CN109387640B - Time-resolved fluorescence immunochromatography test strip capable of carrying out primary screening on meningitis types and preparation method thereof - Google Patents
Time-resolved fluorescence immunochromatography test strip capable of carrying out primary screening on meningitis types and preparation method thereof Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a time-resolved fluorescence immunochromatography test strip capable of carrying out primary screening on meningitis types and a preparation method thereof, wherein the time-resolved fluorescence immunochromatography test strip capable of carrying out primary screening on the meningitis types comprises the following components: sample combination pad, detection pad, inhale sample pad and bottom plate, sample combination pad, detection pad and inhale sample pad set gradually on the bottom plate, and sample combination pad and inhale sample pad fold respectively on the both ends of detection pad, are equipped with four detection lines and a matter control line on the detection pad. The invention has the advantages of small required sample size, high sensitivity, low detection background signal, strong specificity, wide linear range, simple and convenient operation and reliable result; furthermore, the content of PCT, CRP, ESAT-6-CFP 10 antigen and cryptococcus capsular polysaccharide antigen in cerebrospinal fluid can be detected simultaneously, so that the type of meningitis can be roughly judged, the possibility of misdiagnosis caused by empirical sense in clinical diagnosis can be effectively reduced, and a more accurate immunological basis is provided for the accurate diagnosis of the type of meningitis.
Description
Technical Field
The invention relates to a time-resolved fluorescence immunochromatography test strip capable of performing primary screening on meningitis types and a preparation method thereof, and belongs to the technical field of biology.
Background
Central nervous system infections are common and frequently occurring in paediatrics, most commonly seen as bacterial meningitis and viral meningitis. In recent years, with the relative delay of gene mutation of tubercle bacillus and development of antitubercular drugs and the increase of patients with AIDS, the incidence rate and the death rate of tuberculosis at home and abroad are gradually increased. Meanwhile, with the wide application of antibiotics, immunosuppressants and corticosteroids, the organism dysbacteriosis or the immune function is reduced, so that the incidence of cryptococcosis meningitis is also obviously increased. Because the symptoms shown by the meningitis have similarity, especially tubercular meningitis and cryptococcosis meningitis are similar in clinical manifestations, misdiagnosis is easy, and patients cannot be effectively treated in time, so that the patients are lost. Therefore, a method for early diagnosis and identification of meningitis etiology is needed, and the method has great clinical significance for treatment, prognosis and reduction of sequelae of patients.
Numerous studies have shown that PCT and CRP levels in cerebrospinal fluid are significantly higher in both bacterial and viral meningitis patients than in healthy children, indicating that PCT and CRP in cerebrospinal fluid can be an indicator of clinical diagnosis of viral and bacterial meningitis in children. Since tuberculosis antigen appears first in the body of infected Mycobacterium Tuberculosis (MTU), and tuberculosis antibodies are gradually produced 8 weeks after infection, the detection of MTU secretion specific antigen in cerebrospinal fluid can be used as an effective way for rapid diagnosis of meningiomycosis. In early stages of infection, pathogenic Mycobacterium tuberculosis generally secrete large amounts of specific proteins, namely 6000-early secretory antigen target (ESAT-6) and antigen culture filtrate protein 10 (CFP 10), and these two proteins are not present in BCG, so they can be used as markers of active tuberculosis. For cryptococcosis meningitis, methods such as cerebrospinal fluid picture, alisxin blue staining, ink staining and fungus culture are mainly adopted in China, although fungus culture is a gold standard for diagnosing cryptoencephalis, the time is long, 2-5 d is needed, 10d is needed for some fungi, and the culture positive rate is low; the smear and the ink have higher dyeing positive rate, especially the positive rate can be improved by microscopic examination after the cerebrospinal fluid is centrifuged, but the conditions of missed detection and misdiagnosis still exist, so that patients cannot be treated in time.
