CN113504371A - Separation-free chemiluminescence immunoassay method based on cyclodextrin inclusion - Google Patents
Separation-free chemiluminescence immunoassay method based on cyclodextrin inclusion Download PDFInfo
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- CN113504371A CN113504371A CN202110779736.3A CN202110779736A CN113504371A CN 113504371 A CN113504371 A CN 113504371A CN 202110779736 A CN202110779736 A CN 202110779736A CN 113504371 A CN113504371 A CN 113504371A
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Abstract
The invention belongs to the field of biological detection, and particularly relates to a separation-free chemiluminescence immunoassay method based on cyclodextrin inclusion. The invention uses cyclodextrin to include the acridinium ester analogue to develop a novel separation-free chemiluminescence immunoassay technology with higher sensitivity, stronger specificity and higher automation degree, the cyclodextrin is used to include the acridinium ester analogue as a luminous inclusion compound, and the cavity structure of the cyclodextrin can include a large amount of acridinium ester, so that the luminous signal value is enhanced, and the signal amplification effect is achieved; the method does not need carriers such as a microporous plate, magnetic beads, microspheres and the like, does not need washing steps in the detection process, is simple to operate, requires less time, has high sensitivity and good repeatability, is simple in production process, is easy to amplify and produce, has low requirements on detection instruments in the detection process, and is easy to realize full automation.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to a separation-free chemiluminescence immunoassay method based on cyclodextrin inclusion.
Background
The immunoassay technology utilizes antigen-antibody immunoreaction and utilizes a labeling and tracing technology to complete detection and analysis of a target in a sample, has high sensitivity and high specificity, plays an important role in the fields of biology, medicine and the like, and gradually becomes an important clinical detection means for early disease screening, disease auxiliary diagnosis, curative effect evaluation and the like. The chemiluminescence immune analysis technology is a labeling immune analysis technology which is established by combining luminescence analysis and immune analysis and is used for detecting trace antigens or antibodies. Three components, an oxidizing agent, a luminescent agent and a catalyst, are generally required, and the luminescent agent or the catalyst may be used as the marker. In recent years, with the development and application of acridinium esters and luminol luminescent reagents and the development of ultra-weak light detection technology, the development of chemiluminescence immunoassay technology is rapid. Depending on whether the detection process requires a washing step, it can be divided into non-separation-free chemiluminescence immunoassay and separation-free chemiluminescence immunoassay.
The plate-type chemiluminescence method and the magnetic particle chemiluminescence method which are widely used clinically at present belong to non-separation chemiluminescence immunoassay technologies, solid-phase carriers (such as a microporous plate, magnetic microspheres and the like) are needed in a detection system, washing and other steps are needed in the detection process, the detection is accurate, the error is smaller, quantitative detection can be realized, and the sensitivity is higher. However, the detection is complicated in operation, the detection cost is increased, and the instrument cost is relatively high. To overcome these disadvantages, researchers have developed separation-free chemiluminescent immunoassay techniques. The light-activated chemiluminescence technology is a separation-free chemiluminescence detection technology which generates singlet oxygen energy transfer within a certain distance range through photosensitive microspheres and luminescent microspheres so as to emit a fluorescence signal to detect a sample to be detected. Photosensitive microspheres and luminescent microspheres of coupled antibody are respectively filled with photosensitive compound and luminescent compoundWhen target molecules are added into the reaction system, the photosensitive microspheres and the luminescent microspheres are combined through immunoreaction to form immune complexes. When the photosensitive microsphere is irradiated by exciting light, singlet oxygen ions are released, and singlet oxygen can only reach the luminous microsphere close to the photosensitive microsphere, so that a chemical reaction is generated to emit high-energy fluorescence. The light-activated chemiluminescence has the characteristics of no washing, high sensitivity, simple operation and the like, depends on the light sensitivity and the light emission of the two microspheres, and provides higher requirements on the design and the use of a detection instrument. Fluorescence resonance energy transfer, the reaction mechanism of which was first formed by
First elucidated in 1948. When two fluorescent groups (donor and acceptor) are close to each other and the distance is 1-10 nm, and the fluorescence emission spectrum of the donor and the fluorescence excitation spectrum of the acceptor have certain overlap, the phenomenon that fluorescence energy is transferred from the donor to the acceptor can be observed. The fluorescence resonance energy transfer technology or the time-resolved fluorescence resonance energy transfer technology developed by the mechanism can reflect the distance between two molecular groups by detecting the efficiency information of fluorescence resonance energy transfer, and can be used for analyzing the intermolecular interaction or immunoassay and is also a separation-free chemiluminescence immunoassay technology.
However, the existing separation-free chemiluminescence immunoassay technology has the advantages of washing-free, but has the defects of low detection limit, narrow detection range, high cost and the like.
