CN111381022A - Enrichment method of magnetic microspheres for myocardial injury and kidney injury serum markers - Google Patents
Enrichment method of magnetic microspheres for myocardial injury and kidney injury serum markers Download PDFInfo
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Abstract
The invention provides a method for enriching serum markers of myocardial injury and renal injury by magnetic microspheres, which comprises the following steps: step one, preparing carboxyl magnetic microspheres; step two, forming immune magnetic microspheres by activating and coupling specific antibodies corresponding to the markers; step three, removing the marker protein in the enriched serum by using the immunomagnetic microspheres; and step four, eluting the protein by using an eluent, and performing qualitative and quantitative analysis. The invention can provide an effective method for developing traceable standard substances for a metering laboratory, a reference laboratory, an in-vitro diagnostic reagent manufacturer and a clinical examination organization.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for enriching serum markers of myocardial injury and renal injury by magnetic microspheres.
Background
Clinical practice has been to find a variety of markers that respond (characterize) to myocardial tissue injury and renal injury in succession. As associated with myocardial injury: cTnI (troponin I), NT-ProBNP (B-type amino-terminal pro-natriuretic peptide), CRP (human C-reactive protein), etc., associated with kidney injury: cys C (cystatin C), C-Peptide (C Peptide), NGAL (neutrophil gelatinase-associated lipocalin), and the like. Normally, these markers are present in very small amounts in the blood. However, in the case of tissue damage, the levels of these markers in the blood may vary significantly. At present, the markers are detected clinically by a very mature rapid detection method, such as a chemiluminescence immunoassay, a gold-labeled immunoassay, an immunoturbidimetry and the like. The standard substances used in these tests are mostly obtained synthetically.
The detection method which is common in clinic is a rapid detection result, which is very convenient in diagnosis, but is not easy if the standard substance is traced by the method. Currently, there is no application scheme developed for standard substances of diagnostic markers in enriched serum on the market.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the enrichment method of the serum marker of myocardial injury and renal injury by the magnetic microspheres, the invention provides the traceable standard substance by enriching the low-abundance protein and peptide in the serum by aiming at the immune magnetic microspheres of the marker, and provides an effective method for developing the traceable standard substance for a metering laboratory, a reference laboratory, an in-vitro diagnostic reagent manufacturer and a clinical inspection institution.
In order to achieve the technical purpose, the technical scheme of the invention is that the method for enriching the serum markers of myocardial injury and renal injury by using the magnetic microspheres comprises the following steps: step one, preparing carboxyl magnetic microspheres; step two, forming the immune magnetic microsphere by activating and coupling the specific antibody corresponding to the marker on the carboxyl magnetic microsphere; enriching marker protein in serum by using the immunomagnetic microspheres; eluting the marker protein enriched by the immunomagnetic microspheres by using an eluent, and performing qualitative and quantitative analysis; the enrichment method of the magnetic microspheres for the myocardial injury and kidney injury serum markers is not used for disease diagnosis and treatment.
Preferably, the particle size of the carboxyl magnetic microsphere in the first step is 300nm-500 nm.
Preferably, the particle size of the carboxyl magnetic microsphere in the first step is 300 nm.
Preferably, the preparation process of the carboxyl magnetic microsphere in the first step is as follows: first using FeCl3·6H2Preparation of Fe from O3O4Magnetic core, then to Fe3O4Magnetic core coated SiO2Form Fe3O4@SiO2And then MPS (gamma- (methacryloyloxy) propyltrimethoxysilane) is added to react to form Fe3O4@SiO2MPS, followed by coating with GMA (glycidyl methacrylate), MBA (N, N' -methylenebisacrylamide) to form Fe3O4@SiO2-MPS @ P (GMA-MBA), and finally sodium carbonate, sodium bicarbonate and 6-aminocaproic acid, sodium borohydride to form Fe3O4@SiO2-MPS @ P (GMA-MBA) -COOH, the carboxylic magnetic microsphere of step one.
Preferably, the Fe3O4@SiO2MPS coating of GMA, MBA to Fe3O4@SiO2-MPS @ P (GMA-MBA) step, Fe3O4@SiO2MPS is added in a proportion of 3g Fe to GMA, MBA, AIBN3O4@SiO2MPS 9mL GMA, 9.0g MBA, 360mg AIBN were added.
Preferably, in the second step, the carboxyl magnetic microsphere is activated by using an EDC/Sulfo-NHS aqueous phase activation method, and the activated carboxyl magnetic microsphere reacts with a specific antibody solution corresponding to the marker to obtain the immunomagnetic microsphere.
