Chemiluminescence detection kit for interleukin 12P70 as well as preparation and application of chemiluminescence detection kit

Abstract

The invention provides a chemiluminescence detection kit for interleukin 12P70 as well as preparation and application of the chemiluminescence detection kit, aiming at an acridinium ester compound solution connected with an interleukin 12P70 antibody, a chromatographic column is matched with a nucleic acid protein detector to remove free acridinium ester, so that the free acridinium ester and an acridinium ester compound are completely separated, interference of the free acridinium ester is eliminated, and the detection accuracy of the interleukin 12P70 antibody is improved. The purification effect is better; meanwhile, streptavidin antibodies are linked to the magnetic beads, and surface reaction arms of the solid phase carrier are extended, so that mutual interference during reaction of the antibodies and antigens is eliminated, signals are amplified, and the dosage of the antibodies is reduced; when the chemiluminescence detection kit is used for detecting interleukin 12P70, the background value can be reduced, the signal-to-noise ratio can be improved, and the sensitivity and accuracy of chemiluminescence detection of interleukin 12P70 can be remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of chemiluminescence detection, and specifically relates to a chemiluminescence detection kit for interleukin 12P70 and a preparation method and application thereof. Background Art

[0002] Chemiluminescence detection is an immunoassay method that directly labels antigens or antibodies with chemiluminescent agents. Chemiluminescence technology and antibody labeling technology are used to prepare reagent components, and quantitative detection of the analyte is achieved through the specific immune reaction between antigen and antibody. At present, when chemiluminescence detection is used for quantitative detection of analytes, antigens with similar structures to the analyte are prepared into calibrators (liquid or solid) of a certain concentration value using buffer solution, and calibration curves are prepared by measuring the calibrators to calculate the concentration of the analyte.

[0003] Interleukin 12P70 (IL-12P70) is a multi-biologically active cytokine. Its important role in anti-tumor immunity is to establish a connection between natural resistance regulated by macrophages and NK cells and acquired immunity mediated by Th1 cells, NK1.1+TCRint cells, and CTL cells. Interleukin 12P70 regulating the immune response of tumor patients is a new and promising strategy for tumor treatment. Therefore, the development of a highly sensitive interleukin 12P70 protein detection methodology is of great clinical significance.

[0004] The existing chemiluminescence detection method has low sensitivity in detecting interleukin 12P70. In order to improve the sensitivity, a large amount of antibodies are usually used, which increases the cost. Therefore, it is urgent to find a method that can efficiently detect interleukin 12P70. Summary of the invention

[0005] In order to make up for the defects of the prior art, the present invention provides a chemiluminescent detection kit for interleukin 12P70 and its preparation and application. For the acridinium ester complex solution connected with interleukin 12P70 antibody, a chromatography column is used with a nucleic acid protein detector to remove free acridinium ester, so that the free acridinium ester and the acridinium ester complex are completely separated, the interference of free acridinium ester is eliminated, and the purification effect is better; at the same time, streptavidin antibodies are coupled to magnetic beads to extend the surface reaction arms of the solid phase carrier, thereby eliminating the mutual interference between each antibody and antigen during the reaction, amplifying the signal and reducing the amount of antibody; when used for interleukin 12P70 detection, the background value can be reduced, the signal-to-noise ratio can be improved, and the sensitivity and accuracy of chemiluminescent detection of interleukin 12P70 can be significantly improved.

[0006] In one aspect, the present invention provides a method for preparing a chemiluminescent detection kit for interleukin 12P70, the method comprising the following steps:

[0007] 1. Prepare magnetic bead solution connected to the first antibody;

[0008] 2. Prepare an acridinium ester complex solution linked to a second antibody, and remove free acridinium esters through a chromatography column with a nucleic acid protein detector;

[0009] The first antibody and the second antibody are interleukin 12P70 first antibody and interleukin 12P70 second antibody, respectively. Further, the acridinium ester complex solution in step 2 includes a first buffer solution, and the first buffer solution includes NaH2PO4·2H2O, Na2HPO4, and NaCl.

[0010] Furthermore, the acridinium ester solution for linking the second antibody also includes a blocking agent, and the blocking agent includes lysine.

