Biotin-antibody conjugation ratio detection kit and application thereof

Abstract

The application discloses a kit for detecting the coupling ratio of biotin and antibody and application thereof, and comprises an antibody capture carrier, biotinylated antibody quality control L, biotinylated antibody quality control H and chemiluminescence labeled streptavidin, wherein the antibody capture carrier is Protein A / G magnetic beads or a mixture of Protein A and Protein G magnetic beads, and the molar ratio of biotin to antibody in the biotinylated antibody quality control L and H is 1:1-5:1 and 15:1-30:1 respectively. The kit can detect the coupling ratio of biotin and antibody in a wide range, can detect the coupling ratio of biotin and antibody in a range of 1-40, has high sensitivity, and the sample consumption is less than 5 muL, and thus the kit can be used for controlling the coupling ratio of biotin and antibody in each batch of process.

Description

Technical Field

[0001] This invention relates to a biotin-antibody conjugate ratio detection kit and its application. Background Technology

[0002] Biotin, also known as vitamin H, has a molecular weight of 244.31. The molecule has two ring structures. Ring I is an imidazolide ring, which is the main site of binding to streptavidin. Ring II is a thiazole ring with a valeric acid side chain. The terminal carboxyl group is often chemically modified or altered to increase the hydrophilicity of biotin or reduce steric hindrance, transforming it into long-chain hydrazide biotin or N-hydroxysuccinimide ester biotin, which is used to label the thiol and amino groups of antibodies to increase the sensitivity and specificity of the biotin-streptomycin affinity system.

[0003] Generally, one antibody molecule can conjugate 1-20 biotin molecules. The reaction steps are simple, but the process is difficult to control. In direct chemiluminescence systems, a biotin-to-antibody conjugation ratio that is too low directly affects the binding efficiency of the biotinylated antibody to SA microparticles (one SA molecule can bind to four biotin molecules), resulting in excess antibody and a lower signal value for the entire reaction system. Conversely, a biotin-to-antibody conjugation ratio that is too high makes it difficult to completely remove free biotin and also affects antibody stability. Therefore, selecting an appropriate conjugation ratio is particularly important for biotin-streptavidin systems. To control the biotin-to-antibody conjugation ratio in each batch of the process and to confirm the parameters of the chemiluminescence reagent kit, specialized techniques are needed to accurately quantify the biotin-to-antibody conjugation ratio.

[0004] Thermo Scientific is the primary manufacturer of biotin-antibody conjugation ratio assay kits on the market. Their kit works by mixing 4′-hydroxyazobenzene-2-carboxylic acid (HABA) with a biotinylated antibody solution. Because biotin has a higher affinity for avidin, it replaces 4′-hydroxyazobenzene-2-carboxylic acid, resulting in a proportional decrease in absorbance at 500 nm. This method quantifies the proportion of unknown amounts of biotinylated antibody present in a single cuvette or 96-well plate. The advantages are ease of use, simple equipment (colorimetric method), and the ability to determine the conjugation ratio of any protein to biotin. The disadvantages are inaccurate measurement and a narrow range; furthermore, due to the high cost of some proteins or antibodies and the small volume of labeled conjugate (especially around 100 μL), this method requires 20-100 μL of sample, leading to unnecessary cost losses. Based on the above analysis, this invention develops a biotin-antibody conjugation ratio detection kit with a wide detection range, high sensitivity, and a sample volume of less than 5 μL, for controlling the biotin-antibody conjugation ratio in each batch of processes. Summary of the Invention

[0005] The purpose of this invention is to provide a biotin-antibody conjugation ratio detection kit with a wide detection range, high sensitivity, and a sample volume of less than 5 μL, for controlling the biotin-antibody conjugation ratio in each batch of processes.

[0006] The technical solution adopted in this invention is as follows: a biotin-antibody conjugation ratio detection kit, comprising an antibody capture carrier, a biotinylated antibody quality control L, a biotinylated antibody quality control H, and chemiluminescently labeled streptavidin. The antibody capture carrier is Protein A / G magnetic beads or a mixture of Protein A and Protein G magnetic beads. The biotin-antibody conjugation molar ratios of biotinylated antibody quality control L and H are 1:1-5:1 and 15:1-30:1, respectively.