Preferably, the specific method of the second step is as follows: and (2) washing the carboxyl magnetic microspheres by using a MEST buffer, adding EDC and Sulfo-NHS for activation, adding a corresponding antibody solution for reaction, sealing by using a BSA solution, and finally washing and storing to form the immunomagnetic microspheres.
Preferably, the enrichment process of step three is to mix and incubate the immunomagnetic microspheres with the target serum sample.
Preferably, the eluent of the fourth step is selected from glycine-hydrochloric acid solution with pH2.5-3.1 and 0.1M, citric acid solution with pH2.5-3.1 and 0.1M, acetic acid solution with 2.5 percent, ammonium hydroxide solution with pH10.0-11.0 and 0.1M.
Preferably, the myocardial injury serum marker is cTnI, NT-proBNP and CRP, and the kidney injury serum marker is Cys C, C-Peptide and NGAL.
The basic steps of the method are firstly preparing a carboxyl magnetic microsphere with short magnetic response time, long suspension time, uniform particle size and high enough petunidin content, then forming a unique immunomagnetic microsphere by activating and coupling a specific antibody corresponding to the myocardial injury and kidney injury serum markers, then removing the marker protein in enriched serum by the immunomagnetic microsphere, finally eluting the marker protein by eluent, and qualitatively and quantitatively determining.
The tool used in the method is carboxyl magnetic microsphere with the grain diameter of 300nm-500nm (especially 300nm), and the preparation method is as follows, firstly FeCl is used3·6H2Preparation of Fe from O3O4Magnetic core, then coating the magnetic core with SiO2Form Fe3O4@SiO2And then MPS reacts to form Fe3O4@SiO2MPS, subsequent coating with GMA, MBA to Fe3O4@SiO2-MPS @ P (GMA-MBA), and finally sodium carbonate, sodium bicarbonate and 6-aminocaproic acid, sodium borohydride to form Fe3O4@SiO2MPS @ P (GMA-MBA) -COOH, a carboxylic magnetic microsphere used in the present invention. And (3) after the reaction is finished, removing the reaction device, separating the magnetic microspheres by using a magnetic separator, repeatedly washing the magnetic microspheres for more than 5 times by using purified water, finally storing the magnetic microspheres in the purified water, and testing the mass concentration. After the concentration of the magnetic microspheres is calibrated, taking the magnetic microspheres for an electron microscope, observing the coating effect of the polymer, and testing whether residual magnetism exists or not, wherein the particle size is uniform. And testing the magnetic attraction time and the suspension time. And after all quality tests reach the standard, putting the product into the next step for use.
The activation of the carboxyl magnetic microspheres adopts a method of EDC/Sulfo-NHS aqueous phase activation. EDC is a water-soluble carbodiimide. Can be used as activating agent for carboxyl in amide synthesis, and can be used for activating phosphate group, crosslinking protein and nucleic acid, and preparing immune conjugate. The pH range of use is 4.0-6.0, and is often used in combination with N-hydroxysuccinimide (NHS) or N-hydroxythiosuccinimide (Sulfo-NHS) to improve coupling efficiency. NHS is an additive in the improved amidation and peptide coupling reactions to activate the carbonyl group upon amide bond formation. Sulfo-NHS is a derivative of NHS, has better water solubility than NHS, and has negative charge to prevent polymerization of the attached substrate (such as protein). The reaction can be selected from NHS and Sulfo-NHS, and the effect of the Sulfo-NHS is better. The preparation method comprises the steps of washing 10mg/mL carboxyl magnetic microspheres by using MEST buffer (pH5.0-6.0), adding half volume of 20mg/mL EDC and 20mg/mL Sulfo-NHS to MEST, activating for 15min, adding 0.1-1.0 mg/mL antibody solution for reacting for two hours, then sealing for two hours by using 1% BSA solution, and finally washing and storing to form the immunomagnetic microspheres.
The antibody is a rabbit anti-human polyclonal antibody, the purity of SDS-PAGE detection is not lower than 90%, the lower limit of ELISA detection color development is lower than 5ng/ml, and the antibody has no non-specific combination with unrelated antigens. The standard control of the serum marker is recombinant protein, the purity is qualified, the concentration of the serum marker used as a plate coating antigen in an ELISA experiment can be successfully coated at 1-3 mu g/ml, the serum marker specifically recognizes a corresponding antibody, and the serum marker is not in non-specific combination with an unrelated antibody.
After the preparation of the immunomagnetic microspheres is finished, the marker protein in the serum begins to be enriched, and the enrichment process is the mixing incubation of the immunomagnetic microspheres and the serum sample. Immediately after the incubation, the elution is carried out, and the eluate is neutralized. The eluent used was 0.1M glycine-HCl, pH2.5-3.1 (glycine-hydrochloric acid), and an alternative eluent was 0.1M citric acid, pH2.5 to 3.1 (citric acid), 2.5% acetic acid (acetic acid), 0.1M ammonium hydroxide, pH10.0-11.0 (ammonium hydroxide). If a low pH elution buffer is used, a more basic 1M phosphate neutralization buffer or 1M Tris, pH7.5-9.0 neutralization buffer is required and added at a ratio of 1: 10.
