Preparation and application of chemiluminescence detection kit for Abeta1-40

Abstract

The invention provides a chemiluminiscence detection kit for A beta 1-40 and application of the chemiluminiscence detection kit. The invention provides a preparation method of an acridinium ester labeled A beta 1-40 antibody compound solution for chemiluminescence detection of A beta 1-40, according to the method, an antibody preservation solution is replaced, factors such as glycerol or some preservatives, antibody fragments, residues and the like which are not beneficial to labeling in the original preservation solution can be removed, and the stability of an antibody to be labeled is improved; the invention provides a labeling buffer solution formula for improving the labeling efficiency of an acridinium ester antibody complex. The invention provides a formula of a blocking buffer solution for improving the stability of an acridinium ester antibody complex. The invention provides a separation buffer solution formula for column separation of an acridinium ester antibody compound and free acridinium ester. The labeling efficiency of the acridinium ester antibody compound can be improved by eluting a chromatographic column with the separation buffer solution according to the formula. The kit has the advantages of simple operation process, high sensitivity, high accuracy, strong stability, short detection time, reliable result, low cost and the like.

Description

Technical Field

[0001] The invention relates to the field of chemiluminescent immunoassay and discloses the preparation and application of a chemiluminescent detection kit for Aβ1-40. Background Art

[0002] Amyloidβ-Protein (1-40), referred to as Aβ1-40, is the most important component of neuronal structural proteins and a sensitive marker of axonal damage. Aβ (β-amyloid protein) is a transmembrane glycoprotein formed by the cleavage of the APP (amyloid precursor protein) sequence. APP can be decomposed by α-, β-, and γ-proteinases. Aβ is mainly produced through the amyloid degradation pathway. APP is hydrolyzed by β-secretase and γ-secretase to release Aβ fragments with 39-43 amino acid residues. This process is the amyloid degradation pathway. The most common residue Aβ fragments are Aβ40 and Aβ42. Studies have shown that in the state of neurodegenerative diseases, Aβ (β-amyloid protein) is enzymatically hydrolyzed by β-secretase and γ-secretase in turn to produce insoluble oligomers of amyloid precursor protein (APP). The amyloid plaques formed by the accumulation of proteins in the patient's brain eventually lead to neuronal death. Therefore, Aβ (β-amyloid protein) deposition is one of the main pathological features of Alzheimer's disease (AD) and plays an important role in the pathological development of AD. Since β-amyloid protein (Aβ40 / 42) plays a key role in the onset of AD, it has become one of the most studied proteins in protein immunotherapy projects for AD.

[0003] Acridinium ester is a flash-type labeling substance commonly used in chemiluminescent immunoassay. Under alkaline H2O2 conditions, acridinium ester compounds can emit light rapidly with a high quantum yield. For example, the quantum yield of acridinium aromatic ester can reach 0.05. Acridinium ester is used as a label for immunoassay. The luminescence system is simple and rapid, no catalyst is required, and the labeling efficiency is high and the background is low. It has been widely used in the field of in vitro diagnosis. At present, before acridinium ester labeling protein, the labeled antibody is generally not desalted. The storage solution of the labeled antibody is generally tris buffer or PBS buffer. Some antibodies are stored in 50% glycerol or in preservatives such as NaN3 that are not conducive to labeling. If the antibody purity or stability is low, the storage solution will also contain some antibody fragments and residues. These salt ions, glycerol, preservatives, antibody fragments and residues will reduce the efficiency of acridinium ester labeling antibodies. These factors will indirectly affect the sensitivity and accuracy of the test kit in detecting the analyte in the sample, because the specificity of the impure acridinium ester antibody complex binding to the analyte in the test sample is reduced, while non-specific adsorption increases. After the acridinium ester is labeled with the protein, impurities are mainly removed by dialysis to achieve the purification effect, which generally takes at least 6 to 8 hours, requires a large amount of dialysis fluid and dialyzes overnight, which increases time and material costs. There is no specified method for Aβ1-40 detection in the industry, so the sensitivity, stability, and accuracy of various detection methods vary greatly.

