Electrochemiluminescence kit for rapidly detecting tumor markers

Abstract

The invention relates to the technical field of biological medicine detection, and particularly discloses an electrochemical luminescence kit for rapidly detecting tumor markers, which comprises a capture molecule, an electrochemical luminescence ECL labeled molecule, a biotin labeled detection antibody, a streptavidin fixed plate and a liquid reagent, according to the present invention, the capture molecule, the ECL labeling molecule, the biotin labeling detection antibody and the streptavidin fixation platform interact to form the novel multi-protein complex, such that each target antigen can initiate multiple signal conversion during the reaction process; theoretically, the tetramolecular structure can obviously improve the signal transmission efficiency, so that the low-abundance tumor marker can be quickly and accurately detected, and the high-specificity recognition of the target tumor marker is realized by capturing molecules and detecting antibodies. The high affinity between biotin and streptavidin is utilized, so that non-specific binding is greatly reduced, background signals are reduced, and the accuracy and reliability of detection are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical detection, and in particular relates to an electrochemiluminescence kit for rapid detection of tumor markers. Background Art

[0002] At present, the detection of tumor markers is of great significance in the early diagnosis of tumors and the monitoring of therapeutic effects, but traditional methods often have problems such as insufficient sensitivity, cumbersome operation or long detection time. Electrochemiluminescence detection technology has been widely used in the field of immunoassay due to its wide dynamic range and high signal-to-noise ratio. Based on the ECL detection technology, the present invention realizes dual signal amplification by constructing a new multi-molecular complex, and realizes the stable construction of the detection platform with the help of the biotin-streptavidin system.

[0003] In response to this, the inventors proposed an electrochemiluminescence kit for rapid detection of tumor markers to solve the above problems. Summary of the invention

[0004] The purpose of the present invention is to provide an electrochemiluminescence kit for rapid detection of tumor markers to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] Electrochemiluminescence kit for rapid detection of tumor markers, including:

[0007] The kit includes the following reagents:

[0008] A capture molecule, used to specifically bind to the tumor marker in the sample to be tested to form a complex A;

[0009] An electrochemiluminescent (ECL) labeling molecule is used to perform a labeling reaction with the capture molecule in the complex A or directly with the tumor marker to be detected to generate a complex B with an ECL signal;

[0010] A biotin-labeled detection antibody is used to bind to the complex B to form a biotin bridge to obtain a complex C;

[0011] A streptavidin fixed plate is used to capture and fix the complex C to form a stable complex D, thereby facilitating detection of the amplified luminescent signal under electrochemical excitation;

[0012] Liquid reagents include buffer, blocking solution, washing solution and readout buffer for adjusting the reaction environment. The liquid reagents are used to ensure the specificity, stability and repeatability of the above reaction process.

[0013] Preferably, the capture molecule is a monoclonal antibody against a tumor marker fixed on the surface of a plate, which is used to specifically capture the tumor marker to be detected to obtain complex A.

[0014] Preferably, the electrochemiluminescent labeling molecule is Sulfo-TAG or other labels that can generate electrochemiluminescent signals, which are used to combine with the capture molecule or directly with the tumor marker to obtain complex B and achieve signal amplification.

[0015] Preferably, the biotin-labeled detection antibody is a secondary antibody against a tumor marker, which is used to bind to complex B after biotinylation to form complex C, and is further fixed on the plate through the high affinity binding of biotin and streptavidin.

[0016] Preferably, the streptavidin fixed plate is a solid phase material pre-coated with streptavidin, which is used to efficiently bind to the biotin in the complex C to obtain the complex D, thereby achieving signal transmission and amplification.

[0017] Preferably, the buffer system comprises:

[0018] An antigen buffer, used to allow the capture molecule to fully react with the tumor marker to obtain complex A;

[0019] Washing solution, used to remove unbound or non-specifically bound components in each reaction step;

[0020] Blocking solution, used to block nonspecific binding sites;

[0021] The readout buffer is used to provide an optimal luminescence environment under electrochemical excitation conditions, so as to accurately read the luminescence signal of the complex D.

