Preparation method and application for solving low coating amount aggregation of carboxyl magnetic beads

Abstract

The invention relates to the technical field of chemiluminescence immunoassay, in particular to a preparation method and application for solving low coating amount aggregation of carboxyl magnetic beads. Activating agents EDC and NHS are added into a first magnetic bead resuspension; cleaning the activated first magnetic bead resuspension with a coupling buffer solution to obtain a second magnetic bead resuspension with the concentration of 5-20 mg / mL; adding activators EDC and NHS and an antibody into the second magnetic bead resuspension; a first sealing agent with the pH being 6-8 and containing 50-400 mmol / L of ethanolamine is added into the second magnetic bead resuspension for primary sealing; removing the supernatant of the second magnetic bead resuspension after primary sealing, and adding a second sealing agent for secondary sealing; and washing the second magnetic bead resuspension after secondary sealing by using a magnetic bead working solution, and fixing the concentration of carboxyl magnetic beads to 0.4 mg / mL. The carboxyl magnetic bead coating process is optimized, the magnetic bead activity is improved, and the test background signal value is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of chemiluminescent immunoassay, and in particular to a preparation method and application for solving the aggregation of carboxyl magnetic beads with low coating amount. Background Art

[0002] Chemiluminescence immunoassay (CLIA) is a labeling immunoassay technology that combines chemiluminescence with immune response and is based on the specific reaction of antigen and antibody to detect trace amounts of antigen or antibody. Magnetic particle chemiluminescence is the mainstream technology for immunodiagnosis. Compared with other immunodiagnostics, it has the characteristics of rapidity, sensitivity, strong specificity, and automation. According to the principle, it is mainly divided into double antibody sandwich method and competitive method. Currently, the commonly used magnetic beads are carboxyl magnetic beads, carboxyl magnetic beads, Tosyl magnetic beads, etc. The double antibody sandwich method is to couple the antibody to the magnetic beads, and the alkaline phosphatase-labeled secondary antibody reacts specifically with the antigen in the sample to form a solid phase antibody-antigen-antibody sandwich complex, wash away the unbound substances, and react with the substrate 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoacyl)-phenyl-1,2-dioxetane disodium salt (AMPPD), and calculate the antigen content in the sample through the test results.

[0003] At present, the problem of magnetic bead aggregation is a major pain point for IVD. The coating methods of carboxyl magnetic beads are often divided into one-step and two-step methods. The one-step method has the advantages of being fast and efficient, but the activation products during the coating process may have a certain impact on the coupling of antibodies and magnetic beads; although the coupling process of the two-step method avoids this risk, excessive antibodies are often used during coupling to achieve the same titer as the one-step method, so the coupling efficiency is low. The coupling process is generally coated at a ratio of 2ug / mg to 40ug / mg between antibodies and magnetic beads. With the increase in the applicable models of the same kit, in order to improve the universality of the kit on different models and meet the needs of different models, there are certain requirements for the magnetic bead concentration of the working solution. However, as the amount of magnetic bead coating decreases (that is, the amount of coated antibody decreases), the probability of magnetic beads aggregation increases, resulting in a high background signal value, affecting the stability of the reagent, the repeatability of the test, and the sensitivity. The mainstream solution is generally to ultrasonically shake and mix the magnetic bead working solution before use, but ultrasonic treatment will also cause the antibodies captured by the magnetic beads to fall off; adding 0.1% (v / v) non-ionic detergent (such as NP-40, Tween-20 or Triton X-100, etc.) to the magnetic bead storage solution can improve the problem of magnetic bead aggregation. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method and application for solving the aggregation of carboxyl magnetic beads with low coating amount, aiming to solve the problem of high background signal value caused by magnetic bead aggregation.

