Homogeneous chemiluminescence detection method and kit thereof

Abstract

The invention provides a homogeneous chemiluminescence detection method and a kit thereof, the method comprises the following steps: amino oxygen-receiving microspheres firstly react with sulfo-BS (PEG) n, n is an integer greater than or equal to 3, then the amino oxygen-receiving microspheres react with an antibody of a to-be-detected antigen to prepare oxygen-receiving microspheres connected with the antibody of the to-be-detected antigen, and then the amino oxygen-receiving microspheres react with the sulfo-BS (PEG) n to prepare the homogeneous chemiluminescence detection kit. And then detecting the antigen to be detected by a homogeneous chemiluminescence detection method. According to the invention, by optimizing the oxygen-receiving microsphere bio-coupling antibody, that is, through an anti-hydrolysis controllable spacer arm antibody microsphere coupling reaction system, the flexibility of the conjugate is improved, the steric hindrance is reduced, and the homogeneous chemiluminescence high-sensitivity detection of the low-concentration biomarker is realized.

Description

Technical Field

[0001] The present invention relates to the field of homogeneous chemiluminescence, and in particular to a homogeneous chemiluminescence detection method and a kit thereof. Background Art

[0002] As described in CN105758835 B, in recent years, the new homogeneous chemiluminescence technology based on the immunoassay of light-stimulated chemiluminescence oxygen channel (Luminescent Oxygen Channeling Immunoassay, LOCI) has been rapidly developed. It not only takes into account the sensitivity and precision advantages of magnetic particle chemiluminescence immunoassay, showing accuracy and reliability, but also has the characteristics of homogeneous reaction and no need for cleaning, which is an excellent match for the development needs of automated and portable POCT (Point-of-Care Testing, POCT) analysis systems. The principle of LOCI technology is based on the short-distance diffusion of singlet oxygen energy by two nanospheres to stimulate the chemiluminescence reaction of adjacent sites to measure the interaction between molecules. Therefore, each luminescence assay contains two microspheres (oxygen-donating microspheres and oxygen-accepting microspheres) and a pair of antibodies, that is, the detection process adopts a double antibody sandwich method. Usually, the oxygen-donating microspheres are covalently coupled to streptavidin and then combined with the first antibody labeled with biotin; the oxygen-accepting microspheres are directly biocoupled to another antibody. The principle of the homogeneous immunoassay POCT detection method is as follows: the biomolecules to be tested in the test sample react with the oxygen-supplying microspheres and the oxygen-accepting microspheres to form immune complexes. This interaction will bring the oxygen-supplying microspheres and the oxygen-accepting microspheres closer together. Under the irradiation of laser light (wavelength of 680nm), the photosensitizer on the oxygen-supplying microspheres converts the oxygen in the surrounding environment into more active monomeric oxygen. Monomeric oxygen diffuses to the oxygen-accepting microspheres and reacts with the chemiluminescent agent on the oxygen-accepting microspheres, further activating the luminescent groups also on the oxygen-accepting microspheres, causing them to emit light with a wavelength of 520-620nm. The half-life of monomeric oxygen is 4μSec, and the diffusion distance in the solution is about 200nm. If there is no interaction between the biomolecules, monomeric oxygen cannot diffuse to the oxygen-accepting microspheres, and no light signal will be generated. Therefore, by measuring the light intensity emitted by the mixture, the concentration of the biomolecules to be tested in the test sample can be calculated.

[0003] Fibroblast Growth Factors (FGFs) are a class of multifunctional cytokines, of which there are more than 20 types. They are indispensable in the processes of cell proliferation, differentiation, migration and survival. Among them, FGF21 has shown great potential in the treatment of metabolic diseases. However, the accurate detection of FGFs still faces challenges, and both scientific research and clinical applications urgently need highly sensitive, rapid, accurate and easy-to-operate analytical methods or analytical systems. FGFs are usually at very low concentrations, short half-lives (ranging from 0.5 to several hours), and are of many types with high homology. For example, the concentrations of FGF21 and FGF23 in healthy human serum and patient serum are in the order of pg / mL. Conventional detection methods such as ELISA are not sensitive enough, not accurate enough, and are complicated to operate and time-consuming. For example, the detection time is 3-6 hours.

[0004] Bioconjugation is a technical system that covalently links biological macromolecules (antibodies, proteins, nucleic acids, etc.) with functional molecules (fluorescent markers, drugs, nanomaterials, etc.) through controlled chemical reactions. Its core value lies in constructing complexes that combine biological recognition and functional expansion.

