CN106872420B - Kit and method for time-resolved fluorescence quantitative detection of microalbuminuria - Google Patents
Kit and method for time-resolved fluorescence quantitative detection of microalbuminuria Download PDFInfo
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Abstract
the invention relates to a kit and a detection method for quantitatively detecting microalbumin in urine by time-resolved fluorescence. The kit comprises a time-resolved fluorescence test paper card, sample diluent and an SD card containing a calibration curve, wherein the time-resolved fluorescence test paper card comprises a detection test paper strip, the test paper strip comprises a bottom lining, a sample pad, a nitrocellulose membrane and absorbent paper, the sample pad, the nitrocellulose membrane and the absorbent paper are stuck on the bottom lining, a microsphere line, a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane, the microsphere line comprises time-resolved fluorescence microspheres marked with human albumin monoclonal antibodies, the detection line is coated with human albumin recombinant antigens, and the quality control line is coated with goat anti-mouse IgG. The kit can accurately and quantitatively detect the content of the microalbumin in human urine, and the labeling is carried out to connect the microspheres and the antibody through covalent bonds, so that the labeled product is stable. The method has the characteristics of wide detection range (2-300 mg/ml), high sensitivity (detection limit of 2mg/ml), high accuracy, quick and simple detection and the like.
Description
(I) technical field
The invention relates to a kit and a detection method for quantitatively detecting microalbumin in urine by time-resolved fluorescence.
(II) background of the invention
Urine microalbumin refers to the presence of microalbumin in urine. Albumin is a normal protein in blood, and under the condition of normal metabolism of human body, the albumin in urine is very little, and every liter of urine albumin is not more than 20mg (<20mg/L), so that the albumin is called microalbumin. However, early-stage kidney damage such as diabetic nephropathy and hypertensive nephropathy and complications of cardiovascular diseases cause kidney-inherent cell damage, and the structure and function of kidney-inherent cells are changed, so that the albumin content is increased in urine. When the microalbumin in the urine is more than 200mg/L, the trace albumin in the urine proves that a large amount of albumin leaks out, hypoproteinemia possibly occurs, the patient can develop into nephropathy, and the nephropathy can enter the uremia stage if the treatment is not in time. Urine microalbumin is found in diabetic nephropathy, hypertension and preeclampsia and is an early sensitive index of kidney injury.
The prior art mainly comprises an immunoturbidimetry method, a fluorescence immunochromatography method, an enzyme-linked immunosorbent assay, a chemiluminescence method, a colloidal gold method and the like. The colloidal gold method has the advantages of simple and quick operation, but low sensitivity and inaccurate quantification; the enzyme-linked immunosorbent assay has high sensitivity, large sample amount and quantitative detection, but long operation time and low automation; the immunoturbidimetry and chemiluminescence methods are sensitive and accurate, can be applied to full-automatic biochemical instruments, but require instruments and are long in time consumption, suitable for processing a large number of samples and incapable of meeting the aim of rapid detection; the immunofluorescence method is to combine the urine microalbumin antibody on the surface active group of the fluorescent microsphere in a covalent way, and judges the result by judging whether the detection line generates fluorescence after excitation, so that the method is rapid, convenient, accurate and quantitative, has the advantages of high sensitivity, stable marker and the like, and is widely applied to the field of medical immunodetection.
however, many complexes and proteins in biological fluids and blood serum can fluoresce, so that the sensitivity is seriously reduced when fluorescence detection is carried out by using traditional chromophores such as fluorescein and the like, but most of background fluorescence signals exist for a short time, and rare earth fluorescent microspheres (lanthanide chelates) can be used as markers to label antibodies or antigens. Excitation light and emission light can be easily distinguished due to large lanthanide chelate stoke displacement, so that excitation light interference is eliminated, decay time is long, background fluorescence interference can be eliminated through time resolution, and sensitivity can be further improved and background fluorescence can be reduced due to the fact that the lanthanide chelate has a wide excitation spectrum and a narrow emission spectrum. Therefore, the time-resolved fluorescence immunoassay technology is adopted, and two parameters of wavelength and time are detected simultaneously to perform signal resolution, so that the interference of non-specific fluorescence can be effectively eliminated, and the analysis sensitivity is greatly improved.
disclosure of the invention
The invention aims to provide a kit and a detection method for realizing high sensitivity, accurate quantification, convenience and quickness in time-resolved fluorescence quantitative detection of microalbumin by using the sensitivity of a time-resolved fluorescence immunochromatographic technique and combining a dry immunofluorescence analyzer aiming at the defects and defects of the prior art.