Disclosure of Invention
The invention aims to provide a separation-free chemiluminescence immunoassay method based on cyclodextrin inclusion, which develops a novel separation-free chemiluminescence immunoassay technology with higher sensitivity, stronger specificity and higher automation degree by applying cyclodextrin inclusion acridinium ester analogues,
the technical scheme adopted by the invention for solving the technical problems is as follows:
a separation-free chemiluminescence immunoassay method based on cyclodextrin inclusion comprises the steps that an antibody 1 is connected with an acridinium ester analogue included by beta-cyclodextrin to obtain an antibody 1 connected with a luminescent inclusion compound, wherein the acridinium ester analogue is included by the beta-cyclodextrin to obtain the luminescent inclusion compound beta-CDA; connecting the antibody 2 with horseradish peroxidase to obtain an enzyme-connected antibody 2; then adding a sample to be detected and a background inhibitor for incubation, adding an excitation liquid, and immediately detecting a luminescence signal value.
Preferably, the method specifically comprises the following steps:
(1) clathrating acridinium ester analogue with beta-cyclodextrin to obtain luminescent clathrate beta-CDA;
(2) the antibody 1 is connected with a luminous inclusion compound to obtain an antibody 1-beta-CDA;
(3) connecting the antibody 2 with horseradish peroxidase to obtain an antibody 2-HRP;
(4) and (3) detection: adding a detection sample, a background inhibitor, an antibody 1-beta-CDA and an antibody 2-HRP, incubating for 15 minutes at 37 ℃, adding an excitation liquid, and immediately detecting a luminescent signal generated within 1 second.
Preferably, the antibody 1 and the antibody 2 are two specific antibodies of an antigen to be detected, and the antigen to be detected is a common carcinoembryonic antigen, including anti-mullerian hormone (AMH), C-reactive protein (CRP) and alpha-fetoprotein (AFP).
Preferably, the detection sample in step (4) includes a standard substance of the antigen to be detected, serum to be detected, urine to be detected, and the like.
Preferably, the background inhibitor in the step (4) is 20mM hydroxydiethylamine solution.
Preferably, the exciting solution in the step (4) is a mixed solution containing 25mM tris (pH 8.0), 15mM p-hydroxycinnamic acid, 0.5mM EDTA, 0.1% Tween-20 and 200mM carbamide peroxide.
Preferably, in the step (4), the antibody 1- β -CDA and the antibody 2-HRP are diluted respectively, and then the two solutions are mixed according to a volume ratio of 5: 5: 5: 1 adding diluted antibody 1-beta-CDA, diluted antibody 2-HRP, sample to be detected and background inhibitor, incubating for 15 minutes at 37 ℃, adding excitation liquid with 10 times volume of the background inhibitor, and immediately detecting luminescent signals generated within 1 second.
Preferably, the method further comprises the step (5) of determining: and (3) taking the standard substance of the antigen to be detected as a detection sample, preparing a standard substance solution with gradient concentration, carrying out detection in the step (4), and drawing a concentration-luminescence signal curve graph to obtain a concentration-luminescence signal formula of the standard substance. More preferably, the solvent used to configure the gradient is PBS buffer or a negative serum of the antigen to be detected. More preferably, the step (5) further includes performing the detection in the step (4) on the detection sample to be detected, such as serum to be detected, and obtaining the luminescence signal, and then substituting the concentration-luminescence signal formula in the step (5) to obtain the concentration of the antigen to be detected in the sample to be detected.
Preferably, the specific preparation method of the step (1) comprises the following steps: weighing 1mg of acridine ester analogue A, dissolving in 1ml of DMF to obtain acridine ester analogue solution, adding the solution into 1ml of 1% beta-cyclodextrin beta-CD aqueous solution, stirring at 37 ℃ for 5 hours, adding 20ml of absolute ethyl alcohol, standing at room temperature for 2 hours, adding the solution into a dialysis bag with cut-off molecular weight of 1500, placing the dialysis bag in PBS buffer solution for dialysis for 1 day (24 hours) at 25 ℃, replacing 3 times of PBS buffer solution during the dialysis for 1 day, and freeze-drying to obtain beta-CDA, wherein the whole operation process needs to be protected from light.
Preferably, the specific preparation method of the step (2) comprises the following steps: dissolving 1mg of luminescent inclusion compound beta-CDA in 2ml of DMF, adding 50ul of the dissolved beta-CDA into 100ul of antibody 1 with the concentration of 10mg/ml, then adding 800ul of 0.05M boric acid buffer solution and 50ul of 1% glutaraldehyde, and carrying out rotary reaction for 1 hour at room temperature; adding 1ml of the reaction solution into a dialysis bag with cut-off molecular weight of 10000, dialyzing in PBS buffer solution at 4 ℃ for 1 day, replacing the PBS buffer solution for 3 times during the dialysis to obtain the antibody 1-beta-CDA, and finally storing at-20 ℃ by using 1ml of a preservation solution (PBS + 0.05% Proclin-300).
