Fluorescence immunochromatography kit for PCT/MIF/sTREM-1 combined detection and application thereofTechnical Field
The invention belongs to the technical field of medicines, and particularly relates to a fluorescence immunochromatography kit for PCT/MIF/sTREM-1 combined detection and application thereof.
Background
Procalcitonin (Procalcitonin, PCT) is a prohormone of calcitonin, consisting of 116 amino acids, and has a molecular weight of about 12.8kD, which is a hormone-inactive glycoprotein and is also an endogenous non-steroidal anti-inflammatory substance, produced by the thyroid gland in the case of non-infection. When systemic infection occurs, many different cell types of organs secrete procalcitonin after being stimulated by a pro-inflammatory reaction, and particularly when being infected by bacteria, the metabolism is little or not dependent on kidney function, the clearance rate of the patients with renal failure is not affected, and PCT metabolism is not affected by steroid hormones. In clinical diagnosis, procalcitonin concentration of patients with sepsis rises earlier, and early diagnosis and monitoring of doctors are facilitated. The literature reports that PCT in serum begins to rise within 2-4 hours under systemic infection, peaks for 8-24 hours, lasts for days or weeks, and when the PCT is greater than a certain value, patients are considered to possibly develop severe. PCT is currently used mainly in the diagnosis of systemic severe bacterial infections, and can also determine the severity of infection, the effect of treatment, evaluate prognosis and guide the initiation and withdrawal of antibacterial drug therapy based on its dynamic changes. PCT is an important bacterial infection biomarker commonly used clinically at present and having great reference significance.
Macrophage migration inhibitory factor （Macrophage migration inhibitory factor, HYPERLINK"https://www.cusabio.cn/Recombinant-Proteins/Recombinant-Human-Macrophage-migration-inhibitory-factor(MIF)-1179939.html" MIF） is a cytokine with a wide range of functions, which has an irreplaceable role in inflammation and immunity, and which is secreted to limit macrophage migration after the body is stimulated by various inflammatory factors. MIF plays an important role in infectious shock, and its expression and secretion are increased in various acute and chronic inflammatory diseases such as sepsis, arthritis, asthma, and the like. The elevated serum MIF concentration in sepsis patients is closely related to disease severity and prognosis.
TREM1 (TRIGGERING RECEPTOR EXPRESSED ON MYELOID CELLS-1) is one of the immunoglobulin superfamily receptor members associated with infection, and sTREM-1 is its soluble form. sTREM-1 is expressed on the surfaces of neutrophils and mononuclear/macrophages in the acute inflammatory reaction, released in blood or body fluid, and has early appearance and short half-life. The heightening of the traditional Chinese medicine composition can be seen in patients with bacterial meningitis, bacterial pleural effusion, chronic obstructive pulmonary combined infection, sepsis and the like; and little or no expression in non-infectious inflammatory disease, suggesting that it may be a more specific indicator of diagnosing infection.
In the prior art, inflammation infection and sepsis detection are usually carried out by adopting a single inflammation index, but the detection of infection by using PCT alone is not reliable. PCT tends to increase normally or only slightly in focal infections, and PCT does not increase in fever caused by some non-bacterial infections, so that the accuracy of diagnosing infection by separate indicators is to be improved.
Immunochromatography, also called lateral immunochromatography detection (Lateral flow immunoassay), is based on the principle of antigen-antibody reaction, and the detection of a target substance is realized by specific binding of an antibody and a target analyte. Immunochromatography techniques typically employ the form of a test strip or test card to immobilize antibodies on a solid support, such as a nitrocellulose membrane or a microplate. After the target analyte in the sample is combined with the antibody on the solid carrier, the detection result can be intuitively observed and judged through the color development or signal generation of the marker (such as colloidal gold, fluorescent dye and the like). However, many complexes and proteins in biological fluids and serum themselves fluoresce, and thus the sensitivity is severely degraded when fluorescence detection is performed using conventional chromophores such as fluorescein.
In view of the above, there is an urgent need for an immunochromatography method that has high precision, low detection background signal, high sensitivity, and wide linear range when simultaneously detecting a plurality of proteins.
Disclosure of Invention
The invention aims to provide a fluorescence immunochromatography kit for PCT/MIF/sTREM-1 combined detection, which has the advantages of high precision, low detection background signal, high sensitivity, wide linear range and simplicity in operation.