[0007] Protein A contains five Fc receptors that specifically bind to the Fc terminus of mouse antibody IgG. It can also bind to the Fc terminus of antibodies from rabbit, mouse, horse, and human species, but the affinity of antibodies from different species for Protein A varies considerably. Protein G is a type III Fc receptor that can bind not only to the Fc terminus of monoclonal or polyclonal antibodies from rabbit, mouse, sheep, and human species, but also to the F(ab) terminus of antibodies. Therefore, to improve the ability of antibodies to capture different antibody epitopes from different species, it is necessary to bind to both the Fc and F(ab) terminus of antibodies from different species. The antibody traps of the present invention are of two types: one is Protein A / G magnetic beads, and the other is a mixture of Protein A and Protein G magnetic beads. These two antibody traps are prepared by two methods: one is to use artificially expressed Protein A / G complex as the antibody trap; the other is to mix artificially expressed Protein A and Protein G in a certain proportion to prepare an antibody trap, and then use magnetic microspheres as carriers to couple the antibody trap onto the magnetic beads to prepare an antibody trap carrier.

[0008] Preferably, in the preparation of the Protein A / G magnetic beads, the mass ratio of magnetic microspheres to Protein A / G composite is 1:0.05-0.1, more preferably 1:0.1.

[0009] Preferably, in the preparation of the Protein A and Protein G mixture magnetic beads, the mass ratio of the magnetic microspheres to the Protein A and Protein G mixture is 1:0.05-0.1, preferably 1:0.1, wherein the mass ratio of Protein A to Protein G in the Protein A and Protein G mixture is 1:2-2:1, preferably 1:1.

[0010] Preferably, the magnetic microspheres are carboxyl magnetic microspheres or toluene xanthyl magnetic microspheres.

[0011] Preferably, the concentration of the antibody capture carrier is 0.5-2.5 mg / mL.

[0012] Preferably, the luminescent material is selected from one or more of N-(4-aminobutyl)-N-ethyl isoluminol, acridine ester, acridine sulfonamide, horseradish peroxidase, alkaline phosphatase, or any other substance that can react with the chemiluminescent excitation solution to produce chemiluminescence. More preferably, in the preparation of the chemiluminescently labeled streptavidin, the luminescent material is selected from acridine ester, and the molar ratio of streptavidin to acridine ester is 1:1 to 1:20, preferably 1:10.

[0013] Preferably, the concentration of the chemiluminescently labeled streptavidin is 0.1–1 μg / mL.

[0014] Preferably, the concentration range of biotinylated antibody control L and biotinylated antibody control H is 0.05-0.5 μg / mL, preferably 0.1 μg / mL. When using, the concentrations of biotinylated antibody control L and biotinylated antibody control H are the same, and the concentration of the biotinylated antibody to be tested must be equal to the concentrations of biotinylated antibody control L and biotinylated antibody control H.

[0015] This invention also provides the application of the above-mentioned biotin-antibody conjugate ratio detection kit, which is used according to the following steps:

[0016] 1) The biotinylated antibody to be tested, biotinylated antibody quality control L, and biotinylated antibody quality control H were reacted with the antibody capture carrier and chemiluminescently labeled streptavidin under incubation conditions. After washing, chemiluminescent excitation solution was added, and the luminescence values ​​of the biotinylated antibody to be tested, biotinylated antibody quality control L, and biotinylated antibody quality control H were measured and found to be RLU. D RLU L and RLU H ;

[0017] 2) Calculate the molar ratio of biotin to antibody in the biotinylated antibody to be tested: Calculated according to formula (1):

[0018]

[0019] In equation (1), N L For biotinylated antibody quality control product L, the molar ratio of biotin to antibody conjugation is: RLU L The luminescence value of biotinylated antibody quality control product L;

[0020] N H Biotinylated antibody quality control H, biotin to antibody conjugation molar ratio; RLUH The luminescence value of biotinylated antibody quality control product H;

[0021] N D The biotinylated antibody to be tested, with a biotin-to-antibody conjugation molar ratio; RLU D The fluorescence value is the value of the biotinylated antibody to be tested.

[0022] Compared with a commercially available kit, the detection kit of this invention has the following advantages:

[0023] (1) The present invention requires a small sample volume, with a sample concentration of 0.05-0.5 μg / mL, which will not lead to the waste of expensive antibodies. Taking cTnI reagent as an example, the biotinylated antibody to be tested generally needs to be diluted 300-1000 times before use, and 100 μL of biotinylated antibody can prepare approximately 600-2000 reagents.

