trigger Solution (1×) (International) 500mL, catalog number C89959) produced by Shenzhen Yahuilong Biotechnology Co., Ltd. The NaOH solution was selected from the activation solution (TriggerSolution Trigger Solution (1×) (International) 500mL, catalog number C89958) (NaOH solution) produced by Shenzhen Yahuilong Biotechnology Co., Ltd.

[0047] In some embodiments, the mass concentration of reagent R1 in the solution containing reagent R1 is 0.2 mg / mL; The mass concentration of reagent R2 in the solution containing reagent R2 is 0.125~0.5μg / mL; The mass concentration of reagent R3 in the solution containing reagent R3 is 0.2~1.6 μg / mL; The mixing reaction temperature is 37°C, and the mixing reaction time is 10 min; The secondary reaction temperature is 37°C, and the mixing reaction time is 5 minutes. The volume ratio of the sample to be tested to the solution containing reagent R1 and the solution containing reagent R3 is 10:50:50. The volume ratio of the sample to be tested to the solution containing reagent R2 is 10:50; The volume ratio of the sample to be tested to the HNO3-H2O2 solution and the NaOH solution is 10:100:100.

[0048] In some embodiments, the sample to be tested can be a serum sample or a human cerebrospinal fluid sample.

[0049] In some implementations, the above-described method for detecting Aβ1-42 levels is used for the clinical diagnosis of Alzheimer's disease.

[0050] The seventh aspect of the present invention provides the use of the above-described fusion protein, the above-described complex, the above-described reagent combination, or the above-described product in the diagnosis of Alzheimer's disease.

[0051] The following description provides examples of the invention, thereby making the advantages and various effects of the invention more clearly apparent. Those skilled in the art should understand that these examples are illustrative and not intended to limit the invention. Unless otherwise specified, all reagents used herein were purchased from Thermo Fisher Scientific and were of analytical grade. The luminescence instruments, diluents, acridinium esters, and monoclonal antibodies used herein were provided by Shenzhen Yahuilong Biotechnology Co., Ltd.

[0052] Example 1 This embodiment provides a method for preparing a fusion protein-biotin complex, comprising the following steps: Step 1: Design of fusion genes The structural design of fusion proteins needs to take into account the antibody's Aβ1-42 binding activity, Avi-tag biotinylation activity, and mTurquoise2 protein expression activity.

[0053] 1. Obtaining the Aβ1-42 monoclonal antibody gene Access the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), and click "GenBank" or "Nucleotide" at the top to enter the nucleotide database. Enter accession number NC_000014.9 (IGHG1, immunoglobulin heavy constant γ1) to obtain the 107043718 bp heavy chain constant region gene DNA sequence, i.e., the CH1-CH2-CH3 sequence. Enter accession number NC_000002.12 (IGKC, immunoglobulin kappa constant) to obtain the 242193529 bp light chain constant region gene DNA sequence, i.e., the CL sequence. The amino acid sequences of the Aβ1-42 light and heavy chain variable regions are shown in Table 1.

[0054] Table 1

[0055] 2. Obtaining Avi-tags Access the NCBI website and enter the login number NC_000913.3 to obtain the Avi-tag gene that can be stably expressed in E. coli. Its nucleotide and amino acid sequences are shown in Table 2.

[0056] Table 2

[0057] 3. Obtaining the cyan fluorescent protein mTurquoise2 (K206A mutant) gene Access the RCSB PDB website (https: / / www.rcsb.org / structure), enter accession number 3ZTF, and obtain the mature gene sequence encoding 735 amino acids, which can express a protein of 27.68KD. The amino acid and nucleotide sequences of the cyan fluorescent protein mTurquoise2 are shown in Table 3.

[0058] Table 3

[0059] 4. Integrated structural design First, one or two Avi-tag amino acid sequences are linked to the mTurquoise2 amino acid sequence to form Avi-tag-mTurquoise2 and Avi-tag-Avi-tag-mTurquoise2 fusion amino acid sequences, respectively.

[0060] Next, Avi-tag-mTurquoise2 was inserted forward into the amino terminus (N') of the CH3 end of the Fc region of IgG (Aβ1-42 monoclonal antibody), and the antibody expressed was named Aβ1-42 AbN; Avi-tag-mTurquoise2 was inserted forward into the carboxyl terminus (C') of the CH3 end of the Fc region of IgG (Aβ1-42 monoclonal antibody), and the antibody expressed was named Aβ1-42 AbC; Avi-tag-Avi-tag-mTurquoise2 was inserted forward into the carboxyl terminus (C') of the CH3 end of the Fc region of IgG (Aβ1-42 monoclonal antibody), and the antibody expressed was named Aβ1-42 AbC2.

