CN115144585A - ST2 detection method based on magnetic particle time-resolved fluoroimmunoassay - Google Patents
ST2 detection method based on magnetic particle time-resolved fluoroimmunoassay Download PDFInfo
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Abstract
The invention discloses an ST2 detection method based on a magnetic particle time-resolved fluoroimmunoassay method, which comprises the steps of preparing magnetic particles coating ST2 antibodies, preparing europium-labeled ST2 antibodies, mixing and incubating samples to be detected, europium-labeled antibodies and magnetic particles coupled by the antibodies, washing under the action of magnetic force after reaction, removing unconjugated substances, adding enhancement liquid to dissociate and enhance europium, and determining a fluorescence value by using a time-resolved fluoroimmunoassay instrument.
Description
Technical Field
The invention relates to a detection method, in particular to an ST2 detection method based on a magnetic particle time-resolved fluorescence immunoassay, and belongs to the field of biotechnology detection.
Background
The serum marker is used for clinical diagnosis and prognosis judgment, can quantify more quickly, prompt the possibility of sepsis occurrence and help clinicians to adopt correct treatment schemes in time. Growth-stimulating expression factor 2 (ST 2), also called oncostatin 2 (ST 2), is a member of the leukocyte receptor family, has a TOLL-like receptor domain, and is expressed on the surface of a variety of immune cells. ST2 is associated with the severity of sepsis and when its plasma mass concentration is greater than 35ng/ml, the patient's mortality rate rises dramatically. More and more studies have shown that ST2 may have a higher diagnostic and prognostic value for sepsis than CRP, PCT, and IL-6. ST2 is used as a receptor of IL-33, and is combined with IL-33 to form an IL-33/ST2 signal pathway, which can promote the recruitment of neutrophils, cause I/II type immune response and has proinflammatory effect. Thus, ST2 can be used as a serum marker that suggests sepsis.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides the ST2 detection method based on the magnetic particle time-resolved fluorescence immunoassay, which has the technical characteristics of capability of quickly detecting the content of ST2 in a sample to be detected, strong specificity, high sensitivity, good repeatability and the like.
In order to achieve the purpose, the invention is realized by the following technical scheme:
an ST2 detection method based on a magnetic particle time-resolved fluoroimmunoassay method comprises the following steps: the method comprises the steps of magnetic particle-ST 2 coated antibody coupling and europium-labeled detection antibody, mixing and incubating a sample to be detected, the europium-labeled antibody and the ST2 antibody covalently coupled magnetic particles to form a magnetic particle-ST 2 coated antibody-europium-labeled ST2 detection antibody fluorescent immune complex, adding enhancement liquid to perform dissociation enhancement on europium, and determining a fluorescence value;
wherein the europium-labeled detection antibody comprises:
adding the antibody raw material to be labeled into a 30KD centrifugal tube with a filter membrane, adding 200 mu L of europium-labeled protein buffer solution into the concentrated antibody raw material, centrifuging at 10000rpm for 5min to remove filtrate, adding 200 mu L of elution buffer solution, and repeating the centrifuging step for 8 times; taking out the centrifugal tube after several times of centrifugation, discarding filtrate, turning the filter membrane of the centrifugal tube back, centrifuging at 3000rpm for lmin, and collecting filtrate; then reversing the filter membrane again, adding 100 mu L of labeled buffer solution, standing for 1-5 min to enable the filter membrane to absorb the labeled protein buffer solution, dissolving the residual antibody raw material left on the filter membrane, then reversing the filter membrane again, centrifuging at 3000rpm for 1min, collecting liquid on the filter membrane to enable the antibody raw material on the filter membrane to be recovered as much as possible, and obtaining the concentrated antibody concentrated and decontaminated;
according to the mass ratio of the antibody: europium chelate =5, the mixture is fully mixed and labeled respectively, the mixture is placed in a constant temperature incubator at 25 ℃ and is shaken in the dark for 16 hours, the reaction solution is transferred to a Sephadex-G50 column pre-balanced by elution buffer solution for purification, 1 mL/tube eluent is collected, 5 mu L of each tube is taken and added into a 96 micropore plate, 100 mu L of enhancement solution is added into each hole, the mixture is placed on an oscillator for shaking for 5 minutes and then is measured, a plurality of tubes are selected according to the fluorescence value and are combined and collected and placed in a refrigerator at 4 ℃ for storage.
