CN117949272A

CN117949272A - Pretreatment method of streptavidin magnetic beads for electrochemiluminescence detection

Info

Publication number: CN117949272A
Application number: CN202410137598.2A
Authority: CN
Inventors: 王超儿; 陈媛; 张闻; 燕茹凤; 徐启宇
Original assignee: Ningbo Rui Bio Technology Co ltd
Current assignee: Ningbo Rui Bio Technology Co ltd
Priority date: 2024-01-31
Filing date: 2024-01-31
Publication date: 2024-04-30

Abstract

The invention discloses a pretreatment method of streptavidin magnetic beads, which comprises the following steps: s1, cleaning streptavidin magnetic beads with a sealing liquid, diluting the magnetic beads with the sealing liquid, and then incubating; s2: and (3) washing the blocked streptavidin magnetic beads with a reagent buffer solution, and finally diluting the streptavidin magnetic beads with the reagent buffer solution to prepare a solid-phase reagent. The pretreatment method for the commercial streptavidin magnetic beads can change the surface charge and steric hindrance, improve the sensitivity of the commercial streptavidin magnetic beads applied to electrochemiluminescence detection reagents, and solve the problems of endogenous interference and cross contamination of a measuring cell.

Description

Pretreatment method of streptavidin magnetic beads for electrochemiluminescence detection

Technical Field

The invention relates to the technical field of chemiluminescent immunoassay, in particular to a pretreatment method of streptavidin magnetic beads for electrochemiluminescence detection.

Background

The electrochemiluminescence immunoassay technology uses magnetic beads as a solid phase carrier to connect a specific immune complex, the magnetic beads are adsorbed on the surface of an electrode through electromagnetic action, and the terpyridyl ruthenium marked on an antibody generates oxidation-reduction reaction through the electrode action to generate a specific electrochemiluminescence signal, so that the quantitative detection of a specific antigen is finally realized.

The electrochemiluminescent reagent can be designed in two forms depending on the order in which the magnetic beads are bound to the immunocomplexes. The first form is to complete the combination of magnetic beads and antibodies, the magnetic beads coated with the antibodies are used as solid phase reagents to participate in the antigen-antibody reaction of the next step, and the first form is a form commonly adopted by the chemiluminescent reagents at present. The reagent in the form has two defects that the immune reaction is influenced by steric hindrance and the stability of the solid phase reagent is poor. In the other form, a traditional solid-phase reagent is designed into two independent components of streptavidin magnetic beads and biotin labeled antibodies by introducing a biotin-streptavidin system, the biotin labeled antibodies and antigens complete immune reaction in the first detection step, and the streptavidin magnetic beads are added to capture immune complexes in the second detection step, so that the immune reaction efficiency and the reagent stability can be improved.

There are many magnetic bead manufacturers on the market to provide commercial streptavidin magnetic beads, but there are still many problems to be solved in the use of these streptavidin magnetic beads as independent reagent components for the electrochemiluminescence detection reagent of the above reaction system. Firstly, the surface of the magnetic bead is not subjected to sealing treatment, nonspecific adsorption is easy to generate, and the detection result is easily influenced by endogenous interferents such as hemoglobin, bilirubin, triglyceride and the like; secondly, the electrochemiluminescence reaction is carried out in a measuring cell, and magnetic beads are easy to remain on the surface of an electrode, so that the detection background is raised and even cross contamination is caused to the detection of a later sample; in addition, the combination of streptavidin magnetic beads and biotin antibodies is also affected by steric hindrance, which affects the reaction signal value and ultimately the detection sensitivity.

