Cortisol chemiluminescence detection kit and magnetic microsphere coating method

Abstract

The invention discloses a cortisol chemiluminescence detection kit and a magnetic microsphere coating method, and the method comprises the following steps: adding an amino macromolecular polymer and a cortisol antigen analogue into activated magnetic microspheres, and carrying out a coupling reaction to obtain a coupling compound; and blocking the coupling compound by using a first blocking agent to obtain the cortisol antigen analogue coated magnetic microspheres. When the magnetic microsphere is subjected to a coating reaction, an amino macromolecular polymer is mixed into a small-molecule antigen analogue, and the amino macromolecular polymer can occupy part of sites which do not participate in the reaction after activation on the surface of the magnetic bead and can play a role in supporting a structure similar to a small-molecule antigen; the flexible structure of the small-molecule antigen analogue is relatively fixed, and the stability and sensitivity of the prepared magnetic microspheres are obviously improved.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnosis, and in particular to a cortisol chemiluminescence detection kit and a magnetic microsphere coating method. Background Art

[0002] At present, the commonly used methods for detecting cortisol in clinics and laboratories include chemiluminescence, enzyme-linked immunosorbent assay, colloidal gold method, immunofluorescence method, etc. The coupling reactions in the above methods are generally small molecule antigen analogs connected to proteins, such as small molecule antigen analogs coupled with alkaline phosphatase or biotin. However, the kits obtained by the above coupling methods have relatively low sensitivity and large low value deviation. Summary of the invention

[0003] In view of the deficiencies of the prior art, the object of the present invention is to provide a cortisol chemiluminescence detection kit and a magnetic microsphere coating method to solve the problems of low sensitivity and large low value deviation in the prior art.

[0004] To solve the above problems, the present invention adopts the following solutions:

[0005] A magnetic microsphere coating method comprises the following steps:

[0006] Adding amino macromolecular polymers and cortisol antigen analogs into activated magnetic microspheres for coupling reaction to obtain coupling complexes;

[0007] The coupling complex is blocked by a first blocking agent to obtain magnetic microspheres coated with the cortisol antigen analog.

[0008] Furthermore, the mass ratio of the cortisol antigen analog, the amino macromolecular polymer, and the magnetic microspheres is 0.05:(0.1-0.05):1.

[0009] Furthermore, the amino macromolecular polymer includes poly-lysine, gelatin, human serum albumin or BSA.

[0010] Furthermore, the first blocking agent is poly-lysine.

[0011] Furthermore, the preparation method of the activated magnetic microspheres is as follows:

[0012] The magnetic microspheres were washed and resuspended with coupling buffer;

[0013] An activator is added to the washed and resuspended magnetic microspheres to perform an activation reaction to obtain activated magnetic microspheres.

[0014] A cortisol chemiluminescence detection kit comprises a reagent L1 and a reagent L2; the reagent L1 comprises a magnetic microsphere coated with a cortisol antigen analogue and a first buffer; the reagent L2 comprises an acridinium ester-labeled cortisol monoclonal antibody and a second buffer; the magnetic microsphere coated with a cortisol antigen analogue is prepared by the method described above.

[0015] Furthermore, the preparation method of the acridinium ester-labeled cortisol monoclonal antibody is as follows:

[0016] The acridinium ester solution is added to the cortisol monoclonal antibody solution to perform a labeling reaction;

[0017] adding a second blocking agent to the solution after the labeling reaction to perform a blocking reaction;

[0018] The solution after the blocking reaction is purified to obtain acridinium ester labeled cortisol monoclonal antibody.

[0019] Furthermore, the second blocking agent is a Tris solution.

[0020] Furthermore, the first buffer is MES buffer.

[0021] Furthermore, the second buffer is PBS buffer.

[0022] The present invention adopts the above technical solution and has the following advantages:

[0023] During the coating reaction of the magnetic microspheres of the present invention, amino macromolecular polymers are mixed into the small molecule antigen analogs. The amino macromolecular polymers can occupy partial sites on the surface of the magnetic beads that do not participate in the reaction after activation, and can support the structure of the small molecule antigen analogs, so that the flexible structure of the small molecule antigen analogs is relatively fixed. The stability and sensitivity of the prepared magnetic microspheres are significantly improved, thereby improving the sensitivity of the test kit and reducing the low value deviation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a comparison chart of stability test data between the control group and the experimental group in the present invention;

[0025] Figure 2 It is a comparison chart of the concentration batch difference test data of the control group and the experimental group in the present invention. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0027] Experimental reagents: The cortisol monoclonal mouse antibody, acridinium ester, EDC (carbodiimide), MES (morpholineethanesulfonic acid), Tween 20 (Tween 20), NaCl, BSA (bovine serum albumin), Sulfo-NHS (N-hydroxysulfosuccinimide) and other reagents and consumables used in the present invention are all purchased reagents; the magnetic particles and cortisol antigen analogs are all produced by the applicant.