[0005] To achieve the above object, in a first aspect, a preparation method for solving the aggregation of carboxyl magnetic beads with low coating amount comprises the following steps:

[0006] After ultrasonic treatment of the carboxyl magnetic beads, the beads are washed with a pre-coating buffer to obtain a first magnetic bead resuspension solution with a concentration of 5 to 20 mg / mL;

[0007] Adding activators EDC and NHS to the first magnetic bead resuspension solution;

[0008] The activated first magnetic bead resuspension is washed with a coupling buffer to obtain a second magnetic bead resuspension with a concentration of 5 to 20 mg / mL;

[0009] Adding activators EDC and NHS and antibodies to the second magnetic bead resuspension;

[0010] Adding a blocking agent having a pH of 6 to 8 and containing 50 to 400 mmol / L ethanolamine to the second magnetic bead resuspension solution for primary blocking;

[0011] removing the supernatant of the second magnetic bead resuspension solution after the first blocking, and adding blocking agent 2 for secondary blocking;

[0012] The second magnetic bead resuspension after secondary blocking was washed with magnetic bead working solution, and the concentration of carboxyl magnetic beads was fixed to 0.4 mg / mL.

[0013] Among them, in "after ultrasonically treating the carboxyl magnetic beads, use a pre-coating buffer to perform segmentation to obtain a first magnetic bead resuspension with a concentration of 5 to 20 mg / mL", the pre-coating buffer is a PB buffer, MES buffer or CB buffer with a pH of 5 to 7, preferably a 50 mM MES buffer with a pH of 6.0.

[0014] Among them, in "adding activators EDC and NHS to the first magnetic bead resuspension solution", the mass ratio of the EDC to the magnetic beads is 1:2 to 1:20, the mass ratio of the EDC to the NHS is 1:1 to 1:5, and preferably the mass ratio of EDC:magnetic beads:NHS is 1:10:4.

[0015] Among them, in "using a coupling buffer to wash the activated first magnetic bead resuspension to obtain a second magnetic bead resuspension with a concentration of 5 to 20 mg / mL", the coupling buffer refers to a PB buffer, MES buffer, CB buffer, etc. containing 0.02 to 0.1% BSA and 0.02 to 0.1% surfactant (HLB>15), a pH of 5 to 7, preferably a 50 mM MES buffer with a pH of 6.0, 0.04% BSA and 0.1% sodium lauryl sulfate.

[0016] Among them, in "adding activators EDC and NHS and antibodies to the second magnetic bead resuspension", the mass ratio of EDC to magnetic beads is 1:1 to 1:40, the mass ratio of EDC to NHS is 1:1 to 1:3, the ratio of antibodies to magnetic beads is 2ug / mg to 40ug / mg, and preferably the mass ratio of EDC: magnetic beads: NHS is 1:20:1, and the antibody is 5ug / mg.

[0017] Among them, in "adding a blocking agent with a pH of 6 to 8 and containing 50 to 400 mmol / L ethanolamine to the second magnetic bead resuspension for a blocking", the ratio of the blocking agent to the carboxyl magnetic beads is 100 ul / mg, preferably a 250 mmol / mL ethanolamine solution with a pH of 7.4 for blocking.

[0018] Among them, in "removing the supernatant of the second magnetic bead resuspension after the first blocking, and adding blocking agent 2 for secondary blocking", the blocking agent 2 is PB buffer, MES buffer, Tris buffer, etc. with a pH of 6-8 and containing 0.5-2% BSA, preferably Tris buffer with a pH of 7.4 and 1% BSA.

[0019] Among them, in "using magnetic bead working solution to wash the second magnetic bead resuspension after secondary blocking, and adjusting the concentration of carboxyl magnetic beads to 0.4 mg / mL", the magnetic bead working solution is 10mM Tris buffer, pH 7-8, containing 0.5% sodium chloride, 0.2%-2% glucose, 0.1%-1% casein, 0.1%-1% Triton X-100, 0.02%-0.5% preservative, wherein the preservative is preferably 2 of PC300, PC950, and BND-10.

[0020] In the second aspect, an application of a preparation method for solving the aggregation of carboxyl magnetic beads with low coating amounts is provided, wherein the preparation method for solving the aggregation of carboxyl magnetic beads with low coating amounts described in the first aspect is used to solve the aggregation of carboxyl magnetic beads with low coating amounts.