The enriched protein solution was preliminarily identified by enzyme-linked immunoassay. A standard antigen detection kit, a double-antibody sandwich method detection and a standard substance. Coating an antibody plate, sealing with a confining liquid, incubating a standard substance and a sample, detecting a detection antibody marked by HRP, developing TMB, and reading after termination. The concentration of the recovered protein was calculated in comparison to the standard curve. This step can also be performed using a chemiluminescent immunoassay kit, which is further characterized by chromatography.
The particle size of the selected magnetic microspheres is 300nm-500nm, the particle size of common immune magnetic microspheres in the market is more than or equal to 1 mu m, and the magnetic microspheres with larger particle size generally have the problems of short suspension time and inconvenience for subsequent reaction. In patent documents, microspheres smaller than 150 nm are used, but if the particle size is too small, the magnetism of the magnetic microspheres cannot be guaranteed, so that the magnetic response time is too long, or the magnetic microspheres are taken away by liquid during magnetic attraction, thereby affecting the experimental efficiency and the experimental result. The 300nm magnetic microsphere has very good suspension performance through testing, the suspension time is more than three hours, the magnetic response time is short, and the complete magnetic absorption time is less than 10 s.
Magnetic core coated SiO2The temperature control is very important, the water is changed for the ultrasonic instrument every 30-40 minutes in the reaction process, the reaction temperature is prevented from being higher than 50 ℃, the magnetic core oxidation is easily caused due to overhigh reaction temperature, the magnetism of the magnetic microsphere is reduced due to the magnetic core oxidation, and the magnetic microsphere with weaker magnetism is slowly lost in the magnetic attraction process, so that the use standard cannot be met.
The 300nm carboxyl magnetic microsphere is coated with two layers of polymers in the preparation process, so that on one hand, a net structure can be formed, magnetic core leakage is reduced, on the other hand, the acid resistance of the magnetic microsphere is higher, the magnetic microsphere can be used under an acidic condition, and the structure of the magnetic microsphere is not damaged.
The added polymers in the preparation process of the magnetic microspheres are various, and byproducts which are difficult to clean are easily formed, so that the performance of the magnetic microspheres is directly influenced, and the adding amount, concentration and proportion of GMA, MBA and AIBN, such as 3g Fe, are strictly controlled when a polymer layer is coated3O4@SiO2The MPS magnetic microsphere reaction system is 9mL GMA, 9.0g MBA and 360mg AIBN, and when the preparation amount of the magnetic microspheres is increased, the amplification is carried out according to the proportion. And the temperature of the step is also very critical, and strict temperature control is required. After the time of deoxidization and stirring is finished, an oil bath pot switch and a cooling circulating water switch are opened, the temperature is raised to 75 ℃, the consistency between the temperature displayed by the instrument and the actual temperature needs to be concerned, and a thermometer needs to be used for self-detection and verification if necessary. The magnetic microspheres with uniform particle size and no by-products can be prepared only by ensuring that the whole system is uniformly heated.
NHS is an additive in improved amidation and peptide coupling reactions in magnetic microsphere activation, for activating carbonyl groups upon amide bond formation. Sulfo-NHS is a sulfonated product of NHS, is more water soluble than NHS, and has a negative charge that is less likely to cause polymerization of the attached substrate (e.g., protein). And not only NHS but also both organic activation and aqueous activation can be selected in the manner of activation. DMF (N, N-dimethylformamide) is selected as a solvent for organic activation, water is strictly removed from the DMF (N, N-dimethylformamide) before use, and water is quickly prevented from entering the DMF as much as possible during the activation process, so that the intermediate product is hydrolyzed. Through repeated experiments and comparison of the activation mode and the activation condition, the invention finds an optimal activation coupling mode, namely a method for aqueous phase activation of EDC/Sulfo-NHS. Firstly, the use of organic solvents with pungent odor and slight toxicity, such as DMF (N, N-dimethylformamide), DMAC (dimethylacetamide) and the like, is avoided in the experimental process, so that the method is more friendly to the health and environment of operators; secondly, the water phase activation can greatly improve the amount of the protein coated by the carboxyl magnetic microspheres, so that the carrying capacity of the immune magnetic microspheres is obviously improved; the step of water phase activation is simple and easy to operate, the preparation of an intermediate product NHS magnetic microsphere is avoided, and the immune magnetic microsphere is directly prepared and formed; and the phenomena of magnetic microsphere adhesion and agglomeration and the like can not occur in the activation process, and the physical and chemical properties of the magnetic microsphere are well maintained.