[0028] Example 1

[0029] A cortisol chemiluminescence detection kit comprises a reagent L1 and a reagent L2; the reagent L1 comprises a magnetic microsphere coated with a cortisol antigen analogue and a first buffer; the reagent L2 comprises an acridinium ester-labeled cortisol monoclonal antibody and a second buffer; and the kit also comprises an exciting solution and a cleaning solution.

[0030] The preparation method of each component in the above detection kit is as follows:

[0031] 1.1 The method for preparing magnetic microspheres coated with cortisol antigen analogs comprises the following steps:

[0032] The magnetic microspheres were washed and resuspended with coupling buffer;

[0033] adding an activator to the washed and resuspended magnetic microspheres to perform an activation reaction, separating the activated magnetic microspheres, and resuspending the activated magnetic microspheres with a coupling buffer;

[0034] The amino macromolecular polymer, the cortisol antigen analog and the activated magnetic microspheres are coupled to obtain a coupling complex;

[0035] The coupling complex is blocked by a first blocking agent, and then centrifuged to obtain blocked magnetic microspheres;

[0036] The blocked magnetic microspheres are dissolved by a storage buffer (ie, the first buffer) to obtain a magnetic microsphere solution coated with a cortisol antigen analog.

[0037] In this embodiment, MES buffer is used as the coupling buffer, the concentration of MES is 5-20 mM, and the pH is 4.5-6.5.

[0038] The specific steps of the above preparation method are as follows:

[0039] a. Washing and resuspension of carboxyl magnetic microspheres

[0040] Take 1 mL of carboxyl magnetic microspheres with a particle size of 1 μm into a centrifuge tube, place the centrifuge tube on a micromagnetic separation rack for magnetic separation, remove the clarified liquid after about 1 minute, retain the carboxyl magnetic microspheres, add 1 mL of 10 mM pH = 5.0 MES buffer to the carboxyl magnetic microspheres, disperse them again by blowing, repeat the separation twice according to the above steps to obtain washed carboxyl magnetic microspheres, and resuspend the washed carboxyl magnetic microspheres with MES buffer to obtain suspended carboxyl magnetic microspheres.

[0041] b. Activation of carboxyl magnetic microspheres

[0042] According to the mass ratio of carboxyl magnetic microspheres to activator, carboxyl magnetic microspheres: EDC: Sulfo-NHS = 1:1:1.5, first add Sulfo-NHS to the suspended carboxyl magnetic microspheres, mix evenly, then add EDC, react at 37°C, 200rpm for 30 minutes, mix evenly, after the reaction, perform magnetic separation to obtain activated carboxyl magnetic microspheres, use 10mM pH = 5.0MES buffer to wash the activated carboxyl magnetic microspheres, remove excess mixed activator that does not participate in the reaction, and resuspend the washed activated carboxyl magnetic microspheres with MES buffer to obtain suspended activated carboxyl magnetic microspheres.

[0043] c. Coupling reaction of carboxyl magnetic microspheres

[0044] The mass of the cortisol antigen analog required for coupling was calculated based on the mass of the carboxyl magnetic microspheres. The cortisol antigen analog and BSA mixture were slowly added to the activated carboxyl magnetic microspheres at a mass ratio of cortisol antigen analog: BSA: carboxyl magnetic microspheres = 0.05:0.05:1. The mixture was reacted at 200 rpm in a constant temperature shaker at 37°C for 3 hours and mixed evenly to obtain a coupling complex.

[0045] d. Add polylysine (the first blocking agent) to the coupling complex reaction system to block the activated sites on the carboxyl magnetic microspheres that do not participate in the reaction. The proportion of polylysine in the reaction solution is 3%. The reaction is carried out at 37°C shaker at 200 rpm for 1.5 hours. The beads are washed three times with magnetic bead washing solution (PBS-T) to separate the blocked carboxyl magnetic microspheres.

[0046] e. Add storage buffer (50 mM MES, 1% BSA, 0.9% NaCl, 0.2% Tween20, pH 6.0) to the blocked carboxyl magnetic microspheres, adjust the working concentration of the blocked carboxyl magnetic microspheres to 2.5 mg / mL, and obtain a magnetic microsphere solution coated with a cortisol antigen analog.