[0021] The invention discloses a preparation method for solving the problem of aggregation of carboxyl magnetic beads at low coating amount, comprising the following steps: ultrasonically treating the carboxyl magnetic beads and then performing segmentation using a pre-coating buffer to obtain a first magnetic bead resuspension with a concentration of 5-20 mg / mL; adding activators EDC and NHS to the first magnetic bead resuspension; using a coupling buffer to wash the activated first magnetic bead resuspension to obtain a second magnetic bead resuspension with a concentration of 5-20 mg / mL; adding activators EDC and NHS and antibodies to the second magnetic bead resuspension; adding a blocking agent 1 with a pH of 6-8 and containing 50-400 mmol / L ethanolamine to the second magnetic bead resuspension for primary blocking; removing the supernatant of the second magnetic bead resuspension after the primary blocking, and adding a blocking agent 2 for secondary blocking; using a magnetic bead working solution to wash the second magnetic bead resuspension after the secondary blocking, and fixing the carboxyl magnetic beads to a concentration of 0.4 mg / mL. The invention optimizes the coating process of carboxyl magnetic beads, improves the activity of magnetic beads, and solves the problem of magnetic bead aggregation; provides a magnetic bead coupling buffer to improve the antibody coating efficiency; improves the stability of reagents and reduces the test background signal value; improves the test repeatability, sensitivity and clinical relevance, thereby solving the problem of high background signal value caused by magnetic bead aggregation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] Figure 1 Is the correlation between control group 1 and Roche clinical

[0024] Figure 2 It is the clinical relevance of control group 2 and Roche.

[0025] Figure 3 It is clinically relevant to control group 3 and Roche.

[0026] Figure 4 It is the clinical relevance of experimental group 1 and Roche.

[0027] Figure 5 It is the clinical relevance of experimental group 2 and Roche.

[0028] Figure 6 It is the clinical relevance of experimental group 3 and Roche.

[0029] Figure 7 It is the clinical relevance of experimental group 4 and Roche.

[0030] Figure 8The present invention provides a flow chart of a preparation method for solving the aggregation of carboxyl magnetic beads with low coating amount. DETAILED DESCRIPTION

[0031] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0032] See also Figures 1 to 8 In the first aspect, a preparation method for solving the aggregation of carboxyl magnetic beads with low coating amount comprises the following steps:

[0033] S1 ultrasonically treats the carboxyl magnetic beads and then washes them with a pre-coating buffer to obtain a first magnetic bead resuspension solution with a concentration of 5 to 20 mg / mL;

[0034] The pre-coating buffer is a PB buffer, a MES buffer or a CB buffer with a pH of 5 to 7, preferably a 50 mM MES buffer with a pH of 6.0.

[0035] Specifically, the carboxyl magnetic beads are taken and subjected to ultrasonic treatment; the carboxyl magnetic beads treated with ultrasonic treatment are washed three times with a pre-coating buffer and then resuspended to obtain a magnetic bead resuspension solution with a concentration of 5 to 20 mg / mL; the pre-coating buffer refers to a PB buffer, MES buffer, or CB buffer with a pH of 5 to 7, preferably a 50 mM MES buffer with a pH of 6.0

[0036] S2 adding activators EDC and NHS to the first magnetic bead resuspension solution;

[0037] The mass ratio of the EDC to the magnetic beads is 1:2 to 1:20, the mass ratio of the EDC to the NHS is 1:1 to 1:5, and preferably the mass ratio of EDC:magnetic beads:NHS is 1:10:4.

[0038] Specifically, the prepared activating agents EDC and NHS are added to the magnetic bead resuspension in step 1, wherein the mass ratio of EDC to magnetic beads is 1:2 to 1:20, and the mass ratio of EDC to NHS is 1:1 to 1:5, preferably the mass ratio of EDC: magnetic beads: NHS is 1:10:4;

[0039] S3: washing the activated first magnetic bead resuspension with a coupling buffer to obtain a second magnetic bead resuspension with a concentration of 5 to 20 mg / mL;

[0040] The coupling buffer refers to PB buffer, MES buffer, CB buffer, etc. containing 0.02-0.1% BSA and 0.02-0.1% surfactant (HLB>15), pH 5-7, preferably 50mM MES buffer with pH 6.0, 0.04% BSA and 0.1% sodium lauryl sulfate.