[0022] Preferably, the kit also includes preset positive controls and negative controls for calibrating and verifying the accuracy and stability of the luminescent signal during the detection process, thereby ensuring the reliability of the final detection result.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention uses capture molecules to interact with ECL labeled molecules, biotin labeled detection antibodies and streptavidin fixed platforms to form a new multi-protein complex, so that each target antigen can trigger multiple signal conversions during the reaction process. In theory, this four-molecule structure can significantly improve the signal transmission efficiency, so that low-abundance tumor markers can also be detected quickly and accurately.

[0025] (2) The present invention achieves highly specific recognition of target tumor markers through carefully selected capture molecules and detection antibodies. The high affinity between biotin and streptavidin is utilized to greatly reduce nonspecific binding, thereby reducing background signals and improving the accuracy and reliability of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The present invention is a block diagram of the electrochemiluminescence kit for rapid detection of tumor markers. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Embodiment 1:

[0029] See also Figure 1 As shown, carcinoembryonic antigen (CEA) detection based on ECL technology:

[0030] Sample collection and processing:

[0031] Sample source:

[0032] Peripheral blood samples were collected from healthy controls (n=50) and patients with clinically suspected tumors (n=30), and all subjects signed informed consent.

[0033] Pre-treatment conditions:

[0034] After blood collection, serum was separated by centrifugation and stored at -70°C. Before testing, serum samples were taken out and thawed at room temperature, gently mixed and used in the experiment.

[0035] Reagents and equipment configuration

[0036] Capture molecules:

[0037] A high-affinity monoclonal antibody against CEA was used and fixed on an ECL plate pre-coated with streptavidin by chemical cross-linking.

[0038] Fixed concentration: optimized to 100 ng / cm 2 , ensuring sufficient capture efficiency.

[0039] ECL marker molecules:

[0040] Another anti-CEA mAb was labeled with Sulfo-TAG.

[0041] Marking conditions:

[0042] The antibody and Sulfo-TAG were incubated at room temperature in the dark for 1 hour with the aid of 3mmol / L NHS-PEG4-Biotin at a molar ratio of 1:15, and then purified using a Zeba column. The concentration was adjusted to 50ng / mL after labeling.

[0043] Biotinylated detection antibody:

[0044] The detection antibody against CEA was selected, biotinylated, and the concentration was optimized to 0.0625 μg / μL, which was verified to have the highest signal-to-noise ratio.

[0045] Streptavidin immobilized plate:

[0046] The ECL plate coated with streptavidin was selected, and its surface uniformity and binding efficiency met the international testing standards.

[0047] Buffer system:

[0048] Antigen buffer: 1× PBS containing 5% fetal bovine serum (FBS), used to optimize immune reaction conditions;

[0049] Washing solution: 1× PBS containing 0.25% Tween-20;

[0050] Blocking solution: 3% Blocker A (designed specifically for ECL platform);

[0051] Read buffer: 2×Read Buffer (recommended by the instrument manufacturer) to ensure the best electrochemical reaction environment.

[0052] Detection process capture reaction:

[0053] The anti-CEA monoclonal antibody was fixed on the streptavidin plate, and 50 μL of antigen buffer was added to allow the capture molecules to be fully adsorbed. The plate was incubated at room temperature for 1 hour, and then non-specific sites were blocked with blocking solution.

[0054] Sample incubation:

[0055] Take 30 μL of the serum sample to be tested, add the pre-prepared 50 ng / mL labeled Sulfo-TAG anti-CEA antibody and biotin-labeled detection antibody (5 μL / well, concentration 0.0625 μg / μL), mix and add to the capture plate, incubate at room temperature with horizontal shaking at 450 rpm for 2 hours to ensure the formation of a four-molecular complex (complex D) consisting of the capture antibody, labeled antibody and detection antibody.

[0056] Washing and signal reading:

[0057] After the incubation, wash the cells three times with washing buffer to remove nonspecific binding substances.

[0058] 150 μL of read buffer was added and the signal counts were read by MESO QuickPlex SQ 120 electrochemiluminescence instrument.

[0059] Data processing and quality control:

[0060] The CEA concentration of the sample was calculated using the pre-set positive (standard containing known CEA concentration) and negative control calibration signals.