Preferably, the specific preparation method of the step (3) is as follows: weighing 0.5mg HRP, dissolving in 100ul deionized water, adding 200ul of newly-prepared 0.1M sodium periodate solution, and stirring at room temperature in a dark place for 30 minutes; adding the reaction solution into a dialysis bag with the molecular weight cutoff of 7000, and dialyzing in 1mM sodium acetate buffer solution with pH4.4 at 4 ℃ overnight; adding 20ul of 0.2M carbonate buffer solution with pH of 9.5, immediately adding 100ul of antibody 2 with concentration of 10mg/ml, then adding 600ul of 0.01M carbonate buffer solution, and lightly stirring for 2 hours at room temperature in a dark place; adding 100ul of newly prepared 4mg/ml sodium borohydride solution, uniformly mixing, and placing at 4 ℃ for 2 hours; purifying the reaction solution with SUPERDEX 200 gel filtration column, collecting purified antibody 2-HRP, and storing at-20 deg.C with storage solution (PBS + 0.05% Proclin-300).
Preferably, the DMF used in the above step may be replaced with DMSO.
Cyclodextrin is a cyclic oligosaccharide compound formed by connecting 6-12D-glucose molecules by 1, 4-glycosidic bonds, has a hollow cylindrical structure, is water-soluble non-reducing white crystal powder at normal temperature and normal pressure, and is one of the most common loading materials for preparing the inclusion compound. Commonly used cyclodextrins are three types of alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin and modified cyclodextrin derivatives. The cyclodextrin inner cavity can include micromolecular drugs or luminescent compounds with certain molecular weight to form an inclusion compound. The inclusion compound formed by the cyclodextrin can obviously improve the performance of the compound, and has good safety, biocompatibility, storage property and the like. Besides inclusion of small molecular compounds, cyclodextrin can also be used for preparing inclusion compound liposome, inclusion compound nanoparticles and the like, and has wide application prospect.
The invention develops a novel separation-free chemiluminescence immunoassay technology with higher sensitivity, stronger specificity and higher automation degree by applying cyclodextrin to include the acridinium ester analogue, the method does not need carriers such as a micropore plate, a magnetic bead and a microsphere, washing steps are not needed in the detection process, the operation is simple, the time is short, the sensitivity is high, the repeatability is good, and the requirement on a detection instrument is lower.
The invention provides a separation-free chemiluminescence immunoassay method based on cyclodextrin inclusion, which is used for quickly and highly sensitively detecting target molecules and is simpler and more efficient compared with the known method. The technology is characterized in that the cyclodextrin is used for including the acridinium ester analogue as a luminescent inclusion compound, and the cavity structure of the cyclodextrin can include a large amount of acridinium ester, so that the luminescent signal value is enhanced, and the function of signal amplification is achieved; the luminous inclusion compound combined with the antibody 1 can generate immune sandwich reaction with horseradish peroxidase combined with the antibody 2 in a system with target molecules to form immune complexes. The luminescent substrate and the horseradish peroxidase in the compound are close to each other, then the luminescent inclusion is catalyzed and cracked by the enzyme in the compound under the action of the excitation liquid to form an unstable excited state intermediate, when the excited state intermediate returns to the ground state, photons are emitted, and the chemiluminescence intensity of the photons is in direct proportion to the number of target molecules. The uncombined excessive antibody 1 luminous inclusion compound and the antibody 2 catalase are not required to be washed and removed, and the chemiluminescence reaction and the detection result are not influenced under the action of a background inhibitor. Therefore, the method for detecting the target molecules does not need supports such as magnetic beads, microspheres and solid phase carriers, a separation-free reaction system does not need washing operation, so that the immunoreaction is more sufficient, an excitation light source does not need to be provided for the reaction, the instrument design is simpler, and the operation process is more efficient.
Compared with the prior art, the invention has the beneficial effects that:
1. the detection method does not need to use a microporous plate, magnetic beads or microspheres and the like as carriers to coat antibodies, the detection antibodies can fully carry out immunoreaction with analytes to be detected, and washing operation is not carried out, so that no washing waste liquid is generated in the detection process, and the method is a separation-free chemiluminescence technology.
2. The detection method adopts cyclodextrin and acridinium ester analogues to form a luminescent inclusion compound, and can greatly improve the strong scheduling of luminescent signals. The background inhibitor is added to eliminate interference and reduce the background signal value, thereby improving the signal-to-noise ratio, improving the detection sensitivity and enlarging the detection linear range.
3. The detection method has the advantages of small antibody consumption, low cost, simple production process, easy amplification production, convenient detection process, low requirement on detection instruments and easy realization of full automation.
Drawings
FIG. 1 is a schematic diagram of separation-free chemiluminescence method for detecting target molecules.
FIG. 2 is a standard curve for separation-free chemiluminescence detection of AMH, diluted in PBS buffer and serum, respectively.
FIG. 3 is a standard curve for split-free chemiluminescence assay for CRP, diluted in PBS buffer and serum, respectively.
FIG. 4 is a standard curve for AFP detection by apheresis chemiluminescence, wherein AFP is diluted in PBS buffer and serum, respectively.