The technical scheme adopted by the invention is as follows:
A kit for quantitatively detecting microalbumin in urine by time-resolved fluorescence comprises a time-resolved fluorescence test paper card, a sample diluent and an SD card containing a calibration curve;
The time-resolved fluorescence test paper card comprises a detection test paper strip, wherein the test paper strip consists of a bottom lining, and a sample pad, a nitrocellulose membrane and absorbent paper which are stuck on the bottom lining, and the sample pad, the nitrocellulose membrane and the absorbent paper are sequentially lapped and stuck on the bottom lining;
the nitrocellulose membrane is sequentially provided with a microsphere line, a detection line and a quality control line, the microsphere line, the detection line and the quality control line are parallel to each other, the spacing distance is 3-5 mm, the microsphere line is close to the sample adding hole, the quality control line is far away from the sample adding hole, the microsphere line is composed of time-resolved fluorescent microspheres marked with human albumin monoclonal antibodies, the detection line is coated with human albumin recombinant antigens, and the quality control line is coated with goat anti-mouse IgG antibodies.
the time-resolved fluorescent microsphere is doped with rare earth europium element with the solid content of 1% (w/w), the particle size of the microsphere is 100-300 nm, the microsphere is stable under a ground state, and the time-resolved fluorescent microsphere emits fluorescence with the wavelength range of 600-620 nm under the action of an excitation light source of 350-380 nm.
The content of the human albumin monoclonal antibody in the microsphere line is 50-200 mug antibody/200 mug fluorescent microsphere, the coating concentration of the human albumin recombinant antigen in the detection line is 0.5-2 mg/ml, the dosage is 0.5-1.5 mug coating liquid amount/cm membrane, and the coating concentration of the goat anti-mouse IgG antibody in the quality control line is 0.5-2 mg/ml, the dosage is 0.5-1.5 mug coating liquid amount/cm membrane.
The detection test strip is prepared by the following method:
(1) Activation of time-resolved fluorescent microspheres
After the fluorescent microspheres are treated by ultrasonic waves for 2min, 200 mul of fluorescent microspheres are taken and centrifuged at 14000rpm for 5-15 min at a high speed, precipitates are washed to 1ml by MES solution with 10-100 mM and pH value of 5.0-7.0, and the ultrasonic waves are treated for 2 min; adding 10-100 mu l of 20-100 mg/ml carbodiimide, uniformly mixing for 5-10 min, adding 50-200 mu l of 20-100 mg/ml N-hydroxy thiosuccinimide, uniformly mixing for 10-20 min, then centrifuging at 14000rpm for 5-15 min at a high speed, and washing the precipitate to 1ml by using MES solution with the pH of 5.0-7.0;
(2) Preparation of time-resolved fluorescent microsphere labeled human albumin monoclonal antibody
and (2) carrying out ultrasonic treatment on the activated fluorescent microspheres for 2min, adding a human albumin monoclonal antibody according to 50-200 mug/200 mul, uniformly mixing for 1-3 h, sealing for 0.5-1 h by using 10-50 mM of 0.5% BSA and 10-50 mM of pH 7.5-8.5 Tris-HCl sealing solution, then carrying out high-speed centrifugation for 5-15 min at 14000rpm, washing by using 10-50 mM of 1% NaCl, 0.5% BSA and 0.1% Tween20 and 10-50 mM of pH 7.5-8.5 Tris-Hcl preservation solution, and carrying out heavy suspension to 200 mul and storing in the dark at 4 ℃.