[0061] Fifty healthy controls and 30 patient samples were tested repeatedly, and the intra-batch coefficient of variation (CV) was 3.5%-5.0%, and the inter-batch CV was controlled at 6.0%-7.0%, proving that the system has good repeatability and stability.

[0062] The luminescence counts of each well were automatically recorded by an electrochemiluminescence instrument, and the sample concentration was obtained by standard curve fitting (using a multi-point method with a concentration range of 0.1–100 ng / mL).

[0063] As can be seen from the above, the detection limit reaches 0.1 ng / mL, and the signal-to-noise ratio is significantly improved (S / N can reach more than 400);

[0064] ROC curve analysis showed that the system had a sensitivity of 82%, a specificity of 98% and an AUC of 0.92 for CEA detection in clinical tumor patients;

[0065] The system operation time from sample addition to result acquisition is only about 3 hours, which greatly improves clinical testing efficiency.

[0066] As can be seen from the above, the capture molecule interacts with the ECL labeled molecule, the biotin labeled detection antibody and the streptavidin fixed platform to form a new multi-protein complex, so that each target antigen can trigger multiple signal conversions during the reaction. In theory, this four-molecule structure can significantly improve the signal transmission efficiency, so that low-abundance tumor markers can also be detected quickly and accurately.

[0067] Embodiment 2:

[0068] Carbohydrate antigen 125 (CA125) detection based on ECL technology:

[0069] Sample collection and processing Sample source:

[0070] Peripheral blood specimens were collected from healthy women (n=40) and patients clinically diagnosed with ovarian tumors (n=25).

[0071] Pre-treatment conditions:

[0072] After separation, the serum was stored at -70°C and slowly thawed at 4°C before use to ensure that the activity of the sample was not affected.

[0073] Reagents and equipment configuration Capture molecules:

[0074] A highly specific monoclonal antibody against CA125 was used and fixed on an ECL solid phase plate pre-coated with streptavidin.

[0075] Fixed concentration: optimized to 80 ng / cm 2 , ensuring capture efficiency and antigen affinity.

[0076] ECL marker molecules:

[0077] Another anti-CA125 monoclonal antibody was labeled with Sulfo-TAG, and the reaction conditions were similar to those in Example 1.

[0078] Labeling conditions: 1:12 molar ratio, 1 hour incubation time, and 40 ng / mL concentration after labeling.

[0079] Biotinylated detection antibody:

[0080] The detection antibody for CA125 was biotinylated and its concentration was optimized to 0.05 μg / μL. The gradient experiment determined that the optimal signal condition was an addition amount of 5 μL / well.

[0081] Streptavidin immobilized plate:

[0082] A streptavidin pre-coated plate similar to that in Example 1 was used to ensure efficient binding with the biotin-labeled antibody.

[0083] Buffer system:

[0084] Antigen buffer: 1× PBS containing 5% BSA;

[0085] Washing solution: 1× PBS containing 0.25% Tween-20;

[0086] Blocking solution: 3% Blocker A;

[0087] Read buffer: 2×Read Buffer (to ensure the best ECL signal environment).

[0088] Detection process capture reaction:

[0089] After the anti-CA125 monoclonal antibody was fixed on the plate, non-specific sites were blocked with blocking solution and incubated at room temperature for 1 hour.

[0090] Sample incubation:

[0091] In a 96-well plate, 35 μL of pre-prepared antigen buffer, 15 μL of 1× Tris-HCl (pH 7.4 buffer), and 25 μL of serum to be tested were added in sequence, followed by 40 ng / mL labeled Sulfo-TAG anti-CA125 antibody and 5 μL of biotin-labeled detection antibody, and incubated at room temperature with shaking at 450 rpm for 2 hours.

[0092] Washing and signal reading:

[0093] After incubation, the cells were washed three times with PBS containing Tween-20 to remove unbound substances;

[0094] Add 150 μL of read buffer and read the luminescence counts by an electrochemiluminescence instrument.

[0095] Data processing and quality control:

[0096] A CA125 concentration standard curve (concentration range 0.5–200 U / mL) was constructed using standards, and positive and negative controls were used for signal correction;

[0097] After multiple batches of testing, the intra-batch CV was maintained at 3.8%-5.2%, and the inter-batch CV was controlled at 6.5%-7.5%, ensuring the reliability of the test results.