[0061] Three types of "heavy chain-Avi-tag-mTurquoise2" fusion genes: The heavy chain structure of Aβ1-42 AbN is as follows: VH-CH1-CH2-CH3-(N')Avi-tag-mTurquoise2; The heavy chain structure of Aβ1-42 AbC is as follows: VH-CH1-CH2-CH3-(C')Avi-tag-mTurquoise2; The heavy chain structure of Aβ1-42 AbC2 is as follows: VH-CH1-CH2-CH3-(C')Avi-tag-Avi-tag-mTurquoise2; Light chain genes: IgG light chain is a constant chain: VL-CL Step 2: Fusion gene synthesis and plasmid extraction The three "heavy chain-Avi-tag-mTurquoise2" fusion genes and light chain genes were optimized for humanization based on the gene preference codons of mammalian expression systems, and then ligated into the pCDNA3.1 vector. The fusion genes were synthesized and sequenced by Sangon Biotech (Shanghai) Co., Ltd. Four positive clones were obtained with correct sequencing and transformation: the heavy chain plasmid pCDNA3.1-Aβ1-42 AbN IgG-H, the heavy chain plasmid pCDNA3.1-Aβ1-42 AbC IgG-H, the heavy chain plasmid pCDNA3.1-Aβ1-42 AbC2 IgG-H, and the light chain plasmid pCDNA3.1-Aβ1-42 IgG-L.

[0062] The biotin ligase (BPL) gene was optimized for humanization based on the gene preference codons of mammalian expression systems, then ligated into the pCDNA3.1 vector. The resulting fusion gene was synthesized by General Biotechnology (Anhui) Co., Ltd., and sequenced. Positive clones of the correctly sequenced and transformed heavy chain plasmid pCDNA3.1-BPL were obtained.

[0063] After overnight culture of positive clones in ampicillin-resistant liquid LB medium, plasmids were extracted using a large-scale extraction kit, and DNA concentration (A260) and purity (A260 / A280) were detected using a micro spectrophotometer.

[0064] Step 3: HEK293F cell transfection and stable cell line selection 1. Cell pretreatment (1) 3-4 hours in advance, passage HEK293F cells into three 500mL cell culture flasks (200mL of cells were cultured), and culture them in SMM 293-TII medium until the cell viability reaches more than 95% at the time of transfection; 2. Instantaneous co-transfection The three heavy chain plasmids obtained in step 2 (pCDNA3.1-Aβ1-42 AbN IgG-H, pCDNA3.1-Aβ1-42AbC IgG-H, and pCDNA3.1-Aβ1-42 AbC2 IgG-H) were transiently co-transfected with the light chain plasmid (pCDNA3.1-Aβ1-42 IgG-L) obtained in step 2 to obtain pCDNA3.1-Aβ1-42 AbN IgG-H + pCDNA3.1-Aβ1-42 IgG-L vector DNA, pCDNA3.1-Aβ1-42 AbC IgG-H + pCDNA3.1-Aβ1-42 IgG-L vector DNA, and pCDNA3.1-Aβ1-42AbC2 IgG-H + pCDNA3.1-Aβ1-42 IgG-L vector DNA. The specific steps are as follows: (1) Take 6 sterile 15mL centrifuge tubes and label them as tube A, tube B1, tube B2, and tube B3. Add 7mL of PBS solution to each tube. Add 1.5mL of PEI MAX (1 mg / mL) to tube A and mix thoroughly. Add 133.3 μg of the light chain plasmid (pCDNA3.1-Aβ1-42 IgG-L) from step 2, 100μg of the heavy chain plasmid pCDNA3.1-Aβ1-42 AbN IgG-H from step 2, and 100μg of the pCDNA3.1-BPL plasmid (BPL represents biotin ligase, which is responsible for covalently coupling biotin in cells in Avi-tag technology) from step 2 and mix thoroughly. Add 133.3 μg of the light chain plasmid (pCDNA3.1-Aβ1-42 IgG-L) from step 2, 100μg of the heavy chain plasmid pCDNA3.1-Aβ1-42 AbC from step 2, and mix thoroughly. Mix 100 μg of IgG-H and pCDNA3.1-BPL plasmid thoroughly. Add 133.3 μg of the light chain plasmid (pCDNA3.1-Aβ1-42 IgG-L) from step 2, 66.7 μg of the heavy chain plasmid pCDNA3.1-Aβ1-42 AbC2 IgG-H from step 2, and 100 μg of pCDNA3.1-BPL plasmid to tube B3, and mix thoroughly. Slowly add the solutions from tubes A to tubes B1, B2, and B3 dropwise, mixing continuously. After adding all the solutions, gently invert each tube 3-5 times to mix thoroughly. Let the tubes stand at room temperature for 10 minutes.

[0065] (2) Add the mixed solution from tubes B1, B2, and B3 to the transferred cells until the number of HEK293F cells reaches 2.0 × 10⁻⁶. 6 cells / mL; (3) After transfection, the cells were placed back into the carbon dioxide shaker and cultured for another 48 hours. The temperature of the shaker was 36.9℃, the carbon dioxide concentration was 5%, and the rotation speed was 120 rpm. (4) Cell feeding: After 48 h of cell transfection culture, take out the conical flask and add 7 mL of SMS 293-SUPI feeding solution under a clean bench. Then put it back into the carbon dioxide shaker for culture. The culture conditions remain unchanged. After 48 h of culture, add another 7 mL of feeding solution and put it back into the carbon dioxide shaker for culture. Culture for another 48 h under the same conditions.