Disclosure of Invention
The invention provides a time-resolved fluorescence immunochromatography test strip capable of carrying out primary screening on meningitis types and a preparation method thereof, and the test strip can conveniently, accurately and highly sensitively detect the contents of PCT, CRP, ESAT-6-CFP 10 antigens and cryptococcus capsular polysaccharide antigens in cerebrospinal fluid simultaneously by adopting an immunochromatography method and a fluorescence immunoassay analyzer, thereby providing a certain theoretical basis for primary judgment of meningitis types.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a time-resolved fluoroimmunochromatographic test strip capable of performing primary screening for meningitis types, comprising: sample combination pad, detection pad, inhale sample pad and bottom plate, sample combination pad, detection pad and inhale sample pad set gradually on the bottom plate, and sample combination pad and inhale sample pad fold respectively on the both ends of detection pad, are equipped with four detection lines and a matter control line on the detection pad.
The detection indexes of the test strip comprise PCT, CRP, ESAT-6-CFP 10 antigen and cryptococcus capsular polysaccharide antigen in cerebrospinal fluid; the sample binding pad is internally coated with an antibody marked by time-resolved fluorescence microspheres, and the antibody marked by the time-resolved fluorescence microspheres is used as an antibody of a detection index.
In order to improve the detection accuracy, the sample binding pad is coated with a time-resolved fluorescence microsphere marked anti-PCT monoclonal antibody, an anti-CRP monoclonal antibody, an anti-ESAT-6-CFP 10 polyclonal antibody and an anti-cryptococcus capsular polysaccharide monoclonal antibody.
In order to facilitate the manufacture and ensure the detection accuracy, the time-resolved fluorescent microsphere is a modified polystyrene microsphere; the surface modification functional group of the polystyrene microsphere is one of carboxyl, hydroxyl or epoxy, and the particle size of the polystyrene microsphere is 100-500 nm, preferably 180-300 nm.
In order to improve the detection sensitivity, the time-resolved fluorescence microsphere is internally filled with a chelate of 1% (w/w) lanthanide, wherein the lanthanide is one of europium, samarium, zinc or dysprosium.
The applicant finds that the content of cryptococcus capsular polysaccharide antigen in cerebrospinal fluid by an immunoassay method has important clinical application value for diagnosis and curative effect observation of cryptococcus, has high sensitivity and simple operation, and is suitable for popularization; the time-resolved fluoroimmunoassay (TRFIA) is a non-isotope immunoassay technology, which uses lanthanide to mark antigen or antibody, uses time-resolved technology to measure fluorescence according to the luminescence characteristics of lanthanide chelate, and simultaneously uses two parameters of detection wavelength and time to make signal resolution, so that it can effectively eliminate interference of non-specific fluorescence, greatly raise analysis sensitivity, and at the same time has the advantages of simple preparation of marker, long storage time, no radioactive pollution, good detection repeatability, simple operation, wide standard curve range, and free from interference of natural fluorescence of sample, etc.
The method for marking the time-resolved fluorescence microsphere comprises the following steps: the method comprises the following steps of:
1) Dissolving time-resolved fluorescent microspheres in buffer solution, adding an activating agent, and oscillating and activating at constant temperature;
2) Adding an organic solvent into the material obtained in the step 1), uniformly mixing, centrifuging, washing to remove a activator, re-suspending microspheres by using a buffer solution, then respectively adding an anti-PCT monoclonal antibody, an anti-CRP monoclonal antibody, an anti-ESAT-6-CFP 10 polyclonal antibody and an anti-cryptococcus capsular polysaccharide monoclonal antibody, vibrating and marking at constant temperature, re-suspending by using a re-dissolving buffer solution after centrifuging, adding a blocking agent for blocking, and obtaining a time-resolved fluorescent microsphere marked anti-PCT monoclonal antibody complex, an anti-CRP monoclonal antibody complex, an anti-ESAT-6-CFP 10 polyclonal antibody complex and an anti-cryptococcus capsular polysaccharide monoclonal antibody complex, and evaluating the antigen performance of PCT, CRP, ESAT-6-CFP 10 antigens and cryptococcus capsular polysaccharide.
Preferably, the activators in step 1) and step 2) are 1 to (3-dimethylaminopropyl) to 3-ethylcarbodiimide hydrochloride or/and N-hydroxysuccinimide.