(3) Preparation of coating film
respectively adjusting the concentration of a human albumin recombinant antigen and a goat anti-mouse IgG antibody to 0.5-2 mg/ml by using a coating buffer solution (10 mM PBS buffer solution containing 2.5% of sucrose), wherein the amount of the coating solution is 0.5-1.5 mul per cm of membrane, respectively serving as a detection line and a quality control line which are parallelly scratched on a nitrocellulose membrane for coating, diluting a time-resolved fluorescent microsphere for marking the human albumin monoclonal antibody by 3-6 times by using a microsphere diluent, wherein the amount of the coating solution is 0.5-1.5 mul per cm of membrane, scratching the microsphere line on the nitrocellulose membrane in a direction close to a sample pad for coating, and placing the quality control line, the detection line and the microsphere line in an oven at an interval of 3-7 mM and drying at 45 ℃ overnight.
(4) And (3) sequentially attaching a sample pad, a coating film and absorbent paper to the bottom lining in a mutually overlapped manner to obtain a test paper board, and cutting to obtain the test paper strip.
The time-resolved fluorescence test paper card is fixed on the plastic bottom card by a detection test paper strip, the surface of the test paper is tightly pressed by a card surface, and a sample adding hole and an observation window are respectively reserved on the card surface corresponding to the sample pad and the nitrocellulose membrane.
The sample dilutions were composed as follows: 0.5% of NaCl, 0.5% of BSA, 1% of Tween, 10-50 mM of solvent and 7.5-8.5 of Tris-HCl buffer solution, and packaging the buffer solution into centrifugal tubes in 270 mul/tube. Sample buffer was used for the chromatography samples.
The SD card containing the standard curve is obtained by measuring calibrators with different concentrations through a time-resolved fluorescence test strip, drawing the standard curve by taking the concentration of the calibrators as an abscissa and taking the ratio of fluorescence signals as an ordinate, writing and generating corresponding two-dimensional code information, and storing the two-dimensional code information in the SD card. The corresponding two-dimensional code information on the reagent card can be read by a dry-type fluorescence immunoassay instrument, and the corresponding concentration can be measured.
the invention also relates to a method for quantitatively detecting the urinary microalbumin by using the kit, which comprises the following steps:
(1) Placing the detection kit and the sample at room temperature, and using the detection kit and the sample after the detection kit and the sample are recovered to the room temperature;
(2) Starting a dry type fluorescence immunoassay analyzer, preheating for 5min, and then inserting a corresponding SD card;
(3) Accurately absorbing 30 mu L of urine to be detected, adding the urine to the sample diluent, and uniformly mixing;
(4) Sucking 80 mu L of the mixed sample, and adding the sample into a sample adding hole of the detection card;
(5) And inserting the detection card into the detection slot, and detecting, reading and printing the detection result after 10 min. The detection range is 2-300 mg/L.
The detection principle of the kit for the time-resolved fluorescence quantitative detection of the microalbumin in urine is a competition method, and the content of the microalbumin in human urine is detected. The urine sample diluent containing urine microalbumin is dripped into a sample adding area, is chromatographed to a nitrocellulose membrane microsphere line through capillary action, is combined with a human albumin monoclonal antibody marked by a time-resolved fluorescent microsphere to form a microsphere antibody-antigen complex, is chromatographed to a detection area, and because the human albumin recombinant antigen fixed by the detection line and the microalbumin in the sample are competitively combined with the human albumin monoclonal antibody marked by the microsphere, the more albumin in the sample is, the less microsphere antibody is combined at the detection line, and the redundant fluorescent microsphere marker is continuously chromatographed forwards and is combined with a goat anti-mouse fixed on a quality control line. After the reaction is finished, the detection area is scanned and detected by an ultraviolet light source (365nm), the time-resolved fluorescent microspheres on the detection line and the quality control line emit fluorescence (615nm), and the decay time is longer. By using the delayed measurement time, after the naturally occurring short-life fluorescence (1-10 ns) in the sample matrix is completely decayed, the specific fluorescence of the rare earth elements is measured, so that the interference of the specific background fluorescence can be completely eliminated. The concentration of the substance to be detected in the sample can be analyzed through the strength and the ratio of the fluorescence intensity of the detection line and the fluorescence intensity of the quality control line.
