CN111948390A - Time-resolved fluoroimmunoassay kit for detecting t-PAI-C and application thereof - Google Patents
Time-resolved fluoroimmunoassay kit for detecting t-PAI-C and application thereof Download PDFInfo
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Abstract
The invention relates to a biological detection kit, in particular to a time-resolved fluorescence immunoassay kit for detecting tissue plasminogen activator/plasminogen activator inhibitor-1 complex (t-PAI-C) and application thereof, belonging to the field of immunoassay and nano-biotechnology. A time-resolved fluoroimmunoassay kit for detecting tissue plasminogen activator/plasminogen activator inhibitor-1 complex (t-PAI-C), comprising: reaction buffer solution, cleaning solution, enhancement solution, magnetic particle solution coated with t-PAI-C antigen, europium-labeled goat anti-human IgG antibody solution and freeze-dried calibrator of t-PAI-C integrated on the reagent strip. The kit can provide a reaction system close to homogeneous phase, detect the content of t-PAI-C, shorten the detection time, improve the efficiency and provide convenience for clinical use.
Description
Technical Field
The invention relates to a biological detection kit, in particular to a time-resolved fluorescence immunoassay kit for detecting tissue plasminogen activator/plasminogen activator inhibitor-1 complex (t-PAI-C) and application thereof, belonging to the field of immunoassay and nano-biotechnology.
Background
The key component of venous thrombosis is thrombin generation, which acts on the conversion of fibrinogen to fibrin to form a thrombus. Since the thrombin generated has a very short half-life in blood, only a few seconds, and is quickly neutralized by the anticoagulant, it is very difficult to directly measure thrombin, and instead, thrombin and its inhibitor, Antithrombin (AT)1:1, are bound to thrombin-antithrombin complex (TAT) are detected. The production of TAT directly confirms the activation of the coagulation system. Thrombomodulin (TM) is expressed on the surface of vascular endothelial cells and can assist thrombin to activate a protein C system to generate an anticoagulation effect, and the TM represents the activation of the endothelial cells. When coagulation is activated and fibrin is formed, fibrin activates t-PA, which further activates plasminogen to plasmin, which degrades fibrin to form D-dimers and FDP. Activated t-PA is inhibited by PAI-I1: 1 to form the tissue plasminogen activator/plasminogen activator inhibitor-1 complex (t-PAI-C), while activated plasmin is inhibited by α 2-antiplasmin inhibitor 1:1 to form the plasmin- α 2 Plasmin Inhibitor Complex (PIC). Therefore, TAT, TM, PIC, t-PAI-C are important markers in the process of venous thrombosis.
After fibrin formation, the fibrinolytic system is activated to form tissue plasminogen activator (t-PA), which further converts plasminogen to plasmin, degrading it to form D dimer and FDP, PAI-1 is a physiological inhibitor of t-PA, both 1:1 form complex t-PAI-C, and the level of t-PAI-C is positively correlated with t-PA concentration and vascular endothelial injury. Normal reference interval: male: 0-17ng/mL, female: 0-10.5 ng/mL, the increase is usually seen in DIC, vascular endothelial injury, DVT, acute myocardial infarction and the like.
The Tissue Plasminogen activator/Plasminogen activator inhibitor-1 Complex (Tissue Plasminogen activator-Plasminogen activator inhibitor Complex, t-PAI-C) comprehensively reflects molecular markers of damaged fibrinolytic system and vascular endothelial cells, and indicates that the cause is not removed and the thrombosis is in progress. the assay for t-PAI-C has the following clinical significance:
1. one of the best diagnostic indicators of Venous Thromboembolism (VTE);
2. risk indices of myocardial infarction;
3. judging the repair degree of the vascular endothelial system after the operation and monitoring the effect of the thrombus treatment drug.
At present, no reliable commercial t-PAI-C detection method exists, and ELISA detection kits are mostly used in experiments, and the detection principle is as follows: the kit adopts a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). The specimen, the standard, and the detection antibody labeled with HRP were sequentially added to the coated microwells previously coated with the t-PAI-C antibody, incubated, and washed thoroughly. The color is developed with the substrate TMB, which is converted to blue by the catalysis of peroxidase and yellow by the action of an acid. The shade of the color is positively correlated with the t-PAI-C content of the sample.
Disclosure of Invention
The invention aims to provide a time-resolved fluoroimmunoassay kit for detecting tissue plasminogen activator/plasminogen activator inhibitor-1 complex (t-PAI-C), which aims to solve the problems in the prior art.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a time-resolved fluoroimmunoassay kit for detecting tissue plasminogen activator/plasminogen activator inhibitor-1 complex (t-PAI-C), comprising: reaction buffer solution, cleaning solution, enhancement solution, magnetic particle solution coated with t-PAI-C antigen, europium-labeled goat anti-human IgG antibody solution and freeze-dried calibrator of t-PAI-C integrated on the reagent strip.
The invention adopts time-resolved fluoroimmunoassay (TRFIA) to detect t-PAI-C. TRFIA is one ofThe novel technology using lanthanide chelate as a marker has the characteristics of zero background, wide linear range, good stability and the like, and the sensitivity can reach 10-18mol/L, 10 compared with ELISA, CLIA, ECLIA, etc-9～10-15The mol/L is higher, the main innovation of TRFIA lies in that lanthanide atoms with large fluorescence intensity, long decay time and small molecular weight are used for marking immune molecules instead of common macromolecular enzyme or radioactive isotope, so that the sensitivity and the measuring range of the established immune method are multiplied, and no radioactive pollution is caused.