Preferably, the method for labeling the time-resolved fluorescent microsphere comprises the following steps: the method comprises the following steps of:
1) Washing time-resolved fluorescent microsphere with MES buffer with pH6100mM for 2 times, re-suspending, adding activating agent EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) to make their concentrations respectively 0.05% -0.1% and 0.1% -02%, and oscillating and activating at 30+ -5deg.C for 60+ -5 min;
2) Adding ethanol with the volume concentration of 10% into the material obtained in the step 1), uniformly mixing, centrifuging, washing to remove EDC or NHS, adding buffer solution to resuspension microspheres, uniformly mixing, respectively adding anti-PCT monoclonal antibody, anti-CRP monoclonal antibody, anti-ESAT-6-CFP 10 polyclonal antibody and anti-cryptococcus capsular polysaccharide monoclonal antibody, oscillating and marking for 2 hours at 37 ℃, centrifuging, resuspension by using a re-dissolving buffer solution, adding 10% BSA to block for 30 minutes, and obtaining time-resolved fluorescent microsphere marked anti-PCT monoclonal antibody complex, anti-CRP monoclonal antibody complex, anti-ESAT-6-CFP 10 polyclonal antibody complex and anti-cryptococcus capsular polysaccharide monoclonal antibody complex.
The re-dissolving buffer solution comprises Tris-Hcl, BSA, T-20, trehalose and NaN3; preferably, the reconstitution buffer is 10-50 mM Tris-HCl (pH 7.5-8.5), containing 0.05-0.1% BSA, 0.05-0.1% T-20, 3-5% trehalose and 0.05% NaN 3 The percentages are mass percentages.
In order to avoid mutual influence, the four detection lines are parallel to each other, and the four detection lines are respectively coatings formed by paired antibodies capable of being specifically combined with the antibodies marked by the time-resolved fluorescent microspheres, namely, the content of each target object is detected by adopting a double-antibody sandwich method.
Preferably, the four detection lines and the one quality control line are parallel to each other and are sequentially arranged along the length direction of the detection pad, and the four detection lines and the one quality control line are sequentially arranged along the direction from the sample bonding pad to the sample absorbing pad. That is, the four detection lines can be arranged randomly in the front-back order on the detection pad, and the quality control line is nearest to the end part of the sample absorbing pad.
In order to improve the sensitivity and accuracy of detection, preferably, the quality control line is coated with goat anti-mouse IgG, goat anti-chicken IgY or goat anti-rabbit IgG.
As another preferred scheme of this application, detect and fill up including 2 ~ 4 detection divides the pad, four detection lines distribute at random and divide to fill up at the detection, and every detects to divide to fill up and all is equipped with a matter control line, and same detection divides to fill up, compares in detection line matter control line and is closest from the tip that inhales the sample pad.
In order to reduce the cost and ensure the sensitivity and accuracy of detection, the detection pad is a nitrocellulose membrane and is a porous membrane with the aperture of 5-12 um; the sample bonding pad is made of a glass cellulose film or a non-woven fabric; the sample absorbing pad is made of water absorbing filter paper.
The preparation method of the time-resolved fluorescence immunochromatography test strip capable of carrying out primary screening on the meningitis type comprises the following steps of:
(1) Treatment of the detection pad:
attaching a detection pad on a bottom plate, taking 10-50 mM phosphate buffer solution with pH7.4 containing 1-5wt% of sucrose, respectively diluting PCT monoclonal antibody, CRP monoclonal antibody, ESAT-6-CFP 10 polyclonal antibody and cryptococcus capsular polysaccharide monoclonal antibody of the other epitope to 1+/-0.02 mg/ml, and preparing four detection lines; diluting goat anti-mouse IgG antibody to 1+/-0.02 mg/ml by using 10-50 mM phosphate buffer solution containing 1-5 wt% of sucrose, and preparing a quality control line; uniformly marking the 5 diluted antibodies on a detection pad by using a biolot film marking instrument according to the liquid marking amount of 1+/-0.02 mu l/cm to prepare a detection line and a quality control line; placing the marked detection pad in a drying oven at 37+/-3 ℃ and drying for 3+/-0.2 h;
(2) Sample binding pad treatment:
the sample bonding pad is soaked in buffer solution containing surfactant for pre-sealing, and then dried for 3 hours plus or minus 0.2 hours at 37 plus or minus 3 ℃; ultrasonically spraying the antibody marked with the time-resolved fluorescence microsphere onto a sample binding pad according to the amount of 5+/-0.02 mu l/cm through an airjet nozzle of a Biodot instrument, and drying for 3 hours+/-0.2 hours at 37+/-3 ℃ to prepare the sample binding pad;
(3) And (3) assembling:
and (3) fixedly laminating the sample bonding pad obtained in the step (2) on one end of the detection pad obtained in the step (1), fixedly laminating the sample absorbing pad on the other end of the detection pad, wherein the lamination length is 1-4 mm, and cutting by a film cutting instrument according to the width of 4-6 mm to obtain the time resolution fluorescent immunochromatography test strip capable of performing primary screening on the meningitis type.