[0005] The coupling efficiency is regulated by three factors: the activity of the reactive group, the solution environment, and the spatial conformation of the molecule. For different target functional groups, a dedicated reaction system must be established: amino modification preferentially utilizes NHS esters (pH 8.0-8.5, 4-25°C) or imidoesters; sulfhydryl-directed coupling relies on maleimide (pH 6.5-7.5) or disulfide exchange systems. Taking the most widely used NHS ester as an example, its reaction kinetics exhibit significant pH dependence—when the buffer pH increases from 7.2 to 8.5, the rate of amino nucleophilic attack increases approximately 5-fold, but the rate of ester bond hydrolysis simultaneously increases 8-fold, necessitating precise control of the reaction time (30 minutes to 4 hours).

[0006] Accurate determination of FGFs concentration plays an irreplaceable role in understanding the mechanisms of related diseases, guiding clinical treatment and evaluating disease prognosis; rapid detection of FGFs can significantly improve the detection ability of short-half-life FGFs and improve clinical diagnostic efficiency, which is of great significance for disease mechanism research, early diagnosis of diseases and timely treatment. Summary of the Invention

[0007] The present invention provides a homogeneous chemiluminescence detection method, in which amino oxygen-accepting microspheres are first reacted with sulfo-BS(PEG)n, where n is an integer greater than or equal to 3, and then reacted with an antibody to the antigen to be detected to prepare oxygen-accepting microspheres to which the antibody to the antigen to be detected is linked. The antigen to be detected is then detected by a homogeneous chemiluminescence detection method.

[0008] In one method, n is an integer from 5 to 15.

[0009] In one method, n is an integer from 9 to 15.

[0010] In one method, the antigen to be detected is fibroblast growth factor and / or AD protein.

[0011] In one method, the antigen to be detected is fibroblast growth factor FGF21 or FGF23.

[0012] In one method, the present invention provides a homogeneous chemiluminescent detection kit, which includes the following oxygen-accepting microspheres, which are prepared by the following method: amino oxygen-accepting microspheres are first reacted with sulfo-BS(PEG)n, where n is an integer greater than or equal to 3, and then reacted with antibodies to the antigen to be detected to prepare oxygen-accepting microspheres to which the antibodies to the antigen to be detected are linked.

[0013] This invention, for the first time, achieves the detection of fibroblast growth factor using a homogeneous chemiluminescence method. This method features rapid detection, high sensitivity, a wide dynamic range, high reproducibility, and ease of use. It also significantly extends the shelf life of the reagent. By introducing a sulfonic acid group-modified sulfo-NHS ester, the present invention reduces ineffective hydrolysis losses at low protein concentrations while maintaining reaction activity, thereby effectively extending the reagent's shelf life.

[0014] The spacer arm serves as a bridge connecting the biomolecule and the functional module. Its length determines the flexibility of the conjugate. Longer spacers increase flexibility and minimize steric hindrance. Its physicochemical parameters directly influence conjugate function: ① In terms of length, short arms reduce nonspecific adsorption but increase steric hindrance, while long arms enhance antigen binding efficiency. ② In terms of chemical composition, traditional hydrophobic hydrocarbon chains are prone to protein aggregation, while PEGylation can improve water solubility. However, excessively long spacers should be avoided, as they may create more potential nonspecific binding sites. Therefore, optimal chain length is crucial to enhance antigen-antibody binding efficiency and improve detection sensitivity.

[0015] The present invention optimizes the oxygen-accepting microsphere bioconjugated antibody, that is, through the anti-hydrolysis controllable spacer arm antibody microsphere coupling reaction system, to improve the flexibility of the conjugate, reduce steric hindrance, and achieve high-sensitivity homogeneous chemiluminescence detection of low-concentration biomarkers.

[0016] This invention utilizes a step-by-step sulfo-NHS coupling process: one end of the sulfo-BS(PEG)n molecule is first coupled to the amino surface of the oxygen-accepting microsphere, and the other end is then coupled to the amino group of the antibody, thereby enabling targeted coupling of the antibody to the microsphere surface. Compared to NHS esters, the sulfonic acid group in the sulfo-NHS ester reduces the probability of nucleophilic attack and improves stability. The sulfonic acid group also enhances the surface negative charge, reduces nonspecific adsorption, and significantly improves resistance to hydrolysis.