Patent CN112986349 discloses a method for modifying and labeling magnetic beads, which comprises the steps of modifying the magnetic beads by adding a protecting group, and then carrying out chemical coupling to coat antibodies or streptavidin, thereby controlling residual charges and hydrophobic groups on the surfaces of the magnetic beads, and finally achieving the effect of reducing the magnetic bead residues on the surfaces of electrochemical reaction electrodes. The proposal needs to modify the magnetic beads and then coat the streptavidin, which is ineffective for commercial streptavidin magnetic beads. Patent CN112698025 discloses a method for coating magnetic particles with antigen or antibody, which reduces steric hindrance and improves chemiluminescent detection signal value by using short arm biotin and long arm biotin labeled antigen or antibody to coat streptavidin magnetic beads.

So far, no economically viable solution can simultaneously solve the three problems of endogenous interference, cross-contamination of measuring cells and limited sensitivity of streptavidin magnetic beads applied to electrochemiluminescence immunoassay reagents.

Disclosure of Invention

The invention aims to provide a sealing agent for electrochemiluminescence immunoassay and application thereof, which solve the problems of endogenous interference, cross contamination of a measuring cell and limited sensitivity of streptavidin magnetic beads applied to electrochemiluminescence immunoassay reagents.

The aim of the invention is achieved by the following technical scheme:

A pretreatment method of streptavidin magnetic beads, the pretreatment method comprising the steps of:

S1: washing streptavidin magnetic beads with a sealing liquid, diluting the magnetic beads with the sealing liquid, and then incubating;

s2: and (3) washing the blocked streptavidin magnetic beads with a reagent buffer solution, and finally diluting the streptavidin magnetic beads with the reagent buffer solution to prepare a solid-phase reagent.

Further, the blocking fluid component comprises a buffer, a surfactant, a macromolecular protein mixture and a small molecular amino acid.

Further, the blocking solution comprises 50mM Tris, 0.5% sodium caseinate, 0.1% glycine, 0.02% sodium laurylaminopropionate, and the pH of the blocking solution is 8.0.

Further, the incubation conditions are: incubate at 37℃for 3 hours.

Further, the reagent buffer includes a buffer, a protein, a surfactant, and a preservative.

Further, the reagent buffer also includes a saline solution.

Further, the salt solution is sodium chloride or magnesium chloride.

Further, the buffer solution in the reagent buffer solution is 10-50mM phosphate buffer solution, the protein is BSA, the surfactant is Tween 20, the preservative is PC300, and the pH of the reagent buffer solution is 6.5-7.5.

Further, in the step S1, the streptavidin magnetic beads are washed 3 times by using a sealing liquid; and in the step S2, the blocked streptavidin magnetic beads are washed 3 times by using a reagent buffer solution, and the solid-phase reagent is a liquid mixture containing the streptavidin magnetic beads.

The pretreatment method of any streptavidin magnetic beads is applied to electrochemiluminescence detection.

Commercial streptavidin magnetic beads are not usually blocked or screened by a magnetic bead pretreatment method for application to electrochemiluminescence reagents, so that the streptavidin coated on the surfaces of the magnetic beads is abnormal in morphology, so that a part of binding sites of streptavidin and biotin are blocked, the binding process of the streptavidin magnetic beads and biotin antibodies is affected by steric hindrance, and reagent sensitivity is reduced. In addition, the streptavidin magnetic beads are generally obtained by chemical coupling of carboxyl magnetic beads or toluenesulfonyl magnetic beads and streptavidin, carboxyl or toluenesulfonyl is connected with polystyrene on the surfaces of the magnetic beads through alkane chains, the surfaces of the magnetic beads which are not closed usually show hydrophobicity, so that nonspecific adsorption is easy to occur on the surfaces of the magnetic beads, detection results are easily influenced by endogenous interferents such as hemoglobin, and meanwhile, nonspecific adsorption can also occur between the magnetic beads and electrodes, so that the magnetic beads remain on the surfaces of the electrodes to cause cross contamination of a measuring cell.