The invention has the following beneficial effects: the kit can accurately and quantitatively detect the content of the microalbumin in human urine, can avoid the fluorescence interference of a sample by using a time-resolved fluorescence microsphere detection technology, can be used for connecting the microsphere and an antibody by a covalent bond through labeling, has stable labeling products, and has the characteristics of wide detection range (2-300 mg/L), high sensitivity (detection limit of 2mg/L), high accuracy, quick, simple and convenient detection and the like.
(IV) description of the drawings
FIG. 1 is a schematic structural diagram of a test strip in the kit for the time-resolved fluorescence quantitative determination of microalbuminuria of the present invention;
wherein: 1. a sample pad; 2. a coating film; 3. absorbent paper; 4. a micro-ball line; 5. detecting lines; 6. a quality control line; 7. a bottom lining.
FIG. 2 is a standard curve of the kit prepared in example 1 of the present invention.
FIG. 3 is a graph showing the correlation between the detection results of the kit prepared in example 1 of the present invention and the detection results of the kit for detecting microalbumin in urine by Siemens immunoturbidimetry.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1: preparation of time-resolved fluorescence quantitative detection kit for microalbumin in urine
The kit for detecting the microalbumin in urine by time-resolved fluorescence quantification adopts the competitive immunochromatography principle to detect the microalbumin in human urine. The fluorescent test paper consists of a time-resolved fluorescent test paper card, sample diluent and an SD card containing a calibration curve.
In this embodiment, as shown in fig. 1, a bottom liner 7 is sequentially overlapped with a sample pad 1, a coating film 2 and absorbent paper 3, the sample pad 1 is a sample application area for absorbing a urine test sample to be tested, and the coating film 2 is provided with a microsphere line, a test line and a quality control line.
A human albumin monoclonal antibody (100 mu g antibody/200 mu l fluorescent microsphere) is marked on a microsphere line coated on the membrane 2 by using rare earth europium fluorescent microspheres (the diameter is about 200nm, the solid content of europium is 1%) with specific exciting light (365 nm)/emitting light (615nm) wavelengths, a human albumin recombinant antigen (1mg/ml) is coated on a detection line, and a goat anti-mouse IgG antibody with the concentration of 1mg/ml is coated on a quality control line (wherein the time-resolved fluorescent microspheres are purchased from Bangs Lab company in the United states, the human albumin monoclonal antibody is purchased from Chongqing Protectics technology company Limited, the human albumin recombinant antigen is purchased from Loyang Baitai Biotechnology Limited, and the goat anti-mouse IgG antibody is purchased from Changbo Baozao Yongsheng Biotechnology company). The dosage of the microsphere line, the detection line and the quality control line is 1 mul of coating liquid amount/cm of membrane.
In this example, the preparation of the kit for the time-resolved fluorescence quantitative determination of urinary microalbumin comprises the following steps:
1. activation of time-resolved fluorescent microspheres
after the fluorescent microspheres are treated by ultrasonic waves for 2min, 200 mul of fluorescent microspheres are taken and centrifuged at 14000rpm for 15min at a high speed, and precipitates are washed to 1ml by MES solution with 100mM and pH of 6.0 and treated by ultrasonic waves for 2 min; adding 50 μ l of 100mg/ml carbodiimide, mixing for 5min, adding 100 μ l of 100mg/ml N-hydroxy-thiosuccinimide, mixing for 15min, centrifuging at 14000rpm for 15min, and washing the precipitate with MES solution with pH of 6.0 to 1 ml.
2. Preparation of time-resolved fluorescent microsphere labeled human albumin monoclonal antibody
the activated fluorescent microspheres are subjected to ultrasonic treatment for 2min, then human albumin monoclonal antibody is added according to 100 mug/200 ul, the mixture is mixed for 2 hours, the mixture is blocked by 50mM of 0.5% BSA and pH8.0Tris-HCl blocking solution for 1 hour, then the mixture is centrifuged at 14000rpm for 15min at a high speed, washed twice by buffer solution in 50mM of 0.1% NaCl, 0.5% BSA and 0.1% Tween20 and pH8.0Tris-HCl preservation solution, and then the mixture is subjected to ultrasonic treatment and resuspended to 200 ul and stored in a dark place at 4 ℃.
