Method for detecting marking efficiency of light-emitting marker

Abstract

The invention discloses a method for detecting the marking efficiency of a luminous marker, which comprises the following steps: providing a magnetic bead reagent, the solute of which is a magnetic bead coated by a first binding protein; providing a compound solution, wherein a solute of the compound solution is a coupling compound of the second binding protein and the luminous marker; jointly adding the compound solution and a luminous substrate into a chemiluminescence detection instrument, reading a luminous signal and recording the luminous signal as RLU total; jointly adding the compound solution and a magnetic bead reagent into a chemiluminescence detection instrument, sequentially incubating, separating and washing, adding a luminescent substrate, reading a luminescent signal and recording the luminescent signal as an RLU compound; and calculating the marking efficiency of the luminous marker, wherein the marking efficiency of the luminous marker is equal to RLU compound / RLU total * 100%. Compared with a traditional marking efficiency detection method, the method for detecting the marking efficiency of the luminous marker is easy to operate, low in cost and short in detection time.

Description

Technical Field

[0001] The invention relates to the technical field of chemiluminescence immunoassay, and in particular to a method for detecting the labeling efficiency of a luminescent marker. Background Art

[0002] Chemiluminescent immunoassay technology is the most advanced immunoassay technology in modern times. It uses highly sensitive magnetic beads to detect and diagnose target substances in clinical samples. It has the advantages of high automation, fast detection speed, high sensitivity and good analytical specificity, and plays an increasingly important role in clinical testing. The quality of chemiluminescent detection reagents is affected by many factors. The development of qualified detection reagents often includes paired antibody screening, coating and labeling process development, diluent component development, and cleaning fluid development. However, even after a long development cycle, the production process is confirmed. There are often unpredictable batch differences in conversion and mass production. That is, the reagents produced according to the established production process cannot reproduce the initial results when they are produced again, resulting in a great change in the performance of the reagents. The structural complexity of biological raw materials such as antibodies and antigens is certainly an important factor, but experience has shown that protein labeling process control is often the key link in producing batch differences.

[0003] The commonly used immunoreagent labeling process generally includes the following steps: 1. Preparation: Select a suitable buffer solution to prepare protein and marker reaction solutions respectively; 2. Labeling: Use the optimal process (molar ratio of protein and marker, reaction temperature, volume, rotation speed, time, termination labeling reaction conditions, etc.) for labeling; 3. Purification: Use ultrafiltration, dialysis or desalting methods to remove impurities; 4. Preservation: Add a suitable preservation solution to preserve the labeled complex and determine the final concentration. It can be seen that the entire labeling process is cumbersome and there are many influencing factors. Generally speaking, the method of excessive labeling and adding blocking solution is used to control the quality of the labeled complex, but the actual operation effect is not satisfactory. Protein conjugates play a core role in immunoassays, and their composition differences can significantly affect the performance of immunoassays. Therefore, accurate quantitative coupling ratio is a more effective indicator for controlling the quality of the labeled complex.

[0004] Common methods for detecting the labeling efficiency of luminescent markers include chromatography, fluorescence, and electrochemical methods. However, these methods generally have disadvantages such as expensive equipment, complex pre-treatment procedures, and high requirements for operators. Therefore, the prior art urgently needs a method for detecting the labeling efficiency of luminescent markers that is simple to operate, low in cost, and short in detection time. Summary of the invention

[0005] Based on this, it is necessary to provide a method for detecting the labeling efficiency of luminescent markers with simple operation, low cost and short detection time.

[0006] A method for detecting the labeling efficiency of a luminescent marker comprises the following steps:

[0007] Providing a magnetic bead reagent, wherein the solute of the magnetic bead reagent is a magnetic bead coated with a first binding protein;

[0008] Providing a complex solution, wherein the solute of the complex solution is a coupled complex of a second binding protein and a luminescent marker, wherein the second binding protein specifically binds to the first binding protein;

[0009] The complex solution and the luminescent substrate are added to the chemiluminescent detection instrument, and the luminescent signal is read and recorded as RLU. 总 , wherein the luminescent substrate and the luminescent marker match each other;

[0010] The complex solution and the magnetic bead reagent are added to the chemiluminescence detection instrument. After incubation, separation and washing, the luminescent substrate is added, and the luminescent signal is read and recorded as RLU. 复合物 , wherein the amount of the complex solution added twice is the same, and the amount of the luminescent substrate added twice is the same;

[0011] Calculate the labeling efficiency of the luminescent marker, the labeling efficiency of the luminescent marker = RLU 复合物 ÷RLU 总 ×100%.