Preferably, europium benzyldiethylenetriamine tetraacetate isothiocyanate (DTTA-Eu) is used as the bifunctional chelating reagent, wherein one end of the bifunctional chelating reagent chelates europium and the other end of the bifunctional chelating reagent can be contacted with NH of the antibody 2 Ligation, 0.2mg europium chelate per 1mg antibody.
Preferably, the magnetic particle-ST 2 coated antibody coupling comprises:
adding 10mg of magnetic particles into a 2mL centrifuge tube in one batch, sequentially adding EDC and NHS solution, activating the surfaces of the magnetic particles for 30min under the conditions of room temperature and acidity, then cleaning the magnetic particles by using a magnetic frame, adding 0.1mg of filtered and concentrated antibody, shaking for 16h at room temperature in a dark place, removing the antibody which is not excessive by using the magnetic frame, adding TBST, and preserving the antibody at 2-8 ℃;
preferably, the forming of the magnetic particle-ST 2 coated antibody-europium-labeled ST2 detection antibody fluorescent immune complex comprises:
adding a microsphere-ST 2 coated antibody conjugate into a 96-hole enzyme label plate, and incubating a europium-labeled ST2 detection antibody and a sample to be detected with the microsphere at the same time, wherein the incubation conditions are as follows: and (3) keeping out of the sun, shaking at 37 ℃ for 12min to obtain the microsphere-ST 2 coated antibody-ST 2 antigen-europium label ST2 detection antibody fluorescent immune complex, adding an enhancement solution, shaking for 5min, and detecting the fluorescence intensity on a computer to determine the content of the detection index.
Preferably, 28. Mu.L and 40. Mu.L each of 10mg/mL NHS and 10mg/mL EDC are added per 10mg of magnetic particles.
Preferably, the kit further comprises a kit and a plurality of kits arranged in the kit, wherein the kit is provided with the kit for placing the magnetic microspheres, and the magnetic microspheres comprise magnetic microspheres respectively coated with the ST2 antibody; the kit is provided with a kit for placing detection antibodies, wherein the detection antibodies comprise ST2 antibodies which are respectively marked with europium, the kit for placing ST2 standard solution and the kit for placing buffer solution, and the buffer solution is an analysis buffer solution with pH 7.8 and an enhancement solution with pH 3.2; the kit is internally provided with an expandable polystyrene foam layer and comprises a box body and a box cover, the box body and the box cover are connected by a connecting shaft made of phenolic plastics, and an ice groove for storing crushed ice is prefabricated at the bottom of the kit.
Has the advantages that: the kit can quickly detect the content of ST2 in a sample to be detected, has the characteristics of strong specificity, high sensitivity, good repeatability and the like, can be used for clinical auxiliary diagnosis of sepsis, and has a good application prospect.
Drawings
FIG. 1ST2 shows a standard curve.
Detailed Description
The present invention will be further described with reference to the drawings attached to the specification, but the present invention is not limited to the following examples.