3. Preparation of coating film
Respectively adjusting the concentration of a human albumin recombinant antigen and a goat anti-mouse IgG antibody to 1mg/ml by using a coating buffer solution (10 mM PBS buffer solution containing 2.5% of sucrose), wherein the dosage of the coating buffer solution is 1 mul of coating solution per cm of membrane, the human albumin recombinant antigen and the goat anti-mouse IgG antibody are respectively used as a detection line and a quality control line to be parallelly scratched on a nitrocellulose membrane for coating, a microsphere diluent (10 mM PBS buffer solution containing 0.5% of BSA and 20% of sucrose) is used for diluting time-resolved fluorescent microspheres marked with a human albumin monoclonal antibody by 5 times, the dosage of the coating solution per cm of membrane is 1 mul of coating solution, the microspheres are scratched on the nitrocellulose membrane in the direction close to a sample pad for coating, the quality control line, the detection line and the microspheres are separated by 4mM, and the microspheres are dried in an oven at the humidity of less than 30% and the;
4. A sample pad (30X 300mm in size, made of glass fiber cotton), a coating film (25X 300mm in size, made of nitrocellulose) and a water-absorbent paper (28X 300mm in size) were sequentially stuck to a substrate (80X 300mm in size) in an overlapping manner to obtain a test paper sheet, which was cut into test strips of 4mm in width as required.
The time-resolved fluorescence test paper card for detecting the microalbumin urine comprises a test paper strip, wherein the test paper strip is assembled in a plastic shell formed by buckling an upper plastic shell and a lower plastic shell when in use, the upper plastic shell is provided with two openings, a sample adding hole and an observation window, the sample adding hole corresponds to a test paper strip sample pad 1 for detecting the microalbumin urine, the result observation window corresponds to a detection line 5 and a quality control line 6 of the test paper strip for detecting the microalbumin urine, and the test paper strip for detecting the microalbumin urine can be taken out of the plastic shell.
The kit also comprises a sample diluent which is 50mM, pH8.0Tris-Hcl buffer solution containing 0.5% NaCl, 0.5% BSA and 1% Tween, and the sample diluent is subpackaged in a centrifuge tube by 270 mu l per tube. Sample buffer was used for the chromatography samples.
in the kit, each kit contains an SD card with a standard curve (the standard curves in the same batch are the same), calibrators with different concentrations are measured by a time-resolved fluorescence test strip, the concentration of the calibrator is taken as the abscissa, the ratio of fluorescence signals is taken as the ordinate, the calibrator is drawn into the standard curve and written into the SD card to generate a two-dimensional code, and a dry fluorescence immunoassay analyzer can read corresponding two-dimensional code information on a reagent card and measure the corresponding concentration.
example 2: quantitative detection of time-resolved fluorescence quantitative detection urine microalbumin kit
1. Drawing of standard curve
The albumin antigen quality control samples (eight concentrations including 302.5mg/L, 247.5mg/L, 192.5mg/L, 137.5mg/L, 82.5mg/L, 38.5mg/L, 11.0mg/L and 2.0mg/L, each concentration is set to be three times, and each concentration is obtained by diluting 10mg/ml of human albumin recombinant antigen with 0.85% physiological saline) are added into the time-resolved fluorescence test paper card prepared according to the example 1, a sample diluent is added, after chromatography is carried out for 10min, C, T-line fluorescence signals and C/T values are read by an AFS-1000 dry fluorescence immunoassay analyzer produced by Guangzhou Blobo Biotech limited, and the experimental results and analysis are shown in Table 1:
LOG values are taken according to the concentration of the microalbumin quality control substances, and LOG values are taken according to the T/C average value of sample signals, so that a standard curve is drawn, the data of the curve are shown in table 1, and the standard curve is shown in figure 2. Wherein the R value is 0.9943, and the concentration of the microalbumin contained in the sample is quantitatively determined through the marked line.
