CN113049827A - Time-resolved fluorescence immunochromatography reagent strip, preparation method and hemp concentration quantitative detection method - Google Patents
Time-resolved fluorescence immunochromatography reagent strip, preparation method and hemp concentration quantitative detection method Download PDFInfo
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- CN113049827A CN113049827A CN202110267374.XA CN202110267374A CN113049827A CN 113049827 A CN113049827 A CN 113049827A CN 202110267374 A CN202110267374 A CN 202110267374A CN 113049827 A CN113049827 A CN 113049827A
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- G—PHYSICS
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- 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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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- 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
Abstract
The invention discloses a time-resolved fluorescence immunochromatographic reagent strip, a preparation method and a hemp concentration quantitative detection method. The reagent strip comprises a sample pad, a combination pad, a coating film, an absorption pad and a bottom plate; the sample pad, the combination pad and the coating film are sequentially arranged on the bottom plate, and the absorption pad is arranged on the coating film; the binding pad is provided with a hemp monoclonal antibody marked by a time-resolved fluorescent microsphere, the surface of the time-resolved fluorescent microsphere is modified with a functional group, and the time-resolved fluorescent microsphere is filled with lanthanide; the coating film is provided with a detection line and a quality control line, the detection line is close to the combination pad, the quality control line is close to the absorption pad, the detection line is coated with a hemp-BSA antigen, and the quality control line is coated with an antibody. The reagent strip has high sensitivity, and is simple, convenient and quick.
Description
Technical Field
The invention relates to the technical field of biochemical detection, in particular to a time-resolved fluorescence immunochromatography reagent strip, a preparation method and a hemp concentration quantitative detection method.
Background
Cannabis is one of three well-known drugs. The plant cannabis commonly used for drinking is referred to as "Indian hemp" and includes the leaves and flowers of the cannabis plant. More than 400 chemical substances are known in cannabis plants, more than 60 of which have similar chemical properties, and large doses can cause hallucinations, delusions, and psychotic disorders. The long-term consumption of cannabis sativa can cause dependence on the cannabis sativa, which can lead to impairment of higher cognitive functions of the brain, such as attention, memory, fine cognition and the like. Cannabis can also cause organic damage to the body, including respiratory damage, leading to bronchitis, emphysema and histopathological changes, which are high risk factors for cancer.
At present, the bio-matrix for detecting cannabis is blood, sweat, urine, saliva, hair, fingernail and the like. Urine is the most common detection matrix, and the detection window of the urine matrix hemp is as long as about 30 days, so that the reagent has the advantages of easiness in use, low price and the like; the saliva matrix hemp detection window is generally 24 hours, is mainly used for roadside poison drive detection, and is convenient and quick to sample. The detection window of the hair matrix hemp is as long as 90 days, and meanwhile, the detection window has the advantages of convenience in sampling, easiness in storage, easiness in transportation and the like, and the defect of short detection window of urine and saliva matrixes is overcome. The combination of three detection matrixes, namely urine, saliva and hair, can evaluate the abuse condition of a detected person more comprehensively and objectively.
The current detection method for cannabis mainly comprises two types of primary screening and confirmation. The confirmation method mainly comprises methods such as gas chromatography, liquid chromatography, gas chromatography-mass spectrometry combined method, liquid chromatography-mass spectrometry combined method and the like, and the methods have high sensitivity and specificity, but have the defects of complex operation, time consumption, expensive equipment, difficulty in moving, need of professional operation and the like. The prior internationally recognized preliminary screening method is an enzyme-linked immunosorbent assay and a colloidal gold immunochromatography, has the advantages of simple and convenient operation, low price and the like, but the enzyme-linked immunosorbent assay has stronger operation specialty, needs professional personnel for operation, and is relatively time-consuming and high in cost; the colloidal gold immunochromatography can only carry out qualitative analysis, and has lower sensitivity and larger batch-to-batch difference. The existing immunochromatography reagent strip only aims at one matrix such as urine or saliva, has a short detection window, can only trace the abuse condition of drugs within one month, and is easy to miss detection.
Disclosure of Invention
Therefore, the time-resolved fluorescence immunochromatographic reagent strip which is high in sensitivity, simple, convenient and quick and can be simultaneously used for quantitatively detecting the concentration of the hemp in three matrixes of urine, saliva and hair, the preparation method and the quantitative detection method of the concentration of the hemp need to be provided.