The technical solution of the invention is as follows:
firstly, coating an ST2 coated antibody on the surface of a magnetic microsphere to form an immunomagnetic bead-antibody compound, coating 0.1mg of antibody on 10mg of magnetic bead, and rotating the antibody and the microsphere at room temperature in a dark place for 16h. And washing and separating the magnetic microspheres by using a centrifugal tube and a magnetic rack to remove the unbound antibodies. Europium is labeled to the ST2 detection antibody, the using amount of the antibody is optimized, the appropriate using amount of the antibody is determined, and the antibody is diluted by an analysis buffer solution to avoid excessive detection antibody. And (3) incubating the magnetic beads, the europium-labeled detection antibody and the sample to be detected with optimized doses at 37 ℃ and oscillating for 12min to finally form a magnetic bead-ST 2 coated antibody-ST 2 antigen-europium-labeled ST2 detection antibody fluorescent immune complex, and performing fluorescence detection analysis by using a time resolution instrument. The coated antibody refers to an ST2 coated antibody. The coated antibody magnetic beads are formed by activating carboxyl on the surfaces of immunomagnetic beads [ 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxythiosuccinimide (NHS) ] to form covalent bonds with amino on ST2 coated antibodies, so as to form a complex. The detection antibody is europium-labeled ST2 monoclonal antibody. The centrifugal tube and the magnetic frame in the sample preparation process can be replaced by an ELISA plate and a 96-pore plate magnetic frame.
The fluorescence detection and analysis means that by utilizing the characteristic that lanthanide rare earth chelate has long-life fluorescence, after a sample is excited by pulsed light and before a fluorescence signal is collected, a certain time is delayed according to different fluorescence decay times of different fluorescent substances contained in the sample, and after short-life background fluorescence in a sample to be detected is completely quenched in the time period, the long-life rare earth chelate specific fluorescence signal is collected and detected. Interference from other non-specific fluorescence of samples, reagents, instruments and the like can be effectively eliminated through time resolution delay, so that the detection sensitivity is greatly improved.
Sepsis is a disease characterized by an imbalance in the metabolic and immune response abilities caused by infection of the body. As the condition worsens, multiple organ dysfunction and failure may occur, exacerbating systemic inflammation, and in severe cases presenting as life-threatening complications, one of the leading causes of death worldwide. Sepsis is high in fatality rate and complex in pathogenesis, and causes great harm and economic burden to human health. Early diagnosis of sepsis can increase the cure rate of sepsis and is key to reducing mortality. The invention creatively adopts ST2 as a serum marker for prompting the ST2 to be sepsis, and combines with the immune magnetic particles and europium to detect the ST2 concentration of a sample to be detected, so that the ST2 content in human serum can be quickly and accurately detected. The magnetic particle time resolution technology utilizes immune magnetic particles as a carrier, takes time resolution as a detection platform, and can carry out large-scale rapid detection on a sample in a short time. The system is mainly based on immune magnetic particles, has high detection sensitivity and rapid response, can realize rapid evaluation of sepsis, and has wider application space.
The present application will be described in further detail below with reference to fig. 1 and examples 1 to 4.
Example 1 magnetic microparticle-ST 2 coated antibody conjugation
Adding 10mg microspheres into a 2mL centrifuge tube, sequentially adding EDC and NHS solution, activating the surfaces of the microspheres for 30min at room temperature under acidic conditions, cleaning the microspheres by using a magnetic frame, adding 0.1mg of filtered and concentrated antibody, shaking at room temperature in a dark place for 16h, removing the antibody which is not excessive by using the magnetic frame, adding TBST, and storing the antibody at 2-8 ℃.
Example 2 europium-labeled detection antibody
Adding the antibody raw material to be labeled into a 30KD centrifugal tube with a filter membrane, adding 200 mu L of europium-labeled protein buffer solution into the concentrated antibody raw material, centrifuging at 10000rpm for 5min to remove filtrate, adding 200 mu L of elution buffer solution, and repeating the centrifugation step for 8 times. Taking out the centrifugal tube after several times of centrifugation, discarding the filtrate, turning the filter membrane of the centrifugal tube back, centrifuging at 3000rpm for lmin, and collecting the filtrate. And then, reversing the filter membrane again, adding 100 mu L of labeled buffer solution, standing for a moment to enable the filter membrane to absorb the labeled protein buffer solution, dissolving the residual antibody raw material left on the filter membrane, reversing the filter membrane again, centrifuging at 3000rpm for 1min, collecting liquid on the filter membrane, and recycling the antibody raw material on the filter membrane as much as possible, namely the required concentrated antibody raw material. The optimal volume for antibody collection throughout the procedure is around 200. Mu.L. After the antibody is concentrated and decontaminated, the antibody is prepared by the following steps of: europium chelate =5, the mixture is fully mixed and labeled respectively, and the mixture is put into a constant temperature incubator at 25 ℃ and is shaken in the dark for 16 hours. The reaction was transferred to a Sephadex-G50 column pre-equilibrated with elution buffer for purification, and the eluate was collected (1 mL/tube). Adding 5 μ L of the obtained product into 96 micro-porous plate, adding 100 μ L of enhancing solution into each well, placing on oscillator, oscillating for 5min, measuring, selecting several tubes according to fluorescence value, collecting, and storing in refrigerator at 4 deg.C.