[0011] Furthermore, the filler in the chromatography column in step 2 is G25 / G50 dextran, and the filler needs to be washed with a second buffer solution first, wherein the second buffer solution includes NaH2PO4·2H2O, Na2HPO4, NaCl, and ProClinTM300.

[0012] In some embodiments, the preparation process of the acridinium ester complex solution linked to the second antibody is: under incubation conditions of 37° C., the second antibody is linked to the acridinium ester, and the free acridinium ester is removed using a chromatography column with a nucleic acid protein detector to obtain a signal amplification system of the second antibody to be tested linked to the acridinium ester.

[0013] Since the activity of the second antibody is enhanced under 37°C conditions, the binding efficiency of the second antibody and acridinium ester is improved; by using a chromatography column, the acridinium ester (including other impurities) that is not bound to the second antibody can be removed, thereby improving the purity of the second antibody and acridinium ester complex, thereby increasing the signal value, and removing the influence of free acridinium ester, and reducing the background value.

[0014] In some embodiments, the specific preparation process of the acridinium ester complex solution linked to the second antibody is:

[0015] (1) reacting the secondary antibody with acridinium ester and incubating at 37°C for 60 min; adding 3% lysine and reacting for 60 min to achieve a blocking effect;

[0016] (2) filtering the product obtained in (1) using a chromatography column, passing it through a nucleic acid protein detector, collecting the secondary antibody and acridinium ester complex, and removing excess acridinium ester;

[0017] (3) The product of (2) is prepared into a working solution, wherein the working solution includes a buffer solution, wherein the buffer solution is composed of 5.23 g / LMops, 9 g / L NaCl, 20 g / L BSA, and 0.3 mL / L ProClinTM300.

[0018] Furthermore, the method for preparing the magnetic bead solution connected to the first antibody in step 1 is:

[0019] (a) mixing a first antibody solution with a biotin ester to react to prepare a biotinylated antibody;

[0020] (b) Magnetic beads are coupled to streptavidin antibodies and then coated with streptavidin and biotinylated antibodies.

[0021] The present invention designs a signal amplification system for the magnetic bead solution. The magnetic beads are first linked to streptavidin antibodies, and then specifically react with streptavidin to link streptavidin to the magnetic beads; the first antibody is linked to biotin, and the first antibody can be linked to the magnetic beads through the high affinity between biotin and streptavidin. The amplification effect between biotin and avidin can increase the signal value, but because one avidin can bind to four biotinylated antibodies, the space around the solid phase is crowded and the reaction is inhibited, especially when detecting macromolecules. The impact is more obvious. The present invention utilizes streptavidin antibodies coupled to magnetic beads to extend the surface reaction arms of the magnetic beads, thereby eliminating the interference between each antibody and antigen when reacting, which can effectively improve the sensitivity of the reagent and reduce the amount of antibody used. It not only improves the detection performance, but also reduces the cost.

[0022] Furthermore, the acquisition process of the signal amplification system is:

[0023] (1) linking the first antibody to biotin to obtain a biotinylated antibody;

[0024] (2) Streptavidin antibody is linked to the magnetic beads;

[0025] (3) The streptavidin antibody on the magnetic beads reacts with streptavidin, and the biotin on the biotinylated antibody also reacts with streptavidin, thereby obtaining a complex of magnetic beads-streptavidin antibody-streptavidin-biotin-first antibody, and preparing a signal amplification system in which the first antibody is linked to the magnetic beads.

[0026] Furthermore, in step (a), the first antibody solution needs to be dialyzed with PBS buffer.

[0027] Furthermore, after the coating in step (b) is completed, it is necessary to wash with a third buffer solution, and the third buffer solution includes PBS, sodium chloride, bovine serum albumin, glycine, trehalose, gelatin, sodium alginate, PEG 20000, Triton X-100 and ProClinTM300.