The invention provides a fluorescence immunochromatography kit for PCT/MIF/sTREM-1 joint detection, which comprises a fluorescence immunochromatography test strip, wherein the test strip comprises a sample pad 1, a fluorescence pad 2, a bottom plate 3, a coating film 8 and a water absorption pad 9, the sample pad 1, the fluorescence pad 2, the coating film 8 and the water absorption pad 9 are sequentially connected and fixed on the bottom plate 3 in the horizontal direction, a detection line and a quality control line 7 are coated on the coating film 8, the fluorescence pad 2 comprises an antibody-marked immunofluorescence microsphere, and the antibody-marked immunofluorescence microsphere comprises a PCT monoclonal antibody-marked immunofluorescence microsphere, an MIF monoclonal antibody-marked immunofluorescence microsphere and an sTREM-1 monoclonal antibody-marked immunofluorescence microsphere.
In some embodiments, the sample pad 1 is a buffer treated sample pad 1. Preferably, the sample pad is treated with PBS buffer; the concentration of the PBS buffer is preferably 10mM and the pH is preferably 7.0-7.5. Further, the PBS buffer contains 0.5% (w/v%) BSA,0.5% (w/v%) Tween-20; the sample pad treated by the PBS buffer solution containing 0.5% (w/v%) BSA and 0.5% (w/v%) Tween-20 can effectively reduce the interference of detection background signals and improve the sensitivity of PCT, MIF, sTREM-1 detection. More preferably, the PBS buffer comprises 0.5% (w/v%) BSA,0.5% (w/v%) Tween-20,0.03% (w/v%) casein, 0.05% (w/v%) sucrose, 0.01% (w/v%) calcium chloride. The inventor of the present application found that 0.03% (w/v%) casein, 0.05% (w/v%) sucrose and 0.01% (w/v%) calcium chloride were added to the treatment solution to post-treat the sample pad, and the sample pad was more effective for detecting antigens of MIF and sTREM-1, and the interference of detection background signals was effectively reduced, and the sensitivity of both detection was improved.
Further, the fluorescent line 2 further comprises immunofluorescent microspheres marked by rabbit IgG antibodies (when the antibodies on the quality control line are goat anti-rabbit sources).
In some embodiments, the immunofluorescent microsphere has a particle size of 100 to 500nm. If the particle size is higher than 500nm, the microspheres are unstable after being connected with the antibody, are easy to precipitate, and seriously influence the detection linearity and stability of the project; if the particle size is less than 100nm, the antibody connection process becomes complex, the centrifugation time is greatly prolonged, and the detection linearity and stability of the project are also seriously affected. Preferably, the particle size of the fluorescent microsphere is 200-300 nm, and the detection linearity and stability are further improved.
Further, the marked immunofluorescence microsphere of the sTREM-1 monoclonal antibody is selected from the sTREM-1 immunofluorescence microsphere with the particle size of 100nm and the sTREM-1 immunofluorescence microsphere with the particle size of 300nm, and the weight ratio of the sTREM-1 immunofluorescence microsphere to the sTREM-1 immunofluorescence microsphere is 2:1-4:1 (preferably 3:1). When the sample contains high-concentration sTREM-1, the sTREM-1 in the sample can be combined with the anti-sTREM-1 monoclonal antibody coupled with fluorescent microspheres with two particle sizes (100 nm and 300 nm) and the anti-sTREM-1 monoclonal antibody coated on NC (membrane nitrocellulose membrane) in a specific manner, a double-antibody sandwich structure is formed at a detection line, and the fluorescence intensity at the detection line is the sum of the fluorescent microspheres with the two particle sizes. When the concentration of the sTREM-1 in the sample is low, the sTREM-1 in the sample is mainly combined with the anti-sTREM-1 monoclonal antibody coupled with the fluorescent microsphere with the particle size of 300nm and the anti-sTREM-1 monoclonal antibody coated on the NC film in a specific manner, a double-antibody sandwich structure is formed at the detection line, and the fluorescence intensity at the detection line is the fluorescence of the fluorescent microsphere with the particle size of 300 nm; the technical scheme provided by the invention selects two kinds of microspheres with the particle size to simultaneously detect the sTREM-1 antigen with low concentration and high concentration, thereby further improving the detection linear range of the sTREM-1 and further improving the sensitivity.
In some embodiments, the interior of the immunofluorescent microsphere is filled with a chelate of a lanthanide; the lanthanide is preferably one or more of europium, samarium, zinc and dysprosium; the immunofluorescent microsphere is preferably an europium particle chelate filled immunofluorescent microsphere. Preferably, the solid content of rare earth europium (Eu) is 0.5-2% (w/v%), preferably 1% (w/v%). Compared with the traditional chromophores such as fluorescein, the immunofluorescence microsphere containing rare earth europium (Eu) has higher sensitivity.