[0066] 3. Identification of expression clones Remove the cell culture medium and centrifuge at 9000 rpm, 4℃, for 10 min. After centrifugation, collect the cell supernatant. Detect the expression level of the fusion protein-biotin complex in the supernatant using ELISA. The fusion protein-biotin complex expressed by pCDNA3.1-Aβ1-42 AbN IgG-H+pCDNA3.1-Aβ1-42 IgG-L vector DNA after intracellular biotin covalent coupling is called Aβ1-42 AbN-Bio, the fusion protein-biotin complex expressed by pCDNA3.1-Aβ1-42 AbC IgG-H+pCDNA3.1-Aβ1-42 IgG-L vector DNA after intracellular biotin covalent coupling is called Aβ1-42 AbC-Bio, and the fusion protein-biotin complex expressed by pCDNA3.1-Aβ1-42 AbC2 IgG-H+pCDNA3.1-Aβ1-42 IgG-L vector DNA after intracellular biotin covalent coupling is called Aβ1-42 AbC2-Bio. The structural diagrams of Aβ1-42AbN-Bio, Aβ1-42AbC-Bio, and Aβ1-42AbC2-Bio are shown below. Figure 1 As shown. The specific steps are as follows: (1) Coating Aβ1-42 antigen at a concentration of 2 μg / mL, 100 μL per well, and incubating overnight at 4°C; (2) Block with 5% skim milk and incubate at 37°C for 1 hour; (3) Add cell supernatant (1:10 dilution) and incubate at 37°C for 1 h; (4) Add HRP-SA (1:5000 dilution) and incubate at 37°C for 1 h; (5) TMB color development, absorbance (OD value) measured at 450 nm to verify whether the fusion antibody was successfully expressed.

[0067] Step 4: Purification of the fusion protein-biotin complex The purification of the target fusion protein-biotin complex from HEK293F cell culture supernatant relies on the use of the "antibody Fab fragment".

[0068] 1. Supernatant Pretreatment (1) Centrifuge the cell supernatant from the expanded culture (9000 rpm, 10 min, 4℃) to remove cell debris; (2) Filter the supernatant with a 0.22μm filter membrane to remove macromolecular impurities.

[0069] 2. Protein A affinity chromatography (1) Equilibrate the column: Wash 5 mL of Amsphere A3 affinity chromatography column with 1×PBS (pH 7.4), flow rate: 3 mL / min, run 5CV; (2) Sample loading: The concentrated cell supernatant filtered through a 0.45 μM filter membrane was slowly loaded onto the column at a flow rate of 2 mL / min, and the breakthrough solution was collected; (3) Washing: Equilibrate the system with 1×PBS for 5 CV at a flow rate of 2 mL / min; (4) Elution: Elute the target fusion protein-biotin complex with 0.1 M glycine (pH 2.7), collect 2 mL from each tube, and immediately neutralize to pH 7.0 with 200 μL 1M Tris-HCl (pH 8.0) to avoid protein denaturation; (5) Detection: Use an ultraviolet spectrophotometer to detect the OD280 value of each tube and combine the components with OD280>0.1.

[0070] (6) Dialysis concentration: After centrifuging and filtering the purified sample, dialyze it with 100 times the volume of 1×PB (pH 8.0) and a 30kD dialysis card. The sample concentration should not be less than 1 mg / mL. (The sample volume should not exceed 150 mg).

[0071] Step 5: Identification and concentration of the fusion protein-biotin complex The target fusion protein-biotin complex was verified in terms of both molecular weight and purity.

[0072] 1. Molecular weight determination (SDS-PAGE) (1) Prepare a 10% SDS-PAGE gel; (2) Prepare a 40 μL sample from 40 μg of the protein to be tested using 5 × Loading buffer and PBS; (3) Place the sample in boiling water and boil for 10 min, then briefly separate it at 3000 r / min; (4) Load the samples in sequence, with the program set to 80 V for 30 min and 120 V for 45 min; (5) Stain with Coomassie Brilliant Blue staining solution for 1 h; (6) Use decolorizing solution to decolorize for 2 hours each time until the background color of the PAGE gel is transparent. Then develop the color under a developer and keep the image.

[0073] Aβ1-42 AbN-Bio and Aβ1-42 AbC-Bio have a daily energy of approximately 210 kDa, while Aβ1-42 AbC2-Bio has a daily energy of approximately 215 kDa. SDS-PAGE gel electrophoresis results are as follows... Figure 2As shown, the left side is the SDS-PAGE gel image of the reduced light and heavy chains, and the right side is the SDS-PAGE gel image of the unreduced intact antibody.

[0074] 2. Antibody dialysis (1) Concentrate the SDS-PAGE-validated samples to a concentration of 1.2-1.5 mg / mL.

[0075] (2) Select a 30kD dialysis bag with appropriate volume for dialysis. Place the sample to be dialyzed into a 1L beaker filled with 1×PBS (pH7.4), place it on a magnetic stirrer, stir at 200-250 rpm, and dialyze at 2-8℃ for 5-8 h.

[0076] (3) Transfer the dialysis sample to a new 1L beaker containing 1×PBS (pH 7.4), stir at 200-250 rpm, and continue dialysis at 2-8 ℃ for 12-16 h.

[0077] (4) Take out the dialysis sample, filter it through a 0.22 μm filter membrane, and detect the antibody concentration using the A280 method after zeroing the ultra-micro UV spectrophotometer.

[0078] 3. Purity determination (SEC-HPLC) (1) A size exclusion column MAbPac SEC-1 was used, with PBS (pH 7.4) as the mobile phase and a flow rate of 0.5 mL / min. (2) Load 20 μL of the concentrated fusion protein-biotin complex (1 mg / mL) and detect the absorption peak at 280 nm; (3) Calculate the area ratio of the main peak, purity > 98%, and the test results are as follows: Figure 3 As shown.