According to the invention, macromolecular proteins and small molecular amino acids are simultaneously added into the sealing liquid, so that streptavidin on the surface of the magnetic beads maintains a correct form, and the binding sites of biotin are fully exposed. In addition, a surfactant is added into the sealing liquid to change the hydrophobicity of the surface of the magnetic beads, and meanwhile, the surfactant can also play a role of a detergent to remove nonspecific adsorption, reduce the influence of endogenous interferents on detection results and reduce the magnetic bead residues on the surface of the electrode. In order to better support the streptavidin on the surface of the magnetized beads and to fully expose the biotin binding sites, the invention selects a protein mixture comprising a plurality of molecular weight components, namely sodium caseinate. The sodium caseinate contains a plurality of macromolecular proteins with relative molecular weight of 75-375 kDa, and the proteins with different molecular weights can effectively fill the gaps on the surfaces of the streptavidin magnetic beads. Meanwhile, sodium caseinate has similar properties with a surfactant, and can reduce nonspecific adsorption on the surface of the magnetic beads to a certain extent.

The beneficial effects of the invention are as follows:

1. The pretreatment method of the streptavidin magnetic beads provided by the invention improves various commercialized streptavidin magnetic beads, and the surface steric hindrance of the commercialized streptavidin magnetic beads is changed by sealing the commercialized streptavidin magnetic beads, so that the streptavidin on the surface maintains a correct form, the binding sites of biotin are fully exposed, the detection signal value is improved, and the sensitivity of the streptavidin on the application of the streptavidin to an electrochemiluminescence detection reagent is also improved.

2. According to the pretreatment method of the streptavidin magnetic beads, the commercial streptavidin magnetic beads are subjected to sealing treatment, so that the surface charge and hydrophobicity of the magnetic beads are changed, nonspecific adsorption is removed, and endogenous interference and cross contamination of a measuring pool are eliminated.

3. The pretreatment method of the streptavidin magnetic beads provided by the invention is simple and convenient to operate, and is very suitable for popularization and application in electrochemiluminescence detection.

Drawings

FIG. 1 is a graph showing the comparison of the detection results of FPSA solid phase reagent prepared in comparative example 7 and the comparison reagent FPSA of the present invention;

FIG. 2 is a graph showing the comparison of the detection results of FPSA solid phase reagent and the comparison reagent FPSA prepared in example 4 of the present invention.

Detailed Description

Definition of terms used in connection with the present invention: unless otherwise indicated, the initial definitions provided for terms herein apply to terms throughout the specification; for terms not specifically defined herein, the meanings that one skilled in the art can impart based on the disclosure and the context.

The reagents and equipment used in the present invention are known products or are obtained by purchasing commercially available products.

The following terms in the following examples are explained:

NSE: neuron-specific enolase; AFP: alpha fetoprotein; PSA: a prostate specific antigen; FPSA: free prostate specific antigen; RLU: relative light emitting units.

Solid phase reagent: the liquid mixture containing streptavidin magnetic beads captures the immune complex formed after the antigen-antibody reaction by binding to biotin-labeled antibodies in the electrochemiluminescence detection technique.

Streptavidin magnetic beads a purchase manufacturer: JSR (JSR)

Streptavidin magnetic beads B purchase manufacturer: agilent technologies Co Ltd

Streptavidin magnetic beads C purchase manufacturer: hangzhou Boyue biotechnology Co Ltd

Reaction rationale for electrochemiluminescence detection:

Forming an antigen-antibody immune complex by the sample and the biotin labeling reagent and the ruthenium labeling reagent; the solid phase reagent is added, so that the antigen-antibody immune complex is adsorbed to the surface of the magnetic beads through the combination of biotin and streptavidin to form a reactant; transferring the reactant into a measuring pool, applying an electromagnetic field in the measuring pool, adsorbing the magnetic beads on the surface of the electrode under the action of the electromagnetic field, and removing the terpyridyl ruthenium which is not combined with the magnetic beads by the cleaning liquid flowing in the measuring pool; and an electric field is applied in the measuring cell, the terpyridyl ruthenium realizes electrochemical luminescence reaction through the combined action of the electrode and tripropylamine, generated photons are captured by an instrument and converted into a relative luminescence unit RLU, and finally, the conversion of the concentration of the antigen or antibody to be detected in the sample and an electrochemical luminescence signal is realized, and the concentration measurement of the sample to be detected is realized.