Preferably, the buffer in step (2) is 100mM PB (pH 7.4) containing 2% NaCl,2% BSA, 0.5% casein, 0.1% T-20, and 5% sucrose, the percentages being by mass.
The technology not mentioned in the present invention refers to the prior art.
Compared with the prior art, the invention has the following advantages: the invention has the advantages of small required sample size, high sensitivity, low detection background signal, strong specificity, wide linear range, simple and convenient operation and reliable result; furthermore, the content of PCT, CRP, ESAT-6-CFP 10 antigen and cryptococcus capsular polysaccharide antigen in cerebrospinal fluid is detected simultaneously, so that the type of meningitis can be roughly judged, the possibility of misdiagnosis caused by empirically during clinical diagnosis can be effectively reduced, and a more accurate immunological basis is provided for the accurate diagnosis of the type of meningitis; the preparation method is simple and has been popularized.
Drawings
FIG. 1 is a schematic structural diagram of a time-resolved fluorescence immunochromatographic strip capable of performing primary screening on meningitis types. Wherein: 1 is a sample binding pad; 2 is a detection pad; 3 is a sample absorbing pad; 4 is a bottom plate; t1, T2, T3 and T4 are respectively four detection lines; c is a quality control line.
FIG. 2 shows the PCT concentration log-fluorescence count log standard curve in the present invention.
FIG. 3 is a plot of CRP concentration versus fluorescence count log standard curve in the present invention.
FIG. 4 shows ESAT-6-CFP 10 antigen concentration vs. fluorescence count vs. log standard curves in the present invention.
FIG. 5 is a graph showing the logarithmic value of the antigen concentration of cryptococcus capsular polysaccharide to the logarithmic value of fluorescence count standard curve in the present invention.
Detailed Description
For a better understanding of the present invention, the following examples are further illustrated, but are not limited to the following examples.
Example 1
As shown in FIG. 1, a time-resolved fluorescence immunochromatographic test strip capable of initially judging a meningitis type comprises a bottom plate 4, wherein an upper sample binding pad 1, a detection pad 2 and a sample absorbing pad 3 are sequentially arranged on the bottom plate 4, and the sample binding pad 1 and the sample absorbing pad 3 are respectively overlapped on two ends of the detection pad 3; the sample binding pad 1 is coated with a time-resolved fluorescence microsphere marked anti-PCT monoclonal antibody, an anti-CRP monoclonal antibody, an anti-ESAT-6-CFP 10 polyclonal antibody and an anti-cryptococcus capsular polysaccharide monoclonal antibody; the detection pad 2 is provided with detection lines T1, T2, T3, T4 and a quality control line C which are parallel to each other, wherein the detection lines T1, T2, T3, T4 and the quality control line C are sequentially arranged along the length direction of the detection pad and are parallel to each other, the quality control line C is nearest to the sample absorbing pad 3, the detection lines T1, T2, T3 and T4 are coated with monoclonal antibodies of another epitope of the detected index, and the quality control line C is coated with goat anti-mouse IgG. The detection pad is a nitrocellulose membrane and is a porous membrane with the aperture of 5-12 um; the sample bonding pad is made of a glass cellulose membrane; the sample absorbing pad is made of water absorbing filter paper.