[0028] In some embodiments, the preparation process of the magnetic bead solution linked to the first antibody is:

[0029] (1) mixing the interleukin 12P70 first antibody dialyzed with PBS buffer overnight with biotin ester in a certain proportion for reaction, and then dialyzing with PBS buffer overnight to obtain a biotinylated antibody;

[0030] (2) After the magnetic beads are washed and activated, they are coupled with streptavidin antibodies, blocked, and magnetically separated;

[0031] (3) coating the magnetic beads, streptavidin, and biotinylated antibody separated by magnetic separation in (2) at 37°C overnight;

[0032] (4) The coated product was blocked at 37°C for 3 h, washed with the third buffer, and the magnetic beads were resuspended to prepare the working solution.

[0033] In some embodiments, the interleukin 12P70 first antibody and biotin ester are mixed and reacted at a ratio of 1 mg antibody: (1.33 uL-13.3 uL) 10 mmol / L biotin ester.

[0034] In some embodiments, the magnetic beads, streptavidin, and biotinylated antibody after magnetic separation are coated in a ratio of 1 mg: 10 ug: 40 ug.

[0035] On the other hand, the present invention provides a chemiluminescent detection kit for interleukin 12P70 prepared by the method described above.

[0036] On the other hand, the present invention provides a chemiluminescent detection method for interleukin 12P70, which comprises incubating a sample to be tested, a magnetic bead solution, and an acridinium ester complex solution together to form an immune complex, washing the complex, and reacting the complex with a substrate to generate a light signal.

[0037] In another aspect, the present invention provides a second buffer for improving the separation effect of free acridinium ester and acridinium ester complex in a chromatography column, wherein the buffer comprises NaH2PO4·2H2O, Na2HPO4, NaCl, and ProClinTM300.

[0038] In another aspect, the present invention provides a use of a blocking agent for preparing a reagent for improving the differentiation effect of free acridinium ester and acridinium ester complex in a chromatography column, wherein the blocking agent comprises lysine and BSA.

[0039] The beneficial effects of the present invention are:

[0040] 1. By using a chromatography column with a nucleic acid protein detector to remove free acridinium esters and impurities, the purification effect of the acridinium ester complex linked to the interleukin 12P70 antibody is significantly improved, thereby increasing the signal value, removing the influence of free acridinium esters, and reducing the background value;

[0041] 2. By optimizing the blocking agent and buffer formulation in the acridinium ester complex solution, the effect of the chromatography column combined with the nucleic acid protein detector in distinguishing free acridinium ester from acridinium ester complex can be improved;

[0042] 3. Through the high affinity between biotin and streptavidin, the first antibody is linked to the magnetic beads, which can not only extend the surface reaction arm of the magnetic beads, solve the steric hindrance problem, eliminate the mutual interference between each antibody and antigen during the reaction, but also amplify the signal;

[0043] 4. When the magnetic bead solution and the purified acridinium ester complex solution are combined for chemiluminescent detection of interleukin 12P70, the detection sensitivity can be effectively improved and the amount of antibody used can be reduced, which not only improves the detection performance but also reduces the cost, and the detection sensitivity reaches 0.105pg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The separation spectrum of acridinium ester complexes is shown in Example 2, showing the effects of different formulations of blocking agents on the separation and detection of acridinium ester complexes;

[0045] Figure 2 This is the acridinium ester complex separation spectrum showing the effect of the second buffer on the differentiation of free acridinium ester and acridinium ester complex in Example 3. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below in conjunction with the accompanying drawings and examples. It should be noted that the following examples are intended to facilitate understanding of the present invention and do not have any limiting effect on it. The reagents not particularly noted in this example are all known products and were obtained by purchasing commercially available products.

[0047] Example 1: Preparation of a chemiluminescent detection kit for interleukin 12P70

[0048] The interleukin 12P70 chemiluminescent detection kit provided in this embodiment includes a magnetic bead solution and an acridinium ester complex solution, which can be prepared by the following steps:

[0049] 1. Preparation of magnetic bead solution, steps are as follows:

[0050] (1) The interleukin 12P70 primary antibody was dialyzed overnight with 0.02M PBS buffer. The interleukin 12P70 primary antibody was purchased from Hangzhou Boyin Biotechnology Co., Ltd. with a catalog number of M080501;

