A homogeneous chemiluminescence kit for determining alpha-fetoprotein content

Abstract

The present invention relates to the technical field of immunoassay detection, and in particular to a homogeneous chemiluminescence kit for determining the content of alpha-fetoprotein. The kit includes an alpha-fetoprotein specific antibody 1 labeled with a probe D and an alpha-fetoprotein specific antibody 2 labeled with a probe K. The preparation method of the alpha-fetoprotein specific antibody 1 labeled with the probe D includes the steps of: mixing a polymer with a nanogel solution to obtain a probe D, mixing the alpha-fetoprotein specific antibody 1 with the probe D, and obtaining the alpha-fetoprotein specific antibody 1 labeled with the probe D; the preparation method of the alpha-fetoprotein specific antibody 2 labeled with the probe K includes the steps of: mixing a polymer with an MOF solution to obtain a probe K, mixing the alpha-fetoprotein specific antibody 2 with the probe K, and obtaining the alpha-fetoprotein specific antibody 2 labeled with the probe K. The kit of the present invention effectively improves the accuracy and sensitivity of the determination.

Description

Technical Field

[0001] The invention relates to the technical field of immunoassay detection, in particular to a homogeneous chemiluminescence kit for determining the content of alpha-fetoprotein. Background Art

[0002] As an important tumor marker, alpha-fetoprotein (AFP) plays a key role in the early screening and diagnosis of various diseases such as liver cancer and gonadal embryonal tumors. Therefore, the accurate and rapid determination of alpha-fetoprotein content has extremely high medical value. Although traditional alpha-fetoprotein determination methods such as enzyme-linked immunosorbent assay (ELISA) are widely used, they have problems such as cumbersome operation, low sensitivity, and susceptibility to interference. Therefore, the development of a simple, accurate, and rapid method for the determination of alpha-fetoprotein has important practical significance. Summary of the invention

[0003] The purpose of the present invention is to provide a homogeneous chemiluminescence kit for determining the content of alpha-fetoprotein. The kit of the present invention effectively improves the accuracy and sensitivity of the determination.

[0004] In order to achieve the above-mentioned object and other related objects, the present invention provides the following technical solutions:

[0005] A homogeneous chemiluminescence kit for measuring alpha-fetoprotein content, comprising an alpha-fetoprotein specific antibody 1 labeled with a probe D and an alpha-fetoprotein specific antibody 2 labeled with a probe K,

[0006] The preparation method of the alpha-fetoprotein specific antibody 1 labeled with probe D comprises the following steps:

[0007] The polymer and the nanogel solution are mixed to obtain a probe D, and the alpha-fetoprotein specific antibody 1 and the probe D are mixed to obtain the alpha-fetoprotein specific antibody 1 labeled with the probe D;

[0008] The preparation method of the probe K-labeled alpha-fetoprotein-specific antibody 2 comprises the following steps:

[0009] The polymer and the MOF solution are mixed to obtain a probe K, and the alpha-fetoprotein specific antibody 2 and the probe K are mixed to obtain the alpha-fetoprotein specific antibody 2 labeled with the probe K.

[0010] Preferably, polyethyleneimine is dissolved in water, stirred, and then glutaraldehyde is added and stirred to obtain the nanogel solution.

[0011] Preferably, the polymer comprises PEG-SH, and the PEG-SH comprises 4arm-PEG-SH.

[0012] Preferably, PEG-SH is dissolved in water to obtain a polymer.

[0013] Preferably, the method for preparing the alpha-fetoprotein-specific antibody 1 labeled with probe D comprises the following steps:

[0014] The polymer and the nanogel solution are mixed at a volume ratio of 1:10 to 50 to obtain the probe D.

[0015] Preferably, the method for preparing the alpha-fetoprotein-specific antibody 1 labeled with probe D comprises the following steps:

[0016] The alpha-fetoprotein specific antibody 1 and the probe D are mixed at a mass ratio of 1 to 8:1, incubated at 10 to 50° C. with shaking at 500 to 1000 rpm for 30 to 90 min, and after incubation, centrifuged at 500 to 1000 rpm for 1 to 5 min to obtain the alpha-fetoprotein specific antibody 1 labeled with the probe D.

[0017] Preferably, ZIF-8 is dissolved in water and stirred until completely dissolved to obtain the MOF solution.