The time-resolved fluorescent microsphere is a modified polystyrene microsphere, the surface modification functional group of the polystyrene microsphere is carboxyl, and the particle size of the polystyrene microsphere is 200nm; chelate of europium of 1% (w/w) is filled in the time-resolved fluorescence microsphere; the method for marking the time-resolved fluorescent microsphere comprises the following steps: 1mg of time-resolved fluorescent microsphere was washed 2 times with 200. Mu.L of 100mM MES buffer pH=6, resuspended, and 1 to (3 to dimethylaminopropyl) to 3 to ethylcarbodiimide hydrochloride and N to hydroxyl were addedSuccinimide (activating agent) with concentration of 0.08% and 0.15% respectively, oscillating and activating at 30 ℃ for 60min, adding 10% ethanol for 20ul, centrifuging and washing to remove the activating agent, adding 200 ul of 60mM boric acid buffer solution with pH of 7.5 again to resuspend microspheres, mixing evenly, adding 80ug of anti-PCT monoclonal antibody, 80ug of anti-CRP monoclonal antibody, 80ug of ESAT-6-CFP 10 polyclonal antibody and 80ug of anti-cryptococcus capsular polysaccharide monoclonal antibody respectively, oscillating and marking for 2h at 37 ℃, centrifuging, resuspending by using a re-dissolving buffer solution, adding 10% BSA for 50ul, and sealing for 30min (percentage is mass percent) to obtain time-resolved fluorescent microsphere marked anti-PCT monoclonal antibody complex, anti-CRP monoclonal antibody complex, anti-ESAT-6-CFP 10 polyclonal antibody complex and anti-cryptococcus capsular polysaccharide monoclonal antibody complex. The reconstitution buffer is 0.03 MTris-HCl (pH 7.5-8.5) and comprises 0.08% BSA, 0.08% T-20, 4% trehalose and 0.05% NaN 3 The percentages are mass percentages.
The test strip can be prepared by the following preparation method:
(1) Nitrocellulose membrane (NC membrane) treatment:
pasting an NC film to a designated position of a bottom plate, and respectively diluting a PCT monoclonal antibody, a CRP monoclonal antibody, an ESAT-6-CFP 10 polyclonal antibody and a cryptococcus capsular polysaccharide monoclonal antibody of the other epitope to 1mg/ml by using 50mM phosphate buffer solution of pH7.4 containing 1% sucrose to prepare 4T lines; sheep anti-mouse IgG antibody was diluted to 1mg/ml with 50mM phosphate buffer containing 1% sucrose for preparing line C; uniformly marking the 5 diluted antibodies on an NC film by a biood film marking instrument according to the liquid marking amount of 1 mul/cm to prepare a T line and a C line; the scratched NC film was placed in a 37℃drying oven and dried for 3 hours.
(2) Sample binding pad treatment:
the glass cellulose membrane is soaked in a buffer solution (formula: 100mM PB (pH 7.4) containing 2% NaCl,2% BSA, 0.5% casein, 0.1% T-20 and 5% sucrose, wherein the percentages are mass percentages) containing a surfactant for pre-sealing, and then dried at 37 ℃ for 3 hours; the antibody labeled with the time-resolved fluorescent microsphere was ultrasonically sprayed onto a glass cellulose membrane in an amount of 5. Mu.l/cm through an airjet nozzle of a Biodot instrument, and dried at 37℃for 3 hours to prepare a sample-binding pad.
(3) And (3) assembling:
and (3) fixedly laminating the sample bonding pad obtained in the step (2) on one end of the nitrocellulose membrane obtained in the step (1), fixedly laminating the sample absorbing pad on the other end of the nitrocellulose membrane, wherein the lamination length of the two ends is 2mm, and cutting by a membrane cutting instrument according to the width of each piece of nitrocellulose membrane of 4mm to obtain a finished product.