[0017] Sulfo-BS(PEG)n is a disuccinimidyl ester-activated PEG compound used for crosslinking primary amines (-NH2) in proteins and other molecules. It is a homobifunctional amine-amine crosslinker. It contains sulfo-N-hydroxysuccinimide (NHS) esters at both ends of the PEG spacer, which react specifically and efficiently with lysine and N-terminal amino groups at pH 7-9 to form stable amide bonds. Therefore, Sulfo-BS(PEG)n is characterized by the reactive groups being sulfo-NHS esters (at both ends), which react with -NH2 groups to form stable amide bonds at pH 7-9. It is a pure compound with a well-defined structure and molecular weight, ensuring reproducible protein modification effects. The polyethylene glycol spacer arm helps maintain the solubility of the conjugate. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 This is a schematic diagram of the reaction principle of the present invention where sulfo BS (PEG) 5 reacts with amino epoxy microspheres and is linked to antibodies;

[0020] Figure 2 It is a schematic diagram of the homogeneous chemiluminescence reaction principle of the present invention. DETAILED DESCRIPTION

[0021] In order to enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below in conjunction with the following embodiments. Obviously, the embodiments described are only part of the embodiments of this application, rather than all the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of this application. The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0022] Example 1: Homogeneous chemiluminescence reaction of anti-FGF21 antibody using DSS, BS(PEG)5 and sulfo-BS(PEG)5 as crosslinking agents to react with amino oxygen acceptor microspheres

[0023]

[0024] like Figure 1 and Figure 2 As shown, the present invention first reacts amino oxygen acceptor microspheres with the three aforementioned crosslinking agents, then binds a primary antibody to the oxygen acceptor microspheres to obtain primary antibody-linked oxygen acceptor microspheres. Subsequently, a sandwich homogeneous chemiluminescence reaction is performed with a biotin-labeled secondary antibody and streptavidin oxygen-donating microspheres to test the concentration of the antigen to be tested in different samples. The specific experimental process of the present invention is shown below.

[0025] 1. Disperse 30 mg of amino oxygen acceptor microspheres evenly in 3 mL of 100 mM PBS buffer (0.1 M pH 7.2) to obtain a final concentration of 10 mg / mL amino microsphere solution;

[0026] 2. Divide the above amino oxygen acceptor microspheres into 1 mL / tube, with a concentration of 10 mg / mL, for a total of 3 amino oxygen acceptor microspheres. The three tubes are designated as tube 1, tube 2 and tube 3 for standby use.

[0027] 3.DSS is used as a crosslinking agent to complete the crosslinking of FGF21 antibody and oxygen-accepting microspheres

[0028] 3.1. Add DSS to tube 1 to a final concentration of 1 mM. Incubate the reaction mixture of amino microspheres and DSS at room temperature for 2 hours.

[0029] 3.2. Add 1 M Tris solution to the above reaction mixture until the final Tris concentration is 25 mM, and then incubate the reaction mixture at room temperature for 15 minutes;

[0030] 3.3. Remove the above reaction solution, centrifuge and purify it, then disperse it in 1 mL of PBS buffer (0.1 M pH 7.2) and set aside;

[0031] 3.4. Add 1 mg of purified anti-FGF21 antibody to the above reaction solution and react at room temperature for 0.5 hours to finally prepare the anti-FGF21 antibody oxygen-accepting microsphere reaction solution. Store it in the dark at 2-8°C until use.

[0032] 4. BS (PEG) 5 as a crosslinker to complete the crosslinking of FGF21 antibody and oxygen acceptor microspheres

[0033] 4.1. Add BS(PEG)5 to tube 2 to a final concentration of 1 mM. Incubate the reaction mixture of amino microspheres and BS(PEG)5 at room temperature for 2 hours.

[0034] 4.2. Add 1 M Tris solution to the above reaction mixture until the final Tris concentration is 25 mM, and then incubate the reaction mixture at room temperature for 15 minutes;

[0035] 4.3. Remove the above reaction solution, centrifuge and purify it, then disperse it in 1 mL of PBS buffer (0.1 M pH 7.2) and set aside;

[0036] 4.4. Add 1 mg of purified anti-FGF21 antibody to the above reaction solution and react at room temperature for 0.5 hours to finally prepare the anti-FGF21 antibody microsphere reaction solution. Store in the dark at 2-8°C until use.

[0037] 5. Sulfo BS (PEG) 5 as a crosslinker to complete the crosslinking of FGF21 antibody and oxygen acceptor microspheres

[0038] 5.1. Add sulfo-BS(PEG)5 to tube 2 to a final concentration of 1 mM. Incubate the reaction mixture of amino microspheres and sulfo-BS(PEG)5 at room temperature for 2 hours.

[0039] 5.2. Add 1 M Tris solution to the above reaction mixture until the final Tris concentration reaches 25 mM, and then incubate the reaction mixture at room temperature for 15 minutes;

[0040] 5.3. Remove the above reaction solution, centrifuge and purify it, then disperse it in 1 mL of PBS buffer (0.1 M pH 7.2) and set aside;

[0041] 5.4. Add 1 mg of purified anti-FGF21 antibody to the above reaction solution and react at room temperature for 0.5 hours to finally prepare the anti-FGF21 antibody microsphere reaction solution. Store in the dark at 2-8°C until use.