[0041] Specifically, the activated magnetic beads are washed three times with a coupling buffer and then resuspended to obtain a magnetic bead resuspension solution with a concentration of 5 to 20 mg / mL; the coupling buffer refers to a PB buffer, MES buffer, CB buffer, etc. containing 0.02 to 0.1% BSA and 0.02 to 0.1% surfactant (HLB>15) and a pH of 5 to 7, preferably a 50 mM MES buffer with a pH of 6.0, 0.04% BSA and 0.1% sodium lauryl sulfate;

[0042] S4 adding activators EDC and NHS and antibodies to the second magnetic bead resuspension solution;

[0043] The mass ratio of EDC to magnetic beads is 1:1 to 1:40, the mass ratio of EDC to NHS is 1:1 to 1:3, the ratio of antibodies to magnetic beads is 2ug / mg to 40ug / mg, preferably the mass ratio of EDC: magnetic beads: NHS is 1:20:1, and the antibody is 5ug / mg.

[0044] Specifically, the prepared activator EDC and NHS and the antibody are added to the magnetic bead resuspension in step 3, wherein the mass ratio of EDC to magnetic beads is 1:1 to 1:40, the mass ratio of EDC to NHS is 1:1 to 1:3, and the ratio of antibody to magnetic beads is 2 ug / mg to 40 ug / mg, preferably the mass ratio of EDC: magnetic beads: NHS is 1:20:1, and the antibody is 5 ug / mg;

[0045] S5. Add a blocking agent having a pH of 6 to 8 and containing 50 to 400 mmol / L ethanolamine to the second magnetic bead resuspension solution for primary blocking;

[0046] The ratio of the blocking agent 1 to the carboxyl magnetic beads is 100ul / mg, and the blocking is preferably performed with a 250mmol / mL ethanolamine solution at pH 7.4.

[0047] Specifically, a blocking agent 1 containing 50 to 400 mmol / L ethanolamine at a pH of 6 to 8 is added for a first blocking, wherein the ratio of the blocking agent 1 to the carboxyl magnetic beads is 100 ul / mg, and preferably a 250 mmol / mL ethanolamine solution at a pH of 7.4 is used for blocking;

[0048] S6: removing the supernatant of the second magnetic bead resuspension solution after the first blocking, and adding blocking agent 2 for a second blocking;

[0049] The second blocking agent is a PB buffer, MES buffer, Tris buffer, etc. with a pH of 6-8 and containing 0.5-2% BSA, preferably a Tris buffer with a pH of 7.4 and 1% BSA.

[0050] Specifically, the supernatant is removed, and a blocking agent 2 is added for secondary blocking to make the magnetic bead concentration 5 to 20 mg / mL, wherein the blocking agent 2 refers to a PB buffer, MES buffer, Tris buffer, etc., with a pH of 6 to 8 and containing 0.5 to 2% BSA, preferably a Tris buffer with a pH of 7.4 and 1% BSA;

[0051] S7 uses magnetic bead working solution to wash the second magnetic bead resuspension solution after secondary blocking, and adjusts the concentration of carboxyl magnetic beads to 0.4 mg / mL.

[0052] The magnetic bead working solution is 10mM Tris buffer with a pH of 7-8, containing 0.5% sodium chloride, 0.2%-2% glucose, 0.1%-1% casein, 0.1%-1% Triton X-100, and 0.02%-0.5% preservative, wherein the preservative is preferably 2 of PC300, PC950, and BND-10.

[0053] Specifically, the beads are washed three times with magnetic bead working solution, and the concentration of carboxyl magnetic beads is fixed to 0.4 mg / mL, wherein the magnetic bead working solution is 10 mM Tris buffer, pH 7-8, containing 0.5% sodium chloride, 0.2%-2% glucose, 0.1%-1% casein, 0.1%-1% Triton X-100, and 0.02%-0.5% preservative. The preservative is preferably 2 of PC300, PC950, and BND-10.