And (3) manufacturing a standard curve: first, fluorescence values (a: 0ng/mL, b:0.5ng/mL, c:2ng/mL, d:10ng/mL, e:20ng/mL, f:40 ng/mL) of PCT 6 standards of different concentrations were measured according to the kit procedure, the logarithm of the standard concentration was taken as the abscissa, the logarithm of the fluorescence value was taken as the ordinate, and were processed by a double-logarithm mathematical model Log-Log function, and the linear regression equation was Y=0.8624X+3.0824, R2=0.9995. The TRFIA standard curve of PCT is shown in FIG. 2.
Similarly, the fluorescence values (a: 1. Mu.g/ml, b: 5. Mu.g/ml, c: 10. Mu.g/ml, d: 50. Mu.g/ml, e: 100. Mu.g/ml, f: 150. Mu.g/ml, g: 200. Mu.g/ml, h: 250. Mu.g/ml) of 8 different concentration standards of CRP were determined according to the kit procedure, with the logarithm of the standard concentration as the abscissa, the logarithm of the fluorescence value as the ordinate, processed by the Log-Log function of the double-logarithm mathematical model, and the linear regression equation was Y=0.8851X+3.9226, R2=0.9994. The TRFIA standard curve of CRP is shown in FIG. 3.
Similarly, the fluorescence values (a: 2.5ng/mL, b:25ng/mL, c:100ng/mL, d:500ng/mL, e:1600 ng/mL) of 5 standards of ESAT-6-CFP 10 antigens at different concentrations were determined according to the kit operation steps, the logarithm of the standard concentration was taken as the abscissa, the logarithm of the fluorescence value was taken as the ordinate, and were processed by a double-logarithm mathematical model Log-Log function, and the linear regression equation was Y=1.2223X+2.6403, R2=0.9999. The TRFIA standard curve of ESAT-6-CFP 10 antigen is shown in FIG. 4.
Similarly, the fluorescence values (a: 1ng/mL, b:10ng/mL, c:50ng/mL, d:100ng/mL, e:500 ng/mL) of 5 standards of different concentrations of cryptococcus capsular polysaccharide antigen were determined according to the kit procedure, the logarithm of the standard concentration was taken as the abscissa, the logarithm of the fluorescence value was taken as the ordinate, and the Log was obtained by a double-logarithm mathematical model LogLog function processing, linear regression equation is Y=0.9689X+3.523, R 2 =0.9997. The TRFIA standard curve for cryptococcus capsular polysaccharide antigen is shown in fig. 5.
And (3) performing precision experiments, namely measuring the concentration of the precisely-quantified high, medium and low three standard substances by adopting the test strip, wherein 10 compound holes are respectively arranged. The results are shown in Table 1.
TABLE 1 precision experiments
As can be seen from table 1: the variation coefficient in the batch and the variation coefficient between batches of the test strips are less than or equal to 10 percent, and meet the requirement of the test strip regulation.
Accuracy experiment: recovery experiments were performed according to conventional methods. The results are shown in Table 2.
TABLE 2 recovery experiments
As can be seen from table 2: the recovery rate of the three concentrations of PCT low, medium and high is 100.39-102.67%, and the average recovery rate is 101.19%; the recovery rate of CRP with low, medium and high concentrations is between 99.09 and 100.20 percent, and the average recovery rate is 99.6 percent; the recovery rate of ESAT-6-CFP 10 antigens with low, medium and high concentrations is 98.89-100.13%, and the average recovery rate is 99.63%; the recovery rate of the cryptococcus capsular polysaccharide antigen with low, medium and high concentrations is between 99.45 and 100.20 percent, and the average recovery rate is 99.87 percent.
Clinical application experiment:
taking 186 meningitis children positive cerebrospinal fluid in a hospital, wherein 58 cases are diagnosed with bacterial meningitis, 54 cases are diagnosed with viral meningitis, 46 cases are diagnosed with tuberculous meningitis, and 28 cases are diagnosed with cryptococcosis meningitis; 114 healthy children were again taken with negative cerebrospinal fluid.
The specific detection steps are as follows: and (3) dripping the cerebrospinal fluid sample on a sample binding pad, standing for 5-20 min, inserting the test strip into a fluorescence analyzer matched with the test strip, and respectively reading fluorescence intensity values of the C line, the T1 line, the T2 line, the T3 line and the T4 line.