[0042] 6. The oxygen-accepting microspheres of the anti-FGF21 antibody prepared with the above three cross-linking agents (DSS, BS(PEG)5, and sulfo-BS(PEG)5) were combined with biotin-labeled anti-FGF21 secondary antibodies and streptavidin oxygen-donating microspheres to test FGF21 samples of different concentrations. The results are shown in Table 1 below.

[0043] Table 1

[0044]

[0045]

[0046] The signal-to-noise ratio (S / N) statistics are shown in Table 2 below:

[0047] Table 2

[0048]

[0049] From the above data, it can be seen that the background value (0 concentration sample) of the anti-FGF21 antibody oxygen-accepting microsphere test prepared by traditional DSS is higher than the signal value of the anti-FGF21 antibody oxygen-accepting microsphere prepared by BS(PEG)5 and sulfo-BS(PEG)5, while the specific signal is relatively low; as the sample concentration increases, the signal of the anti-FGF21 antibody oxygen-accepting microsphere reaction prepared by BS(PEG)5 and sulfo-BS(PEG)5 is significantly higher than the signal of the anti-FGF21 antibody oxygen-accepting microsphere prepared by DSS, and the difference in the signal of the anti-FGF21 antibody oxygen-accepting microsphere reaction prepared by BS(PEG)5 and sulfo-BS(PEG)5 is not obvious.

[0050] When the anti-FGF21 antibody oxygen-accepting microspheres prepared using traditional DSS tested the 40pg / mL FGF21 sample, the signal value was significantly different from that of the 0 concentration sample. The signal-to-noise ratio (S / N) of the 40pg / mL FGF21 sample and the 0 concentration sample was 1.41.

[0051] The reaction signals of the anti-FGF21 antibody oxygen-accepting microspheres prepared with BS(PEG)5 and sulfo-BS(PEG)5 showed a relatively low background value of the test signal and a relatively high specific signal value; when testing the 8pg / mL concentration FGF21 sample, the anti-FGF21 antibody oxygen-accepting microspheres prepared with BS(PEG)5 and sulfo-BS(PEG)5 had a clear distinction from the signal value of the 0 concentration sample, and the signal-to-noise ratio (S / N) reached 1.32 and 1.39, respectively.

[0052] Example 2: Homogeneous chemiluminescence reaction of DSS, BS(PEG)5 and sulfo-BS(PEG)5 as crosslinking agents to react with amino oxygen acceptor microspheres, linked to anti-FGF21 antibodies and stored for different time periods

[0053] 1. Prepare five FGF21 samples of different concentrations (40 pg / mL, 100 pg / mL, 300 pg / mL, 1000 pg / mL, 2000 pg / mL). Similar to Example 1, anti-FGF21 antibody oxygen acceptor microspheres were prepared using three cross-linking agents (DSS, BS(PEG)5, and sulfo-BS(PEG)5) and stored in aliquots at -80°C.

[0054] 2. The three differently modified oxygen-accepting microspheres were tested monthly with biotin-labeled anti-FGF21 secondary antibodies and streptavidin oxygen-donating microspheres. The signal values of FGF21 samples with different concentrations were tested to observe the effects of different cross-linking reagents on the homogeneous chemiluminescence reaction signal over time. The specific results are shown in Tables 3, 4, and 5 below.

[0055] 3.1.1-13 months The real-time tracking stability results of the anti-FGF21 antibody oxygen-accepting microspheres prepared by traditional DSS are as follows: The test signal values of the anti-FGF21 antibody oxygen-accepting microspheres prepared by traditional DSS were relatively stable in the first 6 months, and the sample signal retention rate tested at the 6th month was 76%-91%; the test signal retention rate was 53%-85% at 7 months, which showed a downward trend. By 12 months, the retention rate was only 30-46%.

[0056] Table 3

[0057]

[0058] 3.2.1-13 months The real-time tracking stability results of the anti-FGF21 antibody oxygen-accepting microspheres prepared by BS(PEG)5 are as follows: the test signal values of the anti-FGF21 antibody oxygen-accepting microspheres prepared by BS(PEG)5 were relatively stable in the first 6 months, and the signal retention rate of the tested samples at the 6th month was 86%-91%; at 7 months, the test signal retention rate was 67%-81%, which has shown a downward trend. By 12 months, the retention rate was only 38-61%.