Detailed Description
The technical solution of the present invention is further specifically described below by way of specific examples in conjunction with the accompanying drawings.
In the present invention, the room temperature is 15 to 40 ℃, more specifically 25 ℃; the overnight period is 12 hours, and the 1 day period is 24 hours; no pH PBS buffer was mentioned, the pH being 7.4.
Example one, a luminous inclusion compound (beta-CDA) of beta-cyclodextrin-acridinium ester analogs is prepared, AMH monoclonal antibody 1 is coupled with the luminous inclusion compound beta-CDA, and AMH monoclonal antibody 2 is coupled with horseradish peroxidase, and the luminous inclusion compound is used for separation-free chemiluminescence immune sandwich detection. The following steps are carried out in sequence:
1. preparation of beta-cyclodextrin-acridinium ester analog luminescence clathrate (beta-CDA)
Weighing 1mg of acridine ester analogue (A) and dissolving in 1ml of DMF, adding the solution into 1ml of 1% beta-cyclodextrin (beta-CD) solution, stirring for 5 hours at 37 ℃, adding 20ml of absolute ethyl alcohol, standing for 2 hours at room temperature, adding the solution into a dialysis bag with cut-off molecular weight of 1500, putting the dialysis bag into PBS buffer solution for dialysis for 1 day (24 hours) at 25 ℃, changing the PBS buffer solution for 3 times during the dialysis, and freeze-drying to obtain the beta-CDA, wherein the whole operation process needs to be protected from light. The freeze drying temperature is below 0 deg.C.
AMH monoclonal antibody 1 coupled beta-CDA
1mg of the luminescent inclusion compound beta-CDA is dissolved in 2ml of DMF, 50ul of the dissolved beta-CDA is added into 100ul of anti-mullerian hormone (AMH) monoclonal antibody 1 with the concentration of 10mg/ml, then 800ul of 0.05M boric acid buffer solution and 50ul of 1% glutaraldehyde are added, and the reaction is carried out for 1 hour at room temperature in a rotating way. Adding 1ml of the reaction solution into a dialysis bag with the molecular weight cutoff of 10000, dialyzing in PBS buffer solution at 4 ℃ for 1 day, changing the PBS buffer solution for 3 times during the dialysis, obtaining AMH monoclonal antibody 1(AMH-CDA) connected with the luminous inclusion compound, and finally storing at-20 ℃ by using 1ml of a storage solution (PBS + 0.05% Proclin-300). The preparation method of the preservation solution (PBS + 0.05% Proclin-300) comprises the following steps: weighing 8g NaCl, 0.2g KCl and 1.44g Na2HPO4And 0.24g KH2PO4Dissolving in 800ml of distilled water, adjusting the pH value of the solution to 7.4 by using HCl, adding 0.5ml of Proclin-300, and finally adding distilled water to a constant volume of 1L, wherein the same is shown below.
Coupling of AMH monoclonal antibody 2 with horseradish peroxidase
0.5mg of HRP was weighed out and dissolved in 100ul of deionized water, 200ul of newly formulated 0.1M sodium periodate solution was added, and stirred at room temperature for 30 minutes in the dark. The reaction solution was added to a dialysis bag with a molecular weight cut-off of 7000, and dialyzed overnight at 4 ℃ in a 1mM sodium acetate buffer solution having a pH of 4.4. After 20ul of 0.2M pH9.5 carbonate buffer was added, 100ul of AMH monoclonal antibody 2 was immediately added at a concentration of 10mg/ml, and then 600ul of 0.01M carbonate buffer was added, and the mixture was gently stirred at room temperature for 2 hours in the dark. Adding 100ul of newly prepared 4mg/ml sodium borohydride solution, uniformly mixing, standing at 4 ℃ for 2 hours. Purifying the reaction solution by adding SUPERDEX 200 gel filtration column, collecting the purified AMH monoclonal antibody 2 and horse radish peroxidase coupled product (AMH-HRP), and storing at-20 deg.C with a storage solution (PBS + 0.05% Proclin-300).
4. Detection of
Preparing an exciting liquid: 25mM tris (pH 8.0), 15mM p-hydroxycinnamic acid, 0.5mM EDTA, 0.1% Tween-20 and 200mM carbamide peroxide.
Preparing a background inhibitor: 20mM hydroxydiethylamine in deionized water.
AMH samples: AMH standard substance is diluted with PBS buffer solution (pH 7.4) to different concentration values of 50ng/ml, 5ng/ml, 1ng/ml, 0.15ng/ml, 0.01ng/ml, 0.002ng/ml, and stored at 4 ℃ to be used as calibration substance. And diluting the AMH standard substance to 50ng/ml, 5ng/ml, 1ng/ml, 0.15ng/ml, 0.01ng/ml and 0.002ng/ml by using AMH negative serum.
