CN116165193A - Carcinoembryonic antigen detection reagent and kit based on double-antibody sandwich chemiluminescence method - Google Patents

Carcinoembryonic antigen detection reagent and kit based on double-antibody sandwich chemiluminescence method Download PDF

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CN116165193A
CN116165193A CN202310179713.8A CN202310179713A CN116165193A CN 116165193 A CN116165193 A CN 116165193A CN 202310179713 A CN202310179713 A CN 202310179713A CN 116165193 A CN116165193 A CN 116165193A
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carcinoembryonic antigen
double
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邓勇辉
刘述德
姚群燕
沈锡中
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Zhongshan Hospital Fudan University
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Abstract

The invention discloses a carcinoembryonic antigen detection reagent and a kit based on a double-antibody sandwich chemiluminescence method. The detection reagent comprises a capture probe, a detection probe and a luminescent substrate, wherein the capture probe uses magnetic iron oxide particles coated with silicon dioxide at the outer layer as a carrier to be coupled with a mouse anti-human carcinoembryonic antigen monoclonal antibody for combining carcinoembryonic antigen in a solution to be detected; the detection probe is coupled with horseradish peroxidase and a mouse anti-human carcinoembryonic antigen monoclonal antibody by mesoporous silica; the carcinoembryonic antigen of the capture probe interacts with the antibody of the detection probe to form a double-antibody sandwich complex; adding a luminescent substrate to the double-antibody sandwich complex to form a detection reagent, wherein the luminescent substrate is used for generating a luminescent value for identifying the concentration of the double-antibody sandwich complex. According to the invention, the magnetic beads and the mesoporous material are introduced into the double-antibody sandwich chemiluminescence method for preparing the capture probe and the detection probe, so that the detection precision and accuracy are improved, trace carcinoembryonic antigen can be effectively detected, the operation is convenient and quick, and the time consumption is short.

Description

Carcinoembryonic antigen detection reagent and kit based on double-antibody sandwich chemiluminescence method
Technical Field
The invention relates to a carcinoembryonic antigen detection reagent and a kit based on a double-antibody sandwich chemiluminescence method, and belongs to the technical field of biomedical detection.
Background
Early diagnosis of cancer can effectively reduce the occurrence risk of tumor-related diseases and improve the survival rate of patients. Thus, high sensitivity and specificity analysis of tumor-associated biomarkers is critical for early diagnosis of tumor-associated diseases. Carcinoembryonic antigen (CEA) is a specific tumor-associated biomarker for colon and rectal cancers. In addition, CEA content is higher than that of healthy patients (0-5 ng/mL) in other malignant tumors such as breast cancer and lung cancer. Currently, there are various methods for detecting CEA, such as inductively coupled plasma mass spectrometry, colorimetry, electrochemistry, chemiluminescence, fluorescence, electrochemistry, surface enhanced raman scattering and enzyme-linked immunosorbent assay (ELISA). Compared with other analysis methods, the chemiluminescence method has the advantages of high signal-to-noise ratio, wide detection range, high sensitivity and the like, and is a rapid and economic detection method which is widely accepted.
The chemiluminescence method is one of molecular luminescence spectrometry, wherein an object to be detected is loaded in a chemical detection system, and the content of the object to be detected is determined according to the luminous intensity by detecting the chemiluminescent intensity of the system by an instrument according to the principle that the concentration of the object to be detected in the chemical detection system and the chemiluminescent intensity of the system are in linear quantitative relation under certain conditions.
In the prior art, when the traditional chemiluminescence method detects the luminous intensity, the energy transfer efficiency is low, the output signal intensity and the continuous measurement time are limited, and the requirements of rapid and accurate analysis and detection are difficult to meet due to the dependence of an automatic sampling and signal reading instrument.
Disclosure of Invention
The purpose of the invention is that: aiming at the problems of low detection sensitivity, low accuracy, time consumption and the like in the traditional chemiluminescence method for detecting carcinoembryonic antigen, the invention provides a carcinoembryonic antigen (CEA) detection reagent and a kit based on a double-antibody sandwich chemiluminescence method, and magnetic beads and mesoporous materials are introduced into the double-antibody sandwich chemiluminescence method for preparing a capture probe and a detection probe, so that the detection precision and accuracy are improved, trace carcinoembryonic antigen can be effectively detected, and the kit is convenient to operate and short in time consumption.