[0059] Table 4

[0060]

[0061] 3.3.1-13 months The real-time tracking stability results of the anti-FGF21 antibody oxygen-accepting microspheres prepared with sulfo-BS (PEG) 5 are as follows: The stability of the anti-FGF21 antibody oxygen-accepting microspheres prepared with sulfo-BS (PEG) 5 has been significantly improved. Not only are the test signal values relatively stable in the first 6 months, but the signal retention rate of the sample tested at the 6th month is 88%-107%; it shows relatively stable measured values until 13 months; the test signal retention rate is 87%-101% at 7 months; the retention rate is 87-92% at 12 months; and even at 13 months, the test signal value retention rate is still 84-92%.

[0062] Table 5

[0063]

[0064]

[0065] Example 3: Homogeneous chemiluminescence reaction of anti-FGF21 antibody using sulfo-BS(PEG)5 of different chain lengths as crosslinkers to react with amino oxygen acceptor microspheres

[0066] 1. Disperse 30 mg of amino oxygen acceptor microspheres evenly in 3 mL of PBS buffer (0.1 M pH 7.2) to obtain a 10 mg / mL amino microsphere solution.

[0067] 2. Divide equally into 3 bottles of amino oxygen acceptor microspheres, 1 mL / tube, concentration 10 mg / mL, and set aside;

[0068] 3. Use sulfo BS (PEG) 9、 Sulfo BS(PEG) 15 and sulfo BS (PEG) 24 Complete the cross-linking of FGF21 antibody and oxygen-accepting microspheres;

[0069] 3.1. Add sulfo-BS(PEG)9, sulfo-BS(PEG)9 to tubes 1, 2, and 3, respectively. 15 and sulfo BS (PEG) 24 , until Sulfo BS (PEG) 9, Sulfo BS (PEG) 15 and sulfo BS (PEG) 24 The final concentration was 1 mM, and the cells were incubated at room temperature for 2 hours;

[0070] 3.2. Add 1 M Tris solution to the above reaction mixture until the final Tris concentration reaches 25 mM. Incubate the reaction mixture at room temperature for 15 minutes.

[0071] 3.3. Remove the above reaction solution, centrifuge and purify it, then disperse it in 1 mL of PBS buffer (0.1 M pH 7.2) and set aside;

[0072] 3.4. Add 1 mg of purified anti-FGF21 antibody to the above reaction solution to obtain the final anti-FGF21 antibody microsphere reaction solution, store at 2-8 ° C in the dark until use;

[0073] 4. The above three cross-linking agents (sulfo BS (PEG) 9, sulfo BS (PEG) 15 , sulfo BS (PEG) 24 ) were used to prepare anti-FGF21 antibodies, and were used in combination with biotin-labeled anti-FGF21 antibody 2 and streptavidin oxygen-donating microspheres to test FGF21 samples of different concentrations; at the same time, the anti-FGF21 antibodies prepared by sulfo-BS (PEG) 5 were used in combination with biotin-labeled anti-FGF21 antibody 2 and streptavidin oxygen-donating microspheres to test FGF21 samples of different concentrations. The results are as follows: the four cross-linking agents (sulfo-BS (PEG) 5, sulfo-BS (PEG) 9, sulfo-BS (PEG) 15 , sulfo BS (PEG) 24 ) prepared by oxygen microspheres tested the background (0 concentration sample) signal value is relatively small, but the sulfo-BS (PEG) 9, sulfo-BS (PEG) 15 , sulfo BS (PEG) 24The specific signal of the anti-FGF21 antibody oxygen acceptor microspheres prepared was relatively high, especially sulfo-BS(PEG)9, sulfo-BS(PEG) 15 The prepared anti-FGF21 antibody oxygen-accepting microspheres were particularly obvious, and the specific signal was greatly improved. The specific results are shown in Table 6.

[0074] Table 6

[0075]

[0076]

[0077] The summary statistical signal-to-noise ratio (S / N) is as follows, see Table 7 for details: 4 cross-linking agents (Sulfo-BS(PEG)5, Sulfo-BS(PEG)9, Sulfo-BS(PEG) 15 , sulfo BS (PEG) 24 ) can effectively distinguish between the 0 concentration sample and the 8pg / mL concentration FGF21 sample; sulfo BS (PEG) 9, sulfo BS (PEG) 15 , sulfo BS (PEG) 24 The detection range of the prepared anti-FGF21 antibody oxygen-receptor microspheres was significantly improved; especially sulfo-BS(PEG)9, sulfo-BS(PEG) 15 The anti-FGF21 antibody oxygen-sensitive microspheres prepared showed more obvious improvement in detection. 15 , sulfo BS (PEG) 24 ) The signal-to-noise ratios (S / N) of the anti-FGF21 antibody oxygen-accepting microspheres prepared were 44.59, 175.71, 223.24, and 88.41 when detecting 5000 pg / mL FGF21 samples and 0 concentration samples, respectively.