2. And (5) testing the performance of the time-resolved fluorescence test paper card.
Minimum detection limit: and (3) carrying out repeated measurement for 20 times by using a zero-value sample, calculating the mean value M and the standard deviation SD of 20 times of results, and reporting the detection limit (M +2SD) of the method by adding two times of the standard deviation to the blank mean value, wherein the result is 1.63mg/L and meets the sensitivity of 2 mg/L.
Linear range: eight concentration values of 2-300 mg/L are taken, each concentration is repeatedly measured for three times, the average value of the measured concentration is subjected to linear analysis on the theoretical concentration, and a linear equation y is 1.0199x +0.2911, and r is 0.9965, so that the kit disclosed by the invention is good in correlation in the linear range of 2-300 mg/L.
Precision: 247.5mg/L and 38.5mg/L repetitive quality controls were detected respectively from three batches of the time-resolved fluorescence quantitative urine microalbumin kits prepared in example 1, each batch of the kits was tested 10 times in parallel with the repetitive quality controls, the CV in the three batches at 247.5mg/L concentration was 5.25%, 7.05% and 5.33%, the CV in the three batches at 6.17%, the CV in the three batches at 38.5mg/L concentration was 8.31%, 9.55% and 9.71%, and the CV in the three batches at 9.97%, all were within 10%.
The accuracy is that the quality control material with the concentration of 22mg/L is selected as a detection sample and divided into 3 parts with the same volume, the accuracy quality control materials with the concentration of 220mg/L and 38.5mg/L are respectively added into 2 parts of the sample to prepare 2 recovery samples with different adding concentrations, and the concentration of the added substance to be detected is calculated. The same amount of the analyte-free solution was added to the other sample to prepare a base sample. The samples were analyzed for 3 replicates and the mean was calculated. The recovery rate was calculated as (concentration of analytical sample-concentration of base sample)/concentration of addition x 100%. The recovery rate of 220mg/L of the recovered sample was 106.03%, the recovery rate of 28.5mg/L was 97.58%, and the average recovery rate was 90.92%. The deviation is within 10%.
3. Clinical sample testing
200 parts of urine samples for detecting the microalbumin in hospitals are collected, and the detection and comparison are carried out by using the kit and the kit for detecting the microalbumin by the Siemens immunoturbidimetry. In the kit, 30 mu l of urine is added into a sample diluent, 80 mu l of urine is added into a sample adding hole of a detection card after uniform mixing, the concentration is read by an AFS-1000 dry type fluorescence immunoassay analyzer produced by Guangzhou blue-Bo biological technology company Limited after 10min of chromatography, and the concentration of the same urine is detected by a contrast system Siemens immunoturbidimetry urine detection kit. The detection results of the two methods are subjected to linear analysis, as shown in fig. 3, the correlation is good, r is 0.9893, P is more than 0.05, the average relative deviation is less than 10%, and the results meet the clinical analysis requirements and are suitable for clinical detection.
Claims (4)
1. the utility model provides a kit of time-resolved fluorescence quantitative determination urine microalbumin, includes time-resolved fluorescence test paper card, sample diluent and contains SD card of calibration curve, its characterized in that:
The time-resolved fluorescence test paper card comprises a test paper strip, wherein the test paper strip consists of a bottom lining, and a sample pad, a nitrocellulose membrane and absorbent paper which are stuck on the bottom lining, and the sample pad, the nitrocellulose membrane and the absorbent paper are sequentially lapped and stuck on the bottom lining;
The nitrocellulose membrane is sequentially provided with a microsphere line, a detection line and a quality control line, the microsphere line, the detection line and the quality control line are parallel to each other, the spacing distance is 3-5 mm, the microsphere line is close to the sample adding hole, and the quality control line is far away from the sample adding hole;
The microsphere line consists of time-resolved fluorescent microspheres marked with human albumin monoclonal antibodies; the time-resolved fluorescent microspheres are doped with rare earth europium elements with the solid content of 1%, and the particle size is 100-300 nm; the content of the human albumin monoclonal antibody is 50-200 mug antibody/200 mul fluorescent microsphere;
The detection line is coated with a human albumin recombinant antigen, and the quality control line is coated with a goat anti-mouse IgG antibody; the human albumin recombinant antigen coating concentration is 0.5-2 mg/ml, the dosage is 0.5-1.5 mul coating liquid amount/cm membrane, the goat anti-mouse IgG antibody coating concentration is 0.5-2 mg/ml, the dosage is 0.5-1.5 mul coating liquid amount/cm membrane;
The SD card containing the standard curve is obtained by measuring calibrators with different concentrations through a time-resolved fluorescence test strip, drawing the standard curve by taking the concentration of the calibrators as an abscissa and taking the ratio of fluorescence signals as an ordinate, writing and generating corresponding two-dimensional code information, and storing the two-dimensional code information in the SD card.