A time-resolved fluorescence immunochromatographic reagent strip is characterized by comprising a sample pad, a combination pad, a coating film, an absorption pad and a bottom plate;
the sample pad, the combination pad and the coating film are sequentially arranged on the bottom plate, and the absorption pad is arranged on the coating film and is in contact with the coating film;
the binding pad is provided with a hemp monoclonal antibody marked by a time-resolved fluorescent microsphere, the surface of the time-resolved fluorescent microsphere is modified with a functional group, and the time-resolved fluorescent microsphere is filled with a substance containing lanthanide;
the coating film is provided with a detection line and a quality control line, the detection line is closer to the binding pad than the quality control line, the detection line is coated with a hemp-BSA antigen, and the quality control line is coated with an antibody capable of binding with the hemp-BSA antigen.
In one embodiment, the functional group comprises a carboxyl group.
In one embodiment, the particle size of the time-resolved fluorescence microsphere is 100-500nm, the excitation wavelength is 300-400nm, and the emission wavelength is 600-700 nm.
In one embodiment, the distance between the quality control line and the detection line is 3-8 mm.
In one embodiment, the coating membrane is a nitrocellulose membrane.
A preparation method of a time-resolved fluorescence immunochromatography reagent strip comprises the following steps:
preparing a hemp monoclonal antibody marked by the time-resolved fluorescent microspheres: adding the time-resolved fluorescent microspheres into a coupling buffer solution, adding a hemp monoclonal antibody to be marked, coupling, sealing, centrifuging and storing at 4 ℃ after heavy suspension, wherein the hemp monoclonal antibody is added in the following content: 10-100 μ g of cannabis monoclonal antibody/100 uL of cannabis monoclonal antibody;
sample pad preparation: dropwise adding the sample pretreatment solution on a sample pad, and drying;
preparing a bonding pad: diluting the obtained hemp monoclonal antibody marked by the time-resolved fluorescent microspheres by using a microsphere diluent to obtain an antibody diluent, wherein the content of the time-resolved fluorescent microspheres is as follows: 0.5-1.5uL of hemp monoclonal antibody/1 mL of microsphere diluent, dripping the antibody diluent on a combination pad, and drying;
preparation of coating film: respectively marking hemp-BSA antigen and goat anti-mouse IgG antibody diluted by PBS solution on a coating film to serve as a detection line and a quality control line, wherein the detection line is closer to a binding pad than the quality control line, and storing at 37 ℃;
and sequentially adhering the prepared sample pad, the prepared combination pad and the prepared coating film on a bottom plate, adhering an absorption pad on the bottom plate, and enabling the quality control line to be close to the absorption pad.
In one embodiment, the coupling time is 5-60min, the blocking solution is added for blocking for 5-60min, the suspension is washed and resuspended by the preservation solution, and the preservation is carried out at 4 ℃.
In one embodiment, in the sample pad preparation step and/or the conjugate pad preparation step, the drying temperature is 45 ℃ and the drying time is 3-4 h.
In one embodiment, in the preparation of the coating film, the coating amount of the detection line and/or the quality control line is 0.5-1 μ L/cm.
In one embodiment, the width of the time-resolved fluoroimmunoassay reagent strip is 3-4 mm.
A method for quantitatively detecting the concentration of cannabis sativa comprises the following steps:
placing the time-resolved fluorescence immunochromatographic reagent strip and a sample to be detected at room temperature;
and dropwise adding a sample to be detected to the time-resolved fluorescence immunochromatography reagent strip, detecting the fluorescence intensity of a detection line and a quality control line on the time-resolved fluorescence immunochromatography reagent strip after reaction, obtaining a T value, a C value and a T/C value, and obtaining the concentration of cannabis in the sample to be detected through a standard curve.
A kit comprises the time-resolved fluorescence immunochromatography reagent strip.
The time-resolved fluorescence immunochromatographic reagent strip can detect the cannabis by utilizing three matrixes, namely urine, saliva and hair, makes up the limitation of a single matrix detection window, and reduces the detection missing condition caused by short-time suspension of the detected person because the three matrixes complement each other.