[0012] In one embodiment, the first binding protein and the second binding protein are antigen and antibody that match each other, or the first binding protein and the second binding protein are specific binding proteins that match each other.

[0013] In one embodiment, the second binding protein is an animal antibody, and the first binding protein is a broad-spectrum antibody binding protein or an anti-animal secondary antibody.

[0014] In one embodiment, in the magnetic beads coated with the first binding protein, the magnetic beads are amino-modified magnetic beads, carboxyl-modified magnetic beads or tosyl-modified magnetic beads.

[0015] In one embodiment, in the magnetic beads coated with the first binding protein, the mass ratio of the magnetic beads to the first binding protein is 1:0.05-0.1.

[0016] In one embodiment, the magnetic beads are tosyl-modified magnetic beads with a particle size of 1 μm, and the mass ratio of the magnetic beads to the first binding protein is 1:0.1.

[0017] In one embodiment, the coupling complex of the second binding protein and the luminescent marker is obtained by coupling the second binding protein and the luminescent marker by glutaraldehyde method, Trauts & Sulfo-SMCC method or periodate method and then purifying.

[0018] In one embodiment, the luminescent label and the luminescent substrate are an acridinium ester system or an alkaline phosphatase system.

[0019] In one embodiment, the solvent of the magnetic bead reagent is a magnetic bead diluent, and the magnetic bead diluent is 50 mM PBS+1% BSA+0.1% Tween 20.

[0020] In one embodiment, the solvent of the complex solution is a complex diluent, and the complex diluent is 50 mM MES+1% BSA+0.1% Tween 20+5 mM MgCl2+0.01 mM ZnCl2.

[0021] The method for detecting the labeling efficiency of the luminescent marker of the present invention is to obtain the luminescent signal RLU 总 and luminescence signal RLU 复合物 , so that the labeling efficiency of the luminescent marker can be obtained simply and quickly.

[0022] Compared with traditional methods for detecting labeling efficiency, the method for detecting the labeling efficiency of the luminescent marker of the present invention is simple to operate, low in cost and short in detection time. Combined with specific embodiments, the method for detecting the labeling efficiency of the luminescent marker of the present invention has small batch differences and high consistency, and has good application prospects.

[0023] The chemiluminescence detection instrument can support the direct reading of free luminescent signals in the solution. The present invention utilizes the fully automatic signal reading function of the chemiluminescence detection instrument for free and luminescent substances captured and separated by magnetic bead carriers, and realizes simple, rapid, and fully automatic quantitative measurement of labeling efficiency by measuring the signal intensity before and after magnetic separation. For marker-antibody complexes represented by alkaline phosphatase labeling, this method can simply control the cross-linking ratio of alkaline phosphatase and antibody, thereby controlling the batch difference of the labeled product; for other marker-antibody complexes that can be measured by colorimetric assay (such as acridinium ester), this method can realize the detection of purification effect or nonspecific adsorption, and has good application prospects.

[0024] The method for detecting the labeling efficiency of the luminescent marker of the present invention does not require additional expensive instruments, complicated standard curve preparation, pretreatment and manual operation steps, and can achieve the determination of the labeling complex signal, the evaluation of the purification effect and the simple elimination of the nonspecific adsorption of the marker, thereby providing a simple batch control method for the labeling process, achieving quantitative control of the immune labeling effect and improving the stability of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] in:

[0027] Figure 1 The present invention is a flow chart of a method for detecting the labeling efficiency of a luminescent label according to one embodiment.

[0028] Figure 2 Test results of calibrator samples and clinical specimens were tested for three batches of stock solutions.

[0029] Figure 3 The test results of the batch difference of the reagents were tested for three batches of mother solutions. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Combination Figure 1 The present invention discloses a method for detecting the labeling efficiency of a luminescent marker according to an embodiment of the present invention, comprising the following steps:

[0032] S10. Provide a magnetic bead reagent, wherein the solute of the magnetic bead reagent is magnetic beads coated with a first binding protein.