[0004] Therefore, it is urgent to find a kit that can solve the above problems and improve the detection sensitivity, stability and accuracy. Summary of the invention

[0005] In order to solve the above problems, the method of the present invention provides a chemiluminescent detection kit for Aβ1-40 and its application. The present invention provides a method for preparing an acridinium ester-labeled Aβ1-40 antibody complex solution for chemiluminescent detection of Aβ1-40. The method adjusts the antibody solution to other salt solution environments suitable for labeling, removes factors that are not conducive to labeling, such as glycerol or certain preservatives and antibody fragments and residues in the original preservation solution, and improves the stability of the antibody to be labeled; the present invention provides a labeling buffer formula that improves the labeling efficiency of the acridinium ester antibody complex; the present invention provides a blocking buffer formula that improves the stability of the acridinium ester antibody complex; the present invention provides a separation buffer formula for column separation of the acridinium ester antibody complex and free acridinium ester, and the separation buffer elution chromatography column of this formula can improve the labeling efficiency of the acridinium ester antibody complex. The kit of the present invention has the advantages of simple operation process, high sensitivity, high accuracy, strong stability, short detection time, reliable results, and low cost.

[0006] A method for preparing an acridinium ester labeled Aβ1-40 antibody complex solution for chemiluminescent detection of Aβ1-40 comprises firstly performing a desalting treatment on the Aβ1-40 antibody and then using the desalting treatment to prepare the acridinium ester labeled Aβ1-40 antibody complex.

[0007] Furthermore, the preparation method comprises the following steps:

[0008] (1) Desalting the Aβ1-40 antibody;

[0009] (2) using the Aβ1-40 antibody treated in step (1) to prepare a crude acridinium ester complex solution, and removing free acridinium ester through a chromatography column combined with a nucleic acid protein detector.

[0010] In some methods, the antibody storage solution is generally tris buffer or PBS buffer. Some antibodies are stored in 50% glycerol or in preservatives such as NaN3 that are not conducive to labeling. If the antibody purity or stability is low, the storage solution will also contain some antibody fragments and residues. These salt ions, glycerol, preservatives, antibody fragments, and residues will reduce the efficiency of acridinium ester labeled antibodies. Therefore, replacing the antibody buffer can adjust the antibody solution to other salt solutions suitable for acridinium ester labeling, which can remove the effects of salt ions, glycerol, preservatives, antibody fragments, and residues on the efficiency of acridinium ester labeled antibodies, reduce nonspecific adsorption, and improve labeling efficiency. Incomplete removal of free acridinium esters will lead to increased background and poor stability of the test results, thereby affecting the accuracy of the test.

[0011] Furthermore, in the preparation method, the desalination treatment in step (1) comprises the following steps:

[0012] (a) treating the desalting column with an antibody labeling buffer; the antibody labeling buffer comprises Na2HPO4, NaH2PO 4、 Glycine, BSA;

[0013] (b) Aβ1-40 antibody was diluted with antibody labeling buffer and desalted by a desalting column.

[0014] In some embodiments, the kit can be used to desalt large quantities of Aβ1-40 antibodies, saving time and cost for large-scale antibody desalting operations, and the final test results are accurate and reliable.

[0015] Furthermore, in the preparation method, the crude acridinium ester complex solution in step (2) includes a blocking buffer, and the blocking buffer includes Na2HPO4, NaH2PO4, lysine, BSA, and Tween-20.

[0016] Furthermore, in the preparation method, in the crude solution of the acridinium ester complex in step (2), the mass ratio of the antibody to the acridinium ester is 5 to 20:1.

[0017] Furthermore, in the preparation method, the filler in the chromatography column in step (2) is G25 / G50 dextran, and the filler needs to be washed with a separation buffer first, and the separation buffer includes Na2HPO4, NaH2PO4, and NaCl.

[0018] Furthermore, in the preparation method, the method of removing free acridinium esters by using a chromatography column in combination with a nucleic acid protein detector is as follows: a crude solution of the acridinium ester complex is added to the chromatography column, and the chromatography column is continuously flushed with a separation buffer.

[0019] In some methods, G25 / G50 dextran separates substances based on the molecular size and shape of the substances to be separated. This separation technique is called size exclusion chromatography, and its basic principle is to use the different penetration degrees of substances with different molecular weights in the gel to achieve separation. In the process of separating acridinium ester antibody complexes and free acridinium esters, G25 / G50 dextran screens the molecules passing through through its unique pore structure. Acridinium ester antibody complexes with larger molecular weights cannot enter the internal pores of the dextran gel because of their large size, so they will be retained outside the gel to form a peak shape, so the acridinium ester antibody complex is eluted first; while impurities or solvents with smaller molecular weights can enter the pores of the dextran gel, and are eluted later inside the gel, thereby achieving the separation of acridinium ester antibody complexes and free acridinium esters.