In order to more clearly illustrate the technical scheme of the invention, the specific embodiments are as follows:

Example 1

S1: taking a certain amount of streptavidin magnetic beads A, cleaning the streptavidin magnetic beads A for 3 times by using a sealing liquid 1, diluting the magnetic beads to 10mg/mL by using the sealing liquid 1, and incubating at 37 ℃ for 3 hours to seal the magnetic beads;

S2: and (3) washing the blocked magnetic beads with a reagent buffer solution for 3 times, and finally diluting the magnetic beads to 0.6mg/mL with an NSE reagent buffer solution to prepare an NSE solid-phase reagent.

Blocking liquid 1:50mM Tris,pH8.0,0.5% sodium caseinate, 0.1% glycine, 0.02% sodium dodecylaminopropionate.

NSE reagent buffer: 50mM phosphate buffer, pH6.5,0.5% BSA,5mM magnesium chloride, 0.05% Tween 20,0.1% PC300.

Comparative example 1:

Comparative example 1 is based on example 1, with the difference that: the streptavidin magnetic beads A are directly prepared into a solid-phase reagent without blocking treatment.

Comparative example 2:

comparative example 1 is based on example 1, with the difference that: streptavidin magnetic beads A are blocked by a blocking liquid 2.

Sealing liquid 2:50mM Tris,pH8.0,2%BSA,0.1% glycine, 0.05% triton X-100.

The solid phase reagents prepared in example 1, comparative example 1 and comparative example 2 were combined with NSE biotin labeling reagent and NSE ruthenium labeling reagent, respectively, to form detection reagents, and RLU values were measured on an electrochemiluminescence immunoassay analyzer, and the RLU value differences are shown in table 1.

Meanwhile, three groups of reagents are subjected to measurement cell cross contamination rate test by adopting a test method of carrying contamination, the high-value samples 3 are parallel in sequence, then the blank samples 3 are parallel, and the cross contamination rate (cross contamination rate= (RLU 3-RLU 6)/(RLU 4-RLU 6) ×100%) is calculated, and the result is shown in table 2. Because the electrochemiluminescence immunoassay analyzer used in the experiment sucks samples and reagents and uses the disposable suction head, no carrying pollution of the suction needle exists, and the abnormal measurement result is caused by cross pollution in the measuring pool.

TABLE 1

Table 1 data shows: compared with comparative examples 1 and 2, the streptavidin magnetic beads A of the example 1 are blocked by the blocking liquid 1, and the increase amplitude of the RLU value of the NSE reagent detection calibrators S1-S7 can reach 50% and 35% on average, and meanwhile, the detection background RLU value is kept unchanged.

TABLE 2

By integrating the data in tables 1 and 2, the RLU value of the blank calibrator S0 was about 600, and the RLU reached 20000 when the blank calibrator S0 was measured after the high value calibrator S7 was measured in comparative example 1, and cross contamination was evident. Table 2 data shows: the cross-contamination rate was significantly reduced in comparative example 2 compared to comparative example 1, but cross-contamination was still present, whereas example 1 completely eliminated the cell cross-contamination.

The results of example 1 and comparative example 1, comparative example 2 show that: the streptavidin magnetic beads A are blocked by a blocking solution mixed by macromolecular proteins, small molecular amino acids and a surfactant, so that the sensitivity of a NSE reagent to a sample can be improved to a certain extent, and the cross contamination of a measuring cell caused by the magnetic bead residue on the surface of an electrode is reduced. Compared with the combination of BSA and non-ionic surfactant triton X-100, the sodium caseinate and amino acid type surfactant sodium dodecyl aminopropionate have more obvious effects of improving the detection sensitivity and reducing the cross contamination of a measuring cell.