[0054] The examples are as follows:

[0055] Taking high-sensitive troponin T (hs-cTnT) as an example, set up a control group and an experimental group, and prepare magnetic bead reagents:

[0056] Take 10 mg of carboxyl magnetic beads, perform ultrasonic treatment, wash the carboxyl magnetic beads three times with 2 mL of pre-coating buffer and resuspend them, add freshly prepared activators EDC and NHS to the magnetic bead resuspension for activation, and the mass ratio of EDC: magnetic beads: NHS is 1:10:4;

[0057] The activated magnetic beads were washed three times with coupling buffer and then resuspended. Freshly prepared activators EDC and NHS and antibodies were added to the magnetic bead resuspension for reactivation and coating. The specific proportions of magnetic beads, antibodies, EDC, and NHS are shown in Table 1. The contents of BSA and surfactant in the coupling buffer are shown in Table 2.

[0058] 1 mL of 250 mmol / mL ethanolamine solution at pH 7.4 was added for one blocking, and after removing the supernatant, blocking agent 2 (Tris buffer at pH 7.4, 1% BSA) was added for a second blocking;

[0059] The beads were washed three times with 2 mL of magnetic bead working solution (10 mM Tris buffer, pH 7.5, 0.5% NaCl, 1% glucose, 0.8% casein, 1% Triton X-100, 0.05% PC300, 0.03% PC950) to dilute the antibody concentration to 2 μg / mL to obtain the final magnetic bead reagent.

[0060] Table 1 Coating ratio of each component

[0061]

[0062] Table 2 BSA and surfactant content in coupling buffer

[0063]

[0064] Example 1

[0065] Blank background and low-end signal-to-noise ratio verification

[0066] The magnetic bead reagents were the hs-cTnT control group and experimental group mentioned above. The above 7 groups of reagents were matched with the same hs-cTnT enzyme marker to form the hs-cTnT reagent. The same group of 0.00 pg / mL concentration samples (blank background) and samples near the reference interval (20.14 pg / mL) were tested. The ratio of the blank background photon quantity and the calculated photon quantity was compared. The test results are shown in Table 3.

[0067] Table 3 Blank background and signal-to-noise ratio test results

[0068]

[0069] From the results of control groups 1-3, it can be seen that as the amount of magnetic beads coated is lower (that is, the amount of coated antibodies is less), the probability of magnetic beads aggregation is higher, resulting in a higher background signal value and a lower signal-to-noise ratio; from the results of control group 1 and experimental group 1, it can be seen that when the amount of carboxyl magnetic beads and antibodies is low, the addition of EDC and NHS for reactivation during coating in experimental group 1 has a greater impact on the blank background and signal-to-noise ratio, which can effectively reduce the blank background and improve the signal-to-noise ratio; from the comparison between experimental group 1 and experimental group 3, it can be seen that under the same coating conditions, the coupling dilution of experimental group 3 is The blank background was further reduced and the signal-to-noise ratio was further improved by adding 0.04% BSA and 0.1% surfactant sodium lauryl sulfate to the release solution; from the comparison of experimental groups 2-4, it can be seen that under the same coupling buffer conditions, as the dosage of EDC and NHS increased, the blank background decreased and the signal-to-noise ratio also increased, but when the coating amount was too high, the blank background showed an increasing trend and the signal-to-noise ratio also decreased; experimental group 3 achieved the best effect when a coupling buffer containing BSA and sodium lauryl sulfate was used in combination with the addition of EDC and NHS for coating.

[0070] Example 2

[0071] Accelerated stability verification

[0072] The magnetic bead reagents were the hs-cTnT control group and experimental group mentioned above. The above 7 groups of reagents were divided into 2 portions, 1 portion was placed at 37°C for 7 days for acceleration, and the other portion was placed at 2-8°C for 7 days at the same time. The same hs-cTnT enzyme marker was used to form the hs-cTnT reagent. The 2 portions of reagents were used to test the same set of gradient samples. The concentrations of samples 1-5 were 0.00 pg / mL, 410.93 pg / mL, 983.20 pg / mL, 2758.57 pg / mL, and 4928.39 pg / mL, respectively. The test results are shown in Table 4.