In order to achieve the above purpose, the invention provides a carcinoembryonic antigen detection reagent based on a double-antibody sandwich chemiluminescence method, which comprises a capture probe, a detection probe and a luminescent substrate, wherein the capture probe uses magnetic iron oxide particles coated with silicon dioxide as a carrier to be coupled with a mouse anti-human carcinoembryonic antigen monoclonal antibody for combining carcinoembryonic antigen in a solution to be detected; the detection probe is coupled with horseradish peroxidase HRP and a mouse anti-human carcinoembryonic antigen monoclonal antibody through mesoporous silica; the carcinoembryonic antigen of the capture probe interacts with the antibody of the detection probe to form a double-antibody sandwich complex; the luminescent substrate consists of separately packaged luminol/p-bromophenol solution with pH=6-13 and hydrogen peroxide solution with pH=7-8. Adding a luminescent substrate to the double-antibody sandwich complex to form a detection reagent, wherein the luminescent substrate is used for generating a luminescent value for identifying the concentration of the double-antibody sandwich complex.
Preferably, the concentration of luminol in the luminol/p-bromophenol solution is 0.001-20 mmol/L, and the concentration of p-bromophenol is 0.001-100 mmol/L; the concentration of the hydrogen peroxide solution is 0.001-15 mmol/L.
Preferably, the preparation method of the capture probe comprises the following steps:
step 1: magnetic bead Fe 3 O 4 @SiO 2 Reacting with 3-aminopropyl triethoxy silane (APTES) to obtain amino magnetic bead Fe 3 O 4 @SiO 2 -NH 2 ；
Step 2: reacting the aminated magnetic beads obtained in the step 1 with carboxylic acid or anhydride to obtain carboxylated magnetic beads Fe 3 O 4 @SiO 2 -COOH；
Step 3: coupling the carboxylated magnetic beads obtained in the step 2 with a mouse anti-human carcinoembryonic antigen monoclonal antibody under the action of EDC and NHS to obtain a capture probe Fe 3 O 4 @SiO 2 -Ab2。
Capturing probe with carboxylated Fe 3 O 4 @SiO 2 As a carrier, the coating can provide quick magnetic response, can meet the functional group requirement of a coupled antibody after carboxylation, and simultaneously coats SiO 2 Can well lead the magnetic core Fe 3 O 4 Isolated from luminescence reaction, thereby avoiding Fe 3 O 4 Background enhancement to luminol chemiluminescent reaction.
Preferably, the preparation method of the detection probe comprises the following steps:
step 1: the mesoporous silica MSNs is reacted with 3-aminopropyl triethoxysilane (APTES) to obtain the aminated mesoporous silica MSNs-NH 2 ；
Step 2: MSNs-NH obtained in step 1 2 Reacting with carboxylic acid or anhydride to obtain carboxylated mesoporous silica MSNs-COOH;
step 3: and (3) coupling the MSNs-COOH obtained in the step (2) with horseradish peroxidase HRP and a mouse anti-human carcinoembryonic antigen monoclonal antibody under the action of EDC and NHS to obtain a detection probe MSNs-HRP/Ab1.
Preferably, the mass ratio of the horseradish peroxidase HRP conjugated with the detection probe and the monoclonal antibody of the mouse anti-human carcinoembryonic antigen in the step 3 is 1:0.5 to 40.
Preferably, the pore diameter of the mesoporous silica ranges from 1 nm to 20nm; the detection probe takes mesoporous silica with the aperture of 1-20 nm as a carrier, so that a large amount of horseradish peroxidase can be loaded in a pore canal, the loading capacity is improved, the amplification of a luminous signal is realized, the antibody can be effectively coupled on the surface of a mesoporous silica sphere, and the antigen-antibody combination efficiency is increased.
The invention also provides application of the carcinoembryonic antigen detection reagent based on the double-antibody sandwich chemiluminescence method in preparation of a kit for detecting carcinoembryonic antigen.
The invention also provides a kit for detecting carcinoembryonic antigen, which at least comprises the carcinoembryonic antigen detection reagent based on the double-antibody sandwich chemiluminescence method as claimed in any one of claims 1 to 5.
Preferably, the kit further comprises a buffer.
Preferably, the buffer also includes a blocking protein.
Compared with the traditional double-antibody sandwich chemiluminescence immunoassay method, the method solves the problems of short luminescence signal time, low signal intensity, complex operation and the like, disperses and enriches the carcinoembryonic antigen by using a magnetic bead coupled antibody with good water dispersibility as a capture probe under an external magnetic field, uses mesoporous silicon with macropore mesopores as a carrier, uses huge specific surface area and good pore channel structure to couple a large amount of horseradish peroxidase and antibody as a detection probe, combines and enriches the capture probe and the antigen to be detected under the action of antigen and antibody when detecting the antigen to be detected, and catalyzes hydrogen peroxide by a large amount of horseradish peroxidase to generate stronger chemiluminescence signals after adding the detection probe and a luminescent substrate solution, thereby realizing lower detection limit and better specificity.
Compared with the prior art, the invention has the beneficial effects that:
according to the carcinoembryonic antigen detection reagent and the kit based on the double-antibody sandwich chemiluminescence method, the magnetic beads and the mesoporous material are combined with the double-antibody sandwich chemiluminescence method, the immune magnetic beads are prepared through coupling of the monoclonal antibodies and the magnetic beads, the carcinoembryonic antigens in a sample to be detected are enriched by the immune magnetic beads, the advantages of high specific surface area and large aperture of the mesoporous material are utilized, higher enzyme and antibody loading capacity is achieved, signal amplification and sensitivity improvement are achieved, and in addition, the operation is more convenient and the time consumption is shorter through the introduction of the magnetic beads.