Example 2:

s1: taking a certain amount of streptavidin magnetic beads B, cleaning the streptavidin magnetic beads B for 3 times by using a sealing liquid 1, diluting the magnetic beads to 10mg/mL by using the sealing liquid 1, and incubating at 37 ℃ for 3 hours to seal the magnetic beads;

s2: and (3) washing the blocked magnetic beads with a reagent buffer solution for 3 times, and finally diluting the magnetic beads to 0.8mg/mL with an AFP reagent buffer solution to prepare the AFP solid-phase reagent.

AFP reagent buffer: 10mM phosphate buffer, pH7.2,0.5% BSA,0.9% sodium chloride, 0.05% Tween 20,0.1% PC300.

Comparative example 3:

Comparative example 3 is based on example 2, with the difference that: the streptavidin magnetic beads B are directly prepared into a solid-phase reagent without blocking treatment.

Comparative example 4:

comparative example 4 is based on example 2, with the difference that: streptavidin magnetic beads B are blocked by a blocking liquid 3.

Sealing liquid 3:50mM Tris,pH8.0,0.5% gamma-globulin, 0.1% glycine, 0.05% tween 20.

The solid phase reagents prepared in example 2, comparative example 3 and comparative example 4 were combined with AFP biotin-labeled reagent and AFP ruthenium-labeled reagent, respectively, to form detection reagents, and the RLU values were measured on an electrochemiluminescence immunoassay analyzer, and the RLU value differences are shown in Table 3.

Three groups of reagents were simultaneously tested for hemoglobin interference. The test method is as follows: a5000 mg/dL hemoglobin solution is prepared by using physiological saline, 5 serum samples are taken, each serum sample is divided into two parts, one part of the hemoglobin solution is added in a volume ratio of 1:9 (the concentration of the hemoglobin added in the final serum sample is 500 mg/dL), the other part of the hemoglobin solution is added in the same proportion to serve as a control group, and the relative deviation of the two groups of test results is the interference ratio of the hemoglobin to the AFP test, and the results are shown in Table 4.

TABLE 3 Table 3

Table 3 data shows: compared with comparative example 3 and comparative example 4, the streptavidin magnetic beads B of example 2 are blocked by the blocking liquid 1, the RLU value of the AFP reagent detection sample can be increased by 80% and 65% on average, and the detection background RLU value is only increased by less than 10%.

TABLE 4 Table 4

Table 4 data shows: comparative example 3 the AFP sample was significantly disturbed by hemoglobin with a disturbance rate as high as 15% and comparative example 4 with a slight decrease in disturbance rate, whereas the modified reagent of example 2 was used to eliminate the hemoglobin disturbance.

The results of example 2 and comparative examples 3, 4 show that: the streptavidin magnetic beads B are blocked by a blocking solution mixed by macromolecular proteins, small molecular amino acids and a surfactant, so that the sensitivity of an AFP reagent to a sample can be improved to a certain extent, and meanwhile, the interference of hemoglobin on a detection result is reduced. Sodium caseinate and sodium dodecylaminopropionate have more pronounced effects on improving detection sensitivity and eliminating hemoglobin interference than the combination of gamma-globulin and tween 20.

Example 3:

S1: taking a certain amount of streptavidin magnetic beads C, cleaning for 3 times by using a sealing liquid 1, diluting the magnetic beads to 10mg/mL by using the sealing liquid 1, and incubating at 37 ℃ for 3 hours to seal the magnetic beads;

s2: and (3) washing the blocked magnetic beads with a reagent buffer solution for 3 times, and finally diluting the magnetic beads to 0.7mg/mL with a PSA reagent buffer solution to prepare the PSA solid-phase reagent.

PSA reagent buffer: 10mM phosphate buffer, pH7.4,0.5% BSA,0.05% Tween 20,0.1% PC300.