[0033] Preferably, in this embodiment, the solvent of the magnetic bead reagent is a magnetic bead diluent, and the magnetic bead diluent is 50 mM PBS+1% BSA+0.1% Tween 20.

[0034] Generally speaking, after the magnetic beads are activated, they are blocked and coupled with the first binding protein, and after being cleaned, the magnetic beads coated with the first binding protein are obtained. The obtained magnetic beads coated with the first binding protein are dispersed in a magnetic bead diluent and stored.

[0035] Preferably, in this embodiment, the magnetic beads are amino-modified magnetic beads, carboxyl-modified magnetic beads or tosyl-modified magnetic beads.

[0036] Preferably, in this embodiment, in the magnetic beads coated with the first binding protein, the mass ratio of the magnetic beads to the first binding protein is 1:0.05-0.1.

[0037] Particularly preferably, in this embodiment, the magnetic beads are tosyl-modified magnetic beads with a particle size of 1 μm, and the mass ratio of the magnetic beads to the first binding protein is 1:0.1.

[0038] It should be pointed out that the specific concentration of the magnetic bead reagent is set according to actual needs, as long as it is within the signal measurement range of the chemiluminescence detection instrument.

[0039] S20, providing a complex solution, wherein the solute of the complex solution is a coupling complex of a second binding protein and a luminescent marker.

[0040] The second binding protein specifically binds to the first binding protein.

[0041] Preferably, in this embodiment, the solvent of the complex solution is a complex diluent, and the complex diluent is 50 mM MES + 1% BSA + 0.1% Tween 20 + 5 mM MgCl2 + 0.01 mM ZnCl2.

[0042] Preferably, in this embodiment, the first binding protein and the second binding protein are an antigen and an antibody that match each other.

[0043] In other embodiments, the first binding protein and the second binding protein can also be specific binding proteins that match each other. For example, the first binding protein is a goat anti-mouse secondary antibody, and the second binding protein is a mouse primary antibody, or the first binding protein is protein A / protein G, and the second binding protein is immunoglobulin IgG. Or the first binding protein is an anti-FITC antibody, and the second binding protein is a FITC labeled antibody, etc.

[0044] More preferably, in this embodiment, the second binding protein is an animal antibody, and the first binding protein is a broad-spectrum antibody binding protein or an animal-derived secondary antibody.

[0045] For example, the second binding protein is mouse antibody, rabbit antibody, goat antibody, etc., and the first binding protein is goat anti-mouse secondary antibody, anti-rabbit secondary antibody, anti-goat secondary antibody.

[0046] Generally speaking, the second binding protein and the luminescent marker are coupled by direct or indirect coupling methods such as the glutaraldehyde method, the Trauts & Sulfo-SMCC method or the periodate method, and then purified (desalting, ultrafiltration, dialysis or other purification methods) to obtain a coupled complex of the second binding protein and the luminescent marker. The obtained coupled complex of the second binding protein and the luminescent marker is stored in a complex diluent.

[0047] It should be pointed out that the specific concentration of the complex solution is set according to actual needs, as long as it is within the signal measurement range of the chemiluminescence detection instrument.

[0048] S30, add the complex solution and the luminescent substrate to the chemiluminescent detection instrument, read the luminescent signal and record it as RLU 总 .

[0049] Wherein, the luminescent substrate and the luminescent marker match each other.

[0050] Preferably, in this embodiment, the luminescent marker and the luminescent substrate are an acridinium ester system or an alkaline phosphatase system.

[0051] At this time, the luminescent label is acridinium ester or alkaline phosphatase acridinium ester, and the luminescent substrate is a corresponding substrate.

[0052] It should be noted that the luminescence signal RLU obtained by S30 总 Includes the sum of cross-linked and uncross-linked luminescence signals.

[0053] Considering that the function name and method of different instruments may be slightly different, it is necessary to repeatedly measure the luminescent signal 5 to 10 times in S30, calculate the average luminescent value and then take the average value.

[0054] It should be pointed out that the specific concentration of the luminescent substrate is set according to actual needs, as long as it is within the signal measurement range of the chemiluminescence detection instrument.

[0055] S40, add the complex solution and magnetic bead reagent to the chemiluminescence detection instrument, incubate, separate and wash in sequence, add the luminescent substrate, read the luminescent signal and record it as RLU 复合物 .