In some embodiments, the preparation steps of the fluorescent pad 2 include: and spraying the mixed solution of immunofluorescent microspheres marked by antibodies (such as PCT monoclonal antibody 1, MIF monoclonal antibody 1, sTREM-1 monoclonal antibody 1 and rabbit IgG antibody) on the fluorescent pad 2, and drying to prepare the fluorescent pad 2 containing the immunofluorescent microspheres marked by the antibodies.
In some embodiments, the amount of spray applied to the fluorescent pad 2 is 4-6. Mu.L/cm. Preferably 5. Mu.L/cm. The inventor finds that the sensitivity lower than 4 is not achieved in the experimental process; no significant change above 6, but the cost would be much higher.
In some embodiments, the drying temperature is 35-40 ℃, preferably 37 ℃, and the drying time is 16-24 hours.
In some embodiments, the antibody (e.g., PCT, MIF, sTREM-1, rabbit IgG) -labeled immunofluorescent-microsphere is dissolved in PBS buffer containing 0.5% (w/v%) BSA to form a mixed solution of immunofluorescent-microsphere at a concentration of 0.001% -0.1% (w/v%). The inventors found that sensitivity below 0.001% (w/v%) was not achieved during the experiment; above 0.1% (w/v%) microsphere release effect is poor and precision is poor.
In some embodiments, the steps of preparing the immunofluorescent microsphere mixed solution labeled with the antibody (such as PCT monoclonal antibody 1, MIF monoclonal antibody 1, sTREM-1 monoclonal antibody 1, rabbit IgG antibody) include: reacting the immunofluorescence microsphere with the antibody, sealing and then performing ultrasonic treatment.
In some embodiments, the immunofluorescent microsphere is activated, and the specific steps include: dissolving immunofluorescent microsphere in buffer solution 1, ultrasonic treating, adding activator, reacting and centrifuging. Preferred buffer 1 is MES buffer; the PH of the MES buffer solution is 6-6.5; the inventors also tried other buffer formulations during the experiment, and overall the signal values were not as good as those of MES.
Further, the activator is NHS and/or EDC; the final concentration of NHS is 0.02-0.1mg/mL, preferably 0.05mg/mL; the final concentration of EDC is 0.02-0.1mg/mL, preferably 0.05mg/mL.
In some embodiments, the immunofluorescent microsphere is reacted with the activator in the absence of light for a period of 20-60 minutes; preferably 30min.
In some embodiments, after the fluorescent microsphere is subjected to activation treatment, the fluorescent microsphere is subjected to redissolution by using a redissolution reagent 1, an antibody reaction is added, then a blocking reagent is added for blocking treatment, and centrifugation and redissolution by using a redissolution reagent 2 are performed.
Further, the re-dissolving agent 1 is PBS buffer solution.
Further, the reaction time of the immunofluorescent microsphere with the antibody is 2-4 hours, preferably 3 hours.
Further, the blocking reagent was BSA at a concentration of 0.5% (w/v%).
Further, the reconstitution reagent 2 was a PBS buffer containing 0.5% BSA. The inventors have also tried to have different ratios of BSA in the reconstitution reagent 2 during the experiment, wherein the BSA content is too high, e.g. 1%, and the signal value is generally less effective than the low concentration.
Further, the rotational speed of the centrifugation is 10000-20000rpm, preferably 12000-15000 rpm.
In some embodiments, the preparation steps of the immunofluorescent microsphere mixed solution comprising an antibody (e.g., PCT monoclonal antibody 1, MIF monoclonal antibody 1, sTREM-1 monoclonal antibody 1, rabbit IgG antibody) label include:
1) Dissolving fluorescent microspheres in MES buffer solution, centrifuging, and removing supernatant;
2) Adding MES buffer solution, adding an activating agent of NHS and/or EDC, reacting, centrifuging, and removing the supernatant;
3) Adding a redissolution reagent 1 for redissolution, adding an antibody for reaction, centrifuging, and removing the supernatant;
4) Adding a blocking reagent, performing blocking treatment, centrifuging, and removing the supernatant;
5) And adding the redissolution reagent 2 for redissolution again to obtain the mixed solution of the antibody-labeled immunofluorescence microsphere.
In some embodiments, buffer 1, buffer 2 is an MES buffer; the PH of the MES buffer solution is 6-6.5; preferably pH 6. The inventors also tried other buffer formulations during the experiment, and overall the signal values were not as good as those of MES.
In some embodiments, the activator is selected from NHS and/or EDC; the final concentration of NHS is 0.02-0.1mg/mL, preferably 0.05mg/mL; the final concentration of EDC is 0.02-0.1mg/mL, preferably 0.05mg/mL.