The antigen-antibody binding is non-covalent bond: electrostatic attraction, van der waals attraction, hydrogen bond bonding force, and hydrophobic force. The principle of the antigen-antibody eluent is that the antigen-antibody complex is changed into an antigen-antibody monomer by using factors such as the destruction of binding force in a strong acid environment. After the immunomagnetic microspheres capture marker proteins, magnetic microsphere-antibody-antigen complexes are formed, and at the moment, only the antigens need to be eluted, and because the antibodies and the magnetic microspheres form stable amido bonds, the antigens can be eluted without changing the protein structure by using strongly acidic 0.1M glycine-HCl with a pH of 2.5-3.1 (glycine-hydrochloric acid). Note that SDS (sodium dodecyl sulfate) cannot be contained in the eluate because SDS is an ionic surfactant and destroys the structure of the protein. In addition, the eluted product should be immediately neutralized with a neutralization buffer to prevent the strong acid from causing irreversible effect on the protein.
The invention takes the carboxyl 300nm magnetic microspheres as a tool, activates and couples corresponding rabbit anti-human polyclonal antibody, enriches markers of myocardial injury and renal injury in serum to obtain traceable natural protein, and solves the problem of difficult tracing of in vitro diagnostic standard substances in a measurement laboratory and a reference laboratory. When the mimic protein is used for testing, the immunomagnetic microspheres can capture serum markers with the concentration ranging from 50ng to 4000 ng/ml. The molecular weight of the mimic protein is 60 KD; the molecular weight of CRP is 115-140KD, and the concentration in normal blood is 0.8-8 mug/mL; cys C has a molecular weight of 13.3KD and a concentration of 0.51-1.09 μ g/mL in normal blood; the molecular weight of C-Peptide is 0.9KD, and the concentration of C-Peptide in normal blood is 1.0 +/-0.23 ng/mL; NGAL has a molecular weight of 25kD and a concentration in normal blood of < 50 ng/ml. Proved by verification, the invention can effectively enrich serum myocardial injury markers CRP and serum kidney injury markers Cys C, C-Peptide and NGAL.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A method for enriching serum markers of myocardial injury and renal injury by magnetic microspheres comprises the following steps: step one, preparing carboxyl magnetic microspheres; step two, the carboxyl magnetic microspheres are activated and coupled with specific antibodies corresponding to markers to form immunomagnetic microspheres; step three, removing the marker protein in the enriched serum by using the immunomagnetic microspheres; eluting the marker protein enriched by the immunomagnetic microspheres by using an eluent, and performing qualitative and quantitative analysis; the enrichment method of the magnetic microspheres for the myocardial injury and kidney injury serum markers is not used for disease diagnosis and treatment.
Example 1 preparation of 300nm carboxyl magnetic microspheres
(1)Fe3O4Preparation of magnetic cores
Weighing 50gFeCl3·6H2Dissolving O in 1000mL of glycol;
sequentially adding 1g of sodium citrate and 55g of sodium acetate into the solution respectively, and continuously stirring until the sodium citrate and the sodium acetate are dissolved to obtain a precursor solution;
the precursor solution is respectively filled into a 250mL hydrothermal reaction kettle;
putting the installed reaction kettle into an oven, setting the reaction temperature to be 200 ℃, and reacting for 20 hours;
collecting the magnetic microspheres after the reaction is finished, and repeatedly washing the magnetic microspheres for more than ten times by using ethanol and purified water;
the washed Fe3O4Stored in purified water and tested for mass concentration.
(2)Fe3O4@SiO2Preparation of
A5-L round-bottomed flask was charged with 2500mL of ethanol, 700mL of purified water, 30mL of aqueous ammonia, and 6g of the above Fe3O4；
Installing a mechanical stirring device on the ultrasonic cleaning instrument, turning on a switch, setting the ultrasonic time to be 6.5h, carrying out ultrasonic treatment on the reaction solution while stirring, wherein the stirring speed is 200 rpm;
diluting 9mL TEOS (tetraethyl orthosilicate) solution in 100mL ethanol, and adding the solution into a 100mL constant-pressure dropping funnel;
after ultrasonic treatment is carried out for 30min, a constant-pressure funnel is arranged on the round-bottom flask, the TEOS/ethanol mixed solution is dropwise added into the reaction system, and the ultrasonic stirring reaction is continuously maintained for 6 h;
in the reaction process, water is changed for the ultrasonic instrument at intervals to prevent overhigh temperature;
after the reaction is finished, the device is dismantled, and a magnetic separator is used for collecting Fe3O4@SiO2Magnetic microsphere, washing with ethanol and purified water for more than 10 times, and washing with Fe3O4@SiO2The magnetic microspheres were stored in purified water and tested for mass concentration.