The fluorescence of the labeled ions by the time-resolved fluorescence immunoassay technology is a quasi-linear spectrum, and is characterized in that the wavelength range of exciting light is wider, and the peak range of an emission spectrum is narrower, so that the background fluorescence intensity is favorably reduced, and the detection resolution is improved; a larger Stokes shift exists between the excitation light and the emission light of the time-resolved fluorescence immune component, which is beneficial to eliminating the interference of non-specific fluorescence, thereby enhancing the specificity of measurement; the fluorescence intensity generated by the labeled ion chelate is high, and the service life is long, so that the influence of non-specific fluorescent substances in a sample and the environment on a detection result can be eliminated; each detection is repeated 1000 times, and the average fluorescence count value is taken as a result, so that the detection accuracy is improved. TRFIA is one of the main development directions of immunoassay at home and abroad.
The invention combines TRFIA and magnetic particle technology, integrates all reagents on one reagent strip, establishes the t-PAI-C-TRFIA method, can be operated by full-automatic equipment, has high accuracy and single-person detection, and is convenient to use.
The reaction principle of the invention is that t-PAI-C antibody is coated on magnetic beads, europium labels anti-human IgG antibody, if t-PAI-C exists in a sample, t-PAI-C antigen-t-PAI-C antibody-europium labels anti-human IgG antibody compound is formed, dissociation enhancement liquid is added after separation to dissociate europium ion, a time resolution fluorometer is adopted to detect fluorescence value, the strength of the fluorescence value is in direct proportion to the content of t-PAI-C in the sample, a standard curve is drawn according to the fluorescence value of a standard t-PAI-C synchronously reacting with the sample, and the content of t-PAI-C in the sample is calculated.
The kit can provide a reaction system close to homogeneous phase, detect the content of t-PAI-C, shorten the detection time, improve the efficiency and provide convenience for clinical use.
Preferably, the t-PAI-C antigen-coated magnetic particle solution is prepared by connecting activated magnetic particles with the diameter of 50-5000 nanometers with the t-PAI-C antigen, washing, sealing and washing.
Preferably, the europium-labeled goat anti-human IgG antibody solution is prepared by mixing Eu3+-N2- [ P-Isocyanato-benzyl group]A sodium diethylenetriamine tetracetate-labeled goat anti-human IgG antibody is prepared, and the goat anti-human IgG antibody and the Eu are3+-N2- [ P-Isocyanato-benzyl group]The mass ratio of the-sodium diethylenetriamine tetracetate is 4-6:1, and the optimal value is 5: 1.
preferably, the reaction buffer is 50mmol/L Tris-HCl pH7.8 containing 8mmol/L NaCl, 0.1% BSA, 50. mu. mol/L DTPA, 0.1ml/L Tween-80 and 0.1% NaN3。
Preferably, the cleaning solution is a buffer solution containing 0.48g/L of Tris, 12.49g/L of NaCl, 1.11g/L of Tween-20 and pH adjusted to 7.8 by hydrochloric acid.
Preferably, the enhancing solution is a mixed solution containing glacial acetic acid with the volume concentration of 3.6 per thousand, sodium acetate with the volume concentration of 0.5g/L, beta-naphthoyl trifluoroacetone with the volume concentration of 0.05g/L, trioctylphosphine oxide with the volume concentration of 0.03g/L and Triton X-100 with the volume concentration of 1 per thousand.
Preferably, the calibrator solution of t-PAI-C is prepared by using a solution containing 2g/L BSA and 1g/L NaN350mmol/L reaction buffer, pH7.8 Tris-HCl, and t-PAI-C was formulated into calibrator solutions of various concentrations and stored by lyophilization.
Preferably, the kit comprises a kit body and a cover covering the opening at the top end of the kit body, a material placing block is arranged on one side inside the kit body, a plurality of material placing holes for storing the freeze-drying cups are formed in the upper surface of the material placing block, a material placing groove is formed in the other side inside the kit body, a reagent rack for storing reagent strips is arranged in the material placing groove, each reagent strip comprises a reagent plate detachably connected with the reagent rack, and a plurality of reagent holes for storing reactants are formed in the upper end surface of each reagent plate in a downward mode. By adopting the technical scheme, the reagent strip in the reagent box encapsulates the required reagent to the reagent hole of the reagent strip in advance, so the problem of adding the detection reagent is not required to be considered in the detection process, the detection procedure is simplified, the pollution possibly brought by repeated liquid adding is avoided, freeze-drying calibrators with different concentrations are arranged in the freeze-drying cup, the required reagent to be detected is integrated in the reagent box, the storage and carrying are convenient, and the operation is simple and practical. As preferred, the lower surface center of box body has inlayed the refrigeration piece, it is equipped with temperature sensor to put the thing inslot, the terminal surface is equipped with the display screen before the box body, temperature sensor's output and refrigeration piece and display screen electricity are connected. Through adopting above-mentioned technical scheme, the refrigeration piece is used for cooling down the box body, measures the inside temperature of box body and shows real-time temperature on the display screen through temperature sensor. Preferably, a battery pack is arranged below the bottom of the box body, and a charging socket and a switch are arranged on the front end face of the battery pack. Through adopting above-mentioned technical scheme, the group battery is used for supplying power to the refrigeration piece, convenient removal, transportation. Preferably, the cover body comprises a top cover and a bottom plate, a lifting handle is arranged in the middle of the top cover, the edge of the top cover extends downwards and vertically to form a surrounding plate, the bottom plate is arranged in a space surrounded by the surrounding plate, a plurality of elastic pieces are distributed between the top cover and the bottom plate in an array mode, and an elastic layer is formed on the lower surface of the bottom plate. Through adopting above-mentioned technical scheme, can avoid in the kit transportation and the top cap between the collision cause seal the membrane fracture, also