[0018] Preferably, the method for preparing the probe K-labeled alpha-fetoprotein-specific antibody 2 comprises the following steps:

[0019] The polymer and the MOF solution are mixed in a volume ratio of 1:10 to 50 to obtain the probe K.

[0020] Preferably, the method for preparing the probe K-labeled alpha-fetoprotein-specific antibody 2 comprises the following steps:

[0021] The alpha-fetoprotein-specific antibody 2 and the probe K are mixed in a mass ratio of 1 to 8:1, and incubated at 10 to 50° C. with shaking at 500 to 1000 rpm for 30 to 90 min. The probe K is added again, and the ratio of the mass of the added probe K to the mass of the alpha-fetoprotein-specific antibody 2 is 0.5 to 1:1. The mixture is then allowed to stand at 10 to 50° C. in the dark for 5 to 10 min. After standing, the mixture is centrifuged at 500 to 1000 rpm for 1 to 5 min to obtain the alpha-fetoprotein-specific antibody 2 labeled with the probe K.

[0022] A homogeneous chemiluminescence kit for determining the content of alpha-fetoprotein, comprising reagent 1, reagent 2, and a sample to be tested;

[0023] Reagent 1 includes an antibody prepared by diluting antibody 1 with a buffer, and antibody 1 includes alpha-fetoprotein-specific antibody 1 labeled with probe D of the aforementioned homogeneous chemiluminescence kit for determining the content of alpha-fetoprotein;

[0024] Reagent 2 includes an antibody prepared by diluting antibody 2 with a buffer, and antibody 2 includes the AFP-specific antibody 2 labeled with probe K of the aforementioned homogeneous chemiluminescence kit for determining the AFP content;

[0025] The reaction system includes 20 to 100 μL of a sample to be tested, 20 to 100 μL of a reagent 1, and 20 to 100 μL of a reagent 2; the incubation temperature is 20 to 50° C., and the incubation time is 10 to 20 minutes.

[0026] The beneficial effects of the present invention are:

[0027] First, the present invention provides a method for determining the content of alpha-fetoprotein based on homogeneous chemiluminescence. The method uses two specific antibodies marked with chemiluminescent markers to bind to the alpha-fetoprotein in the sample to be tested to form a sandwich immune complex, and then the external excitation light is given to induce the occurrence of a chemiluminescent reaction, release a large number of photons and form a chemiluminescent signal, and the purpose of quantitatively analyzing the content of alpha-fetoprotein is achieved by detecting the intensity of the luminescent signal. Specifically, the determination method of the present invention includes the following steps:

[0028] 1. Bind the chemiluminescent markers to two specific antibodies respectively, and disperse them into a suitable storage buffer system to prepare reagent 1 and reagent 2;

[0029] 2. Mix, incubate and combine the labeled reagents 1 and 2 with the sample to be tested to form a "specific antibody 1-alpha-fetoprotein-specific antibody 2" complex;

[0030] 3. Use a chemiluminescence analyzer to provide excitation light and detect the intensity of the luminescent signal. According to the linear relationship between the signal intensity and the concentration of alpha-fetoprotein, calculate the content of alpha-fetoprotein in the sample to be tested.

[0031] Second, the present invention has many advantages over the traditional chemiluminescence method. It does not require washing and separation during the reaction process, avoiding the result deviation caused by the breakage or loss of magnetic beads during the magnetic bead washing process, greatly improving the accuracy and significantly improving the detection efficiency. In addition, the present invention does not contain substances such as biotin and streptavidin, which reduces the detection cost and is energy-saving and environmentally friendly.

[0032] Third, the present invention prepares a new structure: 1. The alpha-fetoprotein-specific antibody 1 labeled with probe D is a nanogel-antibody complex, which is formed by cross-linking polyethyleneimine and glutaraldehyde, and is modified with 4arm-PEG-SH on the surface, which is further combined with the alpha-fetoprotein-specific antibody 1. The nanogel structure provides a large surface area and multifunctional surface chemical properties, which helps to improve the labeling efficiency and stability of the antibody. 2. The alpha-fetoprotein-specific antibody 2 labeled with probe K is a MOF-antibody complex, which is based on a metal organic framework of ZIF-8, and is modified with 4arm-PEG-SH on the surface, which is further combined with the alpha-fetoprotein-specific antibody 2. The MOF structure provides a high specific surface area and a pore structure, which helps to enhance the loading capacity and labeling efficiency of the antibody.