(3)Fe3O4@SiO2Preparation of-MPS
1000mL of ethanol, 500mL of purified water, and 10mL of ammonia were placed in a 2L round-bottom flaskWater and Fe3O4@SiO2Magnetic microspheres;
carrying out ultrasonic treatment on the reaction solution for 30 min;
installing a mechanical stirring device, and setting the stirring speed to be 200 rpm;
diluting 9mL MPS in 100mL ethanol, and adding into a 100mL constant pressure dropping funnel;
a constant-pressure funnel is arranged on the round-bottom flask, MPS/ethanol mixed solution is dripped into a reaction system, and the mixture is stirred and reacted for 20 hours;
after the reaction is finished, the reaction device is dismantled, and a magnetic separator is used for collecting Fe3O4@SiO2-MPS magnetic microspheres, washed 5 times with ethanol and finally Fe3O4@SiO2MPS magnetic microspheres were stored in ethanol and tested for mass concentration.
(4)Fe3O4@SiO2Preparation of-MPS @ P (GMA-MBA)
3g of Fe3O4@SiO2Dispersing MPS magnetic microspheres in ethanol in a centrifugal tube, washing for 3 times, and finally dispersing in 50mL of ethanol to obtain a magnetic microsphere dispersion liquid;
respectively adding 3L of purified water and the magnetic microsphere dispersion and 300mg of sodium dodecyl benzene sulfonate into a 5L round-bottom flask, performing ultrasonic treatment for 10min, installing an oil bath reaction device, setting the stirring speed to be 300rpm, and stirring for 1 h;
adding 9mL of GMA, 9.0g of MBA and 360mg of AIBN (azodiisobutyronitrile) into the reaction system, continuously stirring for 1h, and introducing nitrogen into the flask to remove oxygen;
after the time of deoxidization and stirring is finished, opening an oil bath pot switch and a cooling circulating water switch, heating to 75 ℃, continuing introducing nitrogen for protection and stirring, and reacting for 16 hours;
after the reaction is finished, the reaction device is dismantled, the magnetic separator is used for separating the magnetic microspheres, then the ethanol and the purified water are used for repeatedly washing for more than 10 times, and finally the Fe is obtained3O4@SiO2MPS @ P (GMA-MBA) magnetic microspheres were stored in purified water and tested for mass concentration.
(5)Fe3O4@SiO2-MPSPreparation of @ P (GMA-MBA) -COOH
Taking a beaker, respectively adding 20g of sodium carbonate, 3g of sodium bicarbonate and 40g of 6-aminocaproic acid, adding 1200mL of purified water, magnetically stirring until the sodium carbonate, the sodium bicarbonate and the 6-aminocaproic acid are dissolved, then adding 1.9g of sodium borohydride to continue dissolving, finally controlling the pH value of the solution to be about 10.6, and transferring the solution to a 2L round-bottom flask;
mixing Fe3O4@SiO2Adding MPS @ P (GMA-MBA) magnetic microspheres into the reaction system, and performing ultrasonic treatment for 20 min;
installing an oil bath reaction and mechanical stirring device, setting the stirring speed to be 200rpm, introducing nitrogen into the flask, and deoxidizing for 30 min;
after deoxygenation is finished, opening an oil bath pot switch and a cooling circulating water switch, heating to 70 ℃, continuing introducing nitrogen for protection and stirring, and reacting for 16 hours;
after the reaction is finished, the reaction device is dismantled, the magnetic separator is used for separating the magnetic microspheres, then the purified water is used for repeatedly washing for more than 5 times, and finally the Fe is obtained3O4@SiO2-MPS @ P (GMA-MBA) -COOH magnetic microspheres were stored in purified water and tested for mass concentration.