[0051] (2) The dialyzed IL-12P70 primary antibody prepared in (1) was reacted with 10 mmol / L biotin ester at a ratio of 1 mg antibody to 13.3 μL 10 mmol / L biotin ester at 25°C for 120 min;

[0052] (3) After the reaction was completed, the cell was dialyzed against 0.02 M PBS buffer overnight;

[0053] (4) After dialysis overnight, biotinylated antibodies were obtained;

[0054] (5) Magnetic bead washing: 10 mg of carboxyl magnetic beads were placed in a centrifuge tube, subjected to magnetic separation, and the supernatant was removed. 1 mL of 0.25 mM MES buffer was added to the magnetic beads, and after oscillation and uniform dispersion, magnetic separation was performed and the supernatant was removed. The magnetic separation and washing with 0.25 mM MES buffer were repeated twice. The magnetic beads were purchased from Beijing Boerma Biotechnology Co., Ltd., with the product number MS300 / Carboxyl.

[0055] (6) Activation: Add 500 μL 0.25 mM MES buffer and 250 μL 10 mg / mL NHS solution to the washed magnetic beads, mix well, then add 250 μL 10 mg / mL EDC solution, mix well and react at room temperature for 30 minutes on a mixer. After the reaction is completed, perform magnetic separation, remove the supernatant, and wash twice with 0.25 mM MES buffer. The 10 mg / mL NHS solution is prepared by dissolving 10 mg of NHS in 1 mL 0.25 mM MES buffer, and the 10 mg / ml EDC solution is prepared by dissolving 10 mg of EDC in 1 mL 0.25 mM MES buffer;

[0056] (7) Coupling: The activated magnetic beads obtained in step (6) were dispersed with 1000 μL of 0.25 mM MES buffer, 0.1 mg of streptavidin antibody was added, and the mixture was shaken and reacted on a mixer at room temperature for 2 h.

[0057] (8) Blocking: Add 0.5 mL of 10% BSA solution, shake to disperse evenly, and react on a mixer for 2 h.

[0058] (9) After the blocking, the magnetic beads were magnetically separated, the supernatant was removed, and the beads were washed three times with PBS. 1 mL of PBS was added and mixed.

[0059] (10) The magnetic beads separated by magnetic field in step (9), streptavidin, and the biotinylated antibody in step (4) were coated in a ratio of 1 mg: 10 ug: 40 ug, and reacted at room temperature for 60 min. The streptavidin was purchased from Thermo Fisher, catalog number 434302;

[0060] (11) Add 0.05M vitamin H and block at room temperature for 30 minutes. The ratio of the blocking agent to the magnetic beads is 50ul vitamin H: 1mg magnetic beads. Wash with buffer 3 times, add working buffer to resuspend the magnetic beads, and the concentration of the magnetic bead solution is 0.1mg / mL. The washing buffer is prepared by 0.02M PBS, 9g / L sodium chloride, 5g / L bovine serum albumin, 5g / L glycine, 5g / L trehalose, 10g / L gelatin, 5g / L sodium alginate, 1g / L PEG 20000, 1mL / L Triton X-100, and 1mL / L ProClinTM300. The working solution is prepared by 0.24g / L KH2PO4, 1.44g / L Na2HPO4, 8g / L NaCl, 0.2g / L KCl, and 1mL / L ProClinTM300.

[0061] 2. Preparation of acridinium ester complex solution:

[0062] The specific preparation steps are as follows:

[0063] (1) dialyzing the interleukin 12P70 secondary antibody with a first buffer overnight, the interleukin 12P70 secondary antibody was purchased from Hangzhou Boyin Biotechnology Co., Ltd., with a catalog number of M080502, and the first buffer was prepared with pH 8.0, 0.78 g / L NaH2PO4·2H2O, 8.775 g / L Na2HPO4, and 13.445 g / L NaCl;

[0064] (2) The dialyzed interleukin 12P70 secondary antibody was mixed with acridinium ester at a mass ratio of 20:1 and incubated at 37°C for 60 min. The acridinium ester was C01A purchased from Helison (Xiamen) Biotechnology Co., Ltd. with a catalog number of HS-11015005.