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FIG. 1 is a flow chart of a carcinoembryonic antigen detection method based on a double-antibody sandwich chemiluminescence method;
FIG. 2 is a graph showing the luminescence kinetics of carcinoembryonic antigen detected at various concentrations in example 12 of the present invention;
FIG. 3 is a standard curve fitted to the luminescence values of carcinoembryonic antigen detected at various concentrations in example 12 of the present invention.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.
Example 1
The embodiment provides a carcinoembryonic antigen detection reagent based on a double-antibody sandwich chemiluminescence method, wherein the detection reagent comprises a capture probe, a detection probe and a luminescent substrate.
The preparation method of the capture probe comprises the following steps:
1. carboxylation of magnetic particles
(1) 50mg of magnetic bead Fe 3 O 4 @SiO 2 Dispersing in 50mL absolute ethanol, performing ultrasonic treatment for 30min, adding 2mL 3-aminopropyl triethoxysilane into the solution, stirring at 300rpm at room temperature overnight, centrifuging with absolute ethanol and deionized water, washing the product three times after the reaction, and drying in vacuum oven to obtain aminated magnetic beads denoted as Fe 3 O 4 @SiO 2 -NH 2 ；
(2) The obtained aminated magnetic beads Fe 3 O 4 @SiO 2 -NH 2 Dispersing in 50mL DMF, ultrasonic dispersing for 30min, adding 2g succinic anhydride into the dispersed solution, stirring at 300rpm at room temperature overnight, centrifuging and washing the product with acetone and absolute ethanol for three times after the reaction, and drying in a vacuum oven to obtain carboxylated magnetic beads which are marked as Fe 3 O 4 @SiO 2 -COOH；
2. Carboxylated magnetic particle coupled mouse anti-human carcinoembryonic antigen monoclonal antibody
(1) Carboxylated magnetic beads Fe 3 O 4 @SiO 2 -COOH was dispersed in a buffer at ph=5-6, 22mg EDC and 2.2mg NHS were added and stirred at room temperature for 40min;
(2) The resulting solution was magnetically adsorbed for 30s, washed three times with PBS buffer (ph=7.4) and then redispersed in 1mL of PBS buffer (ph=7.4);
(3) Adding 100 mug of the mouse monoclonal antibody diluted by the antibody diluent, stirring overnight in a dark place, and coupling the mouse monoclonal antibody and magnetic beads; after the coupling is completed, the magnetic separation washing is carried out, the supernatant is discarded to obtain a capture probe, and the separation is carried out againBulk to 1mL PBS buffer (pH=7.4), noted as Fe 3 O 4 @SiO 2 -Ab2。
The preparation method of the detection probe comprises the following steps:
1. mesoporous silica carboxylation
(1) Dispersing 50mg of mesoporous silica MSNs with the aperture of 17nm into 50mL of absolute ethyl alcohol, dispersing for 30min by ultrasonic treatment, adding 2mL of APTES into the solution by using a rubber head dropper, stirring the solution at 300rpm at room temperature overnight, centrifuging and washing the product three times by using absolute ethyl alcohol and deionized water after the reaction is finished, and drying in a vacuum oven to obtain the aminated mesoporous silica MSNs-NH 2 ；
(2) The obtained aminated mesoporous silica MSNs-NH 2 Dispersing in 50mL DMF, performing ultrasonic treatment for 30min, adding 2g succinic anhydride into the solution, stirring at 300rpm at room temperature overnight, after the reaction is finished, centrifugally washing the product with acetone and absolute ethyl alcohol for three times, and drying in a vacuum oven to obtain carboxylated mesoporous silica which is marked as MSNs-COOH;
2. mesoporous silica coupled horseradish peroxidase HRP and mouse anti-human carcinoembryonic antigen monoclonal antibody
(1) Dispersing 5mg of carboxylated mesoporous silica MSNs-COOH in a buffer solution with pH=5-6, adding 22mg of EDC and 2.2mg of NHS, and stirring at room temperature for reaction for 40min;
(2) The resulting solution was centrifuged at 10000rpm in a high-speed centrifuge for 10min, washed three times with PBS buffer (ph=7.4), and then redispersed in 1mL of PBS buffer (ph=7.4);
(3) Different preset amounts of horseradish peroxidase HRP and 100 mug of mouse monoclonal antibody diluted by antibody liquid are added, the mixture is stirred overnight in a dark place, coupling of the HRP and the mouse monoclonal antibody with mesoporous silica is carried out, after coupling is completed, centrifugal washing is carried out, the supernatant is discarded, detection probes with different coupling ratios are obtained, the detection probes are redispersed into 1mL PBS buffer (pH=7.4) and marked as MSNs-HRP/Ab1, and Ab1: HRP (mass ratio) =1: x (1:X) is 1:0.5, 1: 4. 1: 8. 1: 16. 1: 24. 1:32.
the luminous substrate is luminol/p-bromophenol solution with pH=6-13 and hydrogen peroxide solution with pH=7-8, wherein the concentration of luminol is 0.001 mmol/L-20 mmol/L, the concentration of p-bromophenol is 0.001 mmol/L-100 mmol/L, and the concentration of hydrogen peroxide solution is 0.001-15 mmol/L.