In some embodiments, the immunofluorescent microsphere is reacted with the activator in the absence of light for a period of 20-60 minutes; preferably 30min.
In some embodiments, the immunofluorescent microsphere is reacted with the antibody for a period of 2-4 hours, preferably 3 hours.
In some embodiments, the blocking reagent is BSA at a concentration of 0.5% (w/v%).
In some embodiments, the reconstitution reagent 1 is a PBS buffer, having a pH of 7-7.5, preferably a pH of 7.2. The reconstitution reagent 2 is a PBS buffer containing 0.5% BSA, and has a pH of 7-7.5, preferably 7.2. The inventors have also tried to have different ratios of BSA in the reconstitution reagent 2 during the experiment, wherein the BSA content is too high, e.g. 2%, and the signal value is generally less effective than the low concentration.
In some embodiments, the preparation steps of the immunofluorescent microsphere mixed solution comprising an antibody (e.g., PCT monoclonal antibody 1, MIF monoclonal antibody 1, sTREM-1 monoclonal antibody 1, rabbit IgG antibody) label include:
1) Taking 0.1mL Eu time-resolved fluorescence microsphere with solid content of 1% (W/V); 0.9mL of 25mM MES buffer solution with pH of 6.0 is added, after vortex mixing, 12000-15000 rpm are centrifuged for 30 minutes, and the supernatant is removed;
2) In the last step, 1mL of 25mM MES buffer solution with pH of 6.0 is added, the mixture is uniformly mixed by 100w of ultrasound, EDC and NHS are added, and the final concentration is 0.05mg/mL; vortex mixing uniformly and carrying out light-shielding reaction for 30 minutes; centrifuging for 30 min from 12000-15000 rpm, and removing supernatant;
3) Adding 1mL of 10mM PBS buffer solution with pH of 7.2 in the previous step, and uniformly mixing by 100w of ultrasound to uniformly disperse the microspheres; then adding 1.1 mg of PCT monoclonal antibody (or MIF monoclonal antibody 1 or sTREM-1 monoclonal antibody 1 or rabbit IgG), mixing uniformly by vortex, standing for reaction for 3 hours, centrifuging in a centrifuge tube 12000-15000 rpm for 30 minutes, and removing the supernatant;
4) 10mM PBS1mL with pH 7.2 containing 0.5% BSA was added to the previous step; after vortex mixing, standing for 30min; centrifuging for 30min from 12000 to 15000 rpm;
5) The microspheres were resuspended in 1mL of 10mM PBS buffer, pH 7.2, containing 0.5% BSA to give a PCT immunofluorescent microsphere solution mixture.
In some embodiments, the detection line is an antibody that detects the one or more antigens. Preferably 3 antibodies, including PCT detection line 4, MIF detection line 5, and sTREM-1 detection line 6. The PCT detection line 4 is a detection line formed by PCT monoclonal antibody 2, the MIF detection line 5) is a detection line formed by MIF monoclonal antibody 2 and the sTREM-1 detection line 6 is an detection line formed by sTREM-1 monoclonal antibody 2.
In some embodiments, the quality control line 7 is a quality control line composed of goat anti-rabbit monoclonal antibodies.
When the sample contains PCT, MIF, sTREM-1 antigen, and the sample solution passes through the fluorescent pad 2, the corresponding immunofluorescence microsphere in the fluorescent pad is redissolved, and is specifically combined with a specific antibody (such as PCT monoclonal antibody 1) on the immunofluorescence microsphere, and then the forward chromatography is continued, the antigen is captured by a capturing monoclonal antibody 2 (such as PCT monoclonal antibody 2) on a corresponding detection line to form a fluorescent microsphere antibody 1-antigen-antibody 2, the sandwich compound and the content of antigen substances in a fluorescent model strong and weak sample are forward closed, and the content of each index is read by a fluorescent immunoassay instrument.
On the other hand, the invention also provides a preparation method of the fluorescent immunochromatography test strip, which comprises the following steps: diluting the mixed solution of immunofluorescent microspheres marked by antibodies (such as PCT monoclonal antibody 1, MIF monoclonal antibody 1, sTREM-1 monoclonal antibody 1 and rabbit IgG antibody) with PBS buffer solution (pH 7.2) of 0.5% BSA, and then spraying the mixed solution on a glass fiber mat to form a fluorescent mat 2; diluting antibody 2 (such as PCT monoclonal antibody 2, MIF monoclonal antibody 2 and sTREM-1 monoclonal antibody 2) with PBS buffer solution (pH 7.2), then streaking on a nitrocellulose membrane (coating film) to form the detection line, streaking the diluted goat anti-rabbit antibody on the nitrocellulose membrane (coating film) to form the quality control line 7, and drying; the glass fiber mat is treated by a sample treatment liquid to form a sample mat 1; the sample pad 1, the fluorescent pad 2, the coating film 8 and the water absorbing pad 9 are adhered on the bottom plate 3 from left to right.