can avoid rocking of kit transportation in the reagent strip, play fine guard action to the reagent strip. Preferably, two opposite side surfaces of the storage groove are provided with support bars, the upper end surfaces of the support bars are provided with lifting plates, and the reagent rack is placed on the upper end surfaces of the lifting plates. Through adopting above-mentioned technical scheme, the support bar is used for supporting the lifter plate, and the reagent frame is convenient for place to the lifter plate. Preferably, a through hole is formed in the side wall of the box body, a push-pull rod is sleeved in the through hole through a bearing, a push-pull handle is arranged at one end of the push-pull rod, a limiting block is arranged at the other end of the push-pull rod, the push-pull handle is positioned on the outer side of the box body, the limiting block is positioned in the object placing groove, the push-pull rod is in sliding connection with a fixing block positioned at the bottom of the object placing groove, and the fixing block is positioned; the upper part of the push-pull rod is rotatably connected with a plurality of rotating rods, and the upper ends of the rotating rods are rotatably connected with the lower end of the lifting plate. Through adopting above-mentioned technical scheme, when needing to take out reagent strips and use, promote the push-and-pull handle, and then drive the push-and-pull rod and remove, the push-and-pull rod moves and then promotes the connecting rod to the left side, makes the dwang that the slope set up straighten gradually, and then makes the upper end of dwang promote the lifter plate and move up, and then with reagent frame ejecting to the box body top, can make quick convenient reagent strips of taking of operating personnel. Preferably, the reagent frame includes that a plurality of that sets up side by side is the support frame of "U" type structure and the fixed plate of connecting the support frame, the support frame includes the spreader and perpendicular to the spreader of spreader both ends downside. Through adopting above-mentioned technical scheme, can set up a plurality of support frames according to the actual demand and be used for depositing the reagent strip to combine side by side support frame through the fixed plate, but support frame, fixed plate, cross bracing board and side brace board integrated into one piece. Preferably, the both sides of fagging all are equipped with the spout, and the reagent board inlays in the spout of two adjacent faggings and can slide along the spout. Through adopting above-mentioned technical scheme, reagent board on the reagent strip is pegged graft in the spout to can follow the spout and slide, make things convenient for depositing and taking of reagent strip, easy operation. Preferably, one side of the reagent strip is provided with a handle, and the middle part of the reagent strip is also provided with a detection hole. Through adopting above-mentioned technical scheme, the setting of handle has made things convenient for taking and placing of reagent strip, and the operation when the inspection hole setting is convenient for to detect at the middle part of reagent strip. Preferably, the upper surfaces of the reagent strip and the freeze-drying cup are both packaged by adopting a film sealing material. By adopting the technical scheme, the upper surfaces of the reagent strip and the freeze-drying cup are encapsulated by the film sealing material, so that a good preservation effect is achieved, and the film sealing material is made of a high polymer film, an aluminum foil or an aluminum-plastic composite material.
A preparation method of the kit comprises the following steps:
preparation of the t-PAI-C calibrator solution: taking high concentration t-PAI-C solution, adding 2g/L BSA and 1g/L NaN350mmol/L ofpreparing Tris-HCl reaction buffer solution with pH of 7.8 into calibrator solutions with different concentrations, subpackaging, lyophilizing, and storing at 2-8 deg.C;
preparing the magnetic particle solution coated with the t-PAI-C antigen: activating ferroferric oxide microspheres with the diameter of 50-5000nm by using glutaraldehyde with the mass concentration of 5-10%, uniformly mixing for 4 hours at room temperature, washing for 1-3 times by using 0.05mol/L PBS buffer solution with the pH value of 7.2, and suspending by using the buffer solution to prepare magnetic particle suspension with the concentration of 100 mg/mL; taking 100 μ L of the magnetic particle suspension, adding 1mL of 0.05mol/L PBS buffer solution with pH of 7.2 and 50 μ g of t-PAI-C antigen, mixing and incubating at 25 deg.C for 2 hr, performing magnetic separation, retaining magnetic particles, washing with 0.05mol/L PBS buffer solution with pH of 7.2 for 1-3 times, blocking with 1mL of 0.0lmol/L PBS buffer solution with 5% BSA at 25 deg.C for 30 min, washing with 0.05mol/L PBS buffer solution with pH of 7.2 for 1-3 times, washing with 0.5% BSA at mass concentration and 0.1% NaN at mass concentration30.05mol/L Tris-HCl buffer solution with pH value of 7.2 is used for resuspension, subpackaging and storing at 2-8 ℃;
preparing the europium-labeled goat anti-human IgG antibody solution: taking 1mL goat anti-human IgG antibody, converting buffer conditions through a PD-10 column, and eluting with 50mmol/L Na containing 0.155 mol/L NaCl and having a pH of 8.52CO3-NaHCO3Buffer solution, collecting protein peak to obtain sheep anti-human IgG antibody solution concentrated to 2g/L, taking 500 microliter of the sheep anti-human IgG antibody solution, adding 0.2mg of benzyldiethylenetriamine tetraacetic acid europium sodium isothiocyanate, magnetically stirring at 25 ℃ for reaction for 20 hours, transferring the reaction solution to Sepharose CL-6B column balanced by Tris buffer solution with the concentration of 80mmol/L and the pH value of 7.8 in advance for chromatography, collecting protein peak, diluting, subpackaging, vacuum freeze drying, and storing at 2-8 ℃;
preparing the enhancement solution: uniformly mixing 3.6mL of glacial acetic acid, 0.5g of sodium acetate, 0.05g of beta-naphthoyl trifluoroacetone, 0.03g of trioctylphosphine oxide and 1mL of Triton X-100, adding deionized water to a constant volume of 1000mL, subpackaging and storing at 2-8 ℃;
the reaction buffer solution is 50mmol/L Tri with pH of 7.8The s-HCl solution contained 8mmol/L NaCl, 0.1% BSA, 50. mu. mol/L DTPA, 0.1ml/L Tween-80, and 0.1% NaN3；
The cleaning solution is a buffer solution containing 0.48g/L of Tris, 12.49g/L of NaCl, 1.11g/L of Tween-20 and hydrochloric acid for adjusting the pH value to 7.8.