[0065] (3) adding 3% lysine and 5 g / L BSA, and incubating at 37°C for 60 min to obtain a complex of the interleukin 12P70 secondary antibody and acridinium ester;

[0066] (4) Passing the complex obtained in step (3) through a chromatography column, collecting the complex of the interleukin 12P70 secondary antibody and the acridinium ester using a nucleic acid protein detector, and removing excess acridinium ester (including other impurities), the specific method is as follows: Preparation of chromatography column: The chromatography column is filled with and used;

[0067] (5) Add Smartdex G-50 medium filler (purchased from Changzhou Tiandi Renhe Biotechnology Mailbox Company; Catalog No.: SEC0171) to the BXK16 / 40 chromatography column (brand model: purchased from: Boglon (Shanghai) Biotechnology Co., Ltd., catalog number: B-1640), and connect the digital display timing constant flow pump (brand: Shanghai Jiapeng Technology Co., Ltd.; model: BT-100SD), chromatography column, and nucleic acid protein detector (brand: Shanghai Jiapeng Technology Co., Ltd.; model: JP-3001) in sequence with plastic catheters;

[0068] (6) The second buffer (composed of pH 6.5, 10.685 g / L NaH2PO4·2H2O, 8.775 g / L Na2HPO4, 4.473 g / L NaCl, and 1.0 mL / L ProClinTM300) was used to clean the equilibrated chromatography column for 3 to 5 column volumes, and the nucleic acid protein detector was adjusted to zero;

[0069] (7) loading the complex obtained in step (3);

[0070] (8) Eluting with a second buffer solution, collecting the liquid when the UV value of the nucleic acid protein detector rises, and stopping collecting after the UV value returns to 0. The collected liquid is the required complex of the interleukin 12P70 second antibody and acridinium ester;

[0071] (9) Continue eluting. When the UV value of the nucleic acid protein detector rises again, collect the liquid. Stop collecting after the UV value returns to 0. The collected liquid is the excess acridinium ester.

[0072] (10) Use MOPS buffer (composed of 5.23 g / L Mops, 9 g / L NaCl, 20 g / L BSA, and 0.3 mL / L ProClinTM300) to dilute the complex of interleukin 12P70 secondary antibody and acridinium ester collected in step d to a concentration of 0.2 mg / L.

[0073] 3. Kit testing process

[0074] The steps for testing samples using the kit are as follows:

[0075] (a) Place the reagents into the fully automatic chemiluminescence immunoassay analyzer, place the plasma sample in the sample rack and place it into the instrument, and set the test task;

[0076] (b) The instrument automatically dispenses 20 μL of standards of different concentrations into a reaction cup, and sequentially adds 50 μL of magnetic bead solution and 100 μL of acridinium ester complex solution, mixes well, and incubates at 37°C for 3 minutes;

[0077] (c) magnetic separation and washing for 3 times, the washing solution being PBST;

[0078] (d) Pre-excitation solution (purchased from Fipeng Biotechnology, catalog number: CE00001F11) was added and reacted for 2 minutes. After adding the excitation solution (purchased from Fipeng Biotechnology, catalog number: CE00001F12), the luminescence signal was automatically read by the fully automatic chemiluminescence immunoassay analyzer.

[0079] 4. Make a song

[0080] A calibration curve is prepared by measuring reference materials of different concentrations, and a fitting formula of concentration-signal value is obtained according to the standard curve. After the analyte is detected, the signal ratio is calculated and substituted into the fitting formula to obtain the content of the analyte.

[0081] The process of making the calibration curve is as follows: set up multiple groups of standard samples with different concentrations, and obtain the luminescent signal value corresponding to the standard sample according to the above-mentioned kit detection process. Prepare the standard curve with the standard sample concentration as the horizontal axis and the signal ratio as the vertical axis, and obtain the concentration-signal value fitting formula. The results are shown in Table 1 below.

[0082] Table 1. Standard test concentration-signal value

[0083] Standard concentration (pg / mL) Signal value (RLU) 0 320 1.5 1041 20 9121 200 70241 500 204504 1000 408680 3000 1218496 5000 1949020

[0084] According to the results in Table 1, a standard curve can be prepared and the fitting formula of concentration-signal value can be calculated: Y=(3564.1754-19297505.9499) / (1+(Log(X) / 5.0360)^7.2579)+19297505.9499; correlation coefficient r=0.9986.