Sample adding detection: adopting a double-antibody sandwich method, respectively diluting the prepared AMH-CDA and AMH-HRP by 3000 times and 13000 times by using a PBS buffer solution, respectively adding 50ul of the AMH-CDA and AMH-HRP to a sample to be detected during detection, and additionally adding 10ul of a background inhibitor into a transparent reaction tube. After incubation for 15min at 37 ℃ 100ul of excitation solution was added to start the assay immediately.
And (3) detection results:
as shown in fig. 2, the AMH linear regression equation formula: 3.72925+0.64652x, r 2 0.99475, wherein y represents the logarithm of the luminescence signal value to the base 10 and x represents the logarithm of the concentration of the standard to the base 10. The separation-free chemiluminescence method is used for detecting AMH, and the minimum detection limit of AMH of PBS gradient dilution is 0.002ng/ml, which is basically the same as the detection sensitivity in serum. The linear range is 4 orders of magnitude. In the prior art, the detection limit is generally 0.02ng/ml, the linear detection range is generally 3 orders of magnitude, and the detection sensitivity and the detection linear range of the invention are both improved by one order of magnitude.
Example two, a luminous inclusion compound (beta-CDA) of the beta-cyclodextrin-acridinium ester analog is prepared, CRP monoclonal antibody 1 is coupled with the luminous inclusion compound beta-CDA, and CRP monoclonal antibody 2 is coupled with horseradish peroxidase, and the CRP monoclonal antibody is used for detection by a separation-free chemiluminescence immune sandwich method. The following steps are carried out in sequence:
1. preparation of beta-cyclodextrin-acridinium ester analog luminescence clathrate (beta-CDA)
Weighing 1mg of acridine ester analogue (A) and dissolving the acridine ester analogue in 1ml of DMF, adding the solution into 1ml of 1% beta-cyclodextrin (beta-CD) solution, stirring for 5 hours at 37 ℃, adding 20ml of absolute ethyl alcohol, standing for 2 hours at room temperature, adding the solution into a dialysis bag with cut-off molecular weight of 1500, putting the dialysis bag into PBS buffer solution for dialysis for 1 day at 25 ℃, replacing 3 times of PBS buffer solution during the dialysis, and freeze-drying to obtain the beta-CDA, wherein the whole operation process needs to be protected from light.
CRP monoclonal antibody 1 conjugated beta-CDA
Dissolving 1mg of luminescent inclusion compound beta-CDA in 2ml of DMF, adding 50ul of the dissolved beta-CDA into 100ul of C-reactive protein (CRP) monoclonal antibody 1 with the concentration of 10mg/ml, then adding 800ul of 0.05M boric acid buffer solution and 50ul of 1% glutaraldehyde, and carrying out rotary reaction for 1 hour at room temperature. Adding 1ml of the reaction solution into a dialysis bag with the molecular weight cutoff of 10000, dialyzing in PBS buffer solution at 4 ℃ for 1 day, replacing the PBS buffer solution for 3 times during the dialysis, obtaining the CRP monoclonal antibody 1(CRP-CDA) connected with the luminescent clathrate compound, and finally storing at-20 ℃ by using 1ml of a preservation solution (PBS + 0.05% Proclin-300).
Coupling of CRP monoclonal antibody 2 to horse radish peroxidase
0.5mg of HRP was dissolved in 100ul of deionized water and 200ul of newly formulated 0.1M sodium periodate solution was added and stirred at room temperature for 30 minutes in the dark. The reaction solution was added to a dialysis bag with a molecular weight cut-off of 7000, and dialyzed overnight at 4 ℃ in a 1mM sodium acetate buffer solution having a pH of 4.4. After 20. mu.l of 0.2MPH9.5 carbonate buffer was added, 100. mu.l of CRP monoclonal antibody 2 at a concentration of 10mg/ml was immediately added, 600. mu.l of 0.01M carbonate buffer was then added, and the mixture was gently stirred at room temperature for 2 hours in the dark. 100ul of newly prepared 4mg/ml sodium borohydride solution is added, mixed evenly and placed at 4 ℃ for 2 hours. Purifying the reaction solution with SUPERDEX 200 gel filtration column, collecting purified CRP monoclonal antibody 2, and coupling with horse radish peroxidase
The product (CRP-HRP) was finally stored at-20 ℃ in a storage medium (PBS + 0.05% Proclin-300).
4. Detection of
Preparing an exciting liquid: 25mM tris (pH 8.0), 15mM p-hydroxycinnamic acid, 0.5mM EDTA, 0.1% Tween-20 and 200mM carbamide peroxide.
Preparing a background inhibitor: 20mM hydroxydiethylamine in deionized water.
CRP samples: CRP standard sample is diluted with PBS buffer (pH 7.4) to different concentration values of 2000ng/ml, 500ng/ml, 100ng/ml, 20ng/ml, 5ng/ml, 2ng/ml, and stored at 4 ℃ as calibration sample. And diluting the CRP standard product with CRP negative serum to 2000ng/ml, 500ng/ml, 100ng/ml, 20ng/ml, 5ng/ml and 2 ng/ml.