[0073] Table 4 Accelerated stability signal retention rate table

[0074]

[0075] From the results of control groups 1-3, it can be seen that with the lower amount of magnetic beads coated, the signal retention rates of 7 days at 37 degrees and 7 days at 2-8 degrees Celsius gradually decreased; from the results of control group 1 and experimental group 1, when the amount of carboxyl magnetic beads and antibodies was the same, the signal retention rate of experimental group 1 with the addition of EDC and NHS was significantly increased; by comparing experimental groups 1 and 3, it can be seen that under the same conditions, when the coupling buffer contained BSA and sodium lauryl sulfate, the signal retention rate was better; from the comparison of experimental groups 2-4, it can be seen that with the increase of the amount of EDC and NHS, the signal retention rate also gradually increased, but when the content was too high, the signal retention rate also began to decrease; experimental group 3 had the best effect when the coupling buffer containing BSA and sodium lauryl sulfate and the addition of EDC and NHS were used for coating.

[0076] Example 3

[0077] Linear gradient verification

[0078] The magnetic bead reagents were the hs-cTnT control group and experimental group mentioned above, and the above 7 groups of reagents were obtained. The hs-cTnT reagents were composed of the same hs-cTnT enzyme marker, and the same group of linear gradient samples were tested. The concentrations of samples 1-6 were 0.00 pg / mL, 49.34 pg / mL, 312.15 pg / mL, 1505.85 pg / mL, 4143.18 pg / mL, and 9830.91 pg / mL, respectively. The test results are shown in Table 5.

[0079] Table 5 Luminescence value table of linear gradient sample test

[0080]

[0081] From the results of control groups 1-3, it can be seen that as the amount of magnetic beads coated decreases, the linear gradient gradually becomes smaller; from the results of control group 1 and experimental group 1, it can be seen that when the amount of carboxyl magnetic beads and antibodies is the same, the gradient of experimental group 1, which adds EDC and NHS during coating, is more obvious, improving the gradient compression caused by the low coating amount; by comparing experimental groups 1 and 3, it can be seen that under the same conditions, when the coupling buffer contains BSA and sodium lauryl sulfate, the gradient is better; from the comparison of experimental groups 2-4, it can be seen that as the coating amount increases, the gradient ratio also gradually increases, but when EDC and NHS are excessive, an inhibitory effect will be produced; experimental group 3 has the best effect when a coupling buffer containing BSA and sodium lauryl sulfate is used in combination with the addition of EDC and NHS for coating.

[0082] Example 4

[0083] Repeatability verification

[0084] The magnetic bead reagents were the hs-cTnT control group and experimental group mentioned above, and the above 7 groups of reagents were obtained. The hs-cTnT reagents were composed of the same hs-cTnT enzyme marker, and the same group of negative samples (12.48 pg / mL) and positive samples (498.37 pg / mL) were tested. Each sample was tested 10 times, and the coefficient of variation (CV) of the test results was calculated and shown in Table 6.

[0085] Table 6 Repeatability verification test results

[0086]

[0087]

[0088]

[0089] From the results of control groups 1-3, it can be seen that as the amount of magnetic beads coated decreases, the repeatability gradually becomes worse; from the results of control group 1 and experimental group 1, it can be seen that when the amount of carboxyl magnetic beads and antibodies is the same, the repeatability of experimental group 1 in which EDC and NHS are added during coating is improved to a certain extent; by comparing experimental groups 1 and 3, it can be seen that under the same conditions, when the coupling buffer contains BSA and sodium lauryl sulfate, the repeatability is better; from the comparison of experimental groups 2-4, it can be seen that as the coating amount increases, the CV gradually becomes smaller, but when the concentrations of EDC and NHS reach a certain level, the repeatability does not change significantly; experimental group 3 has the best effect when a coupling buffer containing BSA and sodium lauryl sulfate is used in combination with the addition of EDC and NHS for coating.