[0024] (2) The reagent kit of the present invention uses a gradient that is 3-4 times that of a commercially available reagent kit, which has high sensitivity and more accurate quantification. It can detect biotin-antibody conjugate ratios of 1-40, with the lowest detectable biotin-antibody conjugate ratio being 1.

[0025] (3) Biotin used for labeling is a hydrophobic substance and is generally diluted with DMF or DMSO. Compared with commercially available kits, this kit is not affected by DMF or DMSO because the sample is diluted before testing.

[0026] Table 1. Performance comparison of the kit of the present invention with the Pierce Biotin Quantitation Kit (Thermo Scientific)

[0027]

[0028] Note: The gradient is the ratio of the luminescence values ​​of a biotinylated antibody with a conjugation ratio of 40 to that of a biotinylated antibody with a conjugation ratio of 1. Attached Figure Description

[0029] To more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0030] Figure 1 This is a schematic diagram of the detection kit for detecting the conjugation ratio of elements and antibodies.

[0031] Figure 2 The relationship between different biotinylated antibody conjugation ratios and luminescence signal values. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The technical solutions provided by various embodiments of this invention will be described in detail below with reference to the accompanying drawings.

[0033] Example 1 A biotin-antibody conjugation ratio detection kit includes an antibody capture carrier, a biotinylated antibody quality control L with a biotin-antibody conjugation molar ratio of 2:1, a biotinylated antibody quality control H with a biotin-antibody conjugation molar ratio of 20:1, and chemiluminescently labeled streptavidin. The antibody capture carrier is Protein A / G magnetic beads or a mixture of Protein A magnetic beads and Protein G magnetic beads.

[0034] Optionally, the biotin-antibody conjugate assay kit further includes a sample diluent, the sample diluent being formulated as: 0.1 mol / L PBS (pH 7.2) + 0.05% (w / w) diazolidinyl urea.

[0035] Preferably, the chemiluminescent labeled streptavidin is acridine ester labeled streptavidin.

[0036] The concentrations of each component in the biotin-antibody conjugate assay kit are as follows:

[0037] 1) Antibody capture vector: final concentration 1 mg / mL;

[0038] 2) Streptavidin signal marker: Diluted to 1 μg / mL using dilution buffer (0.05 mol / L MES (pH 6.0) + 1.5% (w / v) BSA + 0.05% (w / v) diazolidinyl urea);

[0039] 3) Biotinylated antibody quality control product L (molar coupling ratio of biotin to antibody is 2:1), final concentration is 0.1 μg / mL;

[0040] 4) Biotinylated antibody quality control product H (the molar coupling ratio of biotin to antibody is 20:1), with a final concentration of 0.1 μg / mL.

[0041] The preparation methods for each component of the above-mentioned test kit are as follows:

[0042] 1. Preparation of antibody capture vector:

[0043] (1) Preparation of Protein A / G magnetic beads:

[0044] a) First, sonicate the entire bottle of magnetic beads for 1 min to disperse them evenly. Take 5 mL of carboxylated magnetic microspheres (10 mg / mL) into a 10 mL centrifuge tube, centrifuge at 5000 rpm for 5 min, and slowly aspirate with a pipette to remove the buffer solution from the carboxylated magnetic microspheres. Add 5 mL of 100 mM MES (pH 6.0) to redisperse the mixture, sonicate for 1 min, centrifuge at 5000 rpm for 5 min, and slowly aspirate with a pipette to remove the buffer solution. Then add 5 mL of 10-100 mM MES (pH 6.0) buffer solution.

[0045] b) Weigh 25-100 mg of EDC and quickly add it to the magnetic bead suspension, and place it at 25-37℃ and react at 250 r / min for 30 min.

[0046] c) Wash the activated magnetic beads with 5 mL of 10-100 mM MES (pH 6.0); then add 5 mg of Protein A / G complex (from Shanghai Puxin) and react at 220 rpm for 3 h.

[0047] d) Add 20 mL of washing buffer and wash for a total of 3 times. Centrifuge and discard the buffer. Add 50 mL of magnetic bead storage solution (0.1 mol / L PBS (pH 7.5) + 0.5% (w / v) BSA + 0.05% (w / v) sodium azide) to prepare the antibody capture vector with a final concentration of 1 mg / mL.