Sample adding detection: and (2) respectively diluting the prepared CRP-CDA and CRP-HRP by 5000 times and 23000 times by using a PBS buffer solution by adopting a double-antibody sandwich method, respectively adding 50ul of CRP-CDA and CRP-HRP to a sample to be detected during detection, and additionally adding 10ul of background inhibitor into a transparent reaction tube. After incubation for 15min at 37 ℃ 100ul of excitation solution was added to start the assay immediately.
And (3) detection results:
as shown in fig. 3, the CRP linear regression equation formula: 1.94203+0.86444x, r 2 0.99241, wherein y represents the logarithm of the luminescence signal value to the base 10 and x represents the logarithm of the concentration of the standard to the base 10. CRP was detected by separation-free chemiluminescence, and the lowest detection limit of CRP was 2ng/ml with PBS gradient dilution, which was substantially the same as the sensitivity of detection in serum. The linear range is 3 orders of magnitude.
In the third embodiment, a beta-cyclodextrin-acridinium ester analogue luminescent inclusion compound (beta-CDA) is prepared, an AFP monoclonal antibody 1 is coupled with the luminescent inclusion compound beta-CDA, and an AFP monoclonal antibody 2 is coupled with horseradish peroxidase and is used for detection by a separation-free chemiluminescence immune sandwich method. The following steps are carried out in sequence:
1. preparation of beta-cyclodextrin-acridinium ester analog luminescence clathrate (beta-CDA)
Weighing 1mg of acridine ester analogue (A) and dissolving the acridine ester analogue in 1ml of DMF, adding the solution into 1ml of 1% beta-cyclodextrin (beta-CD) solution, stirring for 5 hours at 37 ℃, adding 20ml of absolute ethyl alcohol, standing for 2 hours at room temperature, adding the solution into a dialysis bag with cut-off molecular weight of 1500, putting the dialysis bag into PBS buffer solution for dialysis for 1 day at 25 ℃, replacing 3 times of PBS buffer solution during the dialysis, and freeze-drying to obtain the beta-CDA, wherein the whole operation process needs to be protected from light.
AFP monoclonal antibody 1 coupled beta-CDA
Dissolving 1mg of luminescent clathrate beta-CDA in 2ml of DMF, adding 50ul of the dissolved beta-CDA into 100ul of alpha-fetoprotein (AFP) monoclonal antibody 1 with the concentration of 10mg/ml, then adding 800ul of 0.05M boric acid buffer solution and 50ul of 1% glutaraldehyde, and carrying out rotary reaction for 1 hour at room temperature. Adding 1ml of the reaction solution into a dialysis bag with the molecular weight cutoff of 10000, dialyzing in PBS buffer solution at 4 ℃ for 1 day, replacing the PBS buffer solution for 3 times during the dialysis, obtaining AFP monoclonal antibody 1(AFP-CDA) connected with the luminescent inclusion compound, and finally storing at-20 ℃ by using 1ml of a preservation solution (PBS + 0.05% Proclin-300).
Coupling of AFP monoclonal antibody 2 with Horseradish peroxidase
0.5mg of HRP was dissolved in 100ul of deionized water and 200ul of newly formulated 0.1M sodium periodate solution was added and stirred at room temperature for 30 minutes in the dark. The reaction solution was added to a dialysis bag with a molecular weight cut-off of 7000, and dialyzed overnight at 4 ℃ in a 1mM sodium acetate buffer solution having a pH of 4.4. After 20ul of 0.2MPH9.5 carbonate buffer was added, 100ul of AFP monoclonal antibody 2 was immediately added at a concentration of 10mg/ml, then 600ul of 0.01M carbonate buffer was added, and the mixture was gently stirred at room temperature for 2 hours in the dark. 100ul of newly prepared 4mg/ml sodium borohydride solution is added, mixed evenly and placed at 4 ℃ for 2 hours. Purifying the reaction solution with SUPERDEX 200 gel filtration column, collecting purified AFP monoclonal antibody 2, and coupling with horse radish peroxidase
The product (AFP-HRP) was finally stored at-20 ℃ in a storage solution (PBS + 0.05% Proclin-300).
4. Detection of
Preparing an exciting liquid: 25mM tris (pH 8.0), 15mM p-hydroxycinnamic acid, 0.5mM EDTA, 0.1% Tween-20 and 200mM carbamide peroxide.
Preparing a background inhibitor: 20mM hydroxydiethylamine in deionized water.
AFP samples: AFP standard substance is diluted with PBS buffer (pH 7.4) to different concentration values of 500ng/ml, 200ng/ml, 50ng/ml, 10ng/ml, 2ng/ml and 1ng/ml, and stored at 4 deg.C to be used as calibration substance. Additionally, AFP standard sample is diluted to 500ng/ml, 200ng/ml, 50ng/ml, 10ng/ml, 2ng/ml and 1ng/ml by AFP negative serum.