[0056] The amount of the complex solution added twice is the same, and the amount of the luminescent substrate added twice is the same, that is, the loading amount each time is the same.

[0057] It should be noted that the luminescence signal RLU obtained by S40 复合物 It is the cross-linking luminescent signal.

[0058] Considering that the function name and method of different instruments may be slightly different, it is necessary to repeat the measurement of the luminescent signal 5 to 10 times in S40, calculate the average luminescent value and then take the average value.

[0059] S50. Calculate the labeling efficiency of the luminescent marker. Labeling efficiency of the luminescent marker = RLU 复合物 ÷RLU 总 ×100%.

[0060] The method for detecting the labeling efficiency of the luminescent marker of the present invention is to obtain the luminescent signal RLU 总 and luminescence signal RLU 复合物 , so that the labeling efficiency of the luminescent marker can be obtained simply and quickly.

[0061] Compared with traditional methods for detecting labeling efficiency, the method for detecting the labeling efficiency of the luminescent marker of the present invention is simple to operate, low in cost and short in detection time. Combined with specific embodiments, the method for detecting the labeling efficiency of the luminescent marker of the present invention has small batch differences and high consistency, and has good application prospects.

[0062] The chemiluminescence detection instrument can support the direct reading of free luminescent signals in the solution. The present invention utilizes the chemiluminescence detection instrument to fully automatically read the signal of the luminescent substances that are free and captured and separated by the magnetic bead carrier, and by measuring the signal intensity before and after magnetic separation, a simple, rapid, and fully automatic quantitative measurement of the labeling efficiency is achieved. For the marker-antibody complex represented by the alkaline phosphatase marker, this method can simply control the cross-linking ratio of alkaline phosphatase and antibody, thereby controlling the batch difference of the labeled product. For other marker-antibody complexes that can be measured by colorimetric assay (such as acridinium ester), this method can realize the detection of purification effect or nonspecific adsorption, and has a good application prospect.

[0063] The method for detecting the labeling efficiency of the luminescent marker of the present invention does not require additional expensive instruments, complicated standard curve preparation, pretreatment and manual operation steps, and can achieve the determination of the labeling complex signal, the evaluation of the purification effect and the simple elimination of the nonspecific adsorption of the marker, thereby providing a simple batch control method for the labeling process, achieving quantitative control of the immune labeling effect and improving the stability of the product.

[0064] The following are specific embodiments.

[0065] In a specific embodiment, the GAM (goat anti-mouse) secondary antibody is model M6898 purchased from Sigma, alkaline phosphatase is model 10567752001 purchased from Roche, Protein A is model P6031 purchased from Sigma, tosyl (Tysol) magnetic beads are magnetic beads with a particle size of 1 μm purchased from Thermo, Trauts reagent is model 16256 purchased from Sigma, and Sulfo-SMCC is model M6035 purchased from Sigma.

[0066] Example 1: TPOAb-alkaline phosphatase labeling efficiency determination

[0067] 1. Reagent Preparation

[0068] Prepare the following reagents:

[0069] Labeling buffer A: 100 mM PBS + 2 mM EDTA-2Na, pH = 7.2;

[0070] Labeling buffer B: 100 mM PBS + 2 mM EDTA-2Na, pH = 8.0;

[0071] Antibody buffer: 100 mM PBS, pH = 7.2;

[0072] Complex diluent: 50 mM MES + 1% BSA + 0.1% Tween 20 + 5 mM MgCl2 + 0.01 mM ZnCl2, pH = 6.0;

[0073] Antibody activator: 5 mg / ml Trauts reagent (pre-set at room temperature for 30 minutes, dissolved in ultrapure water)

[0074] Enzyme activator: 5 mg / ml Sulfo-SMCC reagent (pre-incubated at room temperature for 30 min, dissolved in labeling buffer B)

[0075] Magnetic bead diluent: 50 mM PBS + 1% BSA + 0.1% Tween 20, pH = 7.4.