[0078] Table 7

[0079]

[0080] The signal-to-noise ratio (S / N) corresponding to each concentration point was calculated. This is the ratio of the adjacent S / N ratios, which is the ratio of the S / N ratio of the subsequent concentration to the S / N ratio of the preceding concentration. For samples, this ratio is the ratio of the subsequent concentration to the preceding concentration. The S / N ratios corresponding to the concentrations of the anti-FGF21 antibody oxygen-accepting microspheres prepared with sulfo-BS(PEG)9 and sulfo-BS(PEG)15 were significantly improved compared to those prepared with sulfo-BS(PEG)5 and sulfo-BS(PEG)24. The S / N ratios of the anti-FGF21 antibody oxygen-accepting microspheres prepared with sulfo-BS(PEG)9 and sulfo-BS(PEG)15 for different sample concentrations were more consistent with the S / N ratio of the sample concentration itself, indicating better linearity. See Table 8 for details.

[0081] Table 8

[0082]

[0083]

[0084] Example 4 Application of four cross-linking agents in different chemiluminescence detection methods

[0085] 1. Disperse 2 mg of alkaline phosphatase in 0.5 mL of 0.1 M PBS buffer (pH 7.2) to obtain a 4 mg / mL alkaline phosphatase solution. Simultaneously, disperse 2 mg of horseradish peroxidase in 0.5 mL of 0.1 M PBS buffer (pH 7.2) to obtain a 4 mg / mL horseradish peroxidase solution.

[0086] 2. Cross-linking of FGF21 antibody with alkaline phosphatase and horseradish peroxidase was completed using DSS, BS(PEG)5, sulfo-BS(PEG)5, and sulfo-BS(PEG)9, respectively;

[0087] 2.1. Add DSS, BS(PEG)5, sulfo-BS(PEG)5, and sulfo-BS(PEG)9 to the alkaline phosphatase solution and horseradish peroxidase until the final concentration of traditional DSS is 1 mM. Incubate the above reaction solutions at room temperature for 2 hours.

[0088] 2.2. Add 1 M Tris solution to each of the above reaction mixtures until the final Tris concentration reaches 25 mM. Incubate each reaction mixture at room temperature for 15 minutes.

[0089] 2.3. Remove the above reaction solutions, centrifuge and purify them, then disperse them in 1 mL of PBS buffer (0.1 M pH 7.2) and set aside;

[0090] 2.4. Add 1 mg of purified anti-FGF21 antibody to the above reaction solution to obtain anti-FGF21 antibody alkaline phosphatase and horseradish peroxidase reaction solution, store in the dark at 2 to 8 ° C until use;

[0091] 3. Alkaline phosphatase-labeled anti-FGF21 antibodies and horseradish peroxidase-labeled anti-FGF21 antibodies prepared with the above four cross-linkers (DSS, BS(PEG)5, sulfo-BS(PEG)5, and sulfo-BS(PEG)9) were used in combination with biotin-labeled anti-FGF21 secondary antibodies and streptavidin magnetic beads to test FGF21 samples at different concentrations;

[0092] 4. The test results of alkaline phosphatase-labeled anti-FGF21 antibodies prepared with four cross-linking agents (DSS, BS(PEG)5, sulfo BS(PEG)5 and sulfo BS(PEG)9) are shown in Table 9 below. The results show that there is no significant difference between different cross-linking agents.

[0093] Table 9

[0094]

[0095]

[0096] The signal-to-noise ratio (S / N) results are summarized as follows: There is no significant difference in sensitivity and linearity between products prepared with different cross-linking agents. For specific results, see Table 10.

[0097] Table 10

[0098]

[0099] 5. The test results of horseradish peroxidase-labeled anti-FGF21 antibodies prepared with four cross-linking agents (DSS, BS(PEG)5, sulfo-BS(PEG)5 and sulfo-BS(PEG)9) are shown in Table 11: There is no obvious difference in the precision, detection range and other properties of the products prepared with different cross-linking agents.

[0100] Table 11

[0101]

[0102] The signal-to-noise ratio (S / N) results are summarized in Table 12: There is no significant difference in sensitivity and linearity among products prepared with different cross-linking agents.

[0103] Table 12

[0104]

[0105]

[0106] Example 5 Homogeneous Chemiluminescence Detection of FGF23

[0107] 1. Disperse 10 mg of amino oxygen acceptor microspheres evenly in 1 mL of PBS buffer (0.1 M pH 7.2) to obtain a 10 mg / mL amino microsphere solution.