Example 2
The present embodiment provides a carcinoembryonic antigen detection method (flow is shown in fig. 1) based on a double-antibody sandwich chemiluminescence method, wherein the detection method is based on the detection reagent of embodiment 1, and the detection process and parameters are as follows:
taking 10 mu L of capture probe Fe 3 O 4 @SiO 2 Adding 0.0001 mu g CEA antigen into a PBS buffer solution of Ab2 in a test tube, adding the PBS buffer solution (pH=7.4) until the volume of the solution is 100 mu L, incubating for 60min at 37 ℃, magnetically washing the solution with PBS buffer solution (pH=7.4) three times, adding 10 mu L of detection probe MSNs-HRP/Ab1 (1:24), adding PBS buffer solution (pH=7.4) until the volume of the solution is 100 mu L, incubating for 60min at 37 ℃, magnetically washing the solution with PBS buffer solution (pH=7.4) five times, taking three groups of parallel experiments, providing three groups of blank control groups, respectively adding the luminescent substrate solutions A (with the concentration of luminol being 10mmol/L and the concentration of p-bromophenol being 50 mmol/L) of each of the sample solution, washing the sample solution, transferring the sample solution into a 96-well plate after washing, and preparing luminescent substrate solutions A (with the concentration of luminol being 10mmol/L, and the luminescent substrate solutions B (pH=7.4 and 10 mmol/L) of each well, wherein the luminescent substrate solutions A and the luminescent substrate solutions B are respectively added into a 100 mu L of a luminometer.
The present embodiment focuses on obtaining the detection effect of the luminescent substrate solution a at different pH values, and through experiments, the corresponding detection effect can be obtained at each pH value, and compared with the experimental groups of ph=6, 7, 8, 9, 10, 11, 12, 13, the luminescent signal ratio (P/N value) of the experimental group and the blank group detected at ph=10 is the highest.
Example 3
The embodiment provides a carcinoembryonic antigen detection method based on a double-antibody sandwich chemiluminescence method, wherein the detection method is based on the detection reagent of embodiment 1, and the detection process and parameters are as follows:
taking 10 mu L of capture probe Fe 3 O 4 @SiO 2 Ab2 in test tube, adding 0.0001 μg CEA antigen, adding PBS (pH=7.4) buffer to a solution volume of 100 μl, incubating at 37deg.C for 60min, magnetically washing with PBS (pH=7.4) buffer three times, adding 10 μl detection probe MSNs-HRP/Ab1 (1:24), adding PBS (pH=7.4) buffer to a solution volume of 100 μl, incubating at 37deg.C for 60min, magnetically washing with PBS (pH=7.4) buffer five times, taking three parallel experiments, setting three blank control groups, and performing the same steps as the experimental group except that no CEA antigen is added, after washing, transferring the solution to a 96-well plate, preparing luminol solution A (wherein the solution pH=10 and the p-bromophenol concentration is 50 mmol/L) with luminol concentration of 0.001mmol/L, 0.005mmol/L, 0.01mmol/L, 0.05mmol/L, 0.1mmol/L, 0.5mmol/L, 1mmol/L, 5mmol/L, 10mmol/L and 20mmol/L respectively, preparing luminol solution B (hydrogen peroxide solution, pH=7.4 and the hydrogen peroxide concentration is 10 mmol/L), adding 100 mu L luminol solution A and luminol solution B into each well respectively, and detecting the luminescence value in an enzyme-labeling instrument;
the embodiment focuses on obtaining the detection effect of the luminescent substrate solution A under different luminol concentrations, and corresponding detection effects can be obtained under different concentrations through experiments, wherein compared with the experimental groups with luminol concentration of 0.001mmol/L, 0.005mmol/L, 0.01mmol/L, 0.05mmol/L, 0.1mmol/L, 0.5mmol/L, 1mmol/L, 5mmol/L and 20mmol/L, the experimental group with luminol concentration of 10mmol/L has the highest P/N value.