Compared with the prior art, the invention has the following beneficial effects:
(1) the time-resolved fluorescent microspheres are prepared by filling lanthanide (such as europium) into nanoscale polystyrene microspheres, and compared with common fluorescence, the time-resolved fluorescent microspheres have the characteristics of large Stokes displacement, long fluorescence service life and the like, can effectively eliminate the interference of excitation light and nonspecific fluorescence on signal acquisition, and improve the specificity and accuracy of detection.
(2) The time-resolved fluorescence immunochromatographic reagent strip prepared by the invention can carry out quantitative detection, has the advantages of high sensitivity, convenience and quickness in detection, low price, no need of professional operation and the like, and is beneficial to the field rapid detection of a basic layer.
(3) The time-resolved fluorescence immunochromatographic reagent strip prepared by the invention can be used for quantitatively detecting the concentration of the hemp in three matrixes of urine, saliva and hair, one reagent strip can be used for detecting the three matrixes, the defects that the traditional reagent strip is single in matrix detection, narrow in window and easy to leak detection are overcome, and the abuse condition of a detected person can be comprehensively and objectively evaluated.
Drawings
FIG. 1 is a schematic view of a time-resolved fluorescence immunochromatographic reagent strip according to example 1 of the present invention;
FIG. 2 is a standard graph according to example 3 of the present invention;
FIG. 3 is a standard graph according to example 4 of the present invention;
FIG. 4 is a standard graph of the present invention in example 5.
Description of the reference numerals
10. Time-resolved fluorescence immunochromatography reagent strip; 1. a base plate; 2. a sample pad; 3. a bonding pad; 4. a nitrocellulose membrane; 5. an absorbent pad; 6. detecting lines; 7. and (4) quality control line.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
In the description of the present invention, it should be understood that the terms used in the present invention are used in the description of the present invention, and it should be understood that the directions or positional relationships indicated by the terms "center", "upper", "lower", "bottom", "inner", "outer", etc. in the present invention are based on the directions or positional relationships shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the device or element referred to must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention.
In the description of the present invention, it is to be noted that, unless otherwise explicitly specified or limited, the terms "connected" and "connected" are to be interpreted broadly, e.g., as being fixed or detachable or integrally connected; can be mechanically or electrically connected; they may be directly connected or indirectly connected through intervening elements, or they may be in communication within two elements, i.e., when an element is referred to as being "secured to" another element, it may be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Referring to FIG. 1, one embodiment of the present invention provides a time-resolved fluorescence immunochromatographic reagent strip 10.
A time-resolved fluorescence immunochromatographic reagent strip 10 comprises a sample pad 2, a combination pad 3, a coating film, an absorption pad 5 and a bottom plate 1.
The sample pad 2, the bonding pad 3, and the coating film are sequentially disposed on the base plate 1, and the absorbent pad 5 is disposed on the coating film.
The sample pad 2 is treated with a sample pretreatment solution.
The binding pad 3 is provided with a hemp monoclonal antibody marked by a time-resolved fluorescent microsphere, the time-resolved fluorescent microsphere is a polystyrene microsphere, the surface of the time-resolved fluorescent microsphere is modified with functional groups, the functional groups comprise-COOH or-OH and the like, and the functional groups are used for covalent coupling with the antibody, so that a marker is more stable. The time-resolved fluorescent microspheres are filled with a lanthanide-containing substance.
The coating film is provided with a detection line 6 and a quality control line 7, the detection line 6 is closer to the combination pad 3 than the quality control line, the quality control line 7 is closer to the absorption pad 5, the detection line 6 is coated with hemp-BSA antigen, the quality control line 7 is coated with an antibody capable of being combined with the hemp-BSA antigen, and preferably, the quality control line 7 is coated with goat anti-mouse IgG antibody.
In one specific example, the functional group includes a carboxyl group.
In one specific example, the particle size of the time-resolved fluorescent microsphere is 100-500nm, the excitation wavelength is 300-400nm, and the emission wavelength is 600-700 nm. For example, the particle size of the time-resolved fluorescent microspheres is 100nm, 200nm, 300nm, 400nm, 500nm, or other values. The excitation wavelength is 300nm, 350nm, 400nm or other values. The emission wavelength is 600nm, 650nm, 700nm, or other values.
In one specific example, the spacing between the quality control line 7 and the detection line 6 is 3-8 mm. For example, the spacing between the quality control line 7 and the detection line 6 is 3mm, 4mm, 5mm, 6mm, 7mm, 8mm or other values.