[0079] Example 2 This embodiment provides a method for preparing a streptavidin-acridone ester complex and a method for preparing a magnetic bead-Aβ1-42 capture antibody conjugate: The Aβ1-42 capture antibody (β-amyloid 1-42 (Aβ1-42), antibody catalog number AB1-42102) can be purchased from the Boyue Biotechnology website. Streptavidin and NSP-SA-NHS can be purchased from the Thermo Fisher Biotechnology website.

[0080] 1. Preparation of streptavidin-different acridine ester conjugates Due to the wide variety of acridine esters, various streptavidin-acrididine ester complexes were prepared to screen for acridine esters exhibiting a redshift effect. The following are three acridine esters selected from numerous screening examples of this invention that showed significant effects.

[0081] Streptavidin and NSP-SA-NHS acridinium ester coupling The C-9 position of the acridine ring in NSP-SA-NHS acridine ester is linked to a sulfonamide group (-SO2NHR), which enhances water solubility and hydrolytic stability, while also modulating the emission wavelength through a conjugation effect. Furthermore, the NHS active ester can be coupled with biomolecules such as antibodies and antigens.

[0082] The preparation method of streptavidin-NSP-SA-NHS acridinium ester conjugate (SA-AE1) includes the following steps: 1. Take 1 mg of streptavidin and add it to PBS solution (50 mM, pH 7.4) to a concentration of 0.5 mg / mL. Add 30 μL of DMSO solution of acridinium ester NSP-SA-NHS (4 mg / mL). The molar ratio of streptavidin to acridinium ester NSP-SA-NHS is approximately 1:2. React in a constant temperature incubator at 25℃ for 2 h. 2. Add 200 μL of PBS solution containing 10 g / L glycine to quench the reaction; 3. Purify using a 7 kDa desalting column to remove free acridine ester; 4. The acridine label is designated SA-AE1 and is stored by diluting it to 0.1 mg / mL using acridine ester preservation solution.

[0083] Streptavidin and NSP-DMAE-NHS acridinium ester coupling NSP-DMAE-NHS acridine ester introduces a sulfonic acid propyl group (-SOPr) into the DMAE-NHS base, which can slightly adjust the spectral characteristics through the conjugation effect.

[0084] The preparation method of streptavidin- and NSP-DMAE-NHS acridinium ester conjugate (SA-AE2) includes the following steps: 1. Take 1 mg of streptavidin and add it to PBS solution (50 mM, pH 7.4) to a concentration of 0.5 mg / mL. Add 30 μL of DMSO solution of acridinium ester NSP-DMAE-NHS (4 mg / mL). The molar ratio of streptavidin to acridinium ester NSP-DMAE-NHS is approximately 1:2. React in a constant temperature incubator at 25℃ for 2 h. 2. Add 200 μL of PBS solution containing 10 g / L glycine to quench the reaction; 3. Purify using a 7 kDa desalting column to remove free acridine ester; 4. The acridine label is designated SA-AE2 and is stored by diluting it to 0.1 mg / mL using acridine ester preservation solution.

[0085] Streptavidin and Me-DMAE-NHS acridinium ester coupling Me-DMAE-NHS analogs are trifluoromethyl substituents. The electron-withdrawing effect of the trifluoromethyl (CF3) substituent may cause a redshift effect. CF3 is an important fluorine-containing group. Introducing this functional group into the molecule often significantly alters the lipophilicity of the parent compound, enhances the molecule's metabolic stability, and exhibits high sensitivity and stability.

[0086] The preparation method of streptavidin-Me-DMAE-NHS acridinium ester conjugate (SA-AE3) includes the following steps: 1. Take 1 mg of streptavidin and add it to PBS solution (50 mM, pH 7.4) to a concentration of 0.5 mg / mL. Add 30 μL of DMSO solution of acridine ester Me-DMAE-NHS (4 mg / mL). The molar ratio of streptavidin to acridine ester Me-DMAE-NHS is approximately 1:2. React in a constant temperature incubator at 25℃ for 2 h. 2. Add 200 μL of PBS solution containing 10 g / L glycine to quench the reaction; 3. Purify using a 7 kDa desalting column to remove free acridine ester; 4. The acridine label is designated SA-AE3 and is stored by diluting it to 0.1 mg / mL using acridine ester preservation solution.

[0087] 2. Preparation of magnetic bead-Aβ1-42 capture antibody conjugate The preparation method of the magnetic bead-Aβ1-42 capture antibody conjugate includes the following steps: (1) Take 5 mL of carboxyl nanomagnetic beads (particle size 2 μm, 10 mg / mL, 600 nmol / mg) solution, process it in a magnetic separator for 3 minutes, remove the supernatant with a pipette, and wash 3 times with 50 mL of 100 mM MES solution (pH 6.0).

[0088] (2) Add 10 mL of 50 mM MES solution (pH 6.0) to the washed carboxyl nanomagnetic beads, add 1.5 mL of LEDC solution (concentration 10 mg / mL) and 1.5 mL of Sulfo-NHS solution (concentration 8 mg / mL), and react at 25 °C (40 rpm) for 1 h to obtain activated carboxyl magnetic beads.

[0089] (3) Prepare 5 mL of 100 mM phosphate buffer (pH 8.0) for Aβ1-42 capture antibody, with a concentration of 0.1 mg / mL. Heat in a water bath to 37°C and maintain this temperature for 2 h.