Example 2 coupling of 300nm carboxyl magnetic microsphere with Anti-CRP Rabbit Anti-human antibody to form immunomagnetic microsphere
1. Reagent:
washing buffer solution: MEST buffer, TBST buffer (0.05M TBS +0.15M NaCl + 0.05% Tween, pH 7.5)
Activation buffer: MEST buffer (0.1M MES + 0.05% Tween, pH 5.0)
Coupling buffer: MEST buffer (0.1M MES + 0.05% Tween, pH 5.0)
Sealing liquid: TBST buffer containing 10mg/mL BSA, i.e., TBST buffer containing 1% BSA
Preservation solution: PBS buffer
2. The method comprises the following steps:
(1) taking 15mg of carboxyl 300nm magnetic microspheres in a 2mL EP tube, and washing for 3 times by using MEST buffer (1.5 mL);
(2) configuring 20mg/mL EDC in MEST and 20mg/mL Sulfo-NHS in MEST each at 1mL (now prepared for use);
(3) respectively adding 750 mu L of EDC and Sulfo-NHS into a magnetic microsphere centrifuge tube, uniformly mixing, and placing in a vertical mixer for reaction and activation for 15 min;
(4) preparing 0.1mg/mLANti-CRP rabbit anti-human antibody solution: 160 μ L of the antibody stock solution (1mg/mL) was added to 1.44mL of MEST buffer and mixed well for use;
(5) after activation of the magnetic microspheres is finished, magnetically absorbing supernatant, adding 1.5mL of prepared antibody solution, uniformly mixing, and placing on a vertical mixer for mixing reaction for 2 h;
(6) preparing 10mg/mL BSA blocking solution: weighing 16mg BSA, adding into 1.6mL TBST buffer, and uniformly mixing for later use;
(7) after the magnetic microsphere grafting SA reaction is finished, magnetically absorbing the supernatant, adding 1.5mL of BSA confining liquid, uniformly mixing, placing on a vertical mixer, and carrying out mixed reaction for 1 h;
(8) after the blocking reaction was completed, the supernatant was magnetically aspirated, and the magnetic microspheres were repeatedly washed with TBST buffer (10 times or more);
(9) repeatedly washing with SA storage buffer for 1.5mL-3 times;
(10) and (3) removing the supernatant by magnetic attraction, accurately adding 1.5mL of SA storage buffer to perform constant volume, and maintaining the concentration at 10mg/mL to obtain the immunomagnetic microspheres.
Example 3 ELISA detection of CRP and Cys C antigen antibody
1. Reagent
Coating buffer 0.05M carbonate buffer pH9.6
Washing buffer (ph7.4 pbst): 0.15M, containing 0.1% Tween-20
Blocking solution 5% BSA Bovine Serum Albumin (BSA)
Sample diluent PBS (ph7.4 pbst): 0.15M, without Tween
Weighing 0.5g BSA in 5% BSA solution, dissolving with 10mL 1 × PBST, mixing well, storing at 4 deg.C for 24 h;
goat anti-rabbit HRP working solution: diluting with 5% BSA by 10000 times, and storing at 4 deg.C;
TMB reaction stop solution: 2M concentrated H2SO4: 54.5mL of 98% sulfuric acid solution was measured and added dropwise to 350mL of ultrapure water with stirringAfter the mixture is cooled to room temperature, continuously adding ultrapure water to a constant volume of 500 mL;
2. step (ii) of
(1) Coating: (total 4 pieces) both antigens were diluted to specific concentrations with 0.05M pH9.6 coating buffer; mu.l of the solution was added to each well of the polystyrene plate overnight at 4 ℃. Two columns per packet. The original concentration of CRP is 1mg/ml, 2 mul is sucked to be diluted to 2ml, namely 1 mug/ml; cys C original concentration 1mg/ml, 2. mu.l is sucked and diluted to 2ml, namely 1. mu.g/ml;
(2) the next day, the well solution was discarded, and washed 3 times with wash buffer, 200. mu.l per well for 3 minutes each time;
(3) adding 200 mul of 5% BSA blocking solution into each hole, blocking for one hour at 37 ℃, throwing off liquid in the holes, and washing for three times;
(4) sample adding: diluting the polyclonal antibody to a specific concentration, adding 50 μ l of polyclonal antibody to each well, adding polyclonal antibody correspondingly, and alternately adding polyclonal antibody, and observing nonspecific reaction. Incubating for two hours;
diluting a standard product:
(5) after the incubation time is over, the liquid in the wells is spun off, and the wells are washed three times with 200 mul of washing buffer solution, three minutes each time;
(6) adding 1: 10000 HRP-goat anti-rabbit working solution 100 μ L per well, mixing and incubating for 2 hours at 37 ℃;
(7) after the incubation time was over, the liquid in the wells was spun off and washed five times with 200 μ l of wash buffer, three minutes each time;
(8) adding 100 mu L of TMB color development solution, incubating for 15min in a dark place, and then adding 50 mu L of stop solution;
(9) the plate was transferred to a microplate reader OD450 for reading. And (3) operating a microplate reader: newly building, operating, measuring photometry, and operating on a filter of 450 nm.
Lath setting:
the experimental results are as follows:
and (4) analyzing results: the antigen antibody can be specifically identified, and the sensitivity is good.