Example 4
The embodiment provides a carcinoembryonic antigen detection method based on a double-antibody sandwich chemiluminescence method, wherein the detection method is based on the detection reagent of embodiment 1, and the detection process and parameters are as follows:
taking 10 mu L of capture probe Fe 3 O 4 @SiO 2 -Ab2 in a tube, 0.0001 μg CEA antigen was added, PBS (ph=7.4) buffer was added to a solution volume of 100 μl, incubation was performed at 37 ℃ for 60min, PBS (ph=7.4) buffer was magnetically washed three times, 10 μl detection probe MSNs-HRP/Ab1 (1:24) was added, PBS (ph=7.4) buffer was added to a solution volume of 100 μl, incubation was performed at 37 ℃ for 60min, PBS (ph=7.4) buffer was magnetically washed five times, and samples were takenThree groups of parallel experiments are provided with three groups of blank control groups, the steps are the same as those of the experiment groups except that CEA antigen is not added, the solution is washed and then transferred to a 96-well plate, luminescent substrate solutions A with the concentration of p-bromophenol of 0mmol/L, 0.5mmol/L, 5mmol/L, 10mmol/L, 25mmol/L, 50mmol/L and 100mmol/L (wherein the solution pH=10 and the luminol concentration is 10 mmol/L) are respectively prepared, luminescent substrate solutions B (hydrogen peroxide solution, pH=7.4 and the hydrogen peroxide concentration is 10 mmol/L) are prepared, 100 mu L of luminescent substrate solution A and luminescent substrate solution B are respectively added to each well, and luminescent values are detected in an enzyme-labeling instrument.
By experiment, the P/N value of the experimental group with the concentration of 5mmol/L of P-bromophenol is highest compared with the experimental group with the concentration of 0mmol/L, 0.5mmol/L, 10mmol/L, 25mmol/L, 50mmol/L and 100mmol/L of P-bromophenol.
Example 5
The embodiment provides a carcinoembryonic antigen detection method based on a double-antibody sandwich chemiluminescence method, wherein the detection method is based on the detection reagent of embodiment 1, and the detection process and parameters are as follows:
taking 10 mu L of capture probe Fe 3 O 4 @SiO 2 Ab2 was added to a test tube, blocking buffer (0.5% BSA-PBS, 1% BSA-PBS, 5% BSA-PBS, 0.2% casein-PBS, 1% casein-PBS) was added to a solution volume of 200. Mu.L, incubated at 37℃for 60min, 0.0001. Mu.g CEA was added to the test tube, PBS (pH=7.4) buffer was magnetically washed three times, 10. Mu.L detection probe MSNs-HRP/Ab1 (1:24) was added, PBS (pH=7.4) buffer was added to a solution volume of 100. Mu.L, 60min at 37℃was added, PBS (pH=7.4) buffer was magnetically washed five times, three additional sets of blank control sets were set, the rest steps were identical to those of the experimental set up, washed and transferred to a 96-well plate, luminescent substrate solution A (solution pH=10, luminol concentration was 5mmol/L for luminol for each well, luminescent substrate solution B (hydrogen peroxide solution pH=7.4), and luminescent substrate solution B was prepared, and luminescent substrate solution was added to a well for each well, and luminescent substrate solution was detected by a standard enzyme assay.
By experiment, the blocking solution was found to have the highest P/N value in the 0.2% casein experimental group compared to the blocking solution of 0.5% BSA, 1% BSA, 5% BSA, and 1% casein.
Example 6
The embodiment provides a carcinoembryonic antigen detection method based on a double-antibody sandwich chemiluminescence method, wherein the detection method is based on the detection reagent of embodiment 1, and the detection process and parameters are as follows:
taking 10 mu L of capture probe Fe 3 O 4 @SiO 2 Adding 0.2% casein-PBS (phosphate buffer) into a test tube until the volume of the solution is 200 mu L, respectively incubating at 37 ℃ for 30min, 60min, 90min and 120min, adding 0.0001 mu g CEA for 60min, magnetically washing with PBS (pH=7.4) buffer for three times, adding 10 mu L of detection probe MSNs-HRP/Ab1 (1:24), adding PBS (pH=7.4) buffer until the volume of the solution is 100 mu L, magnetically washing with PBS (pH=7.4) buffer for five times, taking three groups of parallel experiments, additionally arranging three groups of blank control groups, respectively, carrying out the same steps as those of the experimental groups except for no CEA antigen, washing, transferring to a 96-well plate, respectively configuring luminescent substrate solution A (solution pH=10, luminol concentration of p-bromophenol is 5 mmol/L), configuring luminescent substrate solution B (hydrogen peroxide solution, pH=7.4, hydrogen peroxide concentration is 10 mmol/L), and adding 100 mu L of each well into the luminescent substrate solution A and the luminescent substrate solution B in a standard luminescence meter;
through experiments, compared with the experimental groups with the blocking time of 30min, 60min and 120min, the P/N value of the experimental group with the blocking time of 90min is highest.