[0048] (2) Preparation of magnetic beads made from a mixture of Protein A and Protein G:

[0049] a) Take 2.5 mg each of Protein A (from Shanghai Puxin) and Protein G (from Shanghai Puxin) and mix them in a 1:1 ratio (mass ratio) to prepare a mixture of Protein A and Protein G.

[0050] b) Sonicate the Tosyl magnetic microspheres (toluenexanthocyanidin magnetic microspheres) for 1 min to disperse them evenly. Take 5 mL of Tosyl magnetic microspheres (10 mg / mL) into a 10 mL centrifuge tube, centrifuge at 5000 rpm for 5 min, and slowly aspirate with a pipette to remove the buffer solution from the Tosyl magnetic microspheres. Add 5 mL of 0.1 M sodium carbonate buffer (pH 9.5) to redisperse the mixture, sonicate for 1 min, centrifuge at 5000 rpm for 5 min, and slowly aspirate with a pipette to remove the buffer solution. Then add 5 mL of 0.1 M sodium carbonate buffer (pH 9.5) buffer solution.

[0051] c) Take 5 mg of the mixture of Protein A and Protein G from step a) and quickly add it to the magnetic bead suspension, and place it at 37°C and react at 100-250 r / min for 12 h.

[0052] d) Add 20 mL of washing buffer and wash for a total of 3 times. Centrifuge and discard the buffer solution. Add 50 mL of magnetic bead storage solution to prepare the antibody capture vector with a final concentration of 1 mg / mL.

[0053] 2. Preparation of acridinium ester-labeled streptavidin:

[0054] a) Turn on the thermostatic oscillator and set the temperature to 25.0℃;

[0055] b) Take 1 mg of streptavidin (murine progeny, monoclonal antibody), add 4.5 μL of acridinium ester (1 mg / mL), then add 50 mM PB (pH 8.0), with a final volume of 1 mL; shake to mix, and react in a shaker at 25 °C for 60 min;

[0056] c) Add 10 μL of blocking ethanolamine to the above reaction tube and mix to stop the excess acridine ester. Then, place the mixture at 25°C for 30 min.

[0057] d) The above reactants were added to a dialysis bag and then placed in a 2L beaker. 1.5L of 0.1mol / L PBS was added for dialysis. The medium was changed once every 1h for a total of 10 times to obtain SA-AE (acridyl ester labeled streptavidin).

[0058] 3. Preparation of antibody-conjugated control samples:

[0059] In the field of in vitro diagnostics, monoclonal or polyclonal antibodies are mainly derived from mouse, rabbit, sheep, and human recombinant antibodies. The commonly used biotin for antibody labeling is biotin-N-hydroxysuccinimide ester biotin. The specific implementation steps are as follows:

[0060] (1) Preparation of biotinylated antibody quality control material L (2:1):

[0061] a) Turn on the thermostatic oscillator and set the temperature to 25.0℃;

[0062] b) Take 2 mg of myoglobin antibody (mouse-derived, monoclonal antibody), add 15 μL of biotin (1 mg / mL), then add 50 mM CB (pH 9.5), with a final volume of 5 mL; shake to mix, and react in a shaker at 25 °C for 30 min;

[0063] c) Then add 10 μL of blocking solution ethanolamine to the above reaction tube and mix to stop the excess biotin. Place the mixture at 25°C for 30 min.

[0064] d) Add the above reactants to a dialysis bag, then place it in a 2L beaker, add 1.5L of 0.1mol / L PBS for dialysis, change the medium once every 1h, for a total of 10 times. The resulting biotinylated antibody is diluted with sample diluent to a final concentration of 0.1μg / mL, which is the biotinylated antibody quality control L (2:1).

[0065] (2) Preparation of biotinylated antibody quality control material H (20:1):

[0066] Take 2 mg of myoglobin antibody (mouse-derived, monoclonal antibody), add 150 μL of biotin (1 mg / mL), then add 50 mM CB (pH 9.5), with a final volume of 5 mL; shake to mix, and react in a shaker at 25 °C for 30 min; other steps are the same as those for preparing biotinylated antibody quality control L (2:1).