Example 3 magnetic microparticles-ST 2 coated antibody-europium labeled ST2 detection antibody fluorescent immune Complex formation
Adding a microsphere-ST 2 coated antibody conjugate into a 96-hole enzyme label plate, incubating a europium-labeled ST2 detection antibody and a sample to be detected with the microsphere at the same time, wherein the incubation condition is dark and oscillating at 37 ℃ for 12min to obtain a microsphere-ST 2 coated antibody-ST 2 antigen-europium-labeled ST2 detection antibody fluorescence immune complex, adding an enhancement solution, oscillating for 5min, and then detecting the fluorescence intensity on a computer to determine the content of a detection index. The non-specific interference in the sample is eliminated by identifying and utilizing a time-resolved fluorescence measurement method and a spectral separation technology, and the fluorescence intensity in the final product is measured, so that the detection sensitivity is effectively improved.
Example 4 linear range, sensitivity, specificity, precision and recovery of ST2 detection.
Preparing an ST2 standard substance, detecting, drawing a standard curve, drawing the standard curve by using a fluorescence value and a concentration value, wherein a linear equation of ST2 detection is as follows: y =3009X-33531, R2=0.9991, linear range 5ng/mL to 500ng/mL, results are shown in fig. 1.
And (3) parallelly measuring the fluorescence value of the 0ng/mL reference standard substance for 10 times, calculating the mean value (mean) and the Standard Deviation (SD), and substituting mean +2SD into a standard curve equation to calculate the detection sensitivity of the kit to the PSP to be 1.446ng/mL.
The antigens of the interference factors heparin-binding protein (HBP), procalcitonin (PCT) and interleukin-6 (IL-6) were measured 2 times and their concentrations were calculated by a standard curve. Cross-reactivity (%) = detected concentration/actual concentration. The results are shown in Table 1, and the kit obtained by calculation has almost no cross reaction rate on HBP, PCT and IL-6 and has good specificity.
TABLE 1PSP detection specificity
The results of testing the low value sample, the median sample and the high value sample of the ST2 10 times respectively are shown in Table 2, and the intra-batch variation coefficient of the ST2 test is 3.63% -8.52% (< 10%), and the inter-batch variation coefficient is 6.09% -10.10% (< 15%).
TABLE 2ST2 analytical precision
100ng/mL,500ng/mL ST2 standards were mixed with serum at a ratio of 1. The concentration of the mixed solution was measured by a standard curve and set as the actually measured concentration. The initial concentration of the serum sample was 168.40ng/mL. After addition of the standard solution, the final theoretical concentration of ST2 in the serum sample was calculated to be 161.56 and 201.56ng/mL, respectively. The recovery rate calculation formula is as follows: recovery (%) = actual concentration/theoretical concentration. The results are shown in Table 3, with recoveries ranging from 96.29% to 97.93%.