[0098] Data acquisition: Use an automated electrochemiluminescence instrument to collect the luminescence counts of each well, and accurately calculate the CA125 content in the sample through standard curve fitting.

[0099] From the above, we can see that the lower limit of detection is 0.5U / mL, and the signal-to-noise ratio reaches about 350 after gradient optimization;

[0100] ROC analysis showed that the system had a sensitivity of 80% and a specificity of 97% in ovarian tumor patients, with an AUC value of approximately 0.90;

[0101] The overall detection process takes about 3.5 hours and is easy to operate, suitable for large-scale clinical rapid screening and dynamic monitoring.

[0102] As can be seen above, highly specific recognition of target tumor markers can be achieved through carefully selected capture molecules and detection antibodies. The high affinity between biotin and streptavidin can greatly reduce nonspecific binding, thereby reducing background signals and improving the accuracy and reliability of detection.

[0103] Embodiment three:

[0104] Taking the biotin-labeled detection antibody concentration gradient optimization experiment as an example, this paper explains how to determine the optimal antibody concentration to achieve the highest signal-to-noise ratio (S / N).

[0105] In the experiment, fixed other reagent conditions were used, and the concentration of the biotin-labeled detection antibody was gradually changed. The luminescence counts of the sample and the negative control at each concentration were compared, the signal-to-noise ratio was calculated, and the optimal detection conditions were determined.

[0106] Gradient Experimental Design

[0107] Purpose

[0108] Optimize the concentration of biotinylated detection antibody to obtain the highest electrochemiluminescent signal on the streptavidin plate after complex formation, while ensuring that the background noise is as low as possible to achieve the best amplification of the signal.

[0109] Experimental conditions: including fixed conditions and variable conditions;

[0110] The fixed conditions are:

[0111] Capture molecules (monoclonal antibodies against target tumor markers) are immobilized on streptavidin pre-coated plates.

[0112] The ECL labeled molecules (such as Sulfo-TAG labeled antibodies) and other buffers, blocking solutions, and washing solutions all use the conditions optimized in the previous step.

[0113] Sample pretreatment, mixing, incubation time, and temperature were kept constant.

[0114] The variable conditions are:

[0115] The concentration of biotin-labeled detection antibody was set as a gradient: 0.125, 0.0625, 0.03125, 0.0156, 0.0078 μg / μL.

[0116] Experimental steps:

[0117] In a 96-well plate, biotin-labeled detection antibodies of preset concentrations are added in sequence and mixed with ECL-labeled antibodies of fixed concentrations and the sample to be tested (including the target tumor marker).

[0118] After incubation for a certain period of time, the mixture was added to a pre-coated streptavidin plate for binding reaction.

[0119] After the incubation, non-specific binding substances are washed with a washing solution, a readout buffer is added, and the luminescence counts of each well are read using an electrochemiluminescence instrument (such as MESO QuickPlex SQ 120).

[0120] Negative controls (samples without target antigen added) were also set up to obtain background luminescence counts.

[0121] The experiment was repeated several times for each concentration condition (for example, 6 replicate wells for each concentration), and the signal-to-noise ratio (S / N = (sample luminescence counts - negative control counts) / negative control counts) was calculated after statistically averaging.

[0122] Tabular Data

[0123] Table 1 below shows the data of a set of gradient experiments, showing the average luminescence counts of samples and negative controls at different detection antibody concentrations and the calculated signal-to-noise ratios.

[0124] Table 1

[0125]

[0126] When the concentration was 0.125 μg / μL, the sample luminescence count was 800, the background was 200, and the S / N was about 3.0.

[0127] When the concentration was 0.0625 μg / μL, the sample luminescence count reached the highest (1500), the background was stable at around 200, and the calculated signal-to-noise ratio was the highest, which was 6.5.

[0128] As the detection antibody concentration was further reduced (0.03125, 0.0156, 0.0078 μg / μL), although the sample signals changed, the optimal signal-to-noise ratio was not reached. At 0.03125 μg / μL, the signal-to-noise ratio was slightly lower than that at 0.0625 μg / μL, and the signal-to-noise ratio decreased significantly at lower concentrations.