In another aspect, the invention also provides the use of a fluorescent immunochromatographic kit as described above for the detection of antigens, preferably proteins, such as PCT, MIF and sTREM-1.
The fluorescent immunochromatography test strip, the reagent and the kit containing the same and the preparation method thereof can simultaneously and rapidly quantify various antigens (for example, simultaneously detect PCT, MIF, sTREM-1) in a short detection time, not only can improve the sensitivity and the linear range of detection, but also can realize the operation of one-step sample loading (for example, 75-100 mu L of sample), greatly improve the precision and the accuracy of the operation, reduce the error of manual operation, and have simpler operation and are beneficial to industrial production. In the embodiment of the invention, the precision is less than 10% when PCT, MIF, sTREM-1 is detected simultaneously. The lowest detection limit of the detection PCT can reach 0.040ng/ml; the minimum detection limit for detecting MIF can reach 0.040ng/ml; the minimum detection limit for detecting sTREM-1 can reach 4.058pg/ml. PCT linear range is 0.1ng/mL-50 ng/mL, MIF linear range is 0.1ng/mL-50 ng/mL, sTREM-1 linear detection range is 10pg/mL-10000 pg/mL, and linear correlation coefficients of PCT, MIF, sTREM-1 are all >0.99.
Drawings
FIG. 1 is a schematic structural diagram of a fluorescence immunochromatographic test strip for simultaneous detection of PCT/MIF/sTREM-1 in the present invention;
FIG. 2 is a standard graph of PCT of the present invention;
FIG. 3 is a standard graph of the MIF of the present invention;
FIG. 4 is a standard graph of the sTREM-1 of the present invention;
Reference numerals:
1: a sample pad; 2: a fluorescent pad; 3: a bottom plate; 4: PCT detection line; 5: MIF detection line; 6: an sTREM-1 detection line; 7: a quality control line; 8: coating a film; 9: a water absorbing pad.
Detailed Description
The following describes in further detail the embodiments of the present invention with reference to the drawings and examples. The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
The reagents and consumables used in the present invention are commercially available unless otherwise specified.
Example 1 PCT/MIF/sTREM-1 Combined test fluorescence immunochromatographic kit (sample pad treatment)
(1) Preparation of immunofluorescent microsphere mixed solution
Labeling commercial PCT, MIF, sTREM-1 monoclonal antibody 1 with fluorescent microspheres respectively to prepare PCT monoclonal antibody 1, MIF monoclonal antibody 1 and sTREM-1 monoclonal antibody 1 fluorescent immunomicrosphere (T line immunofluorescent microsphere); the rabbit IgG raw material is marked by fluorescent microspheres to prepare the rabbit IgG fluorescent immune microspheres (used as C line, namely quality control line) immune microspheres, which are specifically operated as follows:
Taking a 2.0mLEP tube, and taking 0.1mL Eu time-resolved fluorescence microsphere with the solid content of 1% (W/V) and the diameter of 200-300nm by using a pipette; 0.9mL of 25mM MES (2- (N-morpholinoethanesulfonic acid) buffer solution with pH of 6.0 is added, after vortex mixing, 12000-15000 rpm is centrifugated for 30 minutes, and the supernatant is removed;
Adding 25mM MES (2- (N-morpholinoethanesulfonic acid) buffer solution with pH of 6.0 into the centrifuge tube in the upper step, mixing evenly with 100w ultrasound, adding EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) to the final concentration of 0.05mg/mL; vortex mixing uniformly and carrying out light-shielding reaction for 30 minutes; centrifuging for 30min from 12000-15000 rpm, and removing supernatant;
Adding 1mL of 10mM PBS buffer solution with pH of 7.2 into the centrifuge tube in the upper step, and uniformly mixing by 100w of ultrasound to uniformly disperse the microspheres; then adding 1.1 mg of PCT monoclonal antibody, mixing uniformly by vortex, standing and reacting for 3 hours;
centrifuging the centrifuge tube 12000-15000 rpm from the previous step for 30 minutes, removing the supernatant, and adding 10mM PBS1mL containing 0.5% BSA and having a pH of 7.2; after vortex mixing, standing for 30min; centrifuging for 30min from 12000 to 15000 rpm;
The microspheres were resuspended in 1mL of 10mM PBS buffer at pH 7.2 containing 0.5% Bovine Serum Albumin (BSA) to give PCT immunofluorescent microspheres.