[0020] Furthermore, a labeling buffer is used to improve the labeling efficiency of an acridinium ester antibody complex, wherein the labeling buffer comprises Na2HPO4, NaH2PO4, glycine, and BSA.

[0021] In some embodiments, the labeling buffer is prepared with pH 8.0, 12.00 g / L Na2HPO4, 14.20 g / L NaH2PO4, 5 g / L glycine, and 5 g / L BSA. Glycine can maintain the acid-base balance of the solution and provide a stable buffer environment for the process of acridinium ester labeling antibodies. BSA, as a protein stabilizer, can improve the stability of the connection between the antibody and the acridinium ester, thereby improving the detection accuracy.

[0022] Furthermore, a blocking buffer is used to improve the stability of an acridinium ester antibody complex, wherein the labeling buffer comprises Na2HPO4, NaH2PO4, lysine, BSA, and Tween-20.

[0023] In some embodiments, the labeling buffer is prepared with pH 8.0, 12.00 g / L Na2HPO4, 14.20 g / L NaH2PO4, 0.3% lysine, 5 g / L BSA, and 0.5 mL / L Tween-20. Lysine and BSA are used as blocking agents mainly to utilize their interaction with acridinium esters and adsorb on the non-specific binding sites of acridinium esters to block chemiluminescent substances, avoid non-specific interference, improve the sensitivity and specificity of the detection reagent, and thus improve the accuracy of the detection; Tween-20 blocks the non-binding sites of the antibody protein to reduce the background, enhance the specific binding of the antibody, and thus improve the accuracy of the detection.

[0024] Furthermore, a separation buffer is used to improve the separation effect of acridinium ester antibody complex and free acridinium ester, and the separation buffer comprises Na2HPO4, NaH2PO4, and NaCl.

[0025] In some embodiments, the separation buffer is prepared with pH 6.5, 12.00 g / L Na2HPO4, 14.20 g / L NaH2PO4, and 5.85 g / L NaCl. During the elution of the free acridinium ester and the acridinium ester antibody complex through the chromatography column, the acridinium ester antibody complex is eluted by the first increase in the value of the nucleic acid protein detector after elution with the separation buffer. If the distinction effect is not good, the eluted acridinium ester antibody complex will be mixed with free acridinium ester, and the free acridinium ester will undergo hydrolysis and luminescence under the reaction of hydrogen peroxide solution and hydroxide, resulting in a false positive test result.

[0026] The Aβ1-40 chemiluminescent detection kit and application provided by the present invention have the following effects:

[0027] 1. The method of the present invention provides a two-step desalting method for acridinium ester-labeled Aβ1-40 antibodies. The first step of desalting is used to replace the Aβ1-40 second antibody preservation solution, adjust the antibody solution to other salt solution environments suitable for labeling, remove glycerol or certain preservatives and antibody fragments, residues and other factors in the original preservation solution that are not conducive to labeling, and improve the stability of the antibody to be labeled. The second step of desalting is used to remove excess acridinium ester.

[0028] 2. The method of the present invention provides a separation buffer formula combination for column separation of acridinium ester antibody complex and free acridinium ester. The separation effect can be improved by eluting the chromatography column with the separation buffer with this formula combination, and the influence of the background signal of free acridinium ester can be reduced.

[0029] 3. The present invention provides a diluent formula composition for improving the stability and accuracy of detection reagents.

[0030] 4. The kit of the present invention has the advantages of simple operation process, high sensitivity, high accuracy, strong stability, short detection time, reliable results and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a spectrum showing the effect of the desalting treatment on the separation of acridinium ester antibody complex and free acridinium ester in Example 2;

[0032] Figure 2 This is a spectrum showing the effect of different labeling mass ratios of Aβ1-40 antibody and acridinium ester on the separation of acridinium ester antibody complex and free acridinium ester in Example 3;

[0033] Figure 3 The spectrum is the effect of different separation buffers on the separation of acridinium ester antibody complex and free acridinium ester in Example 6;

[0034] Figure 4 This is a spectrum showing the effect of desalting of a large batch of Aβ1-40 antibodies on the separation of acridinium ester antibody complex and free acridinium ester in Example 7. DETAILED DESCRIPTION

[0035] The embodiments of the present invention are described in further detail below. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and do not have any limiting effect thereon. All features disclosed in the embodiments of the present invention, or all steps in the methods or processes disclosed, except for mutually exclusive features and / or steps, can be combined in any manner.