Example 7
The embodiment provides a carcinoembryonic antigen detection method based on a double-antibody sandwich chemiluminescence method, wherein the detection method is based on the detection reagent of embodiment 1, and the detection process and parameters are as follows:
taking 10 mu L of capture probe Fe 3 O 4 @SiO 2 Ab2 in test tube, adding 0.2% casein-PBS as blocking buffer to a solution volume of 200 μl, incubating at 37deg.C for 90min, adding 0.0001 μg CEA, incubating for 60min, magnetically washing with PBS (pH=7.4) buffer for three times, incubating for 60min at 37deg.C, magnetically washing with PBS (pH=7.4) bufferThree times, adding 10 mu L of detection probes MSNs-HRP/Ab1 (1:X, X=0.5, 4, 8, 16, 24 and 32), adding PBS (pH=7.4) buffer solution to a solution volume of 100 mu L, incubating for 60min at 37 ℃, magnetically washing the PBS (pH=7.4) buffer solution for five times, taking three groups of parallel experiments, taking three groups of blank control groups, and transferring the three groups of parallel experiments to a 96-well plate after washing, respectively preparing a luminescent substrate solution A (solution pH=10, luminol concentration of 10mmol/L and p-bromophenol concentration of 5 mmol/L), preparing a luminescent substrate solution B (hydrogen peroxide solution, pH=7.4 and hydrogen peroxide concentration of 10 mmol/L), adding 100 mu L of the luminescent substrate solution A and the luminescent substrate solution B into each hole, and detecting luminescence values in an enzyme-labeling instrument;
by experiment, the P/N value was highest for the MSNs-HRP/Ab1 (1:24) experimental group compared to the MSNs-HRP/Ab1 (1:X, X=0.5, 4, 8, 16, 32) experimental group.
Example 8
The embodiment provides a carcinoembryonic antigen detection method based on a double-antibody sandwich chemiluminescence method, wherein the detection method is based on the detection reagent of embodiment 1, and the detection process and parameters are as follows:
1. Mu.L, 2.5. Mu.L, 5. Mu.L, 7.5. Mu.L, 10. Mu.L of capture probe Fe were taken, respectively 3 O 4 @SiO 2 Adding 0.2% casein-PBS (buffer solution) as a blocking solution to 200 mu L in a test tube, incubating for 90min at 37 ℃, adding 0.0001 mu g CEA for 60min, incubating for 60min at 37 ℃, magnetically washing with PBS (pH=7.4) buffer solution for three times, adding 10 mu L of detection probe MSNs-HRP/Ab1 (1:24), adding PBS (pH=7.4) buffer solution to 100 mu L in a solution volume, incubating for 60min at 37 ℃, magnetically washing with PBS (pH=7.4) buffer solution for five times, taking three parallel experiments, adding three blank control groups, incubating for the same steps as the experimental groups except for CEA antigen, washing, transferring to a 96-well plate, preparing luminescent substrate solution A (solution pH=10, luminol at 10mmol/L, p-bromophenol concentration of 5 mmol/L), preparing luminescent substrate solution B (hydrogen peroxide solution, pH=7.4, 10 mmol/L), adding luminescent substrate solution 100 mu L per hole of luminescent substrate and 100 mu L of luminescent substrate solution respectivelyAnd (3) detecting the luminescence value in the enzyme-labeled instrument.
By experiment, the P/N value was highest for the 7.5. Mu.L capture probe dose experimental group compared to the 1. Mu.L, 2.5. Mu.L, 5. Mu.L, 10. Mu.L capture probe dose experimental group.
Example 9
The embodiment provides a carcinoembryonic antigen detection method based on a double-antibody sandwich chemiluminescence method, wherein the detection method is based on the detection reagent of embodiment 1, and the detection process and parameters are as follows:
7.5 mu L of detection probe Fe is taken respectively 3 O 4 @SiO 2 Ab2 was added to the tube, incubated at 37℃for 90min with 0.2% casein-PBS as blocking buffer to a solution volume of 200. Mu.L, incubated at 60min with 0.0001. Mu.g CEA, magnetically washed three times with PBS (pH=7.4) buffer, magnetically washed three times with 37℃for 60min, magnetically washed five times with PBS (pH=7.4) buffer, corresponding to 1. Mu.L, 2.5. Mu.L, 5. Mu.L, 7.5. Mu.L, 10. Mu.L of detection probe MSNs-HRP/Ab1 (1:24), 100. Mu.L with PBS (pH=7.4) buffer, magnetically washed five times with PBS (pH=7.4) buffer, taking three groups of parallel experiments, setting three groups of blank control groups, washing the blank control groups and the experiment groups, transferring the washed blank control groups into a 96-well plate, respectively preparing a luminescent substrate solution A (solution pH=10, luminol concentration is 10mmol/L, p-bromophenol concentration is 5 mmol/L), preparing a luminescent substrate solution B (hydrogen peroxide solution, pH=7.4, hydrogen peroxide concentration is 10 mmol/L), adding 100 mu L of the luminescent substrate solution A and the luminescent substrate solution B into each well, and detecting luminescent values in an enzyme-labeling instrument;
by experiment, the P/N value was highest for the 2.5. Mu.L test probe dose compared to the 1. Mu.L, 5. Mu.L, 7.5. Mu.L, 10. Mu.L test probe dose.