By adopting the preparation process, 0.1mg of PCT monoclonal antibody is changed into 0.1mg of MIF monoclonal antibody 1, sTREM-1 monoclonal antibody 1 and rabbit IgG to prepare MIF immunofluorescence microsphere, sTREM-1 immunofluorescence microsphere and rabbit IgG immunofluorescence microsphere respectively.
And diluting the PCT, MIF, sTREM-1 and rabbit IgG immunofluorescence microsphere obtained by the preparation with 10mM PBS buffer solution containing 0.5% BSA and having pH of 7.2 for 2-200 times, and uniformly mixing the diluted PBS buffer solution and the PBS buffer solution with the same volume to prepare the immunofluorescence microsphere mixed solution with the concentration of 0.001% -0.1%.
(2) Preparation of fluorescent pad
And (3) spraying the PCT, MIF, sTREM-1 and rabbit IgG immunofluorescence microsphere mixed solution prepared in the step (1) on a 30 x 6mm glass fiber mat according to a parameter of 5 mu L/cm, and drying for 16-24 hours at the temperature of 37 ℃ in an oven to prepare a fluorescence mat 2, wherein the fluorescence mat 2 contains 4 immunofluorescence microspheres.
(3) Preparation of coating film
A 30 x 25mM size nitrocellulose membrane (coating) was attached to PVC substrate 3, and then, commercially available PCT monoclonal antibody 2, MIF monoclonal antibody 2, sTREM-1 monoclonal antibody 2 and goat anti-rabbit monoclonal antibody were diluted to 1.0mg/mL with 10mM PBS buffer at pH 7.2, and then, the films were drawn, which were PCT detection line 4, MIF detection line 5, sTREM-1 detection line 6 and quality control line 7, respectively, as shown in fig. 1. And then placing the mixture in a 45 ℃ oven, and drying the mixture for 16-24 hours to prepare the coating film.
(4) Preparation of sample pad
Preparing a sample pad treatment solution 10mM PBS solution (pH 7.2) containing 0.5% BSA and 0.5% Tween-20;
and (3) coating the glass fiber mat with the sample mat treatment liquid, and drying the glass fiber mat for 16-24 hours in a 45 ℃ oven to obtain the treated sample mat.
(5) Assembling of fluorescent immune test paper strip
As shown in fig. 1, a lap joint method is adopted in the bottom plate 3, firstly, the coating film 8 is fixed on the bottom plate 3, then the fluorescent pad 2 of the sample pad 1 is sequentially fixed on one side of the coating film 8, and the water absorbing pad 9 is fixed on the other side of the coating film 8; specifically, the sample pad 1 presses the fluorescent pad 21-2mm, the fluorescent pad 2 presses the coating film 81-2mm, and the water absorbing pad 9 presses the coating film 1-2mm; then cutting the sample into a test strip with the width of 4mm, namely a fluorescence immunochromatography test strip for combined detection of PCT/MIF/sTREM-1.
(6) Group card
And (3) loading the test strip in the step (5) into a corresponding cartridge to form a detection kit.
Example 2 PCT/MIF/sTREM-1 Co-assay fluorescence immunochromatographic kit (sample pad treatment)
(1) Preparation of immunofluorescent microsphere mixed solution
Labeling commercial PCT, MIF, sTREM-1 monoclonal antibody 1 with fluorescent microspheres respectively to prepare PCT monoclonal antibody 1, MIF monoclonal antibody 1 and sTREM-1 monoclonal antibody 1 fluorescent immunomicrosphere (T line immunofluorescent microsphere); the rabbit IgG raw material is marked by fluorescent microspheres to prepare rabbit IgG fluorescent immune microspheres (used as C line (quality control line) immune microspheres) which are specifically prepared as follows:
Taking a 2.0mLEP tube, and taking 0.1mL Eu time-resolved fluorescence microsphere with the solid content of 1% (W/V) and the diameter of 200-300nm by using a pipette; 0.9mL of 25mM MES (2- (N-morpholinoethanesulfonic acid) buffer solution with pH of 6.0 is added, after vortex mixing, 12000-15000 rpm is centrifugated for 30 minutes, and the supernatant is removed;
Adding 25mM MES (2- (N-morpholinoethanesulfonic acid) buffer solution with pH of 6.0 into the centrifuge tube in the upper step, mixing evenly with 100w ultrasound, adding EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) to the final concentration of 0.05mg/mL; vortex mixing uniformly and carrying out light-shielding reaction for 30 minutes; centrifuging for 30min from 12000-15000 rpm, and removing supernatant;
Adding 1mL of 10mM PBS buffer solution with pH of 7.2 into the centrifuge tube in the upper step, and uniformly mixing by 100w of ultrasound to uniformly disperse the microspheres; then adding 1.1 mg of PCT monoclonal antibody, mixing uniformly by vortex, standing and reacting for 3 hours;
centrifuging the centrifuge tube 12000-15000 rpm from the previous step for 30 minutes, removing the supernatant, and adding 10mM PBS1mL containing 0.5% BSA and having a pH of 7.2; after vortex mixing, standing for 30min; centrifuging for 30min from 12000 to 15000 rpm;
The microspheres were resuspended in 1mL of 10mM PBS buffer at pH 7.2 containing 0.5% Bovine Serum Albumin (BSA) to give PCT immunofluorescent microspheres.