[0036] Example 1. Preparation of acridinium ester antibody complex

[0037] The steps for preparing the acridinium ester antibody complex are as follows:

[0038] (1) Centrifuge a 0.5 mL 7KD desalting column (Zeba Desalting Column from Thermo) at 1500 × g for 1 min and discard the collected liquid;

[0039] (2) Add 350uL of antibody labeling buffer (pH8.0, 12.00g / L Na2HPO4, 14.20g / LNaH2PO4, 5g / L glycine, 5g / L BSA) to the desalting column and centrifuge the desalting column at 1500×g for 1 min, then discard the collected liquid in the tube;

[0040] (3) Repeat step (2) and discard the collected liquid in the tube;

[0041] (4) Repeat step (3) and discard the collected liquid in the tube;

[0042] (5) Dilute 50 μg of Aβ1-40 secondary antibody to 100 μL with antibody labeling buffer and add it to the cleaned desalting column. Centrifuge at 1500×g for 2 min to desalt and collect the desalted antibody solution.

[0043] (6) adding an acridinium ester solution to the desalted Aβ1-40 antibody solution, wherein the acridinium ester is (Helison NSP-SA-NHS acridinium ester), and the mass ratio of the antibody to the acridinium ester is 5 to 20:1, mixing well, and reacting at 25° C. in the dark for 30 minutes;

[0044] (7) Add 100 uL of blocking buffer (pH 8.0, 12.00 g / L Na2HPO4, 14.20 g / L NaH2PO4, 0.3% lysine, 5 g / L BSA, 0.5 mL / L Tween-20) to the antibody acridinium ester labeled solution, mix well, and react at 25°C in the dark for 30 min to obtain the antibody acridinium ester labeled complex solution.

[0045] (8) After assembling an empty chromatography column (Borgron BXK16 / 40 double-layer glass chromatography column), add G50 dextran (Tian Di Ren He G50F dextran) filler, connect the chromatography column and nucleic acid protein detector (Jia Peng Computer Nucleic Acid Protein Chromatograph, Model JP-3001) in sequence with a catheter, and first rinse the chromatography column with 2 to 3 column volumes of water until the baseline is stable;

[0046] (9) Continue to flush the column with 3 to 5 column volumes of separation buffer (pH 6.5, 12.00 g / L Na2HPO4, 14.20 g / L NaH2PO4, 5.85 g / L NaCl) until the baseline is stable;

[0047] (10) Adjust the nucleic acid protein detector to zero and adjust the steady baseline signal value to 0 Abs;

[0048] (11) slowly adding the antibody acridinium ester labeled complex solution to the top of the chromatography column all at once;

[0049] (12) After the solution has completely penetrated into the chromatography column, continue to flush the chromatography column with the separation buffer;

[0050] (13) Observe the signal display screen of the protein detector. When the UV value at 280 nm rises, receive the solution at the sample outlet of the instrument. When the signal value drops to the baseline, stop receiving, and the acridinium ester antibody complex solution is obtained.

[0051] (14) Continue to wash the column with separation buffer until the second peak appears. At this time, the free acridinium ester flows out of the sample outlet. After the signal value drops to the baseline, continue to use 3 column volumes of separation buffer to wash the column. Finally, rinse the column with water and store it.

[0052] Example 2: Configuration and Evaluation Kit

[0053] 1. Kit Configuration

[0054] (1) Prepare reagent R2: pH 7.20±0.1, 5.75 g / L Na2HPO4, NaH2PO4, 9 g / L NaCl, 5 g / L BSA, 5 g / L glycine, 5 g / L lysine, 5 g / L Casein, 10 g / L trehalose, 0.5 mL / L Tween-20, and 0.3 mL / L Proclin300.