[0090] Example 5

[0091] Low-end clinical validation

[0092] The magnetic bead reagents were the hs-cTnT control group and experimental group mentioned above, and the above 7 groups of reagents were obtained. The hs-cTnT reagents were composed of the same hs-cTnT enzyme markers. The same group of clinical samples (<500pg / mL) were tested. The test results were compared with Roche chemiluminescence. Figure 1-7 .

[0093] From the results, it can be seen that the clinical correlations of control groups 1-3 and experimental groups 1-4 were 0.9472, 0.9483, 0.9623, 0.9652, 0.975, 0.9836, and 0.9792, respectively. From the results of control groups 1-3, it can be seen that as the amount of magnetic beads coated is lower, the clinical correlation is worse; from the results of control group 1 and experimental group 1, it can be seen that when the amount of carboxyl magnetic beads and antibodies is the same, the clinical correlation of experimental group 1, which adds EDC and NHS during coating, is significantly improved; by comparing experimental groups 1 and 3, it can be seen that under the same conditions, when the coupling buffer contains BSA and sodium lauryl sulfate, the clinical correlation is better; from the comparison of experimental groups 2-4, it can be seen that as the amount of EDC and NHS increases, the clinical correlation gradually increases, but when the concentration of EDC and NHS reaches a certain level, the clinical correlation is not much different; experimental group 3 has the best effect when the coupling buffer containing BSA and sodium lauryl sulfate and the addition of EDC and NHS are used for coating.

[0094] In the second aspect, an application of a preparation method for solving the aggregation of carboxyl magnetic beads with low coating amounts is provided, wherein the preparation method for solving the aggregation of carboxyl magnetic beads with low coating amounts described in the first aspect is used to solve the aggregation of carboxyl magnetic beads with low coating amounts.

[0095] The present invention provides a method for preparing and applying carboxyl magnetic beads to solve the problem of low coating amount aggregation, which has the following advantages:

[0096] Beneficial effects:

[0097] 1. A preparation method and application of the present invention for solving the aggregation of carboxyl magnetic beads at low coating amount to optimize the coating process of carboxyl magnetic beads, improve the activity of magnetic beads, and solve the problem of magnetic bead aggregation;

[0098] 2. A preparation method and application of the present invention for solving the aggregation of carboxyl magnetic beads at low coating amount provides a magnetic bead coupling buffer to improve the antibody coating efficiency;

[0099] 3. The present invention provides a method for preparing and applying carboxyl magnetic beads to solve the problem of aggregation at low coating amount, improve reagent stability, and reduce the test background signal value;

[0100] 4. The present invention provides a preparation method for solving the aggregation of carboxyl magnetic beads with low coating amount and its application to improve test repeatability, sensitivity and clinical relevance.

[0101] The above disclosure is only a preferred embodiment of the preparation method and application of the present invention for solving the aggregation of carboxyl magnetic beads with low coating amount. Of course, this cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiments and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A preparation method for solving the aggregation of carboxyl magnetic beads with low coating amount, characterized in that: The following steps are involved: After ultrasonic treatment of the carboxyl magnetic beads, the beads are washed with a pre-coating buffer to obtain a first magnetic bead resuspension solution with a concentration of 5 to 20 mg / mL; Adding activators EDC and NHS to the first magnetic bead resuspension solution; The activated first magnetic bead resuspension is washed with a coupling buffer to obtain a second magnetic bead resuspension with a concentration of 5 to 20 mg / mL; Adding activators EDC and NHS and antibodies to the second magnetic bead resuspension; Adding a blocking agent having a pH of 6 to 8 and containing 50 to 400 mmol / L ethanolamine to the second magnetic bead resuspension solution for primary blocking; removing the supernatant of the second magnetic bead resuspension solution after the first blocking, and adding blocking agent 2 for secondary blocking; The second magnetic bead resuspension after secondary blocking was washed with magnetic bead working solution, and the concentration of carboxyl magnetic beads was fixed to 0.4 mg / mL.