[0090] (4) Add the phosphate buffer of Aβ1-42 capture antibody to the activated carboxyl magnetic beads, control the pH of the mixed solution to 7.2, add 400 μL of DMAP aqueous solution (10 mg / mL), and rotate (60 rpm) at 37 °C for 24 h.

[0091] (5) Magnetic separation: Remove the supernatant with a pipette. Wash three times with 10 mL of 50 mM PBS solution containing 0.1% Tween-20 each time. Store the washed Aβ1-42 capture antibody-magnetic nanobeads in 5 mL of storage solution, with a storage concentration of 10 mg / mL based on the magnetic beads.

[0092] Comparative Example 1 This comparative example provides a method for preparing an Aβ1-42 detection antibody-biotin NHS-PEG2-Biotin conjugate, using a conventional labeling method: biotin-labeled Aβ1-42 detection antibody.

[0093] The Aβ1-42 detection antibody was prepared by Shenzhen Yahuilong Co., Ltd. The difference between this antibody and that in Example 1 is that the Fc terminus of the antibody is absent, making it a non-fusion antibody. Biotin (NHS-PEG2-Biotin) was purchased from the Thermo Fisher Scientific website. A schematic diagram of the structure of the Aβ1-42 detection antibody is shown below. Figure 1 As shown in Aβ1-42Ab.

[0094] The preparation method of Aβ1-42 detection antibody-biotin NHS-PEG2-Biotin conjugate (Aβ1-42-Bio) specifically includes the following steps: 1. Take 1 mg of Aβ1-42 detection antibody and add it to PBS solution (50 mM, pH 7.4) to a concentration of 0.5 mg / mL. Add 30 μL of DMSO solution of biotin NHS-PEG2-Biotin (10 mg / mL). The molar ratio of Aβ1-42 detection antibody to biotin NHS-PEG2-Biotin is approximately 1:2. React in a constant temperature incubator at 25℃ for 2 h. 2. Add 200 μL of PBS solution containing 10 g / L glycine to quench the reaction; 3. Purify using a 7 kDa desalting column to remove free biotin; 4. Biotin-labeled Aβ1-42-Bio was diluted to 0.1 mg / mL using acridinium ester preservation solution and stored.

[0095] Comparative Example 2 This comparative example provides a method for preparing an Aβ1-42 detection antibody-NSP-SA-NHS acridine ester conjugate, which uses a conventional labeling method to label the Aβ1-42 detection antibody-acrididine ester complex.

[0096] The preparation method of Aβ1-42 detection antibody-NSP-SA-NHS acridinium ester conjugate (Aβ1-42-AE) specifically includes the following steps: 1. Take 1 mg of Aβ1-42 detection antibody and add it to PBS solution (50 mM, pH 7.4) to a concentration of 0.5 mg / mL. Add 20 μL of DMSO solution of acridinium ester NSP-SA-NHS (4 mg / mL). The molar ratio of Aβ1-42 detection antibody to acridinium ester NSP-DMAE-NHS is approximately 1:2. React in a constant temperature incubator at 25℃ for 2 h. 2. Add 200 μL of PBS solution containing 10 g / L glycine to quench the reaction; 3. Purify using a 7 kDa desalting column to remove free biotin; 4. The acridine label is labeled Aβ1-42-AE, and the acridine ester preservation solution is diluted to (0.1 mg / mL) for storage.

[0097] Formulation combination examples and formulation combination comparative examples Formulation Combination Example 1 This formulation combination embodiment provides a formulation combination including reagent R1, reagent R2 and reagent R3: The reagent R1 includes: the magnetic bead-Aβ1-42 capture antibody conjugate prepared in Example 2 above; The reagent R2 includes: the streptavidin-NSP-SA-NHS acridinium ester conjugate (SA-AE1) prepared in Example 2 above. The reagent R3 includes: the fusion protein-biotin complex Aβ1-42 AbN-Bio prepared in Example 1 above.

[0098] Formulation Combination Example 2 This formulation combination embodiment provides a formulation combination including reagent R1, reagent R2 and reagent R3: The reagent R1 includes: the magnetic bead-Aβ1-42 capture antibody conjugate prepared in Example 2 above; The reagent R2 includes: the streptavidin-NSP-SA-NHS acridinium ester conjugate (SA-AE1) prepared in Example 2 above. The reagent R3 includes: the fusion protein-biotin complex Aβ1-42 AbC-Bio prepared in Example 1 above.

[0099] Formulation Combination Example 3 This formulation combination embodiment provides a formulation combination including reagent R1, reagent R2 and reagent R3: The reagent R1 includes: the magnetic bead-Aβ1-42 capture antibody conjugate prepared in Example 2 above; The reagent R2 includes: the streptavidin-NSP-SA-NHS acridinium ester conjugate (SA-AE1) prepared in Example 2 above. The reagent R3 includes: the fusion protein-biotin complex Aβ1-42 AbC2-Bio prepared in Example 1 above.

[0100] Formulation Combination Example 4 This formulation combination embodiment provides a formulation combination including reagent R1, reagent R2 and reagent R3: The reagent R1 includes: the magnetic bead-Aβ1-42 capture antibody conjugate prepared in Example 2 above; The reagent R2 includes: the streptavidin-NSP-DMAE-NHS acridinium ester conjugate (SA-AE2) prepared in Example 2 above. The reagent R3 includes: the fusion protein-biotin complex Aβ1-42 AbN-Bio prepared in Example 1 above.