Sample adding detection: adopting a double-antibody sandwich method, respectively diluting the prepared AFP-CDA and AFP-HRP by 6000 times and 15000 times by PBS buffer solution, respectively adding 50ul of AFP-CDA and AFP-HRP to a sample to be detected, and additionally adding 10ul of background inhibitor into a transparent reaction tube during detection. After incubation for 15min at 37 ℃ 100ul of excitation solution was added to start the assay immediately.
And (3) detection results:
as shown in fig. 4, the AFP linear regression equation formula: 1.90769+1.01736x, r 2 0.97145, wherein y represents the logarithm of the luminescence signal value to the base 10 and x represents the logarithm of the concentration of the standard to the base 10. AFP is detected by a separation-free chemiluminescence immune sandwich method, the minimum detection limit of AFP diluted by PBS gradient is 1ng/ml, and the detection sensitivity is basically the same as that in serum. The linear range is 1-500 ng/ml.
Experimental example one, the following 5 kinds of sera were used as samples to be tested, and the results were obtained as described in table 1 below, according to the methods described in the above example one and comparative example one, respectively.
Serum sample a: detecting AMH negative serum by chemiluminescence immune analysis method in advance;
serum sample B: adding AMH into the serum which is detected to be AMH negative by a chemiluminescence immunoassay method in advance until the concentration is 0.008 ng/ml;
serum sample C: adding AMH into serum which is detected to be AMH negative by a chemiluminescence immunoassay method in advance until the concentration is 0.15 ng/ml;
serum sample D: serum with AMH concentration of 12.22ng/ml detected by chemiluminescence immunoassay in advance;
serum sample E: serum with AMH concentration of 32.44ng/ml detected by chemiluminescence immunoassay in advance;
comparative example one, the acridinium ester analog in example one is directly coupled to AMH monoclonal antibody 1 without inclusion of beta-cyclodextrin; the rest is equal to the first embodiment (detection judgment is carried out after the corresponding detection standard curve is obtained).
TABLE 1
Experiment example two, the following 5 kinds of sera were used as samples to be tested, and the results were obtained as described in table 2 below by testing according to the methods described in the above example two and comparative example two, respectively.
Serum sample F: CRP negative serum detected by chemiluminescence immunoassay in advance;
serum sample G: CRP is added into serum which is detected as CRP negative by a chemiluminescence immunoassay method in advance to reach the concentration of 2.21 ng/ml;
serum sample H: CRP is added into serum which is detected as CRP negative by a chemiluminescence immunoassay method in advance to reach the concentration of 20.15 ng/ml;
serum sample I: serum with CRP concentration of 124.43ng/ml detected by chemiluminescence immunoassay;
serum sample J: serum with CRP concentration of 632.37ng/ml detected by chemiluminescence immunoassay;
comparative example II, the acridinium ester analogs in example II were directly coupled to CRP monoclonal antibody 1 without inclusion with β -cyclodextrin; the rest is equal to the second embodiment (detection judgment is carried out after obtaining the corresponding detection standard curve).
TABLE 2
Experiment example three, the following 5 kinds of sera were used as samples to be tested, and the results were obtained as described in table 3 below, according to the methods described in example three and comparative example three, respectively.
Serum sample K: AFP negative serum detected by chemiluminescence immunoassay in advance;
serum sample L: CRP is added into serum which is detected to be AFP negative by a chemiluminescence immunoassay method in advance to reach the concentration of 1.75 ng/ml;
serum sample M: CRP is added into serum which is detected to be AFP negative by a chemiluminescence immunoassay method in advance to reach the concentration of 15.32 ng/ml;
serum sample N: serum with AFP concentration of 87.99ng/ml detected by chemiluminescence immunoassay in advance;
serum sample O: serum with AFP concentration of 274.55ng/ml detected by chemiluminescence immunoassay;
comparative example III, the acridinium ester analogs of example III were directly coupled to AFP monoclonal antibody 1 without inclusion with β -cyclodextrin; the rest is equal to the first embodiment (detection judgment is carried out after the corresponding detection standard curve is obtained).
TABLE 3
The above-described embodiments are merely preferred embodiments of the present invention, which is not intended to be limiting in any way, and other variations and modifications are possible without departing from the scope of the invention as set forth in the appended claims.
Claims (10)
1. A separation-free chemiluminescent immunoassay method based on cyclodextrin inclusion is characterized in that an antibody 1 is linked with an acridinium ester analogue included by beta-cyclodextrin to obtain a luminous inclusion compound linked antibody 1; connecting the antibody 2 with horseradish peroxidase to obtain an enzyme-connected antibody 2; and mixing and incubating the antibody 1 connected with the luminous inclusion compound, the antibody 2 connected with the enzyme, the sample to be detected and the background inhibitor, adding an excitation liquid, and detecting a luminous signal value.