[0055] (2) Prepare the reagent RM as follows:

[0056] 1) Take 10 mg of magnetic microspheres (JSR:MS300-Carboxyl) and wash them by magnetic separation three times with Buffer 1 (pH 5.0±0.1, 10.66 g / LMES), 1 mL each time;

[0057] 2) Add 0.9 mL of Buffer 1 and shake to mix;

[0058] 3) Add 10 μL of EDC / NHS solution (100 mg / mL) and mix by shaking;

[0059] 4) Incubate with rotation at room temperature for 30 minutes;

[0060] 5) Use Buffer 1 for magnetic separation and washing 3 times, 1 mL each time;

[0061] 6) Add 0.2 mg Aβ1-40 antibody (PKT1341C) and add Buffer 1 to make up to 1 mL, and shake to mix;

[0062] 7) Incubate with rotation at room temperature for 2 hours;

[0063] 8) Add 0.1 mL of BSA solution (200 mg / mL) and shake to mix;

[0064] 9) Incubate with rotation at room temperature for 2 hours;

[0065] 10) Use Buffer 2 (pH 7.3±0.1, Tris 3.0g / L, NaCl 9.0g / L, Tween-20 1.0mL / L) for magnetic separation and washing 3 times, 1mL each time;

[0066] 11) Add 1 mL of Buffer 3 (pH 7.3±0.1, Tris 3.0 g / L, NaCl 9.0 g / L, Tween-20 1.0 mL / L, BSA 1.0 g / L, Proclin 1.0 mL / L) solution and shake to resuspend the magnetic microspheres. Store at 2-8°C.

[0067] 12) Use reagent R2 to dilute the antibody-coated magnetic spheres to a concentration of 0.4 mg / mL to obtain reagent RM.

[0068] (3) Prepare reagent R1 as follows:

[0069] The acridinium ester antibody complex solution obtained in Example 1 was diluted with reagent R2 to a concentration of 0.4 mg / L, thereby obtaining reagent R1.

[0070] 2. Kit testing process

[0071] (1) Place the sample and reagents into a fully automatic chemiluminescence immunoassay analyzer (purchased from Coleus (Shenzhen) Technology Co., Ltd., model Venus100), place the sample in the sample rack and place it into the instrument, and set the test task;

[0072] (2) The instrument automatically takes 10 μL of the sample into the reaction cup, adds 50 μL of reagent RM and 100 μL of reagent R1 in sequence, mixes well, and incubates at 37°C for 3 minutes;

[0073] (3) Magnetic separation and washing for 3 times, wherein the washing solution is PBS;

[0074] (4) Add pre-excitation solution (hydrogen peroxide, purchased from Feipeng Biotechnology Co., Ltd., catalog number CE00001F11) and react for 2 minutes. After adding the excitation solution (sodium hydroxide, purchased from Feipeng Biotechnology Co., Ltd., catalog number CE00001F12), the luminescence signal (RLU value) is automatically read by the fully automatic chemiluminescence immunoassay analyzer.

[0075] 3. Make a standard curve

[0076] 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.

[0077] The concentrations of the standards were 14.6 pg / mL and 500.5 pg / mL. The standards were diluted into multiple groups of standards with different concentrations. The luminescent signal values ​​corresponding to the standards were obtained according to the above-mentioned kit detection process. The standard curve was prepared with the concentration of the standards as the horizontal axis and the signal ratio as the vertical axis, and the fitting formula of the concentration-signal value was obtained.

[0078] 4. Linear range assessment

[0079] The high-value plasma sample close to the upper limit of the linear interval (800.6 pg / mL) and the low-value plasma sample close to the lower limit of the linear interval (4.8 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 1.

[0080] Table 1. Detection of linear samples 1-5 by the kit

[0081]

[0082] The results in Table 1 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 detection concentration of the kit of the present invention is very close to the theoretical value. It can be seen that the kit has good linear performance within the detection range.

[0083] 5. Minimum detection limit evaluation

[0084] 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 result should be no more than 5pg / mL. The experimental results are shown in Table 2 below.

[0085] Table 2. Blank detection limit results

[0086] Number of tests Test results 1 52 2 37 3 41 4 85 5 53 6 49 7 71 8 46 9 55 10 51 11 63 12 63 13 54 14 51 15 50 16 67 17 53 18 35 19 74 20 81 Mean value M (RLU value / concentration pg / mL) 57 Standard Deviation SD 14 M+2SD 84 Blank limit 4.2 Acceptance criteria ≤5 pg / mL in conclusion Meet the requirements

[0087] The results in Table 2 show that the kit of the present invention is very sensitive, with the lowest detection reaching 5.0 pg / mL.