[0129] As can be seen from the above, according to the results of the gradient experiment, when the detection antibody concentration is 0.0625μg / μL, the best signal-to-noise ratio can be obtained. This shows that at this concentration, the complex formation effect is optimal, the electrochemiluminescence signal is fully amplified, and the background interference is low, thus ensuring that the system detection sensitivity and specificity are optimally balanced. The optimization results provide a theoretical basis and operating parameters for the use of biotin-labeled detection antibodies in subsequent detection processes, further improving the detection efficiency and clinical application value of the entire kit solution.

[0130] The modular design of the components of the kit makes this solution not only suitable for the detection of specific tumor markers, but also allows flexible adjustment and replacement of capture molecules and detection antibodies to meet the detection needs of different types of tumor markers. This not only meets the needs of diversified clinical testing, but also facilitates the subsequent upgrade and expansion of the technology platform.

[0131] The overall process of the kit has been optimized to effectively connect the steps of sample pretreatment, immune response, and signal reading, which theoretically reduces tedious operations and waiting time. The system is designed to achieve rapid conversion from sample addition to signal acquisition, suitable for high-throughput detection of large quantities of clinical samples, and meet the needs of early screening and dynamic monitoring.

[0132] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples, unless they are contradictory.

[0133] In the drawings of the embodiments disclosed in the present invention, only the structures related to the embodiments disclosed in the present invention are involved, and other structures can refer to the general design. In the absence of conflict, the same embodiment and different embodiments of the present invention can be combined with each other.

[0134] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An electrochemiluminescence kit for rapid detection of tumor markers, characterized in that: The kit includes the following reagents: A capture molecule, used to specifically bind to the tumor marker in the sample to be tested to form a complex A; An electrochemiluminescent (ECL) labeling molecule is used to perform a labeling reaction with the capture molecule in the complex A or directly with the tumor marker to be detected to generate a complex B with an ECL signal; A biotin-labeled detection antibody is used to bind to the complex B to form a biotin bridge to obtain a complex C; A streptavidin fixed plate is used to capture and fix the complex C to form a stable complex D, thereby facilitating detection of the amplified luminescent signal under electrochemical excitation; Liquid reagents, including buffers for conditioning the reaction environment, blocking solutions, wash solutions, and readout buffers.

2. The electrochemiluminescence kit for rapid detection of tumor markers according to claim 1, characterized in that: The capture molecule is a monoclonal antibody against a tumor marker fixed on the surface of a plate, and is used to specifically capture the tumor marker to be detected to obtain a complex A.

3. The electrochemiluminescence kit for rapid detection of tumor markers according to claim 1, characterized in that: The electrochemiluminescent labeling molecule is Sulfo-TAG or other labels that can generate electrochemiluminescent signals, which are used to combine with the capture molecule or directly with the tumor marker to obtain complex B and achieve signal amplification.

4. The electrochemiluminescence kit for rapid detection of tumor markers according to claim 1, characterized in that: The biotin-labeled detection antibody is a secondary antibody against tumor markers, which is used to bind to complex B after biotinylation to form complex C, and is further fixed on the plate through the affinity binding of biotin and streptavidin.

5. The electrochemiluminescence kit for rapid detection of tumor markers according to claim 1, characterized in that: The streptavidin fixed plate is a solid phase material pre-coated with streptavidin, and is used to efficiently bind with the biotin in the complex C to obtain the complex D for signal transmission and amplification.

6. The electrochemiluminescence kit for rapid detection of tumor markers according to claim 1, characterized in that: The buffer system comprises: An antigen buffer, used to allow the capture molecule to fully react with the tumor marker to obtain complex A; Washing solution, used to remove unbound or non-specifically bound components in each reaction step; Blocking solution, used to block nonspecific binding sites; The readout buffer is used to provide an optimal luminescence environment under electrochemical excitation conditions to read the luminescence signal of the complex D.

7. The electrochemiluminescence kit for rapid detection of tumor markers according to claim 1, characterized in that: The kit also includes preset positive controls and negative controls for calibrating and verifying the luminescent signal during the detection process.

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