A method for detecting tissue plasminogen activator/plasminogen activator inhibitor-1 complex (t-PAI-C) using said time-resolved fluoroimmunoassay kit, the method comprising the steps of:
(1) respectively taking a calibrator solution of the t-PAI-C and a sample to be detected, mixing the calibrator solution of the t-PAI-C and the sample to be detected with the magnetic particle solution coated with the t-PAI-C antigen, incubating, then washing with the washing solution, performing magnetic separation, adding a europium-labeled goat anti-human IgG antibody solution diluted by the reaction buffer solution into the mixed magnetic particle solution, further incubating, then washing with the washing solution, performing magnetic separation, then sucking and removing a supernatant, repeating the washing steps, and then adding the enhancement solution;
(2) respectively measuring the fluorescence value of europium in the solution added with the enhancement solution by using a time-resolved fluoroimmunoassay analyzer to obtain the fluorescence values of the calibrator solution of the t-PAI-C and the sample to be detected, then respectively drawing a standard curve according to the concentration of the t-PAI-C in the calibrator solution and the respective corresponding fluorescence value, and then substituting the corresponding fluorescence value of the sample to be detected into the corresponding t-PAI-C standard curve to calculate the content of the t-PAI-C in the sample to be detected.
Compared with the prior art, the invention has the advantages that:
1. the time-resolved fluoroimmunoassay kit for detecting t-PAI-C comprises a reaction buffer solution, a cleaning solution, an enhancement solution, a magnetic particle solution coating a t-PAI-C antibody and a europium-labeled goat anti-human IgG antibody solution which are integrated on a reagent strip, is fully automatically operated, and is simple and easy to implement. The content of t-PAI-C in serum can be detected within 12-24 min, the detection result is accurate and reliable, besides the advantages of high sensitivity, long storage time, no radioactive pollution, wide measurement range and the like of the TRFIA technology, the characteristic that the combination surface area is enlarged due to the enrichment effect of immune magnetic particles and the full diffusion of the magnetic particles in liquid can be realized, the reaction time is greatly shortened, the detection sensitivity is improved, meanwhile, the magnetic particles and an antibody are directionally connected through chemical groups, the using amount of antigens is greatly reduced, the detection precision is remarkably improved, the automation is realized, the problem that the traditional microplate type enzyme linked immunosorbent assay (ELISA) technology can detect samples only when the samples are accumulated to a certain amount is solved, the instant detection of the samples is realized, and the efficiency is high;
2. the kit of the invention is a high-efficiency time-resolved fluorescence technology taking nano magnetic particles as carriers: the magnetic particles are connected with free amino groups of the t-PAI-C antibody to form immune magnetic particles coated with the t-PAI-C antibody, a large amount of t-PAI-C can be combined in a short time by means of the immune magnetic particles depending on the overlarge specific surface area of the immune magnetic particles, Eu-labeled anti-human IgG is added for tracing through magnetic separation, Eu is separated from a compound by using enhancement liquid and then is chelated with a chelating agent in the enhancement liquid to form a colloidal molecular group, the molecular group can emit strong fluorescence with emission wavelength under the excitation of ultraviolet light, signals are enhanced by millions of times, and the content of t-PAI-C in serum can be sensitively detected.
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In order to more clearly illustrate the embodiments or technical solutions of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
FIG. 1 is a schematic diagram of an embodiment of the present invention;
FIG. 2 is a schematic view of an exploded structure of the present invention;
FIG. 3 is a schematic view of the internal structure of the cover of the present invention;
FIG. 4 is a schematic view of the internal structure of the cartridge of the present invention;
FIG. 5 is a schematic view of the lower portion of the storage compartment;
FIG. 6 is a schematic view of the structure of the reagent rack of the present invention;
FIG. 7 is a schematic view of the structure of a reagent strip of the present invention;
FIG. 8 is a schematic view of the structure of the reagent strip of the present invention inserted into the reagent rack;
FIG. 9 is a schematic view of the structure of the reagent rack of the present invention placed in the cassette body;
FIG. 10 is a schematic diagram of the structure of the reagent rack extending out of the cassette body according to the present invention.