2. The method for preparing carboxyl magnetic beads for solving the aggregation of low coating amount as claimed in claim 1, characterized in that: In the step of “after ultrasonically treating the carboxyl magnetic beads, the carboxyl magnetic beads are washed with a pre-coating buffer to obtain a first magnetic bead resuspension having a concentration of 5 to 20 mg / mL”, the pre-coating buffer is a PB buffer, MES buffer or CB buffer having a pH of 5 to 7, preferably a 50 mM MES buffer having a pH of 6.

0.

3. The method for preparing a carboxyl magnetic bead with low coating amount as claimed in claim 2, characterized in that: In "adding activators EDC and NHS to the first magnetic bead resuspension solution", the mass ratio of the EDC to the magnetic beads is 1:2 to 1:20, the mass ratio of the EDC to the NHS is 1:1 to 1:5, and preferably the mass ratio of EDC:magnetic beads:NHS is 1:10:

4.

4. The method for preparing a carboxyl magnetic bead with low coating amount aggregation as claimed in claim 3, characterized in that: In "using a coupling buffer to wash the activated first magnetic bead resuspension to obtain a second magnetic bead resuspension with a concentration of 5 to 20 mg / mL", the coupling buffer refers to a PB buffer, MES buffer, CB buffer, etc. containing 0.02 to 0.1% BSA and 0.02 to 0.1% surfactant (HLB>15), a pH of 5 to 7, preferably a 50 mM MES buffer with a pH of 6.0, 0.04% BSA and 0.1% sodium lauryl sulfate.

5. The method for preparing carboxyl magnetic beads for solving the aggregation of low coating amount as claimed in claim 4, characterized in that, In "adding activators EDC and NHS and antibodies to the second magnetic bead resuspension", the mass ratio of EDC to magnetic beads is 1:1 to 1:40, the mass ratio of EDC to NHS is 1:1 to 1:3, and the ratio of antibodies to magnetic beads is 2ug / mg to 40ug / mg. Preferably, the mass ratio of EDC: magnetic beads: NHS is 1:20:1, and the antibody is 5ug / mg.

6. The method for preparing carboxyl magnetic beads for solving the aggregation of low coating amount as claimed in claim 5, characterized in that: In "adding a blocking agent with a pH of 6 to 8 and containing 50 to 400 mmol / L ethanolamine to the second magnetic bead resuspension for a blocking", the ratio of the blocking agent to the carboxyl magnetic beads is 100 ul / mg, preferably a 250 mmol / mL ethanolamine solution with a pH of 7.4 for blocking.

7. The method for preparing carboxyl magnetic beads for solving the aggregation of low coating amount as claimed in claim 6, characterized in that: In "removing the supernatant of the second magnetic bead resuspension after the first blocking, and adding blocking agent 2 for secondary blocking", the blocking agent 2 is PB buffer, MES buffer, Tris buffer, etc. with a pH of 6 to 8 and containing 0.5 to 2% BSA, preferably Tris buffer with a pH of 7.4 and 1% BSA.

8. The method for preparing carboxyl magnetic beads for solving the aggregation of low coating amount as claimed in claim 7, characterized in that, In "using magnetic bead working solution to wash the second magnetic bead resuspension after secondary blocking, and adjusting the concentration of carboxyl magnetic beads to 0.4 mg / mL", the magnetic bead working solution is 10mM Tris buffer, pH 7-8, containing 0.5% sodium chloride, 0.2%-2% glucose, 0.1%-1% casein, 0.1%-1% Triton X-100, 0.02%-0.5% preservative, wherein the preservative is preferably 2 of PC300, PC950, and BND-10.

9. An application of a preparation method for solving the aggregation of carboxyl magnetic beads with low coating amount, using the preparation method for solving the aggregation of carboxyl magnetic beads with low coating amount according to any one of claims 1 to 8, characterized in that: Used to solve the aggregation of carboxyl magnetic beads with low coating amount.

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