TABLE 3ST2 assay recovery
Finally, it should be noted that the present invention is not limited to the above embodiments, and many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (5)
1. An ST2 detection method based on a magnetic particle time-resolved fluoroimmunoassay method is characterized in that the method comprises the following steps: the method comprises the steps of magnetic particle-ST 2 coated antibody coupling and europium-labeled detection antibody, mixing and incubating a sample to be detected, the europium-labeled antibody and the ST2 antibody covalently coupled magnetic particles to form a magnetic particle-ST 2 coated antibody-europium-labeled ST2 detection antibody fluorescent immune complex, adding enhancement liquid to perform dissociation enhancement on europium, and determining a fluorescence value;
wherein the europium labeled detection antibody comprises:
adding the antibody raw material to be labeled into a 30KD centrifugal tube with a filter membrane, adding 200 mu L of europium-labeled protein buffer solution into the concentrated antibody raw material, centrifuging at 10000rpm for 5min to remove filtrate, adding 200 mu L of elution buffer solution, and repeating the centrifuging step for 8 times; taking out the centrifugal tube after several times of centrifugation, discarding filtrate, returning the filter membrane of the centrifugal tube, centrifuging at 3000rpm for lmin, and collecting filtrate; then inverting the filter membrane again, adding 100 mu L of labeled buffer solution, standing for 1-5 min to enable the filter membrane to absorb the labeled protein buffer solution, dissolving the residual antibody raw material left on the filter membrane, inverting the filter membrane again, centrifuging at 3000rpm for 1min, collecting liquid on the filter membrane to enable the antibody raw material on the filter membrane to be recovered as much as possible, and obtaining concentrated antibody concentrated for removing impurities;
according to the mass ratio of the antibody: europium chelate =5, the mixture is fully mixed and labeled respectively, the mixture is placed in a constant temperature incubator at 25 ℃ in a dark place and is vibrated for 16 hours, reaction liquid is transferred to a Sephadex-G50 column pre-balanced by elution buffer for purification, 1 mL/tube of eluent is collected, 5 mu L of the eluent is taken from each tube and is added into a 96 micro-porous plate, 100 mu L of enhancement liquid is added into each hole, the mixture is placed on an oscillator and is vibrated for 5 minutes, then the value is measured, and a plurality of tubes are selected according to the fluorescence value, are combined and collected and are placed in a refrigerator at 4 ℃ for storage.
2. The method for detecting ST2 based on magnetic particle time-resolved fluoroimmunoassay according to claim 1, wherein the method comprises the following steps: europium isothiocyanatobenzyldiethylenetriamine tetraacetate (DTTA-Eu) is used as a bifunctional chelating reagent, one end of the bifunctional chelating reagent chelates europium, and the other end of the bifunctional chelating reagent can be chelated with NH of an antibody 2 Ligation, 0.2mg europium chelate per 1mg antibody.
3. The method for detecting ST2 based on magnetic particle time-resolved fluoroimmunoassay according to claim 1 or 2, wherein: the magnetic particle-ST 2 coated antibody coupling comprises:
adding 10mg of magnetic particles into a 2mL centrifuge tube, sequentially adding EDC and NHS solution, activating the surfaces of the magnetic particles for 30min at room temperature under acidic conditions, cleaning the magnetic particles by using a magnetic frame, adding 0.1mg of antibody after filtering and concentrating, shaking for 16h at room temperature in a dark place, removing the antibody which is not excessive by using the magnetic frame, adding TBST, and preserving the antibody at 2-8 ℃.
4. The method for detecting ST2 based on magnetic particle time-resolved fluoroimmunoassay according to claim 1, wherein:
the formation of the magnetic particle-ST 2 coated antibody-europium-labeled ST2 detection antibody fluorescent immune complex comprises the following steps:
adding a microsphere-ST 2 coated antibody conjugate into a 96-hole enzyme label plate, and incubating a europium-labeled ST2 detection antibody and a sample to be detected with the microsphere at the same time, wherein the incubation conditions are as follows: and (3) keeping out of the sun, shaking at 37 ℃ for 12min to obtain the microsphere-ST 2 coated antibody-ST 2 antigen-europium label ST2 detection antibody fluorescent immune complex, adding an enhancement solution, shaking for 5min, and detecting the fluorescence intensity on a computer to determine the content of the detection index.
5. The method for detecting ST2 based on magnetic particle time-resolved fluoroimmunoassay according to claim 3, wherein: each of 28. Mu.L and 40. Mu.L of NHS at 10mg/mL and EDC at 10mg/mL was added per 10mg of magnetic particles.
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