[0101] Formulation Combination Example 5 This formulation combination embodiment provides a formulation combination including reagent R1, reagent R2 and reagent R3: The reagent R1 includes: the magnetic bead-Aβ1-42 capture antibody conjugate prepared in Example 2 above; The reagent R2 includes: the streptavidin-NSP-DMAE-NHS acridinium ester conjugate (SA-AE2) prepared in Example 2 above. The reagent R3 includes: the fusion protein-biotin complex Aβ1-42 AbC-Bio prepared in Example 1 above.

[0102] Formulation Combination Example 6 This formulation combination embodiment provides a formulation combination including reagent R1, reagent R2 and reagent R3: The reagent R1 includes: the magnetic bead-Aβ1-42 capture antibody conjugate prepared in Example 2 above; The reagent R2 includes: the streptavidin-NSP-DMAE-NHS acridinium ester conjugate (SA-AE2) prepared in Example 2 above. The reagent R3 includes: the fusion protein-biotin complex Aβ1-42 AbC2-Bio prepared in Example 1 above.

[0103] Formulation Combination Example 7 This formulation combination embodiment provides a formulation combination including reagent R1, reagent R2 and reagent R3: The reagent R1 includes: the magnetic bead-Aβ1-42 capture antibody conjugate prepared in Example 2 above; The reagent R2 includes: the streptavidin-Me-DMAE-NHS acridinium ester conjugate (SA-AE3) prepared in Example 2 above. The reagent R3 includes: the fusion protein-biotin complex Aβ1-42 AbN-Bio prepared in Example 1 above.

[0104] Formulation Combination Example 8 This formulation combination embodiment provides a formulation combination including reagent R1, reagent R2 and reagent R3: The reagent R1 includes: the magnetic bead-Aβ1-42 capture antibody conjugate prepared in Example 2 above; The reagent R2 includes: the streptavidin-Me-DMAE-NHS acridinium ester conjugate (SA-AE3) prepared in Example 2 above. The reagent R3 includes: the fusion protein-biotin complex Aβ1-42 AbC-Bio prepared in Example 1 above.

[0105] Comparative Example 1 of Formulation Combinations This formulation combination comparative example provides a formulation combination including reagent R1, reagent R2 and reagent R3: The reagent R1 includes: the magnetic bead-Aβ1-42 capture antibody conjugate prepared in Example 2 above; The reagent R2 includes: the streptavidin-NSP-SA-NHS acridinium ester conjugate (SA-AE1) prepared in Example 2 above. The reagent R3 includes: the Aβ1-42 detection antibody-biotin NHS-PEG2-Biotin conjugate (Aβ1-42-Bio) prepared in Comparative Example 1 above.

[0106] Comparative Example 2 of Formulation Combinations This formulation combination comparative example provides a formulation combination including reagent R1 and reagent R2: The reagent R1 includes: the magnetic bead-Aβ1-42 capture antibody conjugate prepared in Example 2 above; The reagent R2 includes: the Aβ1-42 detection antibody-NSP-SA-NHS acridine ester conjugate (Aβ1-42-AE) prepared in Example 2 above.

[0107] The specific formulation combinations of Examples 1-8 and Comparative Examples 1-2 are shown in Table 3 below: Table 3

[0108] Performance testing 1. Basic performance testing The formulation combinations Examples 1-9 and Comparative Examples 1-2 were applied to the chemiluminescent immunoassay detection of Aβ1-42 using an iFlash3000 chemiluminescent immunoassay analyzer (Yahuilong Biotechnology) and a semi-finished chemiluminescent reagent kit. The reaction modes of Formulation Combinations Examples 1-9 are illustrated using Formulation Combination Example 2 as an example. Figure 5 The novel double-antibody sandwich reaction mode of the Bio-SA conjugation route is shown; the reaction mode of the formulation combination comparative example 1 is derived from... Figure 6 As shown. Comparative Example 2 of the formulation combination employed a traditional double-antibody sandwich reaction mode.

[0109] The specific testing steps include: Preparation of working solution: (1) Preparation of capture antibody-magnetic bead working solution (reagent R1): The magnetic bead component working solution of formulation combination examples 1-9 and formulation combination comparative examples 1 and 2 is the same. The magnetic bead-Aβ1-42 capture antibody conjugate (10 mg / mL) in formulation combination examples 1-9 and formulation combination comparative examples 1 and 2 is diluted to 0.2 mg / mL using a citrate-sodium citrate buffer system (0.1 mol / L); (2) Preparation of working solution of Aβ1-42 detection antibody-NSP-SA-NHS acridinium ester conjugate (reagent R2 of formulation combination comparison example 2): The concentration of the conjugate stock solution was 0.1 mg / mL, and it was diluted to 1:200 using disodium hydrogen phosphate (12 water)-sodium dihydrogen phosphate buffer system (0.2 mol / L); (3) The stock solution concentration of SA-AE1, SA-AE2, SA-AE3 (reagent R2 in formulation combination examples 1-9 and formulation combination comparative example 1) was 0.1 mg / mL, and it was diluted to 1:600 ​​with disodium hydrogen phosphate (12 water)-sodium dihydrogen phosphate buffer system (0.2 mol / L); (4) Preparation of working solutions for Aβ1-42 AbN-Bio, Aβ1-42 AbC-Bio, and Aβ1-42 AbC2-Bio fusion proteins-biotin complexes (reagent R3 in formulation combination examples 1-9): The antibody raw materials were directly diluted to 0.4 μg / mL using a disodium hydrogen phosphate (12 water)-sodium dihydrogen phosphate buffer system (0.2 mol / L) to prepare working solutions R3 for use in formulation combination examples 1-9; (5) Preparation of Aβ1-42-Bio working solution (reagent R3 in formulation combination comparison example 1): The antibody raw material was diluted to 1:200 using a disodium hydrogen phosphate (dodecanoate)-sodium dihydrogen phosphate buffer system (0.2 mol / L).