By adopting the preparation process, 0.1mg of PCT monoclonal antibody is changed into 0.1mg of MIF monoclonal antibody 1, sTREM-1 monoclonal antibody 1 and rabbit IgG to prepare MIF immunofluorescence microsphere, sTREM-1 immunofluorescence microsphere and rabbit IgG immunofluorescence microsphere respectively.
And respectively using 10mM PBS (pH 7.2) solution containing 0.5% BSA and 0.5% Tween-20 to dilute the obtained PCT, MIF, sTREM-1 and rabbit IgG immunofluorescence microsphere for 2-200 times, and uniformly mixing the diluted solution with the same volume to prepare the immunofluorescence microsphere mixed solution with the concentration of 0.001% -0.1%.
(4) Preparation of sample pad
Sample pad treatment was prepared in 10mM PBS buffer (pH 7.2) containing 0.5% BSA,0.5% Tween-20,0.03% casein, 0.05% sucrose, and 0.01% calcium chloride.
And (3) coating the glass fiber mat with the sample mat treatment liquid, and drying the glass fiber mat for 16-24 hours in a 45 ℃ oven to obtain the treated sample mat.
The preparation process of the fluorescent pad in the step (2), the preparation of the coating film in the step (3), the assembly of the fluorescent immune test strip in the step (5) and the preparation process of the group card in the step (6) are the same as those in the embodiment 1.
Comparative example 1 PCT/MIF/sTREM-1 Combined test fluorescent immunochromatographic kit (sample pad untreated)
The preparation of the immunofluorescent microsphere mixed solution in the step (1), the preparation of the fluorescent mat in the step (2), the preparation of the coating film in the step (3), the assembly of the fluorescent immune test strip in the step (5) and the preparation of the group card in the step (6) are the same as those in the example 1, except that the sample mat in the step (4) is not treated by PBS buffer solution.
Test example 1 drawing of a Standard Curve
When the sample contains PCT, MIF, sTREM-1 antigen, and the sample solution passes through the fluorescent pad, the corresponding immunofluorescence microsphere in the fluorescent pad is redissolved, and is specifically combined with a specific antibody (such as PCT monoclonal antibody 1) on the immunofluorescence microsphere, and then the forward chromatography is continued, the immunofluorescence microsphere solution is captured by a capture monoclonal antibody 2 (such as PCT monoclonal antibody 2) on a corresponding detection line to form a fluorescent microsphere antibody 1-antigen-antibody 2, the sandwich compound and the content of antigen substances in a fluorescent model strong and weak sample are in a forward relation, and the content of each index is read by a fluorescent immunoassay instrument.
Preparing quality control products with different concentrations PCT, MIF, sTREM-1:
PCT:0 ng/mL; 0.1 ng/mL;0.25 ng/mL; 0.5 ng/mL; 1 ng/mL; 2 ng/mL; 5 ng/mL; 10 ng/mL; 20 ng/mL; 50 ng/mL;
MIF: 0 ng/mL; 0.1 ng/mL; 0.25 ng/mL;0.5 ng/mL; 1 ng/mL; 2 ng/mL; 5 ng/mL; 10 ng/mL; 20 ng/mL; 50 ng/mL;
sTREM-1:0 pg/mL; 10 pg/mL; 50 pg/mL; 100 pg/mL; 200 pg/mL; 500 pg/mL; 1000 pg/mL; 2000 pg/mL; 5000 pg/mL; 10000 pg/mL
To the sample pad of the kit of example 1, 75. Mu.L of sample was added, 3 replicates were set for each concentration, OD/RLU values were read by a fluorescence analyzer, and PCT, MIF, sTREM-1 standard curves were plotted, respectively, as shown in FIGS. 2, 3, and 4.
Test results: as can be seen from fig. 2,3 and 4, the PCT R2 value is: 0.9989, an R2 value of MIF, 0.9975, and an R2 value of sTREM-1, 0.9993.