[0085] 5. Configure quality control products

[0086] Use the standard to prepare a low-value quality control product 1 and a high-value quality control product 2, with concentrations of 5 pg / mL and 3000 pg / mL respectively.

[0087] 6. Linear range assessment

[0088] The high-value plasma samples close to the upper limit of the linear interval (5000 pg / mL) and the low-value plasma samples close to the lower limit of the linear interval (1.5 pg / mL) were diluted to 5 concentrations at a ratio of 0:1, 1:3, 1:1, 3:1, and 1:0. Each linear sample was tested 3 times, and the average was taken for linear comparison with the theoretical value, and the correlation coefficient (r) of the linear regression was calculated. The experimental results are shown in Table 2 below.

[0089] Table 2. Detection of linear samples 1-5 by the kit

[0090]

[0091] The results in Table 2 show that the linear correlation coefficient between the linear sample concentration detected by the kit and the theoretical concentration can reach above 0.99, indicating that the kit has a good linear relationship and the test results are highly accurate and consistent within the expected concentration range.

[0092] 7. Sensitivity test

[0093] The zero-concentration working calibrator was tested 20 times, and the RLU values ​​of the 20 test results were calculated, and the mean value M and standard deviation SD were calculated. The adjacent concentration working calibrator was tested 3 times, and the RLU mean of the 3 measurement results was obtained. A linear equation was obtained by performing a two-point regression fitting based on the concentration-RLU value results between the zero-concentration working calibrator and the adjacent working calibrator. The RLU value corresponding to (M+2SD) was substituted into the equation to calculate the corresponding concentration. The experimental results are shown in Table 3 below.

[0094] Table 3. Sensitivity test data

[0095]

[0096] According to the results in Table 3, the linear regression equation obtained by fitting is Y=0.002X-0.85, and M+2SD is substituted into the equation to calculate the detection limit of 0.105 pg / mL, indicating that the kit of the present invention has high sensitivity and can detect target substances at lower concentrations.

[0097] 8. Accuracy Assessment

[0098] Two quality control products were tested, and each quality control product was tested three times. The relative deviations were calculated respectively. The experimental results are shown in Table 4 below.

[0099] Table 4. Accuracy evaluation test results

[0100]

[0101] According to the results in Table 4, the relative deviations between the concentrations of control product 1 and control product 2 detected by the kit and the theoretical concentrations are very small and controlled within ±5%, indicating that the detection results of the kit are accurate and reliable.

[0102] 9. Repeatability determination

[0103] The two quality control products were tested 10 times respectively, and their mean values, standard deviations and coefficients of variation were calculated. The experimental results are shown in Table 5 below.

[0104] Table 5. Repeatability test results

[0105]

[0106] From the results in Table 5, it can be seen that the coefficient of variation is controlled within 5%, which shows that although the data has a certain degree of discreteness, this discreteness is reliable from the perspective of the confidence limit specified by the standard deviation, indicating that this kit has strong consistency, high precision and stable detection.

[0107] 10. Comparison with mass spectrometry results

[0108] For 10 samples, the kit and mass spectrometer provided in this embodiment were used for detection, respectively. Each sample was detected 3 times using the kit and the average value was taken. The mass spectrometer (purchased from Shimadzu, model LC-MS / 8040) used 0.1% formic acid water and 0.1% formic acid acetonitrile as the mobile phase, an ESI ion source, atomization pressure (GS1): 45Psi, auxiliary gas pressure: 45Psi, air curtain pressure: 35Psi, temperature: 650°C, spray voltage: 5000V (positive ion mode), gradient elution, and the deviation between the test results of the kit and the mass spectrometer was calculated. The results are shown in Table 6 below.