2. The kit of claim 1, wherein the test strip is prepared by a method comprising:
(1) activation of time-resolved fluorescent microspheres
After the fluorescent microspheres are treated by ultrasonic waves for 2min, 200 mul of fluorescent microspheres are taken and centrifuged at 14000rpm for 5-15 min at a high speed, precipitates are washed to 1ml by MES solution with 10-100 mM and pH value of 5.0-7.0, and the ultrasonic waves are treated for 2 min; adding 10-100 mu l of 20-100 mg/ml carbodiimide, uniformly mixing for 5-10 min, adding 50-200 mu l of 20-100 mg/ml N-hydroxy thiosuccinimide, uniformly mixing for 10-20 min, then centrifuging at 14000rpm for 5-15 min at a high speed, and washing the precipitate to 1ml by using MES solution with the pH of 5.0-7.0;
(2) Preparation of time-resolved fluorescent microsphere labeled human albumin monoclonal antibody
Adding a human albumin monoclonal antibody into the activated fluorescent microspheres for 2min after ultrasonic treatment according to 50-200 mug/200 mul, uniformly mixing for 1-3 h, sealing with 10-50 mM containing 0.5% BSA and 10-50 mM of pH7.5-8.5 Tris-Hcl sealing solution for 0.5-1 h, then centrifuging at 14000rpm for 5-15 min at a high speed, washing with 10-50 mM containing 1% NaCl, 0.5% BSA and 0.1% Tween20 and 10-50 mM of pH7.5-8.5 Tris-Hcl preservation solution, and resuspending to 200 mul and storing in a dark place at 4 ℃;
(3) preparation of coating film
respectively adjusting the concentration of a human albumin recombinant antigen and a goat anti-mouse IgG antibody to 0.5-2 mg/ml by using coating buffer solution, wherein the amount of the coating buffer solution is 0.5-1.5 mul of coating solution per cm of membrane, the human albumin recombinant antigen and the goat anti-mouse IgG antibody are respectively used as a detection line and a quality control line which are parallelly drawn on a nitrocellulose membrane for coating, a microsphere diluent is used for diluting 3-6 times of time-resolved fluorescent microspheres for marking the human albumin monoclonal antibody, the amount of the coating solution is 0.5-1.5 mul of coating solution per cm of membrane, the microsphere line is drawn on the nitrocellulose membrane in the direction close to a sample pad for coating, the quality control line, the detection line and the microsphere line are spaced by 3-7 mm, and the kit is placed in an oven and;
(4) And (3) sequentially attaching a sample pad, a coating film and absorbent paper to the bottom lining in a mutually overlapped manner to obtain a test paper board, and cutting to obtain the test paper strip.
3. The kit of claim 1 or 2, wherein the time-resolved fluorescence test paper card is fixed on the plastic bottom card by a detection test paper, the surface of the test paper is pressed by a card surface, and the card surface is provided with a sample adding hole and an observation window respectively at the parts corresponding to the sample pad and the nitrocellulose membrane.
4. The kit of claim 1 or 2, wherein the sample diluent consists of: 0.5% of NaCl, 0.5% of BSA, 1% of Tween, 10-50 mM of solvent and 7.5-8.5 of Tris-HCl buffer solution, and packaging the buffer solution into centrifugal tubes in 270 mul/tube.
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