Illustration of the drawings: 1-box body, 2-cover body, 3-object placing block, 4-freeze-drying cup, 5-object placing hole, 6-object placing groove, 7-reagent strip, 8-reagent rack, 9-reagent plate, 10-reagent hole, 11-battery pack, 12-charging socket, 13-switch, 14-refrigeration sheet, 15-temperature sensor, 16-display screen, 17-top cover, 18-bottom plate, 19-handle, 20-coaming, 21-elastic piece, 22-elastic layer, 23-support bar, 24-lifting plate, 25-push-pull rod, 26-push-pull handle, 27-limit block, 28-fixed block, 29-rotating rod, 30-support frame, 31-fixed plate, 32-cross support plate, 33-side support plate, 34-chute, 35-handle, 36-detection hole.
Detailed Description
The technical solution of the present invention will be further specifically described below by way of specific examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.
In the present invention, all parts and percentages are by weight, unless otherwise specified, and the equipment and materials used are commercially available or commonly used in the art. The methods in the following examples are conventional in the art unless otherwise specified. The reagents used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.
EXAMPLE 1 preparation of the kit
A time-resolved fluoroimmunoassay kit for detecting tissue plasminogen activator/plasminogen activator inhibitor-1 complex (t-PAI-C), comprising the following components:
preparation of the t-PAI-C calibrator solution: taking high concentration of t-PAI-CThe solution was diluted with 2g/L BSA and 1g/L NaN3Preparing 50mmol/L Tris-HCl reaction buffer solution with pH of 7.8 into calibrator solutions with different concentrations, subpackaging, lyophilizing, and storing at 2-8 deg.C.
The preparation of the magnetic particle solution coated with the t-PAI-C antigen comprises the following steps: activating ferroferric oxide microspheres with the diameter of 50-5000nm by using glutaraldehyde with the mass concentration of 5-10%, uniformly mixing for 4 hours at room temperature, washing for 1-3 times by using 0.05mol/L PBS buffer solution with the pH value of 7.2, and suspending by using the buffer solution to prepare magnetic particle suspension with the concentration of 100 mg/mL; taking 100 μ L of the magnetic particle suspension, adding 1mL of 0.05mol/L PBS buffer solution with pH of 7.2 and 50 μ g of t-PAI-C antibody, mixing and incubating at 25 deg.C for 2 hr, performing magnetic separation, retaining magnetic particles, washing with 0.05mol/L PBS buffer solution with pH of 7.2 for 1-3 times, blocking with 1mL of 0.0lmol/L PBS buffer solution with 5% BSA at 25 deg.C for 30 min, washing with 0.05mol/L PBS buffer solution with pH of 7.2 for 1-3 times, washing with 0.5% BSA at mass concentration and 0.1% NaN at mass concentration30.05mol/L Tris-HCl buffer solution with pH 7.2, subpackaging and storing at 2-8 ℃.
The preparation of the europium-labeled goat anti-human IgG antibody solution comprises the following steps: taking 1mL goat anti-human IgG antibody, converting buffer conditions through a PD-10 column, and eluting with 50mmol/L Na containing 0.155 mol/L NaCl and having a pH of 8.52CO3-NaHCO3And (2) buffering solution, collecting protein peak to obtain sheep anti-human IgG antibody solution concentrated to 2g/L, taking 500 mu L of the sheep anti-human IgG antibody solution, adding 0.2mg of benzyldiethylenetriamine tetraacetic acid europium sodium isothiocyanate, performing magnetic stirring reaction at 25 ℃ for 20 hours, transferring the reaction solution to a Sepharose CL-6B column equilibrated by a Tris buffer solution with the concentration of 80mmol/L and the pH value of 7.8 in advance, performing chromatography, collecting protein peak, diluting, subpackaging, performing vacuum freeze drying, and storing at 2-8 ℃.
Preparing the enhancement solution: 3.6mL of glacial acetic acid, 0.5g of sodium acetate, 0.05g of beta-naphthoyl trifluoroacetone, 0.03g of trioctylphosphine oxide and 1mL of Triton X-100 are uniformly mixed, deionized water is added to the mixture to reach the constant volume of 1000mL, and the mixture is subpackaged and stored at the temperature of between 2 and 8 ℃.
The reaction buffer solution is 50mmol/L Tris-HCl with pH7.8, contains 8mmol/L NaCl, 0.1% BSA, 50 mu mol/L DTPA, 0.1ml/L Tween-80 and 0.1% NaN3。
The cleaning solution is a buffer solution containing 0.48g/L of Tris, 12.49g/L of NaCl, 1.11g/L of Tween-20 and hydrochloric acid for adjusting the pH value to 7.8.
Example 2
A time-resolved fluoroimmunoassay kit for detecting t-PAI-C is shown in figures 1 and 2 and comprises a box body 1, a cover body 2 covering an opening at the top end of the box body 1 and a battery pack 11 positioned below the bottom of the box body 1.