[0047] 1.2 The preparation method of acridinium ester-labeled cortisol monoclonal antibody is as follows:

[0048] a. Acridinium ester was dissolved in DMSO (dimethyl sulfoxide) to a concentration of 1 mg / mL. According to the molar ratio of cortisol monoclonal antibody: acridinium ester = 1:10, 1 mg of cortisol monoclonal antibody (150KD) was diluted to the required concentration of 1 mg / mL with 50 mM carbonate buffer at pH = 9.0, and 45 mL of 1 mg / mL acridinium ester solution was added to the antibody solution, and the reaction was carried out in a water bath at 37°C for 1 hour;

[0049] b. Add 100 mL of Tris solution (second blocking agent) to the above reaction system to block the acridinium ester that does not participate in the reaction. The concentration of the Tris solution is 20 mM.

[0050] c. The unreacted acridinium ester was purified by Zeba™ desalting centrifugal column (wherein Zeba™ is the product trademark) to collect the acridinium ester-labeled cortisol monoclonal antibody; the acridinium ester-labeled cortisol monoclonal antibody was diluted with a second buffer (PBS buffer containing 1% BSA, 0.9% NaCl, 0.2% Tween20) to a working concentration of 0.02 μg / mL.

[0051] 1.3 The components of chemiluminescent excitation solution A and solution B are as follows:

[0052] Chemiluminescent excitation solution A: an aqueous solution containing hydrogen peroxide and nitric acid, wherein the concentration of hydrogen peroxide is 0.5 v / v%, and the concentration of nitric acid is 0.2 M;

[0053] Chemiluminescent excitation solution B: an aqueous solution containing Tween-20 and sodium hydroxide, wherein the concentration of Tween-20 is 1 v / v%, and the concentration of sodium hydroxide is 0.5M.

[0054] 1.4 The components of the chemiluminescent cleaning solution are as follows:

[0055] The cleaning solution is PBS-T buffer, pH 6.96, and the formula is as follows (taking 1L as an example): KH2PO4 3.35g, Na2HPO4-12H2O 17.9g; KCl 0.2g; NaCl 8.77g; Tween-20 0.5g; Proclin-300 1mL.

[0056] The sample is subjected to immunoassay detection using the detection kit provided in this embodiment:

[0057] 1. In a reaction cup, add 50 μl of sample, 100 μl of acridinium ester-labeled cortisol-specific monoclonal mouse antibody, and 10 μl of magnetic microsphere solution coated with cortisol antigen analogs and react under incubation conditions. The cortisol in the sample and the antigen analogs on the magnetic microspheres compete for binding to the acridinium ester-labeled antibody, and the cortisol in the sample is detected by the competition method.

[0058] 2. Detection reading: After the incubation, add magnetic field for precipitation, remove the supernatant, wash the precipitated complex with cleaning solution, and absorb the waste liquid to remove the substances not bound to the magnetic particles, then send the reaction cup into the measurement chamber, the instrument automatically pumps in two excitation solutions to make the complex produce chemiluminescent signals, and measure the luminescence intensity through the photomultiplier. The instrument automatically calculates the test results through the working curve.

[0059] Example 2

[0060] The difference between this embodiment and embodiment 1 is that in the activation reaction of carboxyl magnetic microspheres, carboxyl magnetic microspheres: EDC: Sulfo-NHS are added in a mass ratio of 1:1:1; when preparing magnetic microspheres, a mixture of cortisol antigen analogs and gelatin is slowly added to the activated carboxyl magnetic microspheres in a mass ratio of cortisol antigen analogs: gelatin: carboxyl magnetic microspheres = 0.05:0.1:1; other parts are the same.

[0061] Example 3

[0062] The difference between this embodiment and embodiment 1 is that in the activation reaction of carboxyl magnetic microspheres, carboxyl magnetic microspheres: EDC: Sulfo-NHS are added in a mass ratio of 1:1:2; when preparing magnetic microspheres, a mixture of cortisol antigen analogs and polylysine is slowly added to the activated carboxyl magnetic microspheres in a mass ratio of cortisol antigen analogs: polylysine: carboxyl magnetic microspheres = 0.05:0.07:1; other parts are the same.

[0063] Example 4

[0064] The difference between this embodiment and embodiment 1 is that BSA is replaced with human serum albumin; the other parts are the same.

[0065] Example 5

[0066] 1. Sensitivity, stability, and batch difference verification

[0067] Experimental groups: The kits prepared by the methods provided in Example 1, Example 2, Example 3, and Example 4 were used as Experimental Group 1, Experimental Group 2, Experimental Group 3, and Experimental Group 4.