Example 4 immunomagnetic microspheres capture CRP antigen and elute
1. Reagent
Binding/Washing Buffer: PBST buffer (0.1% Tween20)
Elution Buffer：0.1M glycine-HCl，pH 2.0(0.1％Tween 20)
Neutralization Buffer: 1M Tris-HCl; neutralization buffer at pH8.6
2. Step (ii) of
Preparation of magnetic microspheres
(1) 2 EP tubes with 1.5ml are prepared, 20 mul (0.2mg) immune magnetic microspheres are added into each tube and are respectively marked;
sample preparation
(2) CRP antigen samples were diluted with Binding/Wash Buffer (PBST Buffer). The working concentration is 4 mug/ml;
(3) placing the centrifuge tube on a magnetic frame, collecting the magnetic microspheres and discarding the supernatant;
(4) incubating 20 μ L (0.2mg) of immunomagnetic microspheres and 500 μ L of sample solution on a vertical mixer for 1-2 hours at room temperature, and slightly swirling the magnetic microspheres every 10-15 minutes to ensure the suspension of the magnetic microspheres;
(5) after the reaction was completed, the EP tube was placed in a centrifuge and centrifuged at 3500rpm for 3 min. Separating the magnetic microspheres from the unbound sample with a magnetic stand (unbound sample was collected and labeled with 2 different EP tubes);
washing machine
(6) Add 500. mu.l Binding/Washing Buffer and gently wash. Placing the centrifuge tube on a magnetic frame, collecting the magnetic microspheres and then discarding the supernatant; washing for three times;
(7) adding 500 mul of ultrapure water into an EP tube, mixing lightly, placing the centrifugal tube on a magnetic frame, collecting magnetic microspheres, and discarding supernatant;
elution is carried out
(8) After washing the magnetic microspheres, adding 100 mul of elution buffer (0.1M glycine pH 2.0) into each tube, incubating for 5 minutes at room temperature on a vertical mixer, magnetically separating the magnetic microspheres, and storing supernatant containing target antigen; this step is carried out three times in total;
neutralization
(10) Adding 10. mu.l of 1M Tris-HCl, pH8.6 neutralization buffer to each 100. mu.l of antigen supernatant; 2 tubes were stored in a 4 ℃ freezer and labeled sample 1 and sample 2, respectively. Example 5, the enriched CRP antigens were identified using a chemiluminescent immunoassay kit.
1. Reagent
Wash Buffer: TBST buffer (20mM Tris30mM NaCl0.1% Tween20)
Enzyme-labeled antibody diluent: PBST containing 5% BSA, ALP-labeled antibody 1: 2000 diluted, 1ul diluted to 2ml
Biotinylated antibody diluent: 1 PBS 0.1% Tween20
Antigen dilution: 1 PBS 0.1% Tween20
Free biotin: 2 μ g/ml, diluted with 1 × PBS containing 0.1% Tween20
Substrate solution: shenzhen Meqitta intlus-A04
SA magnetic microspheres: dynal 1um (T1) (ThermoFisher), biotinylated antibody coupled, then free biological closed, magnetic microspheres closed and one-step method;
a chemiluminescence apparatus: the preparation method of the Zhengzhou Antu antigen comprises the following steps:
2. step (ii) of
(1) Preparing 1ml of 10 mu g/ml biotinylated antibody, and diluting 10 mu l of biotinylated CRP antibody to 1 ml;
(2) taking 50 mu l (10mg/ml) of SA magnetic microspheres to magnetically suck the supernatant, adding 1000 mu l of 10 mu g/ml biotinylated antibody and incubating for 1h at room temperature;
(3) after 3 washes, 200. mu.l of 2. mu.g/ml free biotin were added and incubated for 1h at room temperature. After washing for 3 times, 1000. mu.l PBST was added to dilute to 0.5 mg/ml;
(4) diluting the two samples collected in example 4 by 50 times and 100 times respectively, and making auxiliary holes for each sample in each time, wherein the total number of the auxiliary holes is 8;
(5) adding 50 mu L of magnetic microspheres into a 96-well plate, adding 50 mu L of standard substance (3-10) of the substance to be detected and the sample to be detected into each well, adding 100 mu L of enzyme-labeled antibody into each well, fully shaking and resuspending the magnetic microspheres, incubating for 15min in a constant temperature box at 37 ℃, carrying out magnetic separation, sucking the supernatant by using a pipette, and taking down the 96-well plate from the magnetic separator;
(6) adding 200 mu L of Washing buffer into each hole, fully shaking and resuspending the magnetic microspheres, carrying out magnetic separation, sucking the supernatant by using a pipettor, taking down a 96-hole plate from the magnetic separator, repeating the step for 2 times, and Washing for 3 times in total;
(7) adding 150 mu L of substrate solution into each hole, fully shaking and resuspending the magnetic microspheres, and incubating for 5min in a dark place;
(8) the 96 well plate was placed in a chemiluminescence meter for reading, and figure 2.0 was spotted-logged-measured quickly-allowed to oscillate (low intensity 5S, all wells) -measurement started-Ctrl-C-Ctrl-V to the usb disk.