[0110] Sample screening: A sample diluent was selected as a blank control; the sample diluent was a serum matrix diluent with a zero value. Ten samples (samples 1-10) were selected, including positive sera with low, medium, and high Aβ1-42 antigen concentrations measured by Roche in clinical settings and random physical examinations from different individuals. The actual Aβ1-42 antigen concentrations in the samples measured using Roche's gold standard reagent are shown in Table 4. The Aβ1-42 calibrator was purchased from the Boyue Biotechnology website, and three concentrations of calibrator were prepared: 0.5 pg / mL (calibrator CAL1), 15 pg / mL (calibrator CAL2), and 150 pg / mL (calibrator CAL3).

[0111] test: Take 10 μL of the sample to be tested, add 50 μL of R1 working solution and 50 μL of R3 working solution respectively, react at 37℃ for 10 min, and then perform magnetic separation and washing 3 times; add 50 μL of reagent R2, react at 37℃ for 5 min, and then add 100 μL of pre-activation solution (Pre-Trigger Solution (1×) (International) 500mL, catalog number C89959) (HNO3-H2O2 solution) and 100 μL of activation solution (Trigger Solution (1×) (International) 500mL, catalog number C89958) (NaOH solution) produced by Shenzhen Yahuilong Biotechnology Co., Ltd. respectively. Measure the luminescence value using an iFlash3000 chemiluminescence immunoassay analyzer. Perform three parallel measurements and take the average value. The test results are shown in Tables 4 and 5. The sample concentration and luminescence value curve of formulation combination example 2 is shown in Table 5. Figure 4 As shown.

[0112] Table 4

[0113] Table 5

[0114] The results of comparing the test data in Tables 4 and 5 show that the double-antibody sandwich mode of the antibody-biotin and streptavidin-acidine complex in formulation combination comparison example 1 is significantly more effective than the conventional double-antibody sandwich mode in formulation combination comparison example 2. This is because the affinity of biotin and streptavidin is millions of times higher than the antigen-antibody binding affinity, and one SA can bind four biotin molecules. Therefore, the detection sample can be significantly amplified. However, the mode of formulation combination comparison example 1 also caused a high background value, making the luminescence value (background) of the diluted 0-value pure negative sample tend to be similar to that of the low-value positive sample, which easily leads to false positive failures. Therefore, the experimental group of this invention tried using fusion antibody-biotin complexes with different amounts of biotin in a targeted manner, and then combined them with reagent R2 of SA-AE1 and SA-AE2 respectively. The test results showed that although the signal (luminescence value obtained by direct testing) was reduced, the background was significantly reduced, and the signal-to-noise ratio remained high, even reaching above 8. In Formulation Combination Example 2, when combined with reagent R2 of SA-AE1, the Aβ1-42 AbC-Bio complex showed the most significant background reduction effect, with the highest signal-to-noise ratio among the formulation combination example groups and the formulation combination comparison group. The formulation combination provided by the formulation combination examples of the present invention has high sensitivity, accurate detection results, and low false positive failure rate in detecting Aβ1-42 in serum.

[0115] from Figure 4 The correlation analysis results show that the kits combining Aβ1-42 AbC-Bio and NSP-SA-NHS in Example 2 exhibit excellent correlation, R 2 The value reached 0.9996, with no jump values ​​or false positives observed.

[0116] 2. Stability Test The Aβ1-42-AbC-Bio complex reagent R3, the SA-AE reagent R2 from Example 2, and the Aβ1-42-Bio reagent R3 from Comparative Example 1 were placed in a 4°C refrigerator and a 37°C incubator for acceleration, respectively, to verify the stability after opening and the stability after acceleration. All components were accelerated simultaneously, and after acceleration, the methodology and test procedures for the basic performance tests were kept consistent with those described above. The samples were calibrators and clinical serum-mixed samples.

[0117] The test groups are shown in Table 6 below, and the test results are shown in Tables 7 and 8.

[0118] Table 6 Test Groups

[0119] The test results are as follows: Table 7

[0120] Table 8

[0121] The test results above show that the reagent in Experimental Group 2 exhibited a deviation of approximately -5% in stability upon opening at 4°C, while the deviation in Comparative Example 1 was approximately -15%. This indicates that Aβ1-42 AbC-Bio R3 is more stable than the Aβ1-42-Bio detection antibody R3 reagent component with direct biotin labeling, maintaining stability for over 14 days at 4°C. In the accelerated stability test at 37°C for 7 days, Experimental Group 2 showed an average value drop of approximately 5%, while Comparative Example 1 showed an average value drop of 33.2%, indicating that the Aβ1-42 AbC-Bio R3 reagent component has better accelerated stability. Therefore, the preparation method of the fusion protein-biotin complex expressed by genetic engineering technology in Example 1 of this application demonstrates significant advantages.