As shown in fig. 4, the left side inside the box body 1 is provided with a material placing block 3, the upper surface of the material placing block 3 is provided with two rows of material placing holes 5 for storing freeze-drying cups 4, the right side inside the box body 1 is provided with a material placing groove 6, two opposite side surfaces of the material placing groove 6 are provided with support bars 23, the upper end surfaces of the support bars 23 are provided with lifting plates 24, the upper end surfaces of the lifting plates 24 are provided with reagent racks 8 for storing reagent strips 7, as shown in fig. 7, the reagent strips 7 comprise reagent plates 9 detachably connected with the reagent racks 8, the upper end surfaces of the reagent plates 9 are provided with a plurality of reagent holes 10 for storing reactants, according to actual conditions, a plurality of reserved holes, a plurality of cleaning holes, a magnetic particle solution hole, a reaction buffer solution hole, an europium marker hole, a grabbing hole and a reinforcing solution hole are arranged, one side of each reagent strip 7 is provided with a handle 35, the middle part of each reagent strip 7 is, the upper surfaces of the reagent strip 7 and the freeze-drying cup 4 are both packaged by adopting a film sealing material, and the film sealing material is preferably a high polymer film, an aluminum foil or an aluminum-plastic composite material; a refrigerating piece 14 is embedded in the center of the lower surface of the box body 1, a temperature sensor 15 is arranged in the storage groove 6, a display screen 16 is arranged on the front end face of the box body 1, and the output end of the temperature sensor 15 is electrically connected with the refrigerating piece 14 and the display screen 16; the bottom of the box body 1 is also provided with a battery pack 11, and the front end face of the battery pack 11 is provided with a charging socket 12 and a switch 13.
As shown in fig. 5, a through hole is formed in the right side wall of the box body 1, a push-pull rod 25 is sleeved in the through hole through a bearing, a push-pull handle 26 is arranged at one end of the push-pull rod 25, a limit block 27 is arranged at the other end of the push-pull rod 25, the push-pull handle 26 is positioned outside the box body 1, the limit block 27 is positioned in the object placing groove 6, the push-pull rod 25 and a fixed block 28 positioned at the bottom of the object placing groove 6 are connected in a sliding mode, the fixed block 28 is positioned between the push-pull handle 26 and the limit block 27, the upper portion of the push-pull rod 25.
As shown in fig. 3, the cover body 2 is composed of a top cover 17 and a bottom plate 18, a handle 19 is arranged in the middle of the top cover 17, surrounding edges of the top cover 17 are provided with enclosing plates 20 in a downward vertical extending mode, the bottom plate 18 is arranged in a space enclosed by the enclosing plates 20, a plurality of elastic members 21 are distributed between the top cover 17 and the bottom plate 18 in an array mode, the elastic members 21 are springs, an elastic layer 22 is formed on the lower surface of the bottom plate 18, and the elastic layer 22 is made of sponge.
As shown in fig. 6, the reagent rack 8 is composed of a plurality of support frames 30 arranged side by side and having an inverted "U" shape and a fixing plate 31 connected to the support frames 30, each support frame 30 is composed of a cross plate 32 and side plates 33 perpendicular to the lower sides of the two ends of the cross plate 32, sliding grooves 34 are formed on both sides of the cross plate 32, and as shown in fig. 8, the reagent plates 9 are embedded in the sliding grooves 34 of two adjacent cross plates 32 and can slide along the sliding grooves 34.
The invention discloses a working principle of a time-resolved fluorescence immunoassay kit based on nano magnetic particles, which comprises the following steps: respectively placing the prepared reagent liquid and the freeze-drying calibration product into a reagent hole 10 and a freeze-drying cup 4, then sealing the upper surfaces of a reagent strip 7 and the freeze-drying cup 4 by using a sealing material, placing the freeze-drying cup 4 with the sealed membrane into a placing hole 5 of an object placing block 3, inserting the reagent strip with the sealed membrane into a sliding groove 34 of a reagent rack 8, and then placing the reagent rack 8 onto a lifting plate 24 of an object placing groove 6; the cover body 2 is covered, the cover body 2 and the box body 1 are sealed through a sealing ring, and the elastic layer 22 on the cover body 2 firmly fixes the upper surfaces of the freeze-drying cup 4 and the reagent rack 8 through the elastic action of the elastic piece 21; the temperature sensor 15 is used for sensing the temperature in the box body 1 and displaying the temperature on the display screen 16, when the temperature is too high, the battery pack 11 can automatically supply power to the semiconductor refrigeration piece 14 for refrigeration and cooling, when the temperature reaches a set temperature, the semiconductor refrigeration piece 14 stops working, the semiconductor refrigeration piece 14 repeatedly works within the set temperature in a periodic mode by using a high-low temperature limiter arranged in the temperature sensor 15, and the temperature is set to be 2-8 ℃; as shown in fig. 10, when a reagent strip 7 on a reagent rack 8 needs to be taken out for use, the push-pull handle 26 is pushed, the push-pull rod 25 is driven to move, the push-pull rod 25 moves leftwards and then pushes the rotating rod 29, the obliquely arranged rotating rod 29 is gradually straightened, the upper end of the rotating rod 29 pushes the lifting plate 24 to move upwards, the reagent rack 8 is ejected out of the box body 1, an operator can quickly and conveniently take the reagent strip 7, as shown in fig. 9, when the reagent rack 8 needs to be put back into the box body 1, the push-pull handle 26 is pulled, the push-pull rod 25 is driven to move, the position where the push-pull rod 25 moves can be limited by the limiting block 27, the push-pull rod 25 moves rightwards to push the rotating rod 29, the rotating rod in a vertical state is inclined, the lifting plate 24 moves downwards, and the.
The kit was composed as in example 1, with 5 strips per rack, and a kit containing 25-100 strips per rack with 2 vials of the lyophilized calibrator of t-PAI-C attached.
This kit simple structure, reasonable in design not only has the function of depositing, can maintain low temperature environment in use or transportation, can avoid rocking of kit removal in-process reagent strip moreover, plays fine guard action to the reagent strip, can make quick convenient reagent strip of taking of operating personnel in addition, simple and practical.