[0108] 2. Similarly, sulfo-BS(PEG)9 was used to cross-link the FGF23 antibody and the oxygen acceptor microspheres, and finally the anti-FGF21 antibody microsphere reaction solution was prepared;

[0109] 3. The anti-FGF23 antibody prepared with the cross-linker sulfo-BS(PEG)9 was used in combination with a biotin-labeled anti-FGF23 secondary antibody and streptavidin oxygen-donating microspheres to test FGF23 samples of different concentrations. The results are shown in Table 13 below.

[0110] Table 13

[0111] FGF23 sample concentration pg / mL <![CDATA[Preparation using BS(PEG)9 crosslinker]]> Signal-to-noise ratio (S / N) 0 2055 / 5 3743 1.82 10 6231 3.03 40 18806 9.15 100 35604 17.33 300 109610 53.34 1000 316034 153.79 2000 631667 307.38 4000 1224533 595.88

[0112] When detecting FGF23 samples with a concentration of 5 pg / mL, they were clearly distinguished from samples with a concentration of 0; as the concentration of FGF23 samples continued to increase, the detection signal value showed a very good linear trend.

[0113] Example 6 Detection of AD protein (protein closely related to the pathological process of Alzheimer's disease (AD-related protein))

[0114] Alzheimer's disease (AD) is a neurodegenerative disease that usually has an insidious onset and progresses slowly. It is the most common type of dementia. AD drugs such as donepezil and rivastigmine only delay the onset of the disease and have very limited therapeutic effects. The burden of AD is heavy, and early screening for AD is imminent. In terms of diagnostic technology, AD cerebrospinal fluid testing can be achieved using a variety of immunological techniques, but the drawback is that sample collection requires lumbar puncture, which is risky and cannot be used for large-scale screening. The high price is also a major obstacle. Blood testing is an economical, convenient, minimally invasive and highly accessible detection technology that has emerged in recent years. It is suitable for large-scale early screening of AD and is of great significance for early screening, early diagnosis and follow-up of AD. However, the concentration of blood markers is much lower than that of cerebrospinal fluid, and the sensitivity of the detection technology is required to be high.

[0115] Plasma NfL can be used to screen individuals at high risk for dementia, but it is not specific for AD. Plasma Aβ42, the Aβ42 / 40 ratio, p-tau181 alone, and the plasma Aβ42 / 40 ratio combined with p-tau181 can be used to diagnose AD. Combining blood markers with clinical information such as AD risk factors, age, sex, APOE genotype, and neuropsychological assessments can help improve the diagnostic performance of AD.

[0116] 1. Disperse 40 mg of amino oxygen acceptor microspheres evenly in 4 mL of PBS buffer (0.1 M pH 7.2) to obtain a 10 mg / mL amino microsphere solution.

[0117] 2. Add sulfo-BS(PEG)9 until the final concentration of sulfo-BS(PEG)9 reaches 1 mM and incubate the reaction mixture at room temperature for 2 hours;

[0118] 3. Add 1M Tris solution to the above reaction solution until the final concentration of Tris is 25mM, and incubate the reaction solution at room temperature for 15 minutes;

[0119] 4. Remove the reaction solution, centrifuge and purify it, then disperse it in 4 mL of PBS buffer (0.1 M pH 7.2), divide it into 1 mL / tube, and make a concentration of 10 mg / mL for later use;

[0120] 5. Add 1 mg of purified anti-Aβ40 antibody to prepare the anti-Aβ40 antibody microsphere reaction solution, store at 2-8°C in the dark until use;

[0121] 6. Add 1 mg of purified anti-Aβ42 antibody to prepare the anti-Aβ42 antibody microsphere reaction solution, store at 2-8°C in the dark until use;

[0122] 7. Add 1 mg of purified anti-P-tau-181 antibody to prepare the anti-P-tau-181 antibody microsphere reaction solution, store at 2-8°C in the dark until ready to use;

[0123] 8. Add 1 mg of purified anti-NFL antibody to prepare the anti-NFL antibody microsphere reaction solution, store at 2-8°C in the dark until use;

[0124] 9. The anti-Aβ40 antibody microsphere reaction solution was combined with biotin-labeled anti-Aβ40 antibody 2 and streptavidin oxygen-donating microspheres to test Aβ40 samples of different concentrations. The results are as follows:

[0125] Aβ40 sample concentration pg / mL Detection signal value (RLU) Signal-to-noise ratio (S / N) 0 2372 / 25 3917 1.65 50 6756 2.85 100 11334 4.78 300 30473 12.85 1000 89602 37.77 3000 240800 101.52 5000 434944 183.37

[0126] When detecting Aβ40 samples with a concentration of 25 pg / mL, it was clearly distinguished from the sample with a concentration of 0; as the concentration of Aβ40 samples increased, the detection signal value showed a very good linear trend.