[0122] 3. Inter-batch difference test Three batches of Aβ1-42 AbC-Bio complexes were prepared according to the preparation method in Example 1. Three batches of R3 reagents were prepared according to the experimental method in Performance Test 1. Three batches of Aβ1-42-Bio antibody-biotin complexes were labeled according to the preparation method in Comparative Example 1, and three batches of R3 reagents were prepared according to the experimental method in Performance Test 1. The other reagents, R2 SA-AE reagent and R1 magnetic bead reagent, were kept consistent. The testing methodology and procedures were consistent with the above experiments. The test samples were calibrators and clinical samples. The CV value (the ratio of standard deviation to mean) of the three batches of test results for each reagent was calculated. The test results are shown in Tables 9 and 10. Table 9

[0123] Table 10

[0124] The batch-to-batch variation test results above show that the reagent corresponding to experimental group 2 has the smallest batch-to-batch variation, with an average CV value within 5%, while the average CV value of comparative example 1 exceeds 15%. Therefore, the fusion protein-biotin complex antibody expressed using genetic engineering technology in Example 1 of this application has extremely small batch-to-batch variation, resulting in a reagent kit with very small batch-to-batch variation, around 5%. This overcomes the problem of large batch-to-batch differences in reagents prepared by traditional chemical conjugation methods.

[0125] In summary, the fusion protein-biotin complex, recombinantly expressed using genetic engineering techniques to synthesize antibodies and biotin with luciferin, exhibits excellent streptavidin binding activity, antigen binding function, and significantly improved signal-to-noise ratio. In clinical diagnostic applications, compared to traditional diagnostic reagents, it eliminates the need for luminescent labeling, avoiding the site-specific effects and compatibility issues associated with multiple steps in traditional chemical conjugation labeling. This improves reagent sensitivity and signal-to-noise ratio, reduces batch-to-batch variability, and demonstrates good stability.

[0126] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A fusion protein, characterized in that, The fusion protein includes a detection antibody, an avidin tag, and a cyan fluorescent protein; The detection antibody can specifically bind to the antigen Aβ1-42 to be tested, and the detection antibody includes an Fc region; The avidin tag and cyan fluorescent protein are sequentially linked to the carboxyl terminus of the Fc region.

2. The fusion protein as described in claim 1, characterized in that, The avidin tag includes at least one of Avi-tag and BSP-tag; Optionally, the avidin tag is inserted in a forward direction or in a reverse direction; Optionally, the number of avidin tags is 1 to 4, and optionally 1 to 2.

3. The fusion protein as described in claim 1 or 2, characterized in that, The cyan fluorescent protein includes mTurquoise2.

4. The fusion protein according to any one of claims 1 to 3, characterized in that, The detection antibody includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes CDRH1 to CDRH3 as shown in SEQ ID NO: 1 to 3, respectively. The light chain variable region includes CDRL1 to CDRL3. The amino acid sequence of CDRL1 is shown in SEQ ID NO: 4, the amino acid sequence of CDRL2 is TAS, and the amino acid sequence of CDRL3 is shown in SEQ ID NO:

5. Optionally, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 6; Optionally, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 7; Optionally, the detection antibody is a monoclonal antibody; Optionally, the antibody further includes a light chain constant region selected from kappa or lambda isotypes; Optionally, the antibody may further include a heavy chain constant region selected from IgG1, IgG2, IgG3, and IgG4 subtypes.

5. A complex, characterized in that, The protein comprises the fusion protein of any one of claims 1 to 4, and biotin; said biotin is attached to the avidin tag.

6. A reagent combination, characterized in that, include: Reagent R1 comprises a solid support and a capture antibody coupled to the surface of the solid support; Reagent R2 includes streptavidin and an acridine derivative coupled with said streptavidin; Reagent R3 includes the fusion protein of any one of claims 1 to 4 or the complex of claim 5.

7. The reagent combination as described in claim 6, characterized in that, In reagent R1, the solid support comprises magnetic microparticles; Optionally, the capture antibody specifically binds to the test antigen Aβ1-42, and the detection antibody, the test antigen Aβ1-42, and the capture antibody can form a double antibody sandwich structure.

8. The reagent combination as described in claim 6 or 7, characterized in that, In reagent R2, the acridine derivative includes at least one of acridine ester, acridine acid, acridine amide, and acridine sulfonamide; Optionally, the acridine derivative includes at least one selected from the group consisting of: NSP-SA-NHS, NSP-DMAE-NHS, ME-DMAE-NHS, NSP-DMAE-HEG-Glu-NHS, NSP-SA-ADH, and DMAE-NHS.

9. A product for detecting the content of Aβ1-42, characterized in that, The product comprises the fusion protein of any one of claims 1 to 4, the complex of claim 5, or the reagent combination of any one of claims 6 to 8; Optionally, the product includes at least one of the following: reagents, reagent kits, and instrument platforms.

10. The use of the fusion protein of any one of claims 1 to 4, the complex of claim 5, the reagent combination of any one of claims 6 to 8, or the product of claim 9 in the preparation of a product for diagnosing Alzheimer's disease.

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