Results showed that the bacterial meningitis patient had a PCT level of 12.94+ -4.64 ng/ml and a CRP level of 26.52+ -10.15 μg/ml; the PCT level of cerebrospinal fluid of a patient suffering from viral meningitis is 0.98+/-0.24 ng/ml, and the CRP level is 6.78+/-3.2 mug/ml; the antigen level of the cerebrospinal fluid ESAT-6-CFP 10 of the tuberculous meningitis patient is 137+/-48.4 ng/ml; the antigen level of cryptococcus capsular polysaccharide in cerebrospinal fluid of cryptococcus meningitis patient is 645+/-234 ng/ml; the PCT level of the cerebrospinal fluid of the healthy children is 0.14+/-0.11 ng/ml, the CRP level is 1.01+/-0.13 mug/ml, the ESAT-6-CFP 10 antigen level is 2.4+/-1.38 ng/ml, the cryptococcus capsular polysaccharide antigen level is 1.14+/-0.32 ng/ml, and compared with the cryptobrain patients, the ESAT-6-CFP 10 antigen and the cryptococcus capsular polysaccharide antigen concentration are higher and almost negligible. The detection results all accord with the reference values of PCT, CRP, ESAT-6-CFP 10 antigen and cryptococcus capsular polysaccharide antigen in cerebrospinal fluid of bacterial, viral, tubercular and cryptococcus meningitis patients and healthy children, and the test strip has higher detection sensitivity and accuracy, and can be used for primary screening of meningitis types.
Example 2
Substantially the same as in example 1, except that: the quality control line C is coated with goat anti-chicken IgY; the detection pad comprises 2 detection sub-pads, four detection lines are randomly distributed on the 2 detection sub-pads, each detection sub-pad is provided with two detection lines, each detection sub-pad is provided with a quality control line, and the same detection sub-pad is closest to the end part of the sample absorbing pad compared with the quality control line of the detection line. The clinical application results are the same as those of example 1, and will not be described again.
Example 3
Substantially the same as in example 1, except that: the quality control line C is coated with goat anti-rabbit IgG; the detection pad can also comprise four detection sub-pads, the four detection lines are randomly distributed on the four detection sub-pads, each detection sub-pad is provided with a detection line, each detection sub-pad is provided with a quality control line, and the quality control lines of the detection lines are closest to the end part of the sample absorbing pad compared with the quality control lines of the detection lines. The clinical application results are the same as those of example 1, and will not be described again.
Claims (1)
1. A preparation method of a time-resolved fluorescence immunochromatography test strip capable of carrying out primary screening on meningitis types is characterized by comprising the following steps of: the device comprises a bottom plate, wherein a sample combining pad, a detection pad and a sample absorbing pad are sequentially arranged on the bottom plate, and the sample combining pad and the sample absorbing pad are respectively overlapped on two ends of the detection pad; the sample binding pad is coated with a time-resolved fluorescence microsphere marked anti-PCT monoclonal antibody, an anti-CRP monoclonal antibody, an anti-ESAT-6-CFP 10 polyclonal antibody and an anti-cryptococcus capsular polysaccharide monoclonal antibody; the detection pad is provided with detection lines T1, T2, T3 and T4 which are parallel to each other and a quality control line C line, wherein the T1, T2, T3 and T4 and the quality control line C line are sequentially arranged along the length direction of the detection pad and are parallel to each other, the quality control line C line is nearest to the sample absorbing pad, the detection lines T1, T2, T3 and T4 are coated with antibodies of another epitope of the detected index, and the quality control line C is coated with goat anti-mouse IgG; the detection pad is a nitrocellulose membrane and is a porous membrane with the pore diameter of 5-12 mu m; the sample bonding pad is made of a glass cellulose membrane; the sample absorbing pad is made of water absorbing filter paper;