2. The method for separation-free chemiluminescence immunoassay based on cyclodextrin inclusion according to claim 1, wherein the method comprises the following steps:
(1) beta-cyclodextrin clathrating acridine ester analog to obtain luminescent clathrate;
(2) the antibody 1 is connected with a luminous inclusion compound to obtain an antibody 1-beta-CDA;
(3) connecting the antibody 2 with horseradish peroxidase to obtain an antibody 2-HRP;
(4) and (3) detection: mixing the detection sample, the background inhibitor, the antibody 1-beta-CDA and the antibody 2-HRP, incubating for 15 minutes at 37 ℃, adding an excitation liquid, and detecting a luminescent signal generated within 1 second.
3. The method for separation-free chemiluminescence immunoassay based on cyclodextrin inclusion according to claim 2, wherein the background inhibitor of step (4) is 20mM hydroxydiethylamine solution.
4. The method for non-separation chemiluminescence immunoassay based on cyclodextrin inclusion according to claim 2, wherein the excitation solution of step (4) is a mixed solution containing 25mM tris, 15mM p-hydroxycinnamic acid, 0.5mM EDTA, 0.1% Tween-20 and 200mM carbamide peroxide.
5. The method for separation-free chemiluminescence immunoassay based on cyclodextrin inclusion according to claim 2, wherein in step (4), the antibody 1- β -CDA and the antibody 2-HRP are diluted separately and then mixed in a volume ratio of 5: 5: 5: 1 adding diluted antibody 1-beta-CDA, diluted antibody 2-HRP, sample to be detected and background inhibitor, incubating for 15 minutes at 37 ℃, adding excitation liquid with 10 times volume of the background inhibitor, and detecting luminescent signals generated within 1 second.
6. The method for separation-free chemiluminescence immunoassay based on cyclodextrin inclusion according to claim 2, wherein the method further comprises the step (5) of determining: and (3) taking the standard substance of the antigen to be detected as a detection sample, preparing a standard substance solution with gradient concentration, carrying out detection in the step (4), and drawing a concentration-luminescence signal curve graph to obtain a concentration-luminescence signal formula of the standard substance.
7. The method for separation-free chemiluminescence immunoassay based on cyclodextrin inclusion according to claim 6, wherein step (5) further comprises substituting the concentration-luminescence signal formula of step (5) with the detection sample to be tested after the detection of step (4) is performed and the luminescence signal is obtained, to obtain the concentration of the antigen to be tested in the detection sample to be tested.
8. The method for separation-free chemiluminescence immunoassay based on cyclodextrin inclusion according to claim 2, wherein the specific preparation method of step (1) is as follows: weighing 1mg of acridine ester analogue A, dissolving the acridine ester analogue A in 1ml of DMF to obtain acridine ester analogue solution, adding the solution into 1ml of 1% beta-cyclodextrin beta-CD aqueous solution, stirring for 5 hours at 37 ℃, adding 20ml of absolute ethyl alcohol, standing for 2 hours, adding the solution into a dialysis bag with cut-off molecular weight of 1500, putting the dialysis bag into PBS buffer solution, dialyzing for 1 day at 25 ℃, replacing 3 times of PBS buffer solution during dialysis, and freeze-drying to obtain beta-CDA; the operation process of the step (1) is protected from light.
9. The method for separation-free chemiluminescence immunoassay based on cyclodextrin inclusion according to claim 2, wherein the specific preparation method of step (2) is as follows: dissolving 1mg of luminescent inclusion compound beta-CDA in 2ml of DMF, adding 50ul of the dissolved beta-CDA into 100ul of antibody 1 with the concentration of 10mg/ml, then adding 800ul of 0.05M boric acid buffer solution and 50ul of 1% glutaraldehyde, and reacting for 1 hour; adding 1ml of the reaction solution into a dialysis bag with cut-off molecular weight of 10000, dialyzing in PBS buffer solution at 4 ℃ for 1 day, replacing the PBS buffer solution for 3 times during the dialysis to obtain the antibody 1-beta-CDA, and finally storing at-20 ℃ by using 1ml of a preservation solution (PBS + 0.05% Proclin-300).
10. The method for separation-free chemiluminescence immunoassay based on cyclodextrin inclusion according to claim 2, wherein the specific preparation method of step (3) is as follows: weighing 0.5mg HRP, dissolving in 100ul deionized water, adding 200ul of newly-prepared 0.1M sodium periodate solution, and stirring in the dark for 30 minutes; adding the reaction solution into a dialysis bag with the molecular weight cutoff of 7000, and dialyzing in 1mM sodium acetate buffer solution with pH4.4 at 4 ℃ overnight; adding 20ul of 0.2M carbonate buffer solution with pH of 9.5, adding 100ul of antibody 2 with concentration of 10mg/ml, adding 600ul of 0.01M carbonate buffer solution, and stirring for 2 hours in a dark place; adding 100ul of 4mg/ml sodium borohydride solution, uniformly mixing, and placing at 4 ℃ for 2 hours; purifying the reaction solution with SUPERDEX 200 gel filtration column, collecting purified antibody 2-HRP, and storing at-20 deg.C with a storage solution.
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