[0109] Table 6. Comparison of the results between the kit and mass spectrometry

[0110] Sample No. Kit (pg / mL) Mass spectrometry (pg / mL) Relative deviation 1 22.89 23.45 -2.40％ 2 38.09 37.78 0.82％ 3 9.08 9.01 0.73％ 4 131.01 135.26 -3.14％ 5 85.55 86.83 -1.47％ 6 19.64 19.40 1.26％ 7 273.71 267.32 2.39％ 8 63.72 64.79 -1.65％ 9 176.89 178.64 -0.98％ 10 52.66 51.47 2.31％

[0111] According to the results in Table 6, the relative deviations of the results of the 10 samples tested by the kit were compared with the mass spectrometry results, and were all controlled within ±5%, indicating that the kit has high detection accuracy and high reference value.

[0112] 10. Stability test

[0113] According to the Arrhenius formula, 7 days of acceleration at 37°C can simulate storage in a 2-8°C environment for about 12 months, and 14 days of acceleration at 37°C can simulate storage in a 2-8°C environment for about 24 months. The detection reagent magnetic bead solution and acridinium ester complex solution were placed at 37°C for acceleration, and 10 samples were tested on the 0th, 7th, and 14th days of acceleration. The kit was used for testing, and each sample was tested 3 times to take the average value. The mass spectrometry detection concentration was used as a control to calculate the deviation. The experimental results are shown in Table 7 below.

[0114] Table 7. Stability test results

[0115]

[0116] According to the data analysis in Table 7, as the number of accelerated days increases, the deviation between the test results of the kit and the mass spectrometry test results increases, indicating that part of the protein in the magnetic bead solution and the acridinium ester complex solution is inactivated and therefore no longer functions; the relative deviation between the test results of the accelerated kit for 0 to 14 days and the mass spectrometry test results is within ±5%, indicating that the kit has good stability and can be stored in an environment of 2-8°C for about 24 months.

[0117] Example 2: Effects of different blocking agents on the separation and detection of acridinium ester complexes

[0118] The blocking buffer formula in Example 1 was changed, and other conditions remained unchanged. Ten samples were tested using this kit. Each sample was tested 3 times and the average value was taken. The mass spectrometry detection concentration was used as a control to calculate the deviation. Different separation buffer formulas are shown in Table 8 below. The experimental results are shown in Table 9 below. The separation spectrum of acridinium ester complex and free acridinium ester is shown in Figure 1 shown.

[0119] Table 8. Formulas of different sealants

[0120]

[0121] Table 9. Effects of different sealant formulations on detection

[0122]

[0123] like Figure 1 As shown, from left to right, the separation spectra of acridinium ester complexes and free acridinium esters labeled with acridinium esters using blocking agents of formula 1, formula 2, formula 3, and formula 4 are shown. The peaks of the acridinium ester complexes and free acridinium esters of formula 2 are sharp, long, symmetrical, and have the largest area, indicating that they are eluted relatively quickly, concentrated, and have fewer impurities; the peaks of the acridinium ester complexes and free acridinium esters of formulas 3 and 4 are connected together, indicating that the separated acridinium ester complexes contain impurities, which affect subsequent detection and lead to inaccurate test results; the peak area of ​​the acridinium ester complex of formula 1 is not as large as that of the acridinium ester complex of formula 2, indicating that the elution effect of the acridinium ester complex of formula 1 is not as good as that of formula 2. According to the analysis of the test results in Table 9, the test results of the combination of 3% lysine and 5g / L BSA in Formula 2 have the smallest deviation from the mass spectrometry test, which is better than the test results of the other three formula combinations, indicating that after being blocked by the blocking agent in Formula 2, the separation effect of the acridinium ester complex and the free acridinium ester is better, which is consistent with the results of the mass spectrometry test. Figure 1 The results were consistent, so the combination of formula 2 was selected as the blocking agent. Lysine and BSA are used as blocking agents mainly to utilize their interaction with acridinium esters and adsorb on the nonspecific binding sites of acridinium esters to block chemiluminescent substances, avoid nonspecific interference, improve the sensitivity and specificity of the detection reagents, and thus improve the accuracy of the detection.