the time-resolved fluorescent microsphere is a modified polystyrene microsphere, the surface modification functional group of the polystyrene microsphere is carboxyl, and the particle size of the polystyrene microsphere is 200nm; chelate of europium of 1 wt% is filled in the time-resolved fluorescence microsphere; the method for marking the time-resolved fluorescent microsphere comprises the following steps: washing 1mg time-resolved fluorescent microsphere with 200. Mu.L of 100mM MES buffer solution with pH=6 for 2 times, then re-suspending, adding 1 to (3 to dimethylaminopropyl) to 3 to ethylcarbodiimide hydrochloride and N to hydroxysuccinimide to ensure that the concentrations are 0.08 percent and 0.15 percent respectively, oscillating and activating for 60 minutes at 30 ℃, adding 20 mu.L of 10 percent ethanol, mixing uniformly, centrifuging and washing to remove an activator, adding 200 mu.L of 60mM boric acid buffer solution with pH=7.5 again, re-suspending microsphere with 200 mu.L of boric acid buffer solution with pH=7.5, mixing uniformly, then adding 80 mu g of anti-PCT monoclonal antibody, 80 mu g of anti-CRP monoclonal antibody, 80 mu g of anti-ESAT-6 to CFP10 polyclonal antibody and 80 mu g of anti-cryptococcus capsular polysaccharide monoclonal antibody respectively, oscillating and labeling for 2 hours at 37 ℃, re-suspending with a re-dissolving buffer solution, adding 10 percent BSA and 50 mu.L for blocking for 30 minutes to obtain time-resolved microsphere-marked anti-PCT monoclonal antibody complex, anti-ESAT monoclonal antibody complex and anti-ESAT antigen complex respectively after centrifugation6-CFP 10 polyclonal antibody complex and anti-cryptococcus capsular polysaccharide monoclonal antibody complex; the reconstitution buffer is 0.03 MTris-HCl, pH 7.5-8.5, and comprises 0.08% BSA, 0.08% T-20, 4% trehalose and 0.05% NaN 3 The percentages are mass percentages;
the test strip is prepared by the following preparation method:
(1) Nitrocellulose membrane treatment:
pasting an NC film to a designated position of a bottom plate, taking 50mM phosphate buffer solution containing 1% sucrose and pH7.4 to dilute PCT monoclonal antibody, CRP monoclonal antibody, ESAT-6-CFP 10 polyclonal antibody and cryptococcus capsular polysaccharide monoclonal antibody of the other epitope to 1mg/ml respectively, and preparing 4T lines; sheep anti-mouse IgG antibody was diluted to 1mg/ml with 50mM phosphate buffer containing 1% sucrose for preparing line C; uniformly marking the 5 diluted antibodies on an NC film by a biood film marking instrument according to the liquid marking amount of 1 mul/cm to prepare a T line and a C line; placing the scratched NC film in a drying oven at 37 ℃ and drying for 3 hours;
(2) Sample binding pad treatment:
the glass cellulose membrane is soaked in buffer solution containing surfactant for pre-sealing, and then is dried for 3 hours at 37 ℃; ultrasonically spraying the antibody marked with the time-resolved fluorescence microsphere onto a glass cellulose membrane according to the amount of 5 mu l/cm through an airjet nozzle of a Biodot instrument, and drying at 37 ℃ for 3 hours to prepare a sample binding pad; buffer formulation containing surfactant: 100mM PB, pH7.4, containing 2% NaCl,2% BSA, 0.5% casein, 0.1% T-20, and 5% sucrose, the aforementioned percentages being by mass;
(3) And (3) assembling:
fixedly laminating the sample bonding pad obtained in the step (2) on one end of the nitrocellulose membrane obtained in the step (1), fixedly laminating the sample absorbing pad on the other end of the nitrocellulose membrane, wherein the lamination length of the two ends is 2mm, and cutting by a membrane cutting instrument according to the width of each piece of nitrocellulose membrane of 4mm to obtain a finished product;
the linear regression equation for PCT was y=0.8624x+3.0824, r 2 =0.9995；
The linear regression equation for CRP is y=0.8851X+3.9226，R 2 =0.9994；
The linear regression equation for ESAT-6-CFP 10 antigen is y=1.2223x+2.6403, r 2 =0.9999；
The linear regression equation for cryptococcus capsular polysaccharide antigen was y=0.9689x+3.523, r 2 =0.9997。
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