Example 3
A method for detecting tissue plasminogen activator/plasminogen activator inhibitor-1 complex (t-PAI-C) by using the time-resolved fluoroimmunoassay kit described in example 1 comprises the following steps:
1. after calibrating the calibrator solution, diluting the serum to be tested by using a reaction buffer solution of 1:100, taking 100ul, mixing the diluted serum with the 50ul magnetic particle solution coated with the t-PAI-C antibody, reacting for 20 minutes, carrying out magnetic separation, washing for 2 times by using a washing solution, adding the europium-labeled goat anti-human IgG antibody solution diluted by using the reaction buffer solution into the mixed magnetic particle solution, incubating for 20 minutes, then washing by using the washing solution, carrying out magnetic separation, repeating the washing step for 4 times, and then adding the enhancement solution;
2. and respectively measuring the fluorescence value of europium in the solution added with the enhancement solution by using a full-automatic time-resolved fluorescence immunoassay analyzer, and substituting the corresponding fluorescence value of the sample to be detected into the corresponding standard curve according to the calibration curve to calculate the content of t-PAI-C in the sample to be detected.
Application example 1
The detection method (example 3) performed by using the kit of the invention is compared with the ELISA detection method established by the laboratory, and the specific detection data are as follows:
sample 01: the detection value of the method is 34.6ng/mL, the ELISA method is 36.7ng/mL,
sample 02: the detection value of the method is 9.4ng/mL, the ELISA method is 7.9ng/mL,
sample 03: the detection value of the method is 3.6ng/mL, the ELISA method is 4.2ng/mL,
sample 04: the detection value of the method is 1.6ng/mL, the ELISA method is 1.9ng/mL,
sample 05: the detection value of the method is 6.8ng/mL, the ELISA method is 6.4ng/mL,
sample 06: the detection value of the method is 8.5ng/mL, the ELISA method is 8.7ng/mL,
sample 07: the detection value of the method is 98.1ng/mL, the ELISA method is 78.6ng/mL,
sample 08: the detection value of the method is 87.3ng/mL, the ELISA method is 86.3ng/mL,
sample 09: the detection value of the method is 6.9ng/mL, the ELISA method is 7.3ng/mL,
sample 10: the detection value of the method is 4.7ng/mL, the ELISA method is 4.8ng/mL,
sample 11: the detection value of the method is 2.8ng/mL, the ELISA method is 2.6ng/mL,
sample 12: the detection value of the method is 5.9ng/mL, the ELISA method is 5.9ng/mL,
sample 13: the detection value of the method is 8.4ng/mL, the ELISA method is 7.4ng/mL,
sample 14: the detection value of the method is 6.8ng/mL, the ELISA method is 4.7ng/mL,
sample 15: the detection value of the method is 17.9ng/mL, and the ELISA method is 19.6 ng/mL.
The method has the advantages that:
1. each detection of ELISA needs 2.5 hours, and each detection of the method only needs 12 minutes;
2. the ELISA detection of 15 samples needs to consume 21 parts of reagents (15 parts are used for detection, 6 parts are used for establishing a standard curve for subsequent quantification), and the method only consumes 15 parts of reagents;
3. the detection limit of the ELISA detection method is 1-100 ng/mL, and the detection limit of the method is 0.1-200 ng/mL.
The embodiments are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same or similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.
The time-resolved fluoroimmunoassay kit for detecting tissue plasminogen activator/plasminogen activator inhibitor-1 complex (t-PAI-C) and the application thereof provided by the invention are described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (10)
1. A time-resolved fluoroimmunoassay kit for detecting tissue plasminogen activator/plasminogen activator inhibitor-1 complex (t-PAI-C), comprising: reaction buffer solution, cleaning solution, enhancement solution, magnetic particle solution coated with t-PAI-C antigen, europium-labeled goat anti-human IgG antibody solution and freeze-dried calibrator of t-PAI-C integrated on the reagent strip.
2. The kit of claim 1, wherein: the magnetic particle solution coated with the t-PAI-C antigen is prepared by connecting activated magnetic particles with the diameter of 50-5000 nanometers with the t-PAI-C antigen, washing, sealing and washing.
3. The kit of claim 1, wherein: the europium-labeled goat anti-human IgG antibody solution is prepared by using Eu3+-N2- [ P-Isocyanato-benzyl group]A sodium diethylenetriamine tetracetate-labeled goat anti-human IgG antibody is prepared, and the goat anti-human IgG antibody and the Eu are3+-N2- [ P-Isocyanato-benzyl group]The mass ratio of the-sodium diethylenetriamine tetracetate is 4-6: 1.
4. The kit of claim 1, wherein: the reaction buffer solution is 50mmol/L Tris-HCl with pH7.8, contains 8mmol/L NaCl, 0.1% BSA, 50 mu mol/L DTPA, 0.1ml/L Tween-80 and 0.1% NaN3。
5. The kit of claim 1, wherein: the cleaning solution is a buffer solution containing 0.48g/L of Tris, 12.49g/L of NaCl, 1.11g/L of Tween-20 and hydrochloric acid for adjusting the pH value to 7.8.
6. The kit of claim 1, wherein: the enhancement solution is a mixed solution containing glacial acetic acid with the volume concentration of 3.6 per thousand, sodium acetate with the volume concentration of 0.5g/L, beta-naphthoyl trifluoroacetone with the volume concentration of 0.05g/L, trioctylphosphine oxide with the volume concentration of 0.03g/L and Triton X-100 with the volume concentration of 1 per thousand.