Test example 2 test kit Performance test
(1) Precision detection
Sample 1 (PCT, MIF, sTREM-1 concentrations were 0.5ng/mL, 100pg/mL, respectively), sample 2 (PCT, MIF, sTREM-1 concentrations were 2.0ng/mL, 400pg/mL, respectively), and sample 3 (PCT, MIF, sTREM-1 concentrations were 10.0ng/mL, 1000pg/mL, respectively) were tested using the kit of example 1, and the test was repeated 5 times, and the average value of the test results, and the coefficient of variation CV were calculated, and the test results are shown in Table 1 below.
Table 1 test results
As can be seen from the data in Table 1, the precision of PCT, MIF, sTREM-1 using the triple test strips of the present invention is less than 10%, which meets the requirements.
(2) Lowest detection line (LoD)
Sample 1 (PCT, MIF, sTREM-1 concentrations of 0.04 ng/mL, 0.04 ng/mL and 4 pg/mL, respectively), sample 2 (PCT, MIF, sTREM-1 concentrations of 0.08 ng/mL, 0.08 ng/mL and 8 pg/mL), sample 3 (PCT, MIF, sTREM-1 concentrations of 0.10ng/mL, 0.10ng/mL and 10 pg/mL, respectively), sample 4 (PCT, MIF, sTREM-1 concentrations of 0.12ng/mL, 0.12ng/mL and 12 pg/mL, respectively), sample 5 (PCT, MIF, sTREM-1 concentrations of 0.16 ng/mL, 0.16 ng/mL and 16 pg/mL, respectively), were tested by repeating the measurement 4 times for 3 days, and 60 test results were obtained. And (3) carrying out normal examination on the data by adopting SPSS statistical analysis software, and if the sample measurement result accords with normal distribution, carrying out a calculation formula: lod= LoB + kSDz (SDz is the combined standard deviation of the detection values of the low-concentration samples, the calculation formulas are shown in formulas (1) and (2), if the sample measurement results are in non-normal distribution, the detection results of all batches of samples are respectively counted and ordered, and if the number of the low-level sample distribution results is less than LoB and is not more than 5%, loD of the reagent batch is the median of the distribution of the low-level sample detection results.
Wherein:
ni is the number of results for the ith low concentration level sample;
SDi is the standard deviation of the ith low concentration level sample;
k is the multiplier of the 95 th percentile of the normal distribution;
N is the number of low concentration level samples;
l total number of all low concentration level sample results;
Table 2 test results
As can be seen from the data in Table 2, with the triple detection test strip of the present invention, the lowest detection limit of PCT is 0.040 ng/mL, and the lowest detection limit of MIF is 0.048 ng/mL; the minimum detection limit of sTREM-1 is 4.864 pg/mL.
The limits of detection in example 2 and comparative example 1 were measured by the same method as described above, and the results are shown in Table 3 below:
TABLE 3 detection results
(3) Linear range
The kit of example 1 was used to test the above PCT (sample 1:0.1 ng/mL; sample 2:0.25 ng/mL; sample 3:0.5 ng/mL, sample 4:1 ng/mL, sample 5:2 ng/mL, sample 6:5 ng/mL, sample 7:10 ng/mL, sample 8:20 ng/mL, sample 9:50 ng/mL), MIF (sample 1:0.1 ng/mL, sample 2:0.25 ng/mL, sample 3:0.5 ng/mL, sample 4:1 ng/mL, sample 5:2 ng/mL, sample 6:5 ng/mL, sample 7:10 ng/mL, sample 8:20 ng/mL, sample 9:50 ng/mL), sTREM-1 (sample 1:10 pg/mL, sample 2:50 pg/mL, sample 3:100 pg/mL, sample 4:200 pg/mL, sample 5:500 pg/mL, sample 6:1000 pg/mL, sample 7:2000 pg/mL, sample 8:5000/mL, sample 9:10000 pg/mL) and quality control were tested, and the linear coefficients were calculated as shown in the following equation, and the linear coefficients were repeated for the test results, as shown in Table 3.
Table 4 test results
As can be seen from the data in Table 4, with the triple test strip prepared in example 1 of the present invention, the linear range of PCT was 0.1 ng/mL-50 ng/mL, the linear range of MIF was 0.1 ng/mL-50 ng/mL, the linear detection range of sTREM-1 was 10 pg/mL-10000 pg/mL, and the linear correlation coefficients of PCT, MIF, sTREM-1 were all >0.99. The correlation of the embodiment 2 of the invention in the linear range is good, and meets the requirements.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and substitutions can be made by those skilled in the art without departing from the technical principles of the present invention, and these modifications and substitutions should also be considered as being within the scope of the present invention.