In one specific example, the coating film is a nitrocellulose film 4.
An embodiment of the present invention further provides a method for preparing the time-resolved fluorescence immunochromatographic reagent strip 10.
The time-resolved fluorescence immunochromatographic reagent strip 10 can detect the cannabis by utilizing three matrixes, namely urine, saliva and hair, makes up the limitation of a single matrix detection window, and reduces the detection omission caused by short-time suspension of the detected person because the three matrixes complement each other.
A preparation method of a time-resolved fluorescence immunochromatography reagent strip 10 comprises the following steps:
(1) preparing a hemp monoclonal antibody marked by the time-resolved fluorescent microspheres: adding the time-resolved fluorescent microspheres into a coupling buffer solution, adding a hemp monoclonal antibody to be marked for coupling for 5-60min, adding a sealing solution for sealing for 5-60min, centrifuging for 5-20min under the conditions of 16000-: 10-100. mu.g of Cannabis sativa monoclonal antibody per 100uL of Cannabis sativa monoclonal antibody.
(2) Sample pad 2 preparation: dropwise adding the sample pretreatment solution on the sample pad 2, and drying at 45 ℃ for 3-4 h.
(3) Preparation of the conjugate pad 3: diluting the hemp monoclonal antibody marked by the time-resolved fluorescent microspheres obtained in the step (1) by using a microsphere diluent to obtain an antibody diluent, wherein the content of the time-resolved fluorescent microspheres is as follows: 0.5-1.5uL of time-resolved fluorescent microsphere/1 mL of microsphere diluent, dripping the antibody diluent on the bonding pad 3, and drying at 45 ℃ for 3-4 h.
(4) Preparation of coating film: the hemp-BSA antigen and the goat anti-mouse IgG antibody diluted by the PBS solution are respectively marked on the envelope as a detection line 6 and a quality control line 7, the detection line 6 is close to the binding pad 3 compared with the quality control line 7, and the binding pad is stored at 37 ℃. The coating amount of the detection line 6 and/or the quality control line 7 is 0.5-1 muL/cm.
(5) And (3) sequentially sticking the sample pad 2 prepared in the step (2), the bonding pad 3 prepared in the step (3) and the coating film prepared in the step (4) on the base plate 1, and adhering the absorption pad 5 on the base plate 1, wherein the quality control line 7 is close to the absorption pad 5. The width of the time-resolved fluorescence immunochromatography reagent strip 10 is 3-4 mm.
The embodiment of the invention also provides a hemp concentration quantitative detection method.
A method for quantitatively detecting the concentration of cannabis sativa comprises the following steps:
placing the time-resolved fluorescence immunochromatography reagent strip 10 and a sample to be detected at room temperature;
starting an immunofluorescence analyzer to obtain a standard curve of a sample to be detected;
60-100 mu L of a sample to be detected is dripped into the time-resolved fluorescence immunochromatography reagent strip 10, after reaction is carried out for 5-10min, the fluorescence intensity of a detection line 6 and a quality control line 7 when the time-resolved fluorescence immunochromatography reagent strip 10 is inserted into an immunofluorescence analyzer is detected, a T value, a C value and a T/C value are obtained, and the concentration of cannabis in the sample to be detected is obtained through a standard curve.
A kit, comprising the time-resolved fluorescence immunochromatography reagent strip.
The embodiment of the invention also provides a kit.
A kit, comprising the time-resolved fluorescence immunochromatography reagent strip.
Compared with the prior art, the invention has the following beneficial effects:
(1) the time-resolved fluorescent microspheres are prepared by filling lanthanide (such as europium) into nanoscale polystyrene microspheres, and compared with common fluorescence, the time-resolved fluorescent microspheres have the characteristics of large Stokes displacement, long fluorescence service life and the like, can effectively eliminate the interference of excitation light and nonspecific fluorescence on signal acquisition, and improve the specificity and accuracy of detection.
(2) The time-resolved fluorescence immunochromatographic reagent strip 10 prepared by the invention can carry out quantitative detection, has the advantages of high sensitivity, convenience and quickness in detection, low price, no need of professional operation and the like, and is beneficial to the field rapid detection of a basic layer.