[0068] Control group: The difference between the kit used in the control group and the experimental group is that no poly-lysine, gelatin, human serum albumin or BSA is added to the carboxyl magnetic microspheres during the coupling reaction, and the others are the same.

[0069] The sensitivity, stability, and batch-to-batch differences were evaluated from the perspective of changes in luminescence intensity and concentration;

[0070] Among them, the statistical results of sensitivity detection are shown in Table 1.

[0071] Table 1 Comparison of sensitivity test data

[0072]

[0073] According to Table 1, after adding BSA during the coupling reaction, the reagent sensitivity of the experimental group was significantly improved, and the minimum detection limit was reduced from 0.39 in the control group to 0.21, indicating that the addition of amino-containing macromolecules during the coupling reaction had a significant effect on the sensitivity improvement.

[0074] The stability test results are shown in Table 2. Figure 1 shown.

[0075] Table 2 Comparison of stability test data

[0076]

[0077]

[0078] According to the test results in Table 2 and Figure 1 It shows that the variation range of luminescence value and concentration is small in the coating process treated with BSA, indicating that the stability of the reagent is significantly improved in the process treated with BSA;

[0079] The results of batch difference detection are shown in Table 3. Figure 2 As shown;

[0080] Table 3 Comparison of batch difference test data

[0081]

[0082] According to Table 3 and Figure 2 The inter-batch difference experimental test results shown in the figure show that after the coating process using BSA treatment, the inter-batch differences in luminescence values ​​and concentrations were significantly reduced, indicating that the batches are easier to control.

[0083] From the above data, it can be found that when the present invention uses amino macromolecular polymers to participate in the magnetic microsphere coating reaction, the amino macromolecular polymers can support the structure of small molecule antigen analogs, making the flexible structure of small molecule antigen analogs relatively fixed, and the stability and sensitivity of the prepared magnetic microspheres are significantly improved.

[0084] From the comparison of the results of the control group and the experimental group, it can be seen that the stability, batch difference control and sensitivity of the cortisol detection kit of the present invention are significantly improved; the use of the magnetic microspheres coated with the antigen analog provided by the present invention greatly improves the stability and sensitivity of the product, reduces the batch difference, reduces the cost, and solves the problem of instability after the existing kit is scaled up for production.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A magnetic microsphere coating method, characterized in that: The following steps are involved: Adding amino macromolecular polymers and cortisol antigen analogs into activated magnetic microspheres for coupling reaction to obtain coupling complexes; The coupling complex is blocked by a first blocking agent to obtain magnetic microspheres coated with the cortisol antigen analog.

2. A magnetic microsphere coating method according to claim 1, characterized in that: The mass ratio of the cortisol antigen analog, the amino macromolecular polymer and the magnetic microspheres is 0.05:(0.1-0.05):

1.

3. A magnetic microsphere coating method according to claim 1, characterized in that: The amino macromolecular polymer includes poly-lysine, gelatin, human serum albumin or BSA.

4. A magnetic microsphere coating method according to claim 1, characterized in that: The first blocking agent is poly-lysine.

5. A magnetic microsphere coating method according to claim 1, characterized in that: The preparation method of the activated magnetic microspheres is as follows: The magnetic microspheres were washed and resuspended with coupling buffer; An activator is added to the washed and resuspended magnetic microspheres to perform an activation reaction to obtain activated magnetic microspheres.

6. A cortisol chemiluminescence detection kit, characterized in that: The method comprises reagent L1 and reagent L2; the reagent L1 comprises magnetic microspheres coated with a cortisol antigen analog and a first buffer; the reagent L2 comprises acridinium ester-labeled cortisol monoclonal antibody and a second buffer; the magnetic microspheres coated with a cortisol antigen analog are prepared by the method according to any one of claims 1 to 5.

7. A cortisol chemiluminescence detection kit according to claim 6, characterized in that: The preparation method of the acridinium ester-labeled cortisol monoclonal antibody is as follows: The acridinium ester solution is added to the cortisol monoclonal antibody solution to perform a labeling reaction; adding a second blocking agent to the solution after the labeling reaction to perform a blocking reaction; The solution after the blocking reaction is purified to obtain acridinium ester labeled cortisol monoclonal antibody.

8. A cortisol chemiluminescence detection kit according to claim 7, characterized in that: The second blocking agent is Tris solution.

9. A cortisol chemiluminescence detection kit according to claim 6, characterized in that: The first buffer is MES buffer.

10. A cortisol chemiluminescence detection kit according to claim 6, characterized in that: The second buffer is PBS buffer.

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