[0033] Fourth, the mechanism in the preparation of the present invention: 1. Multifunctional surface modification: The surface modification of nanogel and MOF by PEG-SH not only improves the biocompatibility and stability of the probe, but also provides functionalized binding sites to ensure the efficient binding and uniform distribution of antibodies on the probe surface. 2. Synergistic effect: Reagent 1 and reagent 2 need to be incubated during detection. After the nanogel and MOF structure are combined with the antibody, the formed complex exhibits synergistic effect during the detection process. The combination of the flexibility of the nanogel and the rigidity of MOF provides a stable and flexible detection platform, which enhances the specific binding ability of the probe to the target antigen. 3. Enhanced signal amplification effect: Reagent 1 and reagent 2 need to be incubated during detection. The porous structure of the nanogel and MOF helps to capture and concentrate more target antigen molecules and increase the intensity of the chemiluminescent signal. In addition, the high dispersibility of the nanogel and the high specific surface area of ​​MOF further amplify the detection signal, which significantly improves the detection sensitivity. 4. Reduce background interference: PEG-SH modification further improves the accuracy and specificity of the detection by reducing nonspecific adsorption and background noise. The probe with multifunctional surface modification can stably exist in a complex sample environment, ensuring the reliability of the detection results.

[0034] In summary, the present invention provides a homogeneous chemiluminescence method for determining the content of alpha-fetoprotein, which effectively improves the accuracy and sensitivity of the determination by optimizing the reaction conditions and other means, and provides more accurate and reliable support for medical diagnosis and treatment. In short, the homogeneous chemiluminescence kit is an efficient and sensitive tool for determining the content of alpha-fetoprotein, and has broad application prospects in the fields of cancer screening, hepatitis diagnosis, etc. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Unless otherwise specified, the instruments and reagents used in the examples can be easily obtained from commercial companies. The spin column in the present invention is not a key feature affecting the final result, and commercially available spin columns can be used.

[0037] 1. Prepare HEPES buffer:

[0038] In a clean beaker, weigh 11.92g HEPES, 18g potassium fluoride, 5g BSA, 0.5ml PC300, 0.5ml Tween20, and 1.6g NaOH, add 981.73g purified water and stir to dissolve, ensuring that it is completely dissolved. Pour the dissolved buffer into a sterile bottle and seal it for later use.

[0039] 2. Centrifugal column cleaning:

[0040] 300 μl of HEPES buffer was added to the centrifugal column, followed by centrifugation at 700 rpm for 1 min. The washing was repeated 3 times to obtain the washed centrifugal column, which was stored for later use.

[0041] 3. Sample diluent

[0042] Dissolve 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, and 1.8mM KH2PO4 in ultrapure water, adjust the pH to 7.4, make up to the required volume with ultrapure water, and then add 0.1% BSA and 0.1% NaN3 to obtain a sample diluent. Dispense the prepared sample diluent into sterile bottles and store at 4°C. It can be equilibrated at room temperature before use. The aforementioned components can be purchased from Sigma-Aldrich.

[0043] 4. Chemiluminescent immunoassay analyzer is not a key feature that affects the final result. Common instruments on the market can be used, and NCA-3000 chemiluminescent immunoassay analyzer is preferred.

[0044] Example 1

[0045] The present invention provides a homogeneous chemiluminescence kit for measuring the content of alpha-fetoprotein, comprising an alpha-fetoprotein specific antibody 1 labeled with a probe D and an alpha-fetoprotein specific antibody 2 labeled with a probe K, and the preparation steps are as follows:

[0046] 1. Preparation of probe D-labeled alpha-fetoprotein specific antibody 1

[0047] (1) Dissolve 50 ml of polyethyleneimine (Sigma-Aldrich, catalog number: 408727) in ultrapure water and stir evenly. Then add 1 ml of 25% glutaraldehyde and stir at room temperature for 1 hour to obtain a nanogel solution.

[0048] (2) Dissolve 2 mg of PEG-SH (4arm-PEG-SH, Sigma-Aldrich, JKA7039) in ultrapure water. The concentration of the PEG-SH solution is 1 mg / ml.

[0049] (3) Add 2 ml of PEG-SH solution to 50 ml of nanogel solution, stir at room temperature for 2 h, centrifuge at 12000 rpm for 15 min, and resuspend in ultrapure water to obtain probe D.

[0050] (4) 1.5 mg of alpha-fetoprotein-specific antibody 1 (R&D Systems, catalog number: MAB1368) was mixed with 0.3 mg of probe D, and the mixture was incubated at 25°C with shaking at 700 rpm for 60 min.

[0051] (5) Filter

[0052] After incubation of the alpha-fetoprotein-specific antibody 1 and the probe D, the mixed solution of the alpha-fetoprotein-specific antibody 1 with the probe D was transferred to the washed centrifugal column, and then centrifuged at 700 rpm for 2 minutes.

[0053] (6) Collection, calculation and storage

[0054] After centrifugation, collect the liquid after centrifugation and weigh it. Use a spectrophotometer to measure the absorbance value at 280nm, which is recorded as A_280nm. Calculate the dilution multiple of the reagent mother solution according to the formula a=A_280nm / 0.00329. Then store it at 4°C in the dark to obtain the alpha-fetoprotein-specific antibody 1 labeled with probe D.

[0055] 2. Preparation of probe K-labeled alpha-fetoprotein-specific antibody 2

[0056] (1) Dissolve 50 ml of ZIF-8 (Sigma-Aldrich, cas59061-53-9) in 50 ml of ultrapure water and stir with a magnetic stirrer until completely dissolved to obtain a MOF solution.

[0057] (2) Dissolve 2 mg of PEG-SH (4arm-PEG-SH, Sigma-Aldrich, JKA7039) in ultrapure water. The concentration of the PEG-SH solution is 1 mg / ml.

[0058] (3) Add 2 ml of PEG-SH solution to 50 ml of MOF solution, stir at room temperature for 2 h, centrifuge at 12000 rpm for 15 min, and resuspend in ultrapure water to obtain probe K.

[0059] (4) Take 0.4 mg of alpha-fetoprotein (AFP) Antibody 2 (from HYTEST LTD., HYTEST LTD., 4F16-4A3), mix it with 0.1 mg of probe K, incubate it at 25°C with shaking at 700 rpm for 60 min, then add 0.3 mg of probe K, and let it stand at 25°C in the dark for 8 min.

[0060] (5) Filter

[0061] After incubation of the alpha-fetoprotein-specific antibody 2 and the probe K, the mixed solution of the alpha-fetoprotein-specific antibody 2 with the probe K was transferred to the washed centrifugal column, and then centrifuged at 700 rpm for 2 minutes.

[0062] (6) Collection, calculation and storage

[0063] After centrifugation, collect the liquid after centrifugation and weigh it. Use a spectrophotometer to measure the absorbance value at 280nm, which is recorded as A_280nm. Calculate the dilution multiple of the reagent mother solution according to the formula a=A_280nm / 0.00329. Then store it at 4°C in the dark to obtain the probe K-labeled alpha-fetoprotein specific antibody 2.

[0064] Example 2

[0065] In this example, working solutions of probe D-labeled alpha-fetoprotein specific antibody 1 and probe K-labeled alpha-fetoprotein specific antibody 2 were prepared respectively.

[0066] (1) Prepare the AFP-specific antibody 1 working solution labeled with probe D

[0067] The probe D-labeled alpha-fetoprotein specific antibody 1 was diluted using HEPES buffer. The dilution factor = 2*the dilution factor of the mother solution, and the probe D-labeled alpha-fetoprotein specific antibody 1 working solution was obtained.

[0068] (2) Preparation of probe K-labeled alpha-fetoprotein specific antibody 2 working solution

[0069] The probe K-labeled alpha-fetoprotein specific antibody 2 was diluted using HEPES buffer. The dilution multiple = 3*the dilution multiple of the mother solution, and the probe K-labeled alpha-fetoprotein specific antibody 2 working solution was obtained.

[0070] Example 3

[0071] In this embodiment,

[0072] 1. Prepare the calibration solution and quality control solution

[0073] (1) Prepare the AFP Calibrator Working Solution

[0074] AFP calibrator (Seebio, code: EEI0182A) was prepared using HEPES buffer, with a total of 8 concentration points. The concentrations of AFP calibrator were 0 IU / mL, 1 IU / mL, 5 IU / mL, 25 IU / mL, 50 IU / mL, 200 IU / mL, 600 IU / mL, and 1000 IU / mL.

[0075] (2) Prepare the alpha-fetoprotein control working solution

[0076] AFP control (Seebio, code: EEI0182B) was prepared using HEPES buffer. The concentrations of AFP control were 10 IU / mL and 100 IU / mL, respectively.

[0077] 2. Reagent packaging and assembly

[0078] The probe D-labeled alpha-fetoprotein specific antibody 1 working solution, the probe K-labeled alpha-fetoprotein specific antibody 2 working solution, the calibration substance working solution, and the quality control substance working solution in Examples 2 to 3 are packaged separately. 12 ml of the probe D-labeled alpha-fetoprotein specific antibody 1 working solution and 12 ml of the probe K-labeled alpha-fetoprotein specific antibody 2 working solution are respectively placed in corresponding bottles; finally, 1.0 ml of the calibration substance working solution / quality control substance working solution are respectively placed in special small bottles. After the packaging is completed, these reagents are stored in a low temperature environment of 2-8°C to maintain their stability and activity.

[0079] Example 4

[0080] In this embodiment, a homogeneous chemiluminescence kit detection process for determining the content of alpha-fetoprotein is provided.

[0081] Reagent 1: dilute the alpha-fetoprotein-specific antibody 1 labeled with probe D with 50 mmol / L HEPES buffer to obtain reagent 1.

[0082] Reagent 2: Dilute the alpha-fetoprotein-specific antibody 2 labeled with probe K with 50 mmol / L HEPES buffer to obtain reagent 2.

[0083] Reaction system: 30 μL of sample to be tested, 80 μL of reagent 1, 80 μL of reagent 2; incubation temperature 37°C, incubation time 15 min.

[0084] Samples to be tested: Serum samples: Collect venous blood into blood collection tubes and leave at room temperature. Centrifuge, separate the serum part, and store at 2-8°C. Serum samples can be stable for 7 days at 2-8°C and for 1 month at -20°C, but avoid repeated freezing and thawing more than 2 times. Do not use contaminated samples.

[0085] Detection system: The excitation light wavelength is 320nm, and the detection wavelengths are 665nm and 620nm.

[0086] Detect samples of known concentration, calculate the luminescence intensity ratio of the reaction system at wavelengths of 665nm and 620nm as the signal value, and establish a "sample concentration-signal value" standard curve.

[0087] The sample to be tested is detected to obtain the luminescence intensity ratio of the sample to be tested at wavelengths of 665nm and 620nm, and the content of alpha-fetoprotein in the sample to be tested is calculated by comparing with the above standard curve.

[0088] Example 5

[0089] In this embodiment,

[0090] 1. Homogeneous chemiluminescent immunoassay for detection of alpha-fetoprotein antigen

[0091] (1) Analysis process

[0092] First, the alpha-fetoprotein antigen (Seebio, code: EKY0032E) was configured into a concentration gradient of 1-1000 IU / mL using a homemade sample diluent. Next, the test was performed on a chemiluminescent immunoassay analyzer, and the instrument automatically aspirated 30uL of each alpha-fetoprotein antigen solution, 80uL of probe D-labeled alpha-fetoprotein specific antibody 1 working solution (Example 2), and 80uL of probe K-labeled alpha-fetoprotein specific antibody 2 working solution (Example 2), and incubated at 37°C for 15 minutes to form a probe D-antigen-probe K-labeled alpha-fetoprotein antibody-antigen complex. After the incubation, the instrument automatically aspirated the reaction mixture into the chemiluminescent flow detection pool to perform chemiluminescent measurement of the immune complex. The chemiluminescent signal is proportional to the concentration of the alpha-fetoprotein antigen.

[0093] (2) Data processing

[0094] The calibration curve was obtained by performing four-parameter regression fitting with the concentration of the alpha-fetoprotein standard solution as the horizontal axis and the corresponding luminescence value as the vertical axis.

[0095] (3) Analytical performance research

[0096] According to the standard method, the linear range, blank limit, detection limit, quantification limit, precision and accuracy were studied.

[0097] 2. Statistical Analysis

[0098] SPSS23.0 statistical software was used to analyze the results. Deming regression analysis was used for correlation. P < 0.05 was considered statistically significant.

[0099] 3. Results

[0100] (1) Calibration

[0101] Traceability: The calibrator is traceable to the international standard: Alpha-fetoprotein (AFP) National Reference Material (No. 1804).

[0102] New batches of reagents must be calibrated. Use the matching reagent kit and calibrator to perform calibration tests. Before starting the calibration of the reagent kit, the master calibration curve information in the reagent kit's 2D barcode and the calibrator assignment information in the calibrator's 2D barcode should be scanned and imported into the system.

[0103] (2) Linear range of alpha-fetoprotein

[0104] The test was conducted in accordance with industry standards, using a low concentration sample close to the lower limit of the linear range to dilute a high concentration sample close to the upper limit of the linear range. Within the linear range [1.0 IU / mL-1000 IU / mL], a linear relationship was found. The results are shown in Table 1.

[0105] Table 1 Linear range of alpha-fetoprotein detection

[0106]

[0107]

[0108] (3) Limit of quantification of alpha-fetoprotein

[0109] The limit of quantification (LOQ) of alpha-fetoprotein was established according to industry standards, and multiple samples at low concentration levels were measured repeatedly. The concentration corresponding to the precision CV value of the repeated measurements of the sample with the lowest analyte concentration of ≤15% was the limit of quantification, which was 1.2 IU / mL. The results are shown in Table 2.

[0110] Table 2 AFP quantification limit verification results

[0111]

[0112] (4) AFP blank limit

[0113] The blank limit (LoB) of alpha-fetoprotein was established according to the industry standard. The blank samples were tested 60 times, one batch per day, and each batch was tested 12 times for a total of 5 days, and the concentration values ​​were recorded. According to the recommendations of the International Organization for Standardization (ISO), the type I error and type II error levels were set to 5% (α = β = 5%). If the blank value is normally distributed, the blank limit is calculated according to the following formula: LoB = μB + 1.645σB (where μB and σB are the mean and standard deviation of the blank sample test values, respectively). If the blank value is non-normally distributed, the value of the 95th percentile (PctB) is estimated by a non-parametric method, that is, LoB = PctB 100-α, that is, LoB = the result at the position [NB (ρ / 100) + 0.5] = the result at the position [0.95 × NB + 0.5]. Note: In the formula, NB is the number of repeated tests, and ρ is the percentile. The experimental measurement results are shown in Table 3.

[0114] Table 3 AFP blank limit verification results

[0115] Mean M Standard Deviation SD Skewness Kurtosis α LoB Blank Sample 0.152 0.272 -0.2 -0.30 5％ 0.6

[0116] The skewness coefficient of the data distribution is -0.2; the kurtosis coefficient is -0.30; both coefficients are less than 1, so it can be considered that the concentration distribution of the blank sample is close to the normal distribution. The W test further determines that the data conforms to the normal distribution. Therefore, the blank limit of the vWF kit is: LoB = μB + 1.645σB = 0.6IU / mL.

[0117] (5) Detection limit of alpha-fetoprotein

[0118] The detection limit of 1.0 IU / mL was verified as follows. 12 samples near the detection limit were prepared and tested for 5 rounds. The test results are shown in Table 4, which meet the standard requirements.

[0119] Table 4 Detection limit verification results of alpha-fetoprotein

[0120]

[0121]

[0122]

[0123] (6) AFP accuracy

[0124] The national standard or traceable working calibrator was prepared into two samples of different concentrations for testing. The relative deviation of the measurement results was within the range of ±10.0%. The results are shown in Table 5.

[0125] Table 5 AFP accuracy verification results

[0126]

[0127]

[0128] (7) AFP Precision

[0129] According to the requirements of the health industry standard WST 492-2016, precision verification was carried out. The results are shown in Tables 6 and 7. The total coefficient of variation (CV) of the level 1 laboratory was 3.02%, and that of the level 2 laboratory was 3.35%.

[0130] Table 6 AFP level 1 concentration precision verification results

[0131]

[0132] Table 7 AFP level 2 concentration precision verification results

[0133]

[0134]

[0135] Example 6

[0136] The present invention provides a homogeneous chemiluminescent kit for determining the content of alpha-fetoprotein, which is different from Example 1. When preparing the alpha-fetoprotein-specific antibody 1 labeled with probe D,

[0137] (3) 1 ml of PEG-SH solution was added to 50 ml of nanogel solution, and the mixture was stirred at room temperature for 2 h. After stirring, the mixture was centrifuged at 12,000 rpm for 15 min. After centrifugation, the mixture was resuspended in ultrapure water to obtain probe D.