Example 10
The embodiment provides a carcinoembryonic antigen detection method based on a double-antibody sandwich chemiluminescence method, wherein the detection method is based on the detection reagent of embodiment 1, and the detection process and parameters are as follows:
7.5 mu L of capture probe Fe is taken respectively 3 O 4 @SiO 2 Dosage of Ab2Adding 0.2% casein-PBS as a blocking solution buffer to a solution volume of 200 mu L, respectively incubating for 90min at 37 ℃, adding 0.0001 mu g CEA, respectively incubating for 30min, 60min, 90min and 120min at 37 ℃, magnetically washing with PBS (pH=7.4) buffer three times, correspondingly adding 2.5 mu L of detection probe MSNs-HRP/Ab1 (1:24), adding PBS (pH=7.4) buffer to a solution volume of 100 mu L, incubating for 60min at 37 ℃, magnetically washing with PBS (pH=7.4) buffer five times, taking three parallel experiments, additionally arranging three parallel control groups, respectively preparing luminescent substrate solution A (solution pH=10, luminol concentration of 10mmol/L, p-bromophenol concentration of 5 mmol/L), preparing luminescent substrate solution B (hydrogen peroxide solution, pH=7.4, hydrogen peroxide concentration of 10 mmol/L), magnetically washing with PBS (pH=7.4) buffer five times, taking three parallel experiments, respectively arranging three parallel control groups, washing, transferring to a 96-well plate after washing, respectively preparing luminescent substrate solution A, and adding luminescent substrate solution B to each well.
By experiment, the P/N value was highest in the 60min incubation experimental group compared to the 60min, 90min, 120min incubation of capture probes and antigen experimental group.
Example 11
The embodiment provides a carcinoembryonic antigen detection method based on a double-antibody sandwich chemiluminescence method, wherein the detection method is based on the detection reagent of embodiment 1, and the detection process and parameters are as follows:
7.5 mu L of capture probe Fe is taken respectively 3 O 4 @SiO 2 Adding 0.2% casein-PBS as blocking buffer to 200 μL, incubating at 37deg.C for 90min, adding 0.0001 μg CEA, incubating at 37deg.C for 60min, magnetically washing with PBS (pH=7.4) buffer for three times, adding 2.5 μL detection probe MSNs-HRP/Ab1 (1:24), adding PBS (pH=7.4) buffer to 100 μL, magnetically washing with PBS (pH=7.4) buffer for five times at 37deg.C for 30min, 60min, 90min, 120min, incubating with three parallel experiments, adding three blank control groups, incubating with the same steps as the experimental groups except for no CEA antigen, washing, transferring to 96-well plate, respectively preparing luminescent substrate solution A (wherein solution pH=10 and luminol concentration is 10 mmol/L),p-bromophenol concentration was 5 mmol/L), luminescence substrate solution B (hydrogen peroxide solution, pH=7.4, hydrogen peroxide concentration was 10 mmol/L), 100. Mu.L luminescence substrate solution A and luminescence substrate solution B were added to each well, and luminescence values were detected in an enzyme-labeled instrument.
By experiment, the P/N value of the experimental group incubated for 60min was highest compared with the experimental group incubated for 30min, 60min and 120min for the detection probes and the antigens.
Example 12
The embodiment provides a carcinoembryonic antigen detection method based on a double-antibody sandwich chemiluminescence method, wherein the detection method is based on the detection reagent of embodiment 1, and the detection process and parameters are as follows:
7.5 mu L of capture probe Fe 3 O 4 @SiO 2 Ab2 was used in test tubes, 0.2% casein-PBS was added as blocking buffer to a solution volume of 200. Mu.L, each of which was incubated at 37℃for 90min, 0.0001. Mu.g CEA was added, each of which was incubated at 37℃for 60min, PBS (pH=7.4) buffer was magnetically washed three times, 2.5. Mu.L of detection probe MSNs-HRP/Ab1 (1:24) was added in corresponding amounts, PBS (pH=7.4) buffer was added to a solution volume of 100. Mu.L, each of which was incubated at 37℃for 60min, PBS (pH=7.4) buffer was magnetically washed five times, three parallel experiments were performed, each of which was otherwise identical to the experimental steps except for CEA antigen, and after washing, each of which was transferred to 96-well plates, luminescent substrate solution A (wherein pH=10, luminol was prepared, p-bromophenol concentration was 5 mmol/L) was prepared, luminescent substrate solution B (hydrogen peroxide solution, pH=7.4, hydrogen peroxide concentration was 10 mmol/L) was added to each well, and luminescent substrate solution B was prepared in a 96-well plate. As shown in FIG. 2, the detected luminescence value and concentration curve shows that the higher chemiluminescence value generated by the signal amplification effect is detected by different samples, and the detected chemiluminescence intensity gradually decreases along with the decrease of the concentration, but the chemiluminescence intensity still keeps higher value within a few minutes, does not decay rapidly, and is more beneficial to detection. Linear curve fitting is performed based on chemiluminescent intensities, as shown in FIG. 3, by measuring the luminescent intensity of the sample to be testedThe carcinoembryonic antigen was quantitatively analyzed. The linear fitting result shows that the linear relation is better in the concentration range of 0.02-20ng/mL, and the correlation coefficient R 2 The detection limit of carcinoembryonic antigen was 0.004ng/mL and the quantification limit was 0.01ng/mL.