Comparative example 5:

Comparative example 5 is based on example 3, with the difference that: the streptavidin magnetic beads C are directly prepared into a solid-phase reagent without blocking treatment.

Comparative example 6:

comparative example 6 is based on example 3, with the difference that: streptavidin magnetic beads C are blocked by a blocking liquid 4.

Sealing liquid 4:50mM Tris,pH8.0,2% glycine, 0.05% brij35.

The test solid phase reagents prepared in example 3, comparative example 5 and comparative example 6 were combined with a PSA biotin-labeled reagent and a PSA ruthenium-labeled reagent, respectively, to prepare detection reagents, and RLU values were measured on an electrochemiluminescence immunoassay analyzer, and the RLU value differences are shown in table 5.

Three sets of reagents were tested simultaneously for triglyceride interference. The test method is as follows: the triglyceride interference is simulated by using the fat emulsion, the fat emulsion is diluted to 30000mg/dL by using normal saline, then 5 serum samples are taken, each serum sample is divided into two parts, one part of the fat emulsion with the concentration which is added in the volume ratio of 1:19 (the concentration of the fat emulsion added in the final serum sample is 1500 mg/dL), the other part of the fat emulsion with the concentration which is added in the final serum sample is added in the same ratio is used as a control group, and the relative deviation of the two groups of test results is the interference rate of the triglyceride on the PSA test, and the results are shown in Table 6.

TABLE 5

Table 5 data shows: compared with comparative examples 5 and 6, the streptavidin magnetic beads C of the example 3 are blocked by the blocking liquid 1, the average amplitude of the improvement of the RLU value of the detection sample of the PSA reagent can reach 78% and 75%, and the detection background RLU value is basically unchanged.

TABLE 6

Table 6 data shows: the detection of the PSA sample in the comparative example 5 is obviously interfered by triglyceride, the interference rate is about 25%, the interference of the triglyceride in the comparative example 6 on the PSA detection is improved to a certain extent, and the reagent improved in the example 3 is adopted to eliminate the triglyceride interference.

The results of example 3 and comparative example 5, comparative example 6 show that: after the streptavidin magnetic beads C are blocked, the interference of triglyceride on a detection result is reduced to a certain extent, but at the same time, the detection sensitivity of the PSA reagent is not improved by adopting a blocking solution without macromolecular protein, compared with the detection sensitivity of the PSA reagent without macromolecular protein, the combination of sodium caseinate, small molecular amino acid and sodium dodecyl amino propionate has a remarkable effect on improving the sensitivity, and the interference effect of the combination on eliminating triglyceride is more obvious.

Example 4:

s2: and (3) washing the blocked magnetic beads with a reagent buffer solution for 3 times, and finally diluting the magnetic beads to 0.7mg/mL with an FPSA reagent buffer solution to prepare the FPSA solid-phase reagent.

FPSA reagent buffer: 10mM phosphate buffer, pH7.4,0.5% BSA,0.05% Tween 20,0.1% PC300.

Comparative example 7:

comparative example 7 is based on example 4, with the difference that: the streptavidin magnetic beads C are directly prepared into a solid-phase reagent without blocking treatment.

The solid phase reagents prepared in example 4 and comparative example 7 were combined with a FPSA biotin labeling reagent and a FPSA ruthenium labeling reagent, respectively, to prepare detection reagents, and the RLU values were measured on an electrochemiluminescence immunoassay analyzer, and the RLU value differences are shown in Table 7.

Meanwhile, 8 hemolysis samples, 8 lipid turbidity samples and 9 high bilirubin samples were taken, and a total of 25 interference samples were simultaneously detected in concentration by using the FPSA reagent of example 4, the FPSA reagent of comparative example 7 and the purchased Roche comparison reagent. The detection process comprises the following steps: the FPSA reagent of example 4, the FPSA reagent of comparative example 7 and the alignment reagent were respectively calibrated with respective matched calibrators, then 25 interference samples were sequentially tested, and the instrument automatically calculated a sample concentration value according to the RLU value and calibration curve of the sample test. The sample detection results of example 4 and the comparison reagent are compared and analyzed, and the sample detection results of the comparison reagent of comparative example 7 are compared and analyzed, and the comparison results of the 2 groups of reagents are shown in fig. 1 and 2.