[0076] 2. Preparation of Secondary Antibody Magnetic Beads

[0077] a) Activation of magnetic beads: Commercially purchased tosyl (Tysol) magnetic beads (100 mg / mL) were ultrasonically dispersed for 1 min, 0.1 mL of the suspension (i.e., 10 mg) was taken, 0.4 mL of 100 mM boric acid buffer was added, mixed, and then magnetic separation was performed on a magnetic stand and the supernatant was removed. This was repeated twice; 0.5 mL of 100 mM boric acid buffer (pH 9.5) was added and resuspended for later use;

[0078] b) Coupling: Add 0.2 mL of 1.5 mg / mL GAM (goat anti-mouse) secondary antibody to the activation buffer, vortex to mix, then add 0.35 mL of 3 M ammonium sulfate buffer (pH 9.5), mix thoroughly and react at 37°C, 220 r / min for 16 h;

[0079] c) Blocking: After the coupling is completed, the reaction tube is placed on a magnetic rack for separation, the supernatant is removed, 50 μL of 5% BSA solution is added, and the reaction is carried out at 37°C and 220 r / min for 6 hours;

[0080] d) Washing the magnetic beads: The sealed reaction tube was placed on a magnetic stand for separation and the supernatant was removed. A washing solution (10 mM TBS, 0.05% Tween-20) was added to wash the complex three times;

[0081] e) Storage: The secondary antibody magnetic bead suspension was distributed in a PBS solution containing 0.1% BSA (pH=7.4) and stored in a refrigerator at 2-8° C. for future use.

[0082] 3. Preparation of coupling complex (alkaline phosphatase marker)

[0083] a) Enzyme activation: Dissolve 10 mg of alkaline phosphatase in 1 mL of labeling buffer A, and desalt and purify; Take 1 mg of desalted alkaline phosphatase and add enzyme activator solution to 200 μl, mix thoroughly, stand at room temperature for 60 min, and then purify with a desalting column, determine the concentration of the recovered product, and adjust it to 4 mg / mL with labeling buffer A;

[0084] b) Protein A activation: prepare 10 mg / mL antibody solution with antibody buffer, desalt and purify, and determine the concentration of the recovered product; take 0.5 mg of antibody, add antibody activator solution to 100 μl, mix thoroughly, let stand at room temperature for 60 min, then purify with a desalting column, determine the concentration of the recovered product, and adjust its concentration to 2 mg / ml with labeling buffer A;

[0085] c) Cross-linking: Mix activated alkaline phosphatase and activated protein A at different mass ratios (AP: Ab / Ag = 1.0, 1.5, 2.5), mix thoroughly and react at room temperature for 60 minutes;

[0086] d) Desalting and purifying to obtain a coupling complex, measuring the volume of the coupling complex, adding an equal volume of glycerol, and storing at 2-8° C. for future use.

[0087] 4. Testing

[0088] a) Preparation of magnetic bead working solution: dilute the prepared secondary antibody magnetic bead suspension to 0.2 mg / mL using magnetic bead diluent (100 mM PBS, 1% BSA, 0.05% Tween-20);

[0089] b) Preparation of label working solution: dilute the coupling complex with complex diluent at a dilution ratio of 1:10000 and mix thoroughly.

[0090] c) Test the total luminescence signal RLU 总 :Edit the test program on the Cosmai 6500 instrument, take 10μL of the marker working solution and mix it directly with the substrate, and test the RLU 总 If the signal is too high / too low, adjust the dilution ratio appropriately to within the machine test range; repeat the test 5 times and calculate the average luminescence value;

[0091] d) Test the cross-linked complex luminescent signal RLU complex: edit the test program, take 50 μL of magnetic bead working solution, add 10 μL of marker working solution, incubate 5 groups, then perform magnetic separation, 4 washing procedures, directly mix with substrate, and test RLU 复合物 , repeat the test 5 times and calculate the average luminescence value;

[0092] e) Labeling efficiency calculation: Labeling efficiency = RLU 复合物 ÷RLU 总 ×100%.

[0093] 5. Preparation of multiple batches of mother liquor to control cross-linking ratio: Control the selected labeling efficiency and repeat labeling 3 times at different time periods to obtain 3 batches of mother liquor.

[0094] The three batches of mother liquor were diluted into working solution with the same diluent at a dilution ratio of 1:5000, and then matched with reagent magnetic bead working solution (TPO antigen-coated magnetic beads). After the detection reagent was formed with the working solution, 6 calibrator samples and 7 clinical samples were tested respectively.

[0095] The measured signal was fitted with four parameters to establish the master curve, and the clinical sample concentration was calculated to obtain Figure 2 .