[0088] 6. Accuracy Assessment

[0089] A low-concentration quality control product and a high-concentration quality control product were prepared using the standard protein. The two quality control products were used for testing. The concentrations of the quality control products were 43.4 pg / mL and 190.2 pg / mL, respectively. Each quality control product was tested three times, and the relative deviation was calculated. The experimental results are shown in Table 3 below.

[0090] Table 3. Accuracy evaluation test results

[0091]

[0092] The results in Table 3 show that the relative deviation is very small, indicating that the test results of this kit are accurate and reliable.

[0093] 7. Repeatability determination

[0094] 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 4 below.

[0095] Table 4. Repeatability test results

[0096]

[0097] From the results in Table 4, we can see 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.

[0098] 8. Comparison with mass spectrometry results

[0099] For 10 samples, the kit and mass spectrometer provided in this embodiment were used for detection, and each sample was detected three 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 mobile phases, 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 calculated the deviation between the test results of the kit and the mass spectrometer, and the results are shown in Table 5 below.

[0100] Table 5. Comparison of the results between the kit and mass spectrometry

[0101] Sample No. Kit (pg / mL) Mass spectrometry (pg / mL) deviation 1 20.5 20.7 1％ 2 74.2 76.1 3％ 3 340.5 325.8 -4％ 4 235.5 229.5 -3％ 5 30.2 28.9 -4％ 6 177.1 182.9 3％ 7 125.2 118.9 -5％ 8 157.8 161.6 2％ 9 175.2 167.4 -4％ 10 56.2 53.7 -4％

[0102] According to the results analysis in Table 5, the relative deviations between the test results of the kit and the mass spectrometry test results were controlled within ±5%, indicating that the test results of the kit had small errors and high accuracy.

[0103] 9. Stability test

[0104] 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 reagents RM and R1 were placed at 37°C for acceleration. The test kit was used to test 10 samples on the 0th, 7th, and 14th days of acceleration. Each sample was tested three times, and the average value was taken. The relative deviation was calculated using the mass spectrometry detection concentration as a control. The experimental results are shown in Table 6 below.

[0105] Table 6. Stability test results

[0106]

[0107] According to the data analysis in Table 6, the deviation of the accelerated 14-day data 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.

[0108] Example 2: Effect of Aβ1-42 Antibody Desalting Treatment on Acridinium Ester Antibody Complex Labeling and Detection

[0109] Two experimental groups were set up, one of which was Aβ1-40 antibody solution directly labeled with acridinium ester without desalting, and the other was treated according to Example 1. The kit was used to detect 10 samples respectively, each sample was detected three times, and the average value was taken. The control group was a mass spectrometry detection concentration. The experimental results are shown in Table 7 below. The separation spectrum of the acridinium ester antibody complex is shown in Figure 1 shown.

[0110] Table 7. Effect of Aβ1-40 antibody desalting on detection

[0111]

[0112] According to the analysis in Table 7, the detection results of Aβ1-40 antibody after desalting and then acridinium ester labeling are compared with the detection results of Aβ1-40 antibody without desalting and then acridinium ester labeling, indicating that the detection results of Aβ1-40 antibody after desalting in subsequent detection as a detection reagent are closer to the mass spectrometry detection concentration and the detection accuracy is higher, while the detection results of Aβ1-40 antibody without desalting as a detection reagent in subsequent detection have a large deviation from the mass spectrometry detection concentration, especially when the sample concentration is lower than 10pg / mL, the detected sample concentration is lower than the detection limit and has no reference value, indicating that desalting of Aβ1-40 antibody before acridinium ester labeling can significantly improve the detection accuracy of the kit.

[0113] like Figure 1 As shown, from left to right are the acridinium ester antibody complex peak of the Aβ1-42 antibody acridinium ester-labeled column purified without desalting treatment and its free acridinium ester peak, the acridinium ester antibody complex peak of the Aβ1-42 antibody acridinium ester-labeled column purified with desalting treatment and its free acridinium ester peak. The sample peak of the Aβ1-42 antibody acridinium ester-labeled column purified without desalting treatment has tailing and there are impurity peaks.

[0114] Example 3: Effect of the mass ratio of Aβ1-40 antibody and acridinium ester on labeling and detection

[0115] The mass ratios of Aβ1-40 antibody and acridinium ester were set to 5:1, 10:1, 15:1, and 20:1, respectively. The other conditions remained unchanged. Each sample was tested three times, and the average value was taken to calculate the concentration deviation from mass spectrometry detection. The experimental results are shown in Table 8 below. The separation spectrum of the acridinium ester antibody complex is shown in Figure 2 shown.