(3) The time-resolved fluorescence immunochromatographic reagent strip 10 prepared by the invention can be used for quantitatively detecting the concentration of the hemp in three matrixes of urine, saliva and hair, one reagent strip can be used for detecting the three matrixes, the defects that the traditional reagent strip is single in matrix detection, narrow in window and easy to leak detection are overcome, and the abuse condition of a detected person can be comprehensively and objectively evaluated.
Example 1
This example provides a method for preparing a time-resolved fluorescence immunochromatographic reagent strip 10.
A preparation method of a time-resolved fluorescence immunochromatography reagent strip 10 comprises the following steps:
(1) preparing a hemp monoclonal antibody marked by the time-resolved fluorescent microspheres: adding time-resolved fluorescent microspheres with the particle size of 200nm into HEPES coupling buffer solution, adding a hemp monoclonal antibody to be marked, wherein the ratio of the hemp monoclonal antibody to the time-resolved fluorescent microspheres is (40-100ug/100 mu L microspheres), carrying out ultrasonic coupling for 5-30min, adding sealing liquid for sealing, centrifuging for 5-15min under the conditions of 16000-.
(2) Sample pad 2 preparation: the sample pretreatment solution was dropped on the sample pad 2 and baked at 45 ℃ for 3 to 4 hours, and the sample pretreatment solution was an aqueous solution containing 0.5% to 1.5% NaCl and 0.1% to 0.5% Tetronic 1307 (surfactant S9).
(3) Preparation of the conjugate pad 3: taking a 20mM glycine buffer solution containing 0.5-2% (W/W) BSA and 3-8% (W/W) trehalose as a microsphere diluent, diluting the hemp monoclonal antibody marked by the time-resolved fluorescent microsphere obtained in the step (1) by using the microsphere diluent to obtain an antibody diluent, wherein the volume ratio of the microsphere diluent to the time-resolved fluorescent microsphere in the step (1) is 1 mL: (0.9-1.5. mu.L), the antibody dilution was dropped onto the conjugate pad 3 and placed in an oven at 45 ℃ for 3-4 hours.
(4) Preparation of coating film: after the hemp-BSA antigen and the goat anti-mouse IgG antibody diluted by the PBS solution are balanced in the environment with room temperature and humidity of 30% -60%, the hemp-BSA antigen and the goat anti-mouse IgG antibody are marked on a nitrocellulose membrane 4 and respectively used as a detection line 6 and a quality control line 7, the coating amount of the detection line 6 and the quality control line 7 is 0.5-1 muL/cm, the distance between the detection line 6 and the quality control line 7 is 3-6mm, the detection line 6 is close to a binding pad 3, and the hemp-BSA antigen and the goat anti-mouse IgG antibody are stored at 37 ℃.
(5) And (3) sequentially sticking the sample pad 2 prepared in the step (2), the bonding pad 3 prepared in the step (3) and the coating film prepared in the step (4) on the bottom plate 1, adhering the absorption pad 5 on the bottom plate 1, enabling the quality control line 7 to be close to the absorption pad 5, cutting the absorption pad 5 into time-resolved fluorescence immunochromatography reagent strips 10 with the width of 3-4mm, putting the time-resolved fluorescence immunochromatography reagent strips 10 into a reagent card, and sealing the reagent card for storage.
Example 2
The embodiment provides a method for quantitatively detecting the concentration of cannabis.
A method for quantitatively detecting the concentration of cannabis sativa comprises the following steps:
(1) the time-resolved fluoroimmunoassay reagent strip 10 prepared in example 1 and the sample to be measured were placed at room temperature and used after returning to room temperature.
(2) Starting an immunofluorescence analyzer, inserting an ID card corresponding to a sample to be detected, and reading a standard curve;
(3) and (2) dropwise adding 80-100 mu L of a sample to be detected into a sample adding hole of the time-resolved fluorescence immunochromatographic reagent strip 10, reacting for 5-10min, inserting the time-resolved fluorescence immunochromatographic reagent strip 10 into an immunofluorescence analyzer, reading the fluorescence intensity (T value) of the detection line 6 and the fluorescence intensity (C value) of the quality control line 7, obtaining the T value, the C value and the T/C value, obtaining the concentration of the cannabis in the sample to be detected through a standard curve, and judging.
Example 3
The embodiment provides a method for quantitatively detecting the concentration of cannabis in a urine matrix.