[0096] Combination Figure 2 It can be seen that when the molar ratio of AP to protein is less than 1, that is, as the proportion of AP decreases, the labeling efficiency gradually increases, that is, the utilization of AP gradually increases. That is, the cross-linking efficiency can be reflected by the ratio.

[0097] According to the clinical test results, a labeling ratio of 1:1.1 (cross-linking ratio 0.47) was selected, and the cross-linking value was controlled to be 0.47. Three batches of mother solution were prepared, and the batch difference of the reagent was tested. Figure 3 .

[0098] Combination Figure 3 It can be seen that when the three batches of reagents were used to measure the calibration products, the signals of batch 2 and batch 1 had a negative deviation, with an average deviation of -6.2%, and the signals of batch 3 and batch 1 had a positive deviation, with an average deviation of 7.4%. When the three batches of reagents were used to measure the samples, the mean deviation of the calibration back-calculated concentration was within 5%, and the differences between batches were small.

[0099] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for detecting the labeling efficiency of a luminescent marker, characterized in that: The steps include: Providing a magnetic bead reagent, wherein the solute of the magnetic bead reagent is a magnetic bead coated with a first binding protein; Providing a complex solution, wherein the solute of the complex solution is a coupled complex of a second binding protein and a luminescent marker, wherein the second binding protein specifically binds to the first binding protein; The complex solution and the luminescent substrate are added to the chemiluminescent detection instrument, and the luminescent signal is read and recorded as RLU. 总 , wherein the luminescent substrate and the luminescent marker match each other; The complex solution and the magnetic bead reagent are added to the chemiluminescence detection instrument. After incubation, separation and washing, the luminescent substrate is added, and the luminescent signal is read and recorded as RLU. 复合物 , wherein the amount of the complex solution added twice is the same, and the amount of the luminescent substrate added twice is the same; Calculate the labeling efficiency of the luminescent marker, the labeling efficiency of the luminescent marker = RLU 复合物 ÷RLU 总 ×100%.

2. The method for detecting the labeling efficiency of a luminescent marker according to claim 1, characterized in that: The first binding protein and the second binding protein are antigen and antibody that match each other, or the first binding protein and the second binding protein are specific binding proteins that match each other.

3. The method for detecting the labeling efficiency of a luminescent marker according to claim 2, characterized in that: The second binding protein is an animal antibody, and the first binding protein is a broad-spectrum antibody binding protein or an anti-animal secondary antibody.

4. The method for detecting the labeling efficiency of a luminescent marker according to any one of claims 1 to 3, characterized in that: In the magnetic beads coated with the first binding protein, the magnetic beads are amino-modified magnetic beads, carboxyl-modified magnetic beads or tosyl-modified magnetic beads.

5. The method for detecting the labeling efficiency of a luminescent marker according to claim 4, characterized in that: In the magnetic beads coated with the first binding protein, the mass ratio of the magnetic beads to the first binding protein is 1:0.05-0.

1.

6. The method for detecting the labeling efficiency of a luminescent marker according to claim 5, characterized in that: The magnetic beads are tosyl-modified magnetic beads with a particle size of 1 μm, and the mass ratio of the magnetic beads to the first binding protein is 1:0.

1.

7. The method for detecting the labeling efficiency of a luminescent marker according to claim 4, characterized in that: The coupling complex of the second binding protein and the luminescent marker is obtained by coupling the second binding protein and the luminescent marker through glutaraldehyde method, Trauts & Sulfo-SMCC method or periodate method, followed by purification.

8. The method for detecting the labeling efficiency of a luminescent marker according to claim 4, characterized in that: The luminescent marker and the luminescent substrate are an acridinium ester system or an alkaline phosphatase system.

9. The method for detecting the labeling efficiency of a luminescent marker according to claim 6, characterized in that: The solvent of the magnetic bead reagent is a magnetic bead diluent, and the magnetic bead diluent is 50mM PBS+1%BSA+0.1%Tween20.

10. The method for detecting the labeling efficiency of a luminescent marker according to claim 6, characterized in that: The solvent of the complex solution is a complex diluent, and the complex diluent is 50 mM MES+1% BSA+0.1% Tween 20+5 mM MgCl2+0.01 mM ZnCl2.

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