[0116] Table 8. Test results of different mass ratios of Aβ1-40 antibody and acridinium ester

[0117]

[0118] According to the experimental results in Table 8, there was no significant deviation in the mass ratio of Aβ1-40 antibody to acridinium ester within the range of 5 to 20:1, and all were controlled within ±5%.

[0119] like Figure 2 As shown, from left to right are the peaks of the acridinium ester antibody complex after column purification and the free acridinium ester peak of Aβ1-40 antibody and acridinium ester at mass ratios of 5:1, 10:1, 15:1, and 20:1, respectively, and their peak shapes and areas are similar.

[0120] Example 4: Effect of labeling buffer on labeling and detection of acridinium ester antibody complexes

[0121] The formulation of the labeling buffer in Example 1 was changed, and other conditions remained unchanged. Ten samples were tested, and each sample was tested three times using the kit. The average value was taken, and the concentration deviation from mass spectrometry detection was calculated. The formulations of different labeling buffers are shown in Table 9 below, and the experimental results are shown in Table 10 below.

[0122] Table 9 Labeling buffers with different formulations

[0123]

[0124] Table 10. Effects of different formulations of labeled buffer on test results

[0125]

[0126]

[0127] According to the results in Table 10, compared with Formula 1 and Formula 2, Formula 3 has more accurate sample detection results, so it is the best labeling buffer formula. Glycine can provide a stable buffer environment, and BSA is a protein stabilizer, which can improve the stability of the connection between the antibody and the acridinium ester, thereby improving the detection accuracy.

[0128] Example 5. Effect of blocking buffer on labeling and detection of acridinium ester antibody complex

[0129] The blocking buffer formula in Example 1 was changed, and other conditions remained unchanged. 10 samples were tested, and each sample was tested three times using the kit. The average value was taken, and the deviation from the mass spectrometry detection result was calculated. Different separation buffer formulas are shown in Table 11 below, and the experimental results are shown in Table 12 below.

[0130] Table 11. Blocking buffer with different formulations

[0131]

[0132] Table 12. Effects of different blocking buffers on test results

[0133]

[0134] According to the data analysis in Table 12, only the sample test results of the blocking buffer of Formula 2 were controlled within ±5% of the overall deviation from the mass spectrometry detection, so Formula 2 was the best formula for the blocking buffer. Lysine has a better blocking effect than other amino acids. Lysine and BSA are used as blocking agents mainly to utilize their interaction with acridinium esters and adsorb on the non-specific binding sites of acridinium esters to block chemiluminescent substances, avoid non-specific interference, improve the sensitivity and specificity of the detection reagents, and thus improve the accuracy of the detection; Tween-20 blocks the non-binding sites of the antibody protein to reduce the background, enhance the specific binding of the antibody, and thus improve the accuracy of the detection.

[0135] Example 6: Effect of separation buffer formulation on labeling and detection of acridinium ester antibody complexes

[0136] The separation buffer formula in Example 1 was changed, and other conditions remained unchanged. 10 samples were tested, and each sample was tested three times using the kit. The average value was taken, and the deviation from the mass spectrometry detection result was calculated. The different separation buffer formulas are shown in Table 13 below, and the experimental results are shown in Table 14 below. The separation spectrum of the acridinium ester antibody complex is shown in Figure 3 shown.

[0137] Table 13. Separation buffers with different formulations

[0138]

[0139] Table 14. Effects of different separation buffers on test results

[0140]

[0141] According to the results of Table 14, Formula 2 is the best separation buffer. The results of the test sample prepared by the acridinium ester antibody complex eluted by the separation buffer of Formula 2 have the smallest deviation from the mass spectrometry test results, so the separation buffer of Formula 2 has the best effect on distinguishing free acridinium ester from acridinium ester antibody complex. During the elution of free acridinium ester and acridinium ester antibody complex through the chromatography column, after elution by the separation buffer, the first value of the nucleic acid protein detector increases and the acridinium ester antibody complex is eluted. If the distinction effect is not good, the eluted acridinium ester antibody complex will be mixed with free acridinium ester, and the free acridinium ester will undergo hydrolysis and luminescence under the reaction of hydrogen peroxide solution and hydroxide, resulting in a false positive test result and a high test result.