[0124] Example 3: Effect of the Second Buffer on the Discrimination of Free Acridinium Ester and Acridinium Ester Complex

[0125] The second buffer solution formula in Example 1 was changed, and other conditions remained unchanged. Ten samples were tested using this kit. Each sample was tested 3 times and the average value was taken. The mass spectrometry detection concentration was used as a control to calculate the deviation. Different second buffer solution formulas are shown in Table 10 below. The experimental results are shown in Table 11 below. The separation spectrum of acridinium ester complex and free acridinium ester is shown in Figure 2 shown.

[0126] Table 10, different second buffer formulations

[0127]

[0128] Table 11. Effects of different second buffer solutions on test results

[0129]

[0130] like Figure 2 As shown, from left to right are the separation spectra of acridinium ester complex and its free acridinium ester separated by three separation buffers: Formula 1, Formula 2 and Formula 3. Among them, the peaks of the separation spectra of acridinium ester complex and free acridinium ester separated by Formula 1 buffer are relatively sharp, long and symmetrical, indicating that the elution is relatively fast, concentrated and has fewer impurities; the peaks of acridinium ester complex separated by Formulas 2 and 3 are connected with the peaks of free acridinium ester, indicating that the separated acridinium ester complex contains impurities, which affects subsequent detection and causes inaccurate detection results. According to the data analysis in Table 11, the results of the detection samples prepared by the detection reagent composed of the acridinium ester complex eluted by the PBS buffer of Formula 1 have the smallest deviation from the mass spectrometry detection results, and the detection results of Formulas 2 and 3 are not as good as those of Formula 1, indicating that Formula 1 buffer has the best effect in separating acridinium ester complex and free acridinium ester, which is consistent with the results of the mass spectrometry detection. Figure 2 The results were consistent, so formula 1 was selected as the second buffer.

[0131] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. A method for preparing a chemiluminescent detection kit for interleukin 12P70, characterized in that: The following steps are involved: (1) preparing a magnetic bead solution linked to a first antibody; (2) preparing an acridinium ester complex solution linked to a second antibody, and removing free acridinium ester through a chromatography column and a nucleic acid protein detector; The first antibody and the second antibody are interleukin 12P70 first antibody and interleukin 12P70 second antibody respectively.

2. The preparation method according to claim 1, characterized in that The acridinium ester complex solution in step (2) includes a first buffer solution, which includes NaH2PO4·2H2O, Na2HPO4, and NaCl.

3. The preparation method according to claim 2, characterized in that: The acridinium ester solution for connecting the second antibody also includes a blocking agent, and the blocking agent includes lysine.

4. The preparation method according to claim 3, characterized in that: The filler in the chromatography column of step (2) is G25 / G50 dextran, and the filler needs to be washed with a second buffer solution first, wherein the second buffer solution includes NaH2PO4·2H2O, Na2HPO4, NaCl, and ProClinTM300.

5. The preparation method according to claim 1, characterized in that: The method for preparing the magnetic bead solution connected to the first antibody in step (1) is: (a) mixing a first antibody solution with a biotin ester to react to prepare a biotinylated antibody; (b) Magnetic beads are coupled with streptavidin antibody and then coated with streptavidin and biotinylated antibody.

6. The preparation method according to claim 5, characterized in that: After the coating in step (b) is completed, it is necessary to wash with a third buffer solution, and the third buffer solution includes PBS, sodium chloride, bovine serum albumin, glycine, trehalose, gelatin, sodium alginate, PEG 20000, Triton X-100 and ProClinTM300.

7. A chemiluminescent detection kit for interleukin 12P70 prepared by the method according to any one of claims 1 to 6.

8. A chemiluminescent detection method for interleukin 12P70, characterized in that: The sample to be tested, the magnetic bead solution, and the acridinium ester complex solution are incubated together to form an immune complex, which is then washed and reacted with a substrate to generate a light signal.

9. Use of a buffer for preparing a reagent for improving the effect of distinguishing free acridinium ester and acridinium ester complex in a chromatography column, characterized in that: The buffer solution includes NaH2PO4·2H2O, Na2HPO4, NaCl, and ProClinTM300.

10. Use of a blocking agent for preparing a reagent for improving the differentiation effect of free acridinium ester and acridinium ester complex in a chromatography column, characterized in that: The blocking agents include lysine and BSA.

Images