And (4) analyzing results: through comparison with a standard curve, the average recovery rate of the sample 1 is 88.03%, the average recovery rate of the sample 2 is 86.41%, the average recovery rates are both more than 85%, and the recovery rate meets the requirement of 85% -115% of the national medical and pharmaceutical industry standard of the people's republic of China. Note: sample 1 and sample 2 were both eluted in example 4.
The invention enriches low-abundance proteins and peptides in serum by aiming at the immunomagnetic microspheres of the marker, provides a traceable standard substance, and provides an effective method for developing the traceable standard substance in a metering laboratory, a reference laboratory, an in-vitro diagnostic reagent manufacturer and a clinical inspection institution.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (9)
1. The method for enriching the serum markers of myocardial injury and renal injury by using the magnetic microspheres is characterized by comprising the following steps of: step one, preparing carboxyl magnetic microspheres; step two, forming the immune magnetic microsphere by activating and coupling the specific antibody corresponding to the marker on the carboxyl magnetic microsphere; enriching marker protein in serum by using the immunomagnetic microspheres; eluting the marker protein enriched by the immunomagnetic microspheres by using an eluent, and performing qualitative and quantitative analysis;
the enrichment method of the magnetic microspheres for the myocardial injury and kidney injury serum markers is not used for disease diagnosis and treatment.
2. The method for enriching serum markers of myocardial injury and renal injury by using magnetic microspheres of claim 1, wherein the carboxyl magnetic microspheres in the first step have a particle size of 300nm to 500 nm.
3. The method for enriching serum markers of myocardial injury and renal injury by using magnetic microspheres according to claim 1, wherein the carboxyl magnetic microspheres obtained in the first step are prepared by the following steps: first using FeCl3·6H2Preparation of Fe from O3O4Magnetic core, then to Fe3O4Magnetic core coated SiO2Form Fe3O4@SiO2And then MPS reacts to form Fe3O4@SiO2MPS, followed by Fe3O4@SiO2MPS coating of GMA, MBA to Fe3O4@SiO2-MPS @ P (GMA-MBA), and finally sodium carbonate, sodium bicarbonate and 6Formation of Fe from aminocaproic acid, sodium borohydride3O4@SiO2-MPS @ P (GMA-MBA) -COOH, the carboxylic magnetic microsphere of step one.
4. The method for enriching serum markers of myocardial injury and renal injury by using magnetic microspheres of claim 3, wherein the serum markers of myocardial injury and renal injury are enriched by Fe3O4@SiO2MPS coating of GMA, MBA to Fe3O4@SiO2-MPS @ P (GMA-MBA) step, Fe3O4@SiO2MPS is added in a proportion of 3g Fe to GMA, MBA, AIBN3O4@SiO2MPS 9mL GMA, 9.0g MBA, 360mg AIBN were added.
5. The method for enriching serum markers of myocardial injury and renal injury by using magnetic microspheres of claim 1, wherein the second step is to activate the carboxyl magnetic microspheres by using EDC/Sulfo-NHS aqueous phase activation method, and the activated carboxyl magnetic microspheres react with specific antibody solution corresponding to the markers to obtain the immunomagnetic microspheres.
6. The method for enriching serum markers of myocardial injury and renal injury by using magnetic microspheres according to claim 3, wherein the specific method in the second step is as follows: and (2) washing the carboxyl magnetic microspheres by using a MEST buffer, adding EDC and Sulfo-NHS for activation, adding a corresponding antibody solution for reaction, sealing by using a BSA solution, and finally washing and storing to form the immunomagnetic microspheres.
7. The method for enriching serum markers of myocardial injury and renal injury by using magnetic microspheres according to claim 1, wherein the enriching process in the third step is mixing and incubating the immunomagnetic microspheres with the target serum sample.
8. The method for enriching serum markers of myocardial injury and renal injury by using magnetic microspheres according to claim 1, wherein the eluent in step four is selected from glycine-hydrochloric acid solution with pH2.5-3.1 and 0.1M, citric acid solution with pH2.5-3.1 and 0.1M, acetic acid solution with 2.5% concentration, ammonium hydroxide solution with pH10.0-11.0 and 0.1M.
9. The method for enriching serum markers of myocardial injury and renal injury by using magnetic microspheres according to any one of claims 1 to 8, wherein the serum markers of myocardial injury are cTnI, NT-proBNP and CRP, and the serum markers of renal injury are Cys C, C-Peptide and NGAL.
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