[0127] 1. The anti-Aβ42 antibody microsphere reaction solution was combined with biotin-labeled anti-Aβ42 antibody 2 and streptavidin oxygen-donating microspheres to test Aβ42 samples of different concentrations. The results are shown in Table 14 below.

[0128] Table 14

[0129] Aβ42 sample concentration pg / mL Detection signal value (RLU) Signal-to-noise ratio (S / N) 0 1889 / 30 3680 1.95 100 11030 5.84 300 32235 17.06 500 86063 45.56

[0130] When detecting Aβ42 samples with a concentration of 30 pg / mL, it is clearly distinguished from the sample with a concentration of 0; as the concentration of Aβ42 samples increases, the detection signal value shows a very good linear trend

[0131] 2. The anti-P-tau-181 antibody microsphere reaction solution was combined with biotin-labeled anti-P-tau-181 antibody 2 and streptavidin oxygen-donating microspheres to test P-tau-181 samples of different concentrations. The results are shown in Table 15 below.

[0132] Table 15

[0133] P-tau-181 sample concentration pg / mL Detection signal value (RLU) Signal-to-noise ratio (S / N) 0 974 / 5 2347 2.41 10 4884 5.01 30 14297 14.68 100 43057 44.21 300 118847 122.02 500 264481 271.54

[0134] When detecting a P-tau-181 sample with a concentration of 5 pg / mL, it was clearly distinguished from the sample with a concentration of 0; as the concentration of the P-tau-181 sample increased, the detection signal value showed a very good linear trend.

[0135] 3. The anti-NFL antibody microsphere reaction solution was combined with biotin-labeled anti-NFL antibody 2 and streptavidin oxygen-donating microspheres to test NFL samples of different concentrations. The results are shown in Table 16 below.

[0136] Table 16

[0137] NFL sample concentration pg / mL Detection signal value (RLU) Signal-to-noise ratio (S / N) 0 1347 / 10 3131 2.32 30 7994 5.93 100 22642 16.81 300 62514 46.41 1000 187112 138.91 3000 558715 414.78 5000 1000661 742.88

[0138] When detecting NFL samples with a concentration of 10 pg / mL, they were clearly distinguished from samples with a concentration of 0; as the concentration of NFL samples increased, the detection signal value showed a very good linear trend.

[0139] It should be understood that the present invention disclosed is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the term used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0140] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. A homogeneous chemiluminescence detection method, characterized in that: In the method, the amino oxygen acceptor microspheres are firstly reacted with sulfo BS (PEG) n Reaction, wherein n is an integer greater than or equal to 3, followed by reaction with an antibody to the antigen to be detected to prepare oxygen-accepting microspheres connected with the antibody to the antigen to be detected, and then detecting the antigen to be detected by a homogeneous chemiluminescence detection method.

2. The homogeneous chemiluminescence detection method according to claim 1, characterized in that Said n is an integer of 5-15.

3. The homogeneous chemiluminescence detection method according to claim 2, characterized in that Said n is an integer of 9-15.

4. The homogeneous chemiluminescence detection method according to any one of claims 1 to 3, characterized in that The antigen to be detected is fibroblast growth factor and / or AD protein.

5. The homogeneous chemiluminescence detection method according to claim 4, characterized in that: The antigen to be detected is fibroblast growth factor FGF21 or FGF23.

6. A homogeneous chemiluminescence detection kit, characterized in that: The kit includes the following oxygen-accepting microspheres, which are prepared by the following method: amino oxygen-accepting microspheres are first reacted with sulfo-BS(PEG)n, where n is an integer greater than or equal to 3, and then reacted with antibodies to the antigen to be detected to prepare oxygen-accepting microspheres connected with antibodies to the antigen to be detected.

7. The homogeneous chemiluminescence detection kit according to claim 6, characterized in that Said n is an integer of 5-15.

8. The homogeneous chemiluminescence detection kit according to claim 6, characterized in that Said n is an integer of 9-15.

9. The homogeneous chemiluminescence detection kit according to any one of claims 6 to 8, characterized in that The antigen to be detected is fibroblast growth factor and / or AD protein.

10. The homogeneous chemiluminescence detection kit according to claim 9, characterized in that The antigen to be detected is fibroblast growth factor FGF21 or FGF23.

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