TABLE 7

Table 7 data shows: compared with comparative example 7, the streptavidin magnetic beads C of example 4 are blocked by the blocking liquid 1, the average improvement amplitude of the RLU value of the FPSA reagent detection sample can reach 95%, and the detection background RLU value is basically unchanged.

The data in fig. 1 and 2 show that, for the hemolyzed sample, the lipidated sample and the hyperbilirubin sample with the endogenous interferent clinically, the comparison correlation coefficient r2= 0.9407 of the comparative example 7 and the comparison reagent has poor correlation, which indicates that the endogenous interferent has a relatively large influence on the test result of the clinical sample, while the sample with the endogenous interferent has a high consistency with the test result of the comparison reagent by adopting the reagent modified in example 4, and the correlation coefficient R2 is close to 0.99.

The results of examples 1-4 are obviously different from those of comparative examples 1,3, 5 and 7, and indicate that the pretreatment method of the invention is applied to 3 different commercial streptavidin magnetic beads, is applied to NSE, AFP, PSA, FPSA items, can obviously improve the detection sensitivity of reagents, can solve the endogenous interference problem and the cross contamination problem of a measuring cell when the common streptavidin magnetic beads are directly applied to electrochemiluminescence detection reagents, and can improve the accuracy of sample detection.

The results of examples 1 to 3 are different from those of comparative examples 2,4 and 6, and show that compared with the sealing liquid without adding macromolecular proteins and with only adding macromolecular proteins with single molecular weight, the combination of the protein mixture containing various molecular weight combinations, small molecular amino acid and surfactant selected by the invention is more obvious in improving detection sensitivity, improving cross contamination of a measuring cell and improving endogenous interference effects.

Claims

1. A pretreatment method of streptavidin magnetic beads, which is characterized by comprising the following steps:

s1, cleaning streptavidin magnetic beads with a sealing liquid, diluting the magnetic beads with the sealing liquid, and then incubating;

2. The method of claim 1, wherein the blocking solution comprises a buffer, a surfactant, a mixture of macromolecular proteins, and a small amino acid.

3. The method for pretreatment of streptavidin magnetic beads according to claim 2, wherein the blocking solution comprises 50mM Tris, 0.5% sodium caseinate, 0.1% glycine and 0.02% sodium dodecylaminopropionate, and the pH of the blocking solution is 8.0.

4. The method for pretreatment of streptavidin magnetic beads according to claim 1, wherein the incubation conditions are: incubate at 37℃for 3 hours.

5. The method for pretreating streptavidin magnetic beads according to claim 1, wherein the reagent buffer comprises a buffer, a protein, a surfactant, and a preservative.

6. The method for pretreating streptavidin magnetic beads according to claim 5, wherein the reagent buffer further comprises a salt solution.

7. The method for pretreating streptavidin magnetic beads according to claim 6, wherein the salt solution is sodium chloride or magnesium chloride.

8. The method for pretreating streptavidin magnetic beads according to claim 5, wherein the buffer in the reagent buffer is 10-50mM phosphate buffer, the protein is BSA, the surfactant is Tween 20, the preservative is PC300, and the pH of the reagent buffer is 6.5-7.5.

9. The method for pretreating streptavidin magnetic beads according to claim 5, wherein the streptavidin magnetic beads are washed 3 times with a blocking solution in step S1;

and in the step S2, the blocked streptavidin magnetic beads are washed 3 times by using a reagent buffer solution, and the solid-phase reagent is a liquid mixture containing the streptavidin magnetic beads.

10. Use of a pretreatment method of necklace mould avidin magnetic beads according to any of claims 1-9 in electrochemiluminescence detection.