[0142] like Figure 3 As shown, from left to right are the separation spectra of acridinium ester antibody complex and its free acridinium ester separated by three separation buffers, namely, Formula 1, Formula 2 and Formula 3. Among them, the separation spectrum of acridinium ester antibody complex and its free acridinium ester separated by Formula 2 buffer has a relatively sharp and long peak shape, indicating that its elution is relatively fast, concentrated, and has fewer impurities.

[0143] Example 7: Effect of large-scale Aβ1-40 antibody desalting on acridinium ester antibody complex labeling and detection

[0144] The mass of Aβ1-40 antibody added to the desalting column for centrifugation in step (5) of Example 1 was changed to 100 ug, 200 ug, 300 ug, 400 ug, and 500 ug, respectively. Other conditions remained unchanged. Ten samples were tested. Each sample was tested three times using the kit, and the average value was taken. The deviation from the mass spectrometry test result was calculated. The test results are shown in Table 15 below. The separation spectrum of the acridinium ester antibody complex is shown in Figure 4 shown.

[0145] Table 15. Effect of large-scale antibody desalting on detection

[0146]

[0147] According to the results shown in Table 15, there is no significant difference in the effect of the desalting amount of Aβ1-40 antibody in the range of 100ug to 500ug on the labeling process and detection of the acridinium ester antibody complex. Even when the desalting amount of Aβ1-40 antibody reaches 500ug, the deviation between the final kit test result and the mass spectrometry test result is controlled within ±5%, indicating that this kit can be used for large-scale desalting of Aβ1-40 antibodies, saving time and cost for large-scale antibody desalting operations, and the final test results are accurate and reliable.

[0148] like Figure 4As shown, from left to right, they are the separation spectra of 100ug, 200ug, 300ug, 400ug, and 500ug Aβ1-40 antibodies desalted, labeled with acridinium ester, and then purified by column to separate the acridinium ester antibody complex and its free acridinium ester, and the peak shape results are all normal.

[0149] 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 an acridinium ester-labeled Aβ1-40 antibody complex solution for chemiluminescent detection of Aβ1-40, characterized in that: The Aβ1-40 antibody is first desalted and then used to prepare an acridinium ester-labeled Aβ1-40 antibody complex.

2. The preparation method according to claim 1, characterized in that The following steps are involved: (1) Desalting the Aβ1-40 antibody; (2) using the Aβ1-40 antibody treated in step (1) to prepare a crude acridinium ester complex solution, and removing free acridinium ester through a chromatography column combined with a nucleic acid protein detector.

3. The preparation method according to claim 2, characterized in that: The desalination treatment in step (1) comprises the following steps: (a) treating a desalting column with an antibody labeling buffer; the antibody labeling buffer comprises Na2HPO4, NaH2PO4, glycine, and BSA; (b) Aβ1-40 antibody was diluted with antibody labeling buffer and desalted by a desalting column.

4. The preparation method according to claim 2, characterized in that: The crude acridinium ester complex solution in step (2) includes a blocking buffer, and the blocking buffer includes Na2HPO4, NaH2PO4, lysine, BSA, and Tween-20.

5. The preparation method according to claim 4, characterized in that: In the crude solution of the acridinium ester complex in step (2), the mass ratio of the antibody to the acridinium ester is 5 to 20:

1.

6. The preparation method according to claim 2, 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 separation buffer first, and the separation buffer includes Na2HPO4, NaH2PO4, and NaCl.

7. The preparation method according to claim 6, characterized in that: The method for removing free acridinium esters by using a chromatography column in combination with a nucleic acid protein detector is as follows: a crude solution of acridinium ester complex is added to the chromatography column, and the chromatography column is continuously flushed with a separation buffer.

8. A use of a labeling buffer for improving the labeling efficiency of an acridinium ester antibody complex, characterized in that: The labeling buffer comprises Na2HPO4, NaH2PO4, glycine and BSA.

9. A use of a blocking buffer for improving the stability of an acridinium ester antibody complex, characterized in that: The labeling buffer comprises Na2HPO4, NaH2PO4, lysine, BSA and Tween-20.

10. A use of a separation buffer for improving the separation effect of acridinium ester antibody complex and free acridinium ester, characterized in that: The separation buffer comprises Na2HPO4, NaH2PO4, and NaCl.

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