A method for quantitatively detecting the concentration of cannabis in a urine matrix comprises the following steps:
(1) placing the time-resolved fluorescence immunochromatography reagent strip 10 prepared in example 1 at room temperature, and using the reagent strip after the temperature is restored to room temperature; diluting tetrahydrocannabinic acid standard substance with blank artificial urine matrix, wherein the concentrations are as follows: 0ng/mL, 0.5ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, 25ng/mL, 50ng/mL, 75ng/mL, to give nine concentrations of standard urine matrix, each concentration of standard urine matrix set up in three replicates, all standard urine matrix placed in room temperature, after returning to room temperature for use.
(2) Drawing a standard curve: turning on an immunofluorescence analyzer; adding each standard sample urine matrix into a sample adding hole of a time-resolved fluorescence immunochromatography reagent strip 10, reacting for 10min, inserting each time-resolved fluorescence immunochromatography reagent strip 10 into an immunofluorescence analyzer, reading the fluorescence intensity (T value) of a detection line 6 and the fluorescence intensity (C value) of a quality control line 7, obtaining the T value, the C value and the T/C value, establishing an equation and fitting the equation into a standard curve by taking the concentration of the standard sample as a horizontal coordinate and the T/C value as a vertical coordinate, wherein the specific numerical values are shown in Table 1.
TABLE 1
According to the data in Table 1, the concentration of the standard substance in the urine matrix is used as the abscissa and the T/C value is usedOn the ordinate, a standard curve is plotted, the standard curve is shown in FIG. 2 (the ordinate of fig. 2, which is logit (Y), represents the natural logarithm of the signal detected by the instrument, and the ordinate of fig. 2, which is logit (X), represents the natural logarithm of the concentration of the drug detected), and the standard curve has good linearity (R, R)20.9903) can be used to quantitatively detect cannabis in urine matrix by this standard curve. As can be seen from the data in Table 1, the sensitivity of the urine matrix detection of cannabis can reach 0.5ng/mL, and the CV at each concentration point is almost around 15%.
(3) Detection of the simulated sample: adding a hemp standard substance into a blank artificial urine matrix to enable the concentration of the hemp standard substance to be 10ng/mL, adding the prepared solution serving as a sample to be detected into a sample adding hole of a time-resolved fluorescence immunochromatography reagent strip 10, after reacting for 10min, respectively inserting each time-resolved fluorescence immunochromatography reagent strip 10 into an immunofluorescence analyzer, reading the fluorescence intensity (T value) of a detection line 6 and the fluorescence intensity (C value) of a quality control line 7, and obtaining the T/C values which are 2.910 respectively; 3.035, respectively; 2.963, respectively; 2.816, respectively; 3.249, respectively; 2.979, the average value of the T/C values was calculated to be 2.992, and this value was substituted into the standard curve to calculate a concentration of 11.59ng/mL and a recovery rate of 115.91% (recovery rate ═ concentration of test sample/concentration of added sample × 100%).
Example 4
This example provides a method for quantitatively determining the concentration of cannabis in a saliva matrix.
A method for quantitatively detecting the concentration of cannabis in saliva matrix comprises the following steps:
(1) placing the time-resolved fluorescence immunochromatography reagent strip 10 prepared in example 1 at room temperature, and using the reagent strip after the temperature is restored to room temperature; diluting tetrahydrocannabinic acid standard with blank artificial saliva base, wherein the concentrations are as follows: 0ng/mL, 0.1ng/mL, 0.5ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, to give eight concentrations of the standard saliva base, in triplicate for each concentration of the standard saliva base, all of the standard saliva base was placed at room temperature and used after returning to room temperature.
(2) Drawing a standard curve: turning on an immunofluorescence analyzer; adding each standard saliva matrix into a sample adding hole of a time-resolved fluorescence immunochromatographic reagent strip 10, reacting for 10min, inserting each time-resolved fluorescence immunochromatographic reagent strip 10 into an immunofluorescence analyzer, reading the fluorescence intensity (T value) of a detection line 6 and the fluorescence intensity (C value) of a quality control line 7, obtaining the T value, the C value and the T/C value, establishing an equation and fitting the equation into a standard curve by taking the concentration of the standard as a horizontal coordinate and the T/C value as a vertical coordinate, wherein the specific numerical values are shown in Table 2.