[0067] Example 2

[0068] The detection steps using the above biotin-antibody conjugate ratio assay kit are as follows:

[0069] 1) Dilute the biotinylated antibody to be tested to 0.1 μg / mL using sample diluent;

[0070] 2) Add 10 μL of 1 mg / mL antibody capture carrier to 80 μL of 0.1 μg / mL sample (biotinylated antibody to be tested), 0.1 μg / mL biotinylated antibody quality control L, and 0.1 μg / mL biotinylated antibody quality control H, respectively. Incubate for 10 min and wash 3 times to obtain the biotinylated antibody-carrier complex to be tested, the biotinylated antibody quality control L-carrier complex, and the biotinylated antibody quality control H-carrier complex.

[0071] 3) Add 100 μL of 1 μg / mL acridinium-labeled streptavidin to the biotinylated antibody-carrier complex to be tested, the biotinylated antibody quality control L-carrier complex, and the biotinylated antibody quality control H-carrier complex, respectively. Incubate for 10 min, wash 3 times, and pump in activation solution 1 (0.05 mol / L HNO3 + 0.5% (mass fraction) H2O2) and activation solution 2 (0.2 mol / L NaOH) to induce chemiluminescence signals in the complexes. Measure the luminescence intensity using a photomultiplier (PMT) to obtain the luminescence values ​​of the biotinylated antibody to be tested, antibody-conjugated ratio quality control L, and antibody-conjugated ratio quality control H, which are respectively RLU values. D RLU L and RLU H ;

[0072] 4) Calculate the molar ratio of biotin to antibody in the biotinylated antibody to be tested: Calculated according to formula (1):

[0073]

[0074] In equation (1), N L For biotinylated antibody quality control product L, the molar ratio of biotin to antibody conjugation is: RLU L The luminescence value of biotinylated antibody quality control product L;

[0075] N H Biotinylated antibody quality control H, biotin to antibody conjugation molar ratio; RLU H The luminescence value of biotinylated antibody quality control product H;

[0076] N D The biotinylated antibody to be tested, with a biotin-to-antibody conjugation molar ratio; RLU D The fluorescence value is the value of the biotinylated antibody to be tested.

[0077] The detection principle of the biotin-antibody conjugate ratio detection kit of the present invention is as follows: Figure 1 As shown: The biotinylated antibody to be tested, the antibody capture carrier, and the streptavidin signal marker react under incubation conditions. The antibody capture carrier binds to the Fc or F(ab) end of the biotinylated antibody to be tested, and then binds to the streptavidin signal marker to form a reaction complex. After incubation, the precipitated complex is washed with washing solution, and the waste liquid is aspirated to remove unreacted substances. The reaction vessel is then placed into the measurement chamber, and the instrument pumps in excitation solution 1 and excitation solution 2 respectively, causing the complex to generate a chemiluminescent signal. The luminescence intensity is measured by a photomultiplier (PMT). Figure 2 As shown, the biotinylated antibody conjugation ratio is positively correlated with the luminescence signal. Therefore, formula (1) is derived based on the slope, and the conjugation ratio of antibody molecules to biotin is calculated based on the signal values ​​of two biotinylated antibody quality control samples (biotinylated antibody quality control samples L and H).

[0078] Following the steps of Example 2, six samples were prepared for testing using different ratios of myoglobin antibody to biotin. The ratios were 1, 3, 5, 10, 20, and 40. The resulting six samples are Sample 1, Sample 2, Sample 3, Sample 4, Sample 5, and Sample 6. The results are shown in Table 2.

[0079] Table 2 Comparison of the kit of the present invention with the Pierce Biotin Quantitation Kit (Thermo Scientific) kit

[0080]

[0081] The results in Table 2 show that the measured values ​​of the kit of the present invention are basically consistent with the actual values.

[0082] Table 3. Interference comparison between the reagent kit of this invention and the Pierce Biotin Quantitation Kit (Thermo Scientific).