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to be limiting in any way and in nature, and it should be noted that several modifications and additions may be made to those skilled in the art without departing from the invention, which modifications and additions are also intended to be construed as within the scope of the invention.

Claims (9)

1. The carcinoembryonic antigen detection reagent based on the double-antibody sandwich chemiluminescence method is characterized by comprising a capture probe, a detection probe and a luminescent substrate, wherein the capture probe uses magnetic iron oxide particles coated with silicon dioxide on the outer layer as a carrier to be coupled with a mouse anti-human carcinoembryonic antigen monoclonal antibody for combining carcinoembryonic antigen in a solution to be detected; the detection probe is coupled with horseradish peroxidase HRP and a mouse anti-human carcinoembryonic antigen monoclonal antibody through mesoporous silica; the carcinoembryonic antigen of the capture probe interacts with the antibody of the detection probe to form a double-antibody sandwich complex; the luminescent substrate consists of separately packaged luminol/p-bromophenol solution with pH=6-13 and hydrogen peroxide solution with pH=7-8.
2. The carcinoembryonic antigen detection reagent based on the double-antibody sandwich chemiluminescence method of claim 1, wherein the concentration of luminol in the luminol/p-bromophenol solution is 0.001-20 mmol/L, and the concentration of p-bromophenol is 0.001-100 mmol/L; the concentration of the hydrogen peroxide solution is 0.001-15 mmol/L.
3. The carcinoembryonic antigen detection reagent based on the double-antibody sandwich chemiluminescence method of claim 1, wherein the preparation method of the capture probe comprises the following steps:
step 1: magnetic bead Fe 3 O 4 @SiO 2 With 3-aminopropyl triethoxy siliconAlkane reaction to obtain aminated magnetic bead Fe 3 O 4 @SiO 2 -NH 2 ；
Step 2: reacting the aminated magnetic beads obtained in the step 1 with carboxylic acid or anhydride to obtain carboxylated magnetic beads Fe 3 O 4 @SiO 2 -COOH；
Step 3: coupling the carboxylated magnetic beads obtained in the step 2 with a mouse anti-human carcinoembryonic antigen monoclonal antibody under the action of EDC and NHS to obtain a capture probe Fe 3 O 4 @SiO 2 -Ab2。
4. The carcinoembryonic antigen detection reagent based on the double-antibody sandwich chemiluminescence method of claim 1, wherein the preparation method of the detection probe comprises the following steps:
step 1: mesoporous silica MSNs is reacted with 3-aminopropyl triethoxysilane to obtain aminated mesoporous silica MSNs-NH 2 ；
Step 2: MSNs-NH obtained in step 1 2 Reacting with carboxylic acid or anhydride to obtain carboxylated mesoporous silica MSNs-COOH;
step 3: and (3) coupling the MSNs-COOH obtained in the step (2) with horseradish peroxidase HRP and a mouse anti-human carcinoembryonic antigen monoclonal antibody under the action of EDC and NHS to obtain a detection probe MSNs-HRP/Ab1.
5. The carcinoembryonic antigen detection reagent based on the double-antibody sandwich chemiluminescence method of claim 4, wherein the ratio of the horseradish peroxidase HRP conjugated with the detection probe and the mouse anti-human carcinoembryonic antigen monoclonal antibody in the step 3 is 1:0.5 to 40.
6. Use of the carcinoembryonic antigen detection reagent based on the double-antibody sandwich chemiluminescence method according to any one of claims 1-5 in preparing a kit for detecting carcinoembryonic antigen.
7. A kit for detecting carcinoembryonic antigen, comprising at least the carcinoembryonic antigen detection reagent according to any one of claims 1 to 5 based on the double antibody sandwich chemiluminescence method.
8. The kit for detecting carcinoembryonic antigen of claim 7, further comprising a buffer.
9. The kit for detecting carcinoembryonic antigen according to claim 8, further comprising a blocking protein in the buffer.
CN202310179713.8A 2023-02-27 2023-02-27 Carcinoembryonic antigen detection reagent and kit based on double-antibody sandwich chemiluminescence method Pending CN116165193A (en)

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