TABLE 2
According to the data in Table 2, a standard curve is plotted with the concentration of the standard substance in the saliva matrix as the abscissa and the T/C value as the ordinate, the standard curve is shown in FIG. 3 (the ordinate of the log (Y) in FIG. 3 is taken as the natural logarithm of the signal value detected by the instrument, the ordinate of the log (ln), (X) is taken as the natural logarithm of the concentration of the drug detected), and the standard curve is good in linearity (R)20.9939) it is possible to carry out quantitative detection of cannabis in saliva matrix by this curve. As can be seen from the data in Table 2, the sensitivity of the saliva-based assay for cannabis was up to 0.1ng/mL, with CV values around 15% for each concentration point.
(3) Detection of the simulated sample: using blank artificial saliva matrix, hemp standard was added to a concentration of 5 ng/mL. Adding the prepared solution serving as a sample to be detected into a sample adding hole of a time-resolved fluorescence immunochromatographic reagent strip 10, after reacting for 10min, respectively inserting each time-resolved fluorescence immunochromatographic reagent strip 10 into an immunofluorescence analyzer, reading the fluorescence intensity (T value) of a detection line 6 and the fluorescence intensity (C value) of a quality control line 7, and obtaining the T/C values which are 3.449 respectively; 3.386; 3.714, respectively; 4.228, respectively; 3.274, respectively; 4.109, the average value of T/C values was calculated to be 3.693, and this value was substituted into the standard curve to calculate a concentration of 4.73ng/mL and a recovery of 94.63% (recovery ═ concentration of test sample/concentration of added sample × 100%).
Example 5
This example provides a method for quantitatively determining the concentration of cannabis in a hair matrix.
A hemp concentration quantitative detection method of a hair matrix comprises the following steps:
(1) placing the time-resolved fluorescence immunochromatography reagent strip 10 prepared in example 1 at room temperature, and using the reagent strip after the temperature is restored to room temperature; the tetrahydrocannabinic acid standard was diluted with blank hair matrix at concentrations of: 0ng/mL, 0.1ng/mL, 0.5ng/mL, 1ng/mL, 2ng/mL, 10ng/mL, 50ng/mL, to give six concentrations of standard hair base, each concentration of standard hair base is provided in triplicate, all standard hair bases are placed in room temperature and used after returning to room temperature.
(2) Drawing a standard curve: turning on an immunofluorescence analyzer; adding each standard substance hair matrix into a sample adding hole of a time-resolved fluorescence immunochromatography reagent strip 10, reacting for 10min, inserting each time-resolved fluorescence immunochromatography reagent strip 10 into an immunofluorescence analyzer, reading the fluorescence intensity (T value) of a detection line 6 and the fluorescence intensity (C value) of a quality control line 7, obtaining the T value, the C value and the T/C value, establishing an equation and fitting the equation into a standard curve by taking the concentration of the standard substance as a horizontal coordinate and the T/C value as a vertical coordinate, wherein the specific numerical values are shown in Table 3.
TABLE 3
According to the data in Table 3, a calibration curve is plotted with the concentration of the standard in the hair matrix as the abscissa and the T/C value as the ordinate, the calibration curve being shown in FIG. 4 (the ordinate, position (Y), in FIG. 4 is taken as the natural logarithm of the signal value detected by the instrument, and the ordinate, ln, (X), is taken as the natural logarithm of the concentration of the drug detected), and the calibration curve is well-linear (R)20.9898) it is possible to carry out quantitative detection of cannabis in hair matrix by this curve. As can be seen from the data in Table 3, the sensitivity of cannabis in the hair matrix detection was 0.1ng/mL, and the CV was substantially around 15% at each concentration point.
(3) Detection of the simulated sample: the blank hair matrix treatment solution was used and hemp standard was added to a concentration of 2 ng/mL. Adding the prepared solution serving as a sample to be detected into a sample adding hole of a time-resolved fluorescence immunochromatographic reagent strip 10, after reacting for 10min, respectively inserting each time-resolved fluorescence immunochromatographic reagent strip 10 into an immunofluorescence analyzer, reading the fluorescence intensity (T value) of a detection line 6 and the fluorescence intensity (C value) of a quality control line 7, and obtaining the T/C values which are 4.784 respectively; