Homogeneous chemiluminescence kit for determining content of cancer antigen 72-4

Abstract

The invention belongs to the technical field of immunoassay detection, and particularly relates to a homogeneous chemiluminescence kit for determining the content of cancer antigen 72-4. The kit comprises a reagent 1 and a reagent 2, wherein the reagent 1 comprises a functional structure, a cancer antigen 72-4 specific antibody 1 and DOTA-Eu; the reagent 2 comprises a functional structure, a cancer antigen 72-4 specific antibody 2 and DOTA-Eu. The preparation method of the functional structure comprises the following steps: uniformly mixing trialdehyde phloroglucinol, p-phenylenediamine, PVP, DMAc and acetic acid to obtain a solution; heating the solution to obtain a precipitate; washing the precipitate, and performing vacuum drying to obtain dry powder; and resuspending the dry powder to form a dispersion liquid to obtain the functional structure. The kit disclosed by the invention is high in sensitivity, simple and convenient to operate and high in accuracy.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay detection technology, and more specifically, relates to a homogeneous chemiluminescence reagent kit for determining the content of cancer antigen 72-4. Background Technology

[0002] Cancer antigen 72-4 (CA72-4) is an important tumor marker, playing a significant role in the early detection, diagnosis, disease monitoring, and efficacy evaluation of various cancers, including gastric cancer, ovarian cancer, colorectal cancer, pancreatic cancer, gallbladder cancer, breast cancer, and cervical cancer. Currently, methods for measuring CA72-4 mainly include immunoassay and electrochemiluminescence immunoassay. However, these methods still require improvement in terms of sensitivity and ease of operation. Therefore, developing a highly sensitive and easy-to-operate method for measuring CA72-4 has significant clinical application value. Summary of the Invention

[0003] (a) Technical problems to be solved In view of the above problems, the present invention aims to provide a homogeneous chemiluminescence reagent kit for determining the content of cancer antigen 72-4.

[0004] The cancer antigen 72-4 assay kit based on homogeneous chemiluminescence of the present invention has the following advantages: High sensitivity: By optimizing the luminescent label and reaction conditions, this method can achieve trace detection of cancer antigen 72-4, which is of great significance for early cancer screening.

[0005] Simple to operate: The homogeneous reaction system does not require complicated separation and washing steps, which simplifies the operation process and improves detection efficiency.

[0006] High accuracy: Through the specific binding of antigen-antibody reaction and the precise detection of chemiluminescence technology, this method can accurately determine the content of cancer antigen 72-4 in the sample to be tested.

[0007] (II) Technical Solution To achieve the above and other related objectives, the present invention provides the following technical solutions: A homogeneous chemiluminescence reagent kit for determining the content of cancer antigen 72-4 includes reagent 1 and reagent 2. Reagent 1 includes a functional structure, cancer antigen 72-4 specific antibody 1, and DOTA-Eu; reagent 2 includes a functional structure, cancer antigen 72-4 specific antibody 2, and DOTA-Eu.

[0008] Detailed or preferred methods for preparing functional structures include the following steps: Trialdehyde resorcinol, p-phenylenediamine, PVP, DMAc, and acetic acid were mixed thoroughly to obtain a solution; the solution was then heated to obtain a precipitate. The precipitate was washed and vacuum dried to obtain a dry powder; the dry powder was resuspended to form a dispersion to obtain the functional structure.

[0009] Detailed or preferred methods include heating the solution at 80-120°C in a sealed container for 20-26 hours.

[0010] The detailed or preferred method for preparing reagent 1 includes the following steps: Add EDC·HCl and NHS to the functional structure, shake, then add cancer antigen 72-4 specific antibody 1 and DOTA-Eu, and react; wash and block after reaction; incubate, wash, and finally disperse in PBS buffer to obtain reagent 1.

[0011] The detailed or preferred method for preparing reagent 2 includes the following steps: Add EDC·HCl and NHS to the functional structure, shake, then add cancer antigen 72-4 specific antibody 2 and DOTA-Eu, and react; wash and block after reaction; incubate, wash, and finally disperse in PBS buffer to obtain reagent 2.

[0012] For detailed or preferred methods, closure is achieved by adding BSA.

[0013] For detailed or preferred incubation, the incubation period is 30-40 minutes at room temperature.

[0014] Detailed or preferred, cancer antigen 72-4 specific antibody 1 includes mouse anti-human CA72-4 monoclonal antibody B72.3.

[0015] Detailed or preferred, cancer antigen 72-4 specific antibody 2 includes mouse anti-human CA72-4 monoclonal antibody CC49.

[0016] In detail or preferably, before the kit is used for testing, the sample to be tested, reagent 1, and reagent 2 are incubated together to obtain a reaction system at a temperature of 35-40℃ for 10-20 minutes.

[0017] (iii) Beneficial technical effects Advantages of this invention: This invention provides a homogeneous chemiluminescence method for determining the content of cancer antigen 72-4. This method is based on the principle of homogeneous chemiluminescence and achieves rapid and highly sensitive determination of cancer antigen 72-4 through the immune reaction between antibodies labeled with luminescent substances and cancer antigen 72-4.

[0018] Specifically, the method includes the following steps: First, two cancer antigen 72-4 specific antibodies are bound to luminescent substances to form stable luminescent labeled antibodies; then, under appropriate reaction conditions, the luminescent labeled antibodies specifically bind to cancer antigen 72-4 to form a sandwich luminescent complex; finally, the luminescence intensity of the luminescent complex is detected by a chemiluminescence detector, and the content of cancer antigen 72-4 in the sample is determined based on the relationship between the luminescence intensity and the concentration of cancer antigen 72-4.

[0019] The key to this invention lies in the selection of specific antibodies and the optimization of immune reaction conditions. By using highly sensitive luminescent materials and specific antibodies, and conducting the immune reaction under appropriate conditions, rapid and highly sensitive determination of cancer antigen 72-4 was achieved.

[0020] This invention constructs a highly sensitive analytical platform for cancer antigen 72-4 based on a covalent organic framework (COF), suitable for homogeneous chemiluminescence detection. It overcomes the limitations of traditional sandwich immunoassays that require solid-phase carriers and washing operations, and realizes a complete immunoassay process that completes specific recognition, signal amplification and target quantification in a homogeneous system.

[0021] The COF material used in this invention is specifically formed by the condensation of trialdehyde phloroglucinol and p-phenylenediamine in the presence of N,N-dimethylacetamide and glacial acetic acid. This structure possesses good crystal order and a two-dimensional lamellar configuration, and its framework is rich in phenolic hydroxyl and secondary amine groups that have not fully participated in the cross-linking reaction. These functional groups provide the reaction sites required for subsequent coupling reactions.

[0022] To enhance the stability of aqueous dispersion, PVP was added as a surface stabilizer during the synthesis process, enabling the resulting COF microparticles to be stably resuspended in PBS buffer and maintain good homogeneity under immunoassay conditions. Due to its high specific surface area and abundant surface active sites, this COF material can serve as a covalent carrier for antibodies and luminescent ligands.

[0023] In the reagent preparation process, this invention utilizes the native phenolic hydroxyl groups and a small amount of residual primary and secondary amine groups in the COF structure to generate intermediate esters and amide intermediates through an activation reaction in aqueous phase EDC·HCl and NHS, which then react with cancer antigen 72. 4. Specific antibodies (B72.3 and CC49) and DOTA-Eu were covalently linked to form reagent 1 and reagent 2, respectively, which are bifunctional luminescent probes.

[0024] During the testing process, if cancer antigen 72 is present in the sample... 4. It can bind to the two monoclonal antibodies modified in reagents 1 and 2 respectively, forming a double-antibody sandwich structure. Due to the luminescent group Eu... 3+Fixed on the COF surface, when the sandwich structure is formed, the two COF particles approach each other specifically, which can (1) induce COF sheet aggregation leading to enhanced luminescence: the aggregation conformation after cancer antigen bridging makes the luminescent group in a dense arrangement, increasing the probability of excitation light absorption and radiative transition; (2) the low polarity microdomain formed by the proximity of the two COFs can reduce the hydration quenching effect around the Eu ligand; (3) by monitoring the ratio of the two emission wavelengths at 665 nm and 620 nm, the specific signal and the background luminescence of the free probe can be effectively distinguished, and the high correlation between the real luminescence response and the concentration of cancer antigen can be achieved. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0026] It should be noted that some raw material information is as follows: N,N-Dimethylacetamide: DMAc Acetic acid: glacial acetic acid Polyvinylpyrrolidone (PVP): Select polyvinylpyrrolidone K30 Azide-treated Eu complex: DOTA-Eu, available from Xi'an Qiyue Biotechnology, 98% purity. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: EDC·HCl N-hydroxysuccinimide: NHS Cancer antigen 72-4 specific antibody 1: Mouse anti-human CA72-4 monoclonal antibody B72.3, recognizing the TAG-72 main chain epitope. Cancer antigen 72-4 specific antibody 2: Mouse anti-human CA72-4 monoclonal antibody CC49, which recognizes a TAG-72 branched epitope that is different from cancer antigen 72-4 specific antibody 1. Phosphate buffer: PBS buffer, pH=7.4 Bovine serum albumin (BSA) Example 1 The specific steps for preparing the functional structure are as follows: Add 63 mg of trialdehyde phloroglucinol, 32 mg of p-phenylenediamine, 30 mg of PVP, 10 mL of DMAc, and 0.5 mL of acetic acid to a reaction flask, sonicate for 30 minutes, mix well, and obtain a solution; heat the solution at 100°C in a sealed container for 24 hours to obtain a precipitate.

[0027] The precipitate was washed three times in sequence with DMAc, ethanol, and water, and then vacuum dried to obtain a dry powder. The powder was then resuspended in 0.1% PVP PBS to form a dispersion, which is the functional structure.

[0028] Example 2 The specific steps for preparing reagent 1 are as follows: Take 1 mL of the functional structure from Example 1 with a concentration of 2 mg / mL, add 5 mg EDC·HCl and 5 mg NHS, gently shake for 30 min, then add 10 μg of cancer antigen 72-4 specific antibody 1 and 5 μg of DOTA-Eu, and react for 4 h; after the reaction, wash three times with PBS, add 1% BSA for blocking; incubate at room temperature for 30 min, wash three times, and finally disperse in PBS buffer containing 0.1% BSA to obtain reagent 1.

[0029] Example 3 The specific steps for preparing reagent 2 are as follows: Take 1 mL of the functional structure from Example 1 with a concentration of 2 mg / mL, add 5 mg EDC·HCl and 5 mg NHS, gently shake for 30 min, then add 10 μg of cancer antigen 72-4 specific antibody 2 and 5 μg of DOTA-Eu, and react for 4 h; after the reaction, wash three times with PBS, add 1% BSA for blocking; incubate at room temperature for 30 min, wash three times, and finally disperse in PBS buffer containing 0.1% BSA to obtain reagent 2.

[0030] Example 4 A homogeneous chemiluminescence reagent kit for determining the content of cancer antigen 72-4, the specific steps of which are as follows: Preparation of the reaction system: 70 μL of the sample to be tested, 50 μL of reagent 1 from Example 2, and 50 μL of reagent 2 from Example 3; incubation temperature 37℃, incubation time 15 min.

[0031] The sample to be tested is a cancer antigen 72-4 antigen standard solution.

[0032] Detection system: excitation wavelength is 320nm, detection wavelength is 665nm and 620nm.

[0033] For samples with known concentrations, the ratio of luminescence intensity at wavelengths of 665 nm and 620 nm in the reaction system is calculated as the signal value, and a standard curve of sample concentration-signal value is established.

[0034] The sample to be tested was analyzed to obtain the ratio of the luminescence intensity of the sample at wavelengths of 665 nm and 620 nm. By comparing with the above standard curve, the content of cancer antigen 72-4 in the sample to be tested was calculated.

[0035] Example 5 1. Specific experiment: 1.1 Establishing the experimental protocol for blank limits The blank limit was evaluated with reference to "YY / T1789.3-2022 Performance Evaluation Methods for In Vitro Diagnostic Testing Systems Part 3: Limit of Detection and Limit of Quantification". The default α=β=0.05. The design scheme was as follows: 1 instrument (fully automated chemiluminescence immunoassay analyzer (model: NCA-3000)); test sample, 3 test days, 5 blank samples; each sample was measured 4 times.

[0036] Statistical analysis method: ① Statistical outliers are identified by Grubbs' test. One outlier is allowed to be removed; otherwise, the test is repeated. ② Perform statistical analysis on the test results to determine whether the test results follow a normal distribution. ③ If the data is normally distributed, the blank limit is calculated using parametric analysis, according to formulas (1) and (2).

[0037] …………………………………………(1) ……………………………………………(2) In the formula: The mean of the test results for the blank sample; The multiplier of the 95th percentile of a normal distribution; : Standard deviation of the test results for blank samples; The number of tests conducted on the blank samples; The number of blank samples; Estimated The degree of freedom.

[0038] If the data is not normally distributed, nonparametric analysis can be used to calculate the blank limit, and the data should be sorted in ascending order: X （1） X （2） , ..., X （i） Estimate based on the arranged data p The value at the percentile position; if this value is not an integer, linear interpolation is performed. p Percentiles are calculated using formula (3): ………………………………………………(3) In the formula: RP: Ranked Matches; N: The number of test results; P: The appropriate percentile, usually 95.

[0039] 1.2 Establishing the test protocol for the detection limit The detection limit was evaluated with reference to "YY / T1789.3-2022 Performance Evaluation Method for In Vitro Diagnostic Testing Systems Part 3: Limit of Detection and Limit of Quantification". The default α=β=0.05. The design scheme was as follows: 1 instrument (fully automated chemiluminescence immunoassay analyzer (model: NCA-3000)); test sample; 3 test days; 5 low concentration level samples; 4 repeated measurements for each sample.

[0040] Statistical analysis method: ① Statistical outliers are identified by Grubbs' test. One outlier is allowed to be removed; otherwise, the test is repeated. ② Perform statistical analysis on the test results to determine whether the test results are normally distributed, and conduct a homogeneity of variance test.

[0041] ③ If the test data of low concentration level samples are normally distributed and have homogeneous variances, the detection limit is calculated by parametric analysis. Specific method: Calculate the pooled standard deviation SDz from the test results of multiple low concentration level samples. The pooled standard deviation of multiple low concentration level samples is calculated according to equations (4) to (6).

[0042] …………………………………………………(4) ………………………………………………(5) …………………………………………………(6) In the formula: SDz: The pooled standard deviation of N low-concentration level samples; : The number of results for the i-th low-concentration level sample; : The standard deviation of the i-th low concentration level sample; The multiplier of the 95th percentile of a normal distribution; N: Number of samples at low concentration levels; L: The total number of low-concentration level sample results across all reagent batches; LN: Estimated degrees of freedom of SDz.

[0043] If the test results of low-concentration level samples are non-normally distributed, the data can be transformed into other forms (e.g., logarithmic form) before statistical analysis. If the transformed data also shows a non-normal distribution, a non-parametric analysis method is used to calculate the limit of detection (LOD). Specifically, the test results of low-concentration samples from each batch of reagents are statistically analyzed and sorted. If the percentage of results below the LOB value is less than the expected Type II error (β), then the LOD of that reagent batch is the median of the distribution of low-concentration sample test results.

[0044] 1.3 Blank Limit and Detection Limit Validation Test Protocol A simple verification was performed with reference to section 7.3 of YY / T 1789.3-2022 Performance Evaluation Methods for In Vitro Diagnostic Testing Systems Part 3: Limit of Detection and Limit of Quantification. Five low-value samples with concentrations close to the limit of detection were tested, with five tests performed on each sample. The test results were sorted by size.

[0045] Statistical analysis method: Sort the test results and count the number of test results that are below the declared blank limit value.

[0046] 2. Acceptable Standards 1.1 Validation of blank limit and detection limit When the number of detection results below the blank limit is less than or equal to 3, the blank limit and detection limit are successfully verified.

[0047] 3. Results and Statistical Analysis 3.1 Results and Analysis of Blank Limit Establishment 3.1.1 Original Results Table 1. Raw data for establishing blank limits (U / mL)

[0048] 3.1.2 Grubbs Outlier Check Data checks were performed on the data from the three batches to determine if the test results were outliers. The statistical results are shown in the table below. The test results for all three batches were not outliers.

[0049] Table 2. Blank Limit Detection Results and Outlier Check

[0050] 3.1.3 Statistical Analysis Normal distribution analysis was performed to determine whether the test results were normally distributed. The statistical results are shown in the table below. The test results of the three batches did not conform to a normal distribution.

[0051] Table 3 Results of Blank Limit Normal Distribution Analysis

[0052] Because the test results of the test samples do not conform to a normal distribution, a nonparametric method is used to estimate the LOB (Lower Bound Rank): For a given reagent batch number, the test results are sorted from low to high. The p-percentile position is estimated based on the sorted data. If this value is not an integer, linear interpolation is performed. The p-percentile is calculated using the following formula.

[0053]

[0054] In the formula: RP: Ranked Matches; N: The number of test results; p: The appropriate percentile, usually 95.

[0055] Therefore, RP = 60 × (95 / 100) + 0.5 = 57.5, LOB = X (57) +0.5×[X (58) -X (57) The LOB of the test sample was found to be 0.49 U / mL, as shown in the table below. Table 4. Results of Blank Limit Analysis

[0056] The blank limit for this test kit is 0.5 U / mL.

[0057] 3.2 Results and Analysis of Detection Limit Establishment 3.2.1 Original Results Table 5. Raw data for establishing the limit of detection (U / mL)

[0058] 3.2.2 Grubbs Outlier Check The data of the test samples were examined to determine whether the test results were outliers. The statistical results are shown in the table below. The test results of the test samples were all without outliers.

[0059] Table 6 Outlier Check of Detection Limit Detection Results

[0060] 3.2.3 Statistical Analysis Normal distribution analysis was performed to determine whether the test results were normally distributed. The statistical results are shown in the table below. The test results of the three batches did not conform to a normal distribution.

[0061] Table 7 Results of Normal Distribution Analysis of Detection Limits

[0062] Because the test results of the test samples do not conform to a normal distribution, a nonparametric method is used to estimate the LOD: the test results of the test samples are statistically analyzed and sorted. If the percentage of results below the LOB is less than the expected Type II error (β, β=0.05), then the LOD of the reagent batch is the median of the batch's test results. The LOD of the test sample is calculated to be 1.00 U / mL from the table below. Table 8 Results of Limit of Detection Analysis

[0063] After rounding to one decimal place, the detection limit of this test kit is 1.0 U / mL.

[0064] 3.4 Blank Limit and Detection Validation Table 9. Blank limit and detection limit verification data (U / mL)

[0065] 4. Experimental Conclusions The blank limit of this test kit is 0.5 U / mL; the detection limit is 1.0 U / mL. The blank limit and detection limit have been validated and meet the requirements.

[0066] Although the present invention has been illustrated and described with specific embodiments, it should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein, without departing from the spirit and scope of the present invention; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention; therefore, this means that all such substitutions and modifications that fall within the scope of the present invention are included in the appended claims.

Claims

1. A homogeneous chemiluminescence reagent kit for determining the content of cancer antigen 72-4, characterized in that, It includes reagent 1 and reagent 2. Reagent 1 includes functional structure, cancer antigen 72-4 specific antibody 1, and DOTA-Eu; reagent 2 includes functional structure, cancer antigen 72-4 specific antibody 2, and DOTA-Eu.

2. The homogeneous chemiluminescence reagent kit for determining the content of cancer antigen 72-4 according to claim 1, characterized in that, The preparation method of the functional structure includes the following steps: Trialdehyde resorcinol, p-phenylenediamine, PVP, DMAc, and acetic acid were mixed thoroughly to obtain a solution; the solution was then heated to obtain a precipitate. The precipitate was washed and vacuum dried to obtain a dry powder; the dry powder was resuspended to form a dispersion to obtain the functional structure.

3. The homogeneous chemiluminescence reagent kit for determining the content of cancer antigen 72-4 according to claim 1, characterized in that, Heating the solution involves sealing and heating the solution at 80-120°C for 20-26 hours.

4. The homogeneous chemiluminescence reagent kit for determining the content of cancer antigen 72-4 according to claim 1, characterized in that, The preparation method of reagent 1 includes the following steps: Add EDC·HCl and NHS to the functional structure, shake, then add cancer antigen 72-4 specific antibody 1 and DOTA-Eu, and react; wash and block after reaction; incubate, wash, and finally disperse in PBS buffer to obtain reagent 1.

5. The homogeneous chemiluminescence reagent kit for determining the content of cancer antigen 72-4 according to claim 1, characterized in that, The preparation method of reagent 2 includes the following steps: Add EDC·HCl and NHS to the functional structure, shake, then add cancer antigen 72-4 specific antibody 2 and DOTA-Eu, and react; wash and block after reaction; incubate, wash, and finally disperse in PBS buffer to obtain reagent 2.

6. A homogeneous chemiluminescence reagent kit for determining the content of cancer antigen 72-4 according to claim 4 or 5, characterized in that, Closure is achieved by adding BSA.

7. A homogeneous chemiluminescence reagent kit for determining the content of cancer antigen 72-4 according to claim 4 or 5, characterized in that, Incubation is performed at room temperature for 30-40 minutes.

8. The homogeneous chemiluminescence reagent kit for determining the content of cancer antigen 72-4 according to claim 1, characterized in that, Cancer antigen 72-4 specific antibody 1 includes mouse anti-human CA72-4 monoclonal antibody B72.

3.

9. A homogeneous chemiluminescence reagent kit for determining the content of cancer antigen 72-4 according to claim 1, characterized in that, Cancer antigen 72-4 specific antibody 2 includes mouse anti-human CA72-4 monoclonal antibody CC49.

10. A homogeneous chemiluminescence reagent kit for determining the content of cancer antigen 72-4 according to claim 1, characterized in that, Before testing with the kit, incubate the sample to be tested, reagent 1, and reagent 2 together to obtain a reaction system at a temperature of 35-40℃ for 10-20 minutes.

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