[0138] The rest of the contents are the same as those in Example 1.

[0139] Example 7

[0140] The present invention provides a homogeneous chemiluminescent kit for determining the content of alpha-fetoprotein, which is different from Example 1, in that when preparing the alpha-fetoprotein-specific antibody 2 labeled with probe K,

[0141] (3) Add 1 ml of PEG-SH solution to 50 ml of MOF solution, stir at room temperature for 2 h, centrifuge at 12000 rpm for 15 min, and resuspend in ultrapure water to obtain probe K.

[0142] The rest of the contents are the same as those in Example 1.

[0143] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A homogeneous chemiluminescence kit for determining the content of alpha-fetoprotein, characterized in that: The method comprises an alpha-fetoprotein-specific antibody 1 labeled with a probe D and an alpha-fetoprotein-specific antibody 2 labeled with a probe K, The preparation method of the alpha-fetoprotein specific antibody 1 labeled with probe D comprises the following steps: Dissolving polyethyleneimine in water, stirring, then adding glutaraldehyde, stirring, to obtain a nanogel solution; The polymer and nanogel solution are mixed to obtain probe D; The alpha-fetoprotein specific antibody 1 and the probe D are mixed at a mass ratio of 1 to 8:1, and incubated at 10 to 50°C with shaking at 500 to 1000 rpm for 30 to 90 min. After incubation, the mixture is centrifuged at 500 to 1000 rpm for 1 to 5 min to obtain the alpha-fetoprotein specific antibody 1 labeled with the probe D; The preparation method of the probe K-labeled alpha-fetoprotein-specific antibody 2 comprises the following steps: Dissolve ZIF-8 in water and stir until completely dissolved to obtain a MOF solution; Mix the polymer and MOF solution to obtain probe K; The alpha-fetoprotein-specific antibody 2 and the probe K are mixed at a mass ratio of 1 to 8:1, and incubated at 10 to 50°C with shaking at 500 to 1000 rpm for 30 to 90 min, and the probe K is added again, at which time the ratio of the mass of the added probe K to the mass of the alpha-fetoprotein-specific antibody 2 is 0.5 to 1:1, and then allowed to stand at 10 to 50°C in the dark for 5 to 10 min, and after standing, centrifuged at 500 to 1000 rpm for 1 to 5 min to obtain the alpha-fetoprotein-specific antibody 2 labeled with the probe K; Wherein, the polymer is 4arm-PEG-SH.

2. The homogeneous chemiluminescence kit for determining the content of alpha-fetoprotein according to claim 1, characterized in that: 4arm-PEG-SH was dissolved in water to obtain a polymer.

3. The homogeneous chemiluminescence kit for determining the content of alpha-fetoprotein according to claim 1, characterized in that: The preparation method of the alpha-fetoprotein specific antibody 1 labeled with probe D comprises the following steps: The polymer and the nanogel solution are mixed at a volume ratio of 1:10-50 to obtain the probe D.

4. The homogeneous chemiluminescence kit for determining the content of alpha-fetoprotein according to claim 1, characterized in that: The preparation method of the probe K-labeled alpha-fetoprotein-specific antibody 2 comprises the following steps: The polymer and the MOF solution are mixed in a volume ratio of 1:10 to 50 to obtain the probe K.

5. A homogeneous chemiluminescence kit for determining the content of alpha-fetoprotein, characterized in that: It includes reagent 1, reagent 2, and a sample to be tested; Reagent 1 comprises an antibody prepared by diluting antibody 1 with a buffer, and antibody 1 comprises an alpha-fetoprotein specific antibody 1 labeled with probe D of the homogeneous chemiluminescence kit for determining the alpha-fetoprotein content according to any one of claims 1 to 4; Reagent 2 comprises an antibody prepared by diluting antibody 2 with a buffer, and antibody 2 comprises an alpha-fetoprotein-specific antibody 2 labeled with probe K of the homogeneous chemiluminescence kit for determining the alpha-fetoprotein content according to any one of claims 1 to 4; The reaction system includes 20~100μL of a sample to be tested, 20~100μL of reagent 1, and 20~100μL of reagent 2; the incubation temperature is 20~50°C, and the incubation time is 10~20min.

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