[0083]

[0084] Generally, biotin used for labeling needs to be dissolved in DMF or DMSO to prepare a 1-10 mg / mL biotin solution. When labeling antibodies with biotin, a biotin solution (containing DMF or DMSO) needs to be added; the labeled antibody then needs to be purified by dialysis or centrifugation to remove free, unreacted biotin. Centrifugation and column purification are generally difficult to remove DMF or DMSO, or may result in residual DMF or DMSO in the biotinylated antibody to be tested. Table 3 compares the interference tests of the kit of this invention and the Pierce kit by adding different concentrations (volume percentage) of DMF or DMSO to the biotinylated antibody to be tested (without DMF or DMSO). The results in Table 3 show that the signal value of the kit of this invention is not affected by DMF and DMSO interference; the Pierce kit is unaffected when the DMF or DMSO concentration is less than 5%, but the interference is strong when the concentration is greater than 10%, resulting in inaccurate measurements.

[0085] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method of using a biotin-antibody conjugate ratio detection kit, characterized in that, The biotin-antibody conjugation ratio assay kit includes an antibody capture carrier, biotinylated antibody control L, biotinylated antibody control H, and chemiluminescently labeled streptavidin. The antibody capture carrier is Protein A / G magnetic beads or a mixture of Protein A and Protein G magnetic beads. The biotin-antibody conjugation molar ratios of biotinylated antibody control L and H are 1:1-5:1 and 15:1-30:1, respectively. The biotin-antibody conjugate ratio assay kit is used according to the following steps: 1) The biotinylated antibody to be tested, biotinylated antibody control L, and biotinylated antibody control H were reacted with the antibody capture carrier and chemiluminescently labeled streptavidin under incubation conditions. After washing, chemiluminescent excitation solution was added, and the luminescence values ​​of the biotinylated antibody to be tested, biotinylated antibody control L, and biotinylated antibody control H were measured and found to be RLU. D RLU L and RLU H ; 2) Calculate the molar ratio of biotin to antibody in the biotinylated antibody to be tested: Calculated according to formula (1): (1) In equation (1), N L For biotinylated antibody quality control product L, the molar ratio of biotin to antibody conjugation is: RLU L The luminescence value of biotinylated antibody quality control product L; N H Biotinylated antibody quality control H, biotin to antibody conjugation molar ratio; RLU H The luminescence value of biotinylated antibody quality control H; N D The biotinylated antibody to be tested, with a biotin-to-antibody conjugation molar ratio; RLU D The fluorescence value is the value of the biotinylated antibody to be tested.

2. The method of using the biotin-antibody conjugate ratio detection kit according to claim 1, characterized in that, In the preparation of Protein A / G magnetic beads, the mass ratio of magnetic microspheres to Protein A / G composite is 1:0.05-0.

1.

3. The method of using the biotin-antibody conjugate ratio detection kit according to claim 1, characterized in that, In the preparation of the protein A and protein G mixture magnetic beads, the mass ratio of the magnetic microspheres to the protein A and protein G mixture is 1:0.05-0.1, wherein the mass ratio of protein A to protein G in the protein A and protein G mixture is 1:2-2:

1.

4. The method of using the biotin-antibody conjugate ratio detection kit according to claim 2 or 3, characterized in that, The magnetic microspheres are carboxyl magnetic microspheres or toluenesulfonyl magnetic microspheres.

5. The method of using the biotin-antibody conjugate ratio detection kit according to claim 1, characterized in that, The concentration of the antibody capture carrier is 0.5-2.5 mg / mL.

6. The method of using the biotin-antibody conjugate ratio detection kit according to claim 1, characterized in that, In the chemiluminescently labeled streptavidin, the luminescent substance is selected from one of N-(4-aminobutyl)-N-ethyl isoluminol, acridine ester, acridine sulfonamide, horseradish peroxidase, alkaline phosphatase, or any other substance that can react with the chemiluminescent excitation solution to produce chemiluminescence.

7. The method of using the biotin-antibody conjugate ratio detection kit according to claim 6, characterized in that, In the preparation of the chemiluminescent labeled streptavidin, the luminescent substance is selected from acridine ester, and the molar ratio of streptavidin to acridine ester is 1:1-1:

20.

8. The method of using the biotin-antibody conjugate ratio detection kit according to claim 1, characterized in that, The concentration of the chemiluminescently labeled streptavidin is 0.1~1 μg / mL.

9. The method of using the biotin-antibody conjugate ratio detection kit according to claim 1, characterized in that, The concentration of biotinylated antibody quality control L is 0.05-0.5 μg / mL, and the concentration of biotinylated antibody quality control H is 0.05-0.5 μg / mL.

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