7. The kit of claim 1, wherein: the calibrator solution of t-PAI-C was prepared with 2g/L BSA and 1g/L NaN350mmol/L reaction buffer, pH7.8 Tris-HCl, and t-PAI-C was formulated into calibrator solutions of various concentrations and stored by lyophilization.
8. The kit of claim 1, wherein: the reagent kit comprises a kit body and a cover, wherein the cover is arranged at the opening at the top end of the kit body, a material placing block is arranged on one side inside the kit body, a plurality of material placing holes for storing the freeze-drying cups are formed in the upper surface of the material placing block, a material placing groove is formed in the other side inside the kit body, a reagent rack for storing reagent strips is arranged in the material placing groove, the reagent strips comprise reagent plates detachably connected with the reagent rack, and a plurality of reagent holes for storing reactants are formed in the upper end surface of each reagent plate in a downward mode.
9. A method for preparing the kit of claim 1, characterized in that the method comprises the steps of:
preparation of the t-PAI-C calibrator solution: taking high concentration t-PAI-C solution, adding 2g/L BSA and 1g/L NaN3Preparing 50mmol/L Tris-HCl reaction buffer solution with pH of 7.8 into calibrator solutions with different concentrations, subpackaging, lyophilizing, and storing at 2-8 deg.C;
preparing the magnetic particle solution coated with the t-PAI-C antigen: activating ferroferric oxide microspheres with the diameter of 50-5000nm by using glutaraldehyde with the mass concentration of 5-10%, uniformly mixing for 4 hours at room temperature, washing for 1-3 times by using 0.05mol/L PBS buffer solution with the pH value of 7.2, and suspending by using the buffer solution to prepare magnetic particle suspension with the concentration of 100 mg/mL; taking 100 μ L of the magnetic particle suspension, adding 1mL of 0.05mol/L PBS buffer solution with pH of 7.2 and 50 μ g of t-PAI-C antigen, mixing and incubating at 25 deg.C for 2 hr, performing magnetic separation, retaining magnetic particles, washing with 0.05mol/L PBS buffer solution with pH of 7.2 for 1-3 times, blocking with 1mL of 0.0lmol/L PBS buffer solution with 5% BSA at 25 deg.C for 30 min, washing with 0.05mol/L PBS buffer solution with pH of 7.2 for 1-3 times, washing with 0.5% BSA at mass concentration and 0.1% NaN at mass concentration30.05mol/L Tris-HCl buffer solution with pH value of 7.2 is used for resuspension, subpackaging and storing at 2-8 ℃;
preparing the europium-labeled goat anti-human IgG antibody solution: taking 1mL goat anti-human IgG antibody, converting buffer conditions through a PD-10 column, and eluting with 50mmol/L Na containing 0.155 mol/L NaCl and having a pH of 8.52CO3-NaHCO3Buffering solution, collecting protein peak to obtain concentrated 2g/L goat anti-human IgG antibody solution, collectingAdding 0.2mg of benzyldiethylenetriamine tetraacetic acid europium sodium isothiocyanate into 500 mu L of the goat anti-human IgG antibody solution, carrying out magnetic stirring reaction at 25 ℃ for 20 hours, transferring the reaction solution to a Sepharose CL-6B column which is balanced by a Tris buffer solution with the pH value of 7.8 and the concentration of 80mmol/L for chromatography, collecting protein peaks, diluting, subpackaging, carrying out vacuum freeze drying, and storing at 2-8 ℃;
preparing the enhancement solution: uniformly mixing 3.6mL of glacial acetic acid, 0.5g of sodium acetate, 0.05g of beta-naphthoyl trifluoroacetone, 0.03g of trioctylphosphine oxide and 1mL of Triton X-100, adding deionized water to a constant volume of 1000mL, subpackaging and storing at 2-8 ℃;
the reaction buffer solution is 50mmol/L Tris-HCl with pH7.8, contains 8mmol/L NaCl, 0.1% BSA, 50 mu mol/L DTPA, 0.1ml/L Tween-80 and 0.1% NaN3；
The cleaning solution is a buffer solution containing 0.48g/L of Tris, 12.49g/L of NaCl, 1.11g/L of Tween-20 and hydrochloric acid for adjusting the pH value to 7.8.
10. A method for detecting tissue plasminogen activator/plasminogen activator inhibitor-1 complex (t-PAI-C) using the time-resolved fluoroimmunoassay kit of claim 1, comprising the steps of:
(1) respectively taking a calibrator solution of the t-PAI-C and a sample to be detected, mixing the calibrator solution of the t-PAI-C and the sample to be detected with the magnetic particle solution coated with the t-PAI-C antigen, incubating, then washing with the washing solution, performing magnetic separation, adding a europium-labeled goat anti-human IgG antibody solution diluted by the reaction buffer solution into the mixed magnetic particle solution, further incubating, then washing with the washing solution, performing magnetic separation, then sucking and removing a supernatant, repeating the washing steps, and then adding the enhancement solution;
(2) respectively measuring the fluorescence value of europium in the solution added with the enhancement solution by using a time-resolved fluoroimmunoassay analyzer to obtain the fluorescence values of the calibrator solution of the t-PAI-C and the sample to be detected, then respectively drawing a standard curve according to the concentration of the t-PAI-C in the calibrator solution and the respective corresponding fluorescence value, and then substituting the corresponding fluorescence value of the sample to be detected into the corresponding t-PAI-C standard curve to calculate the content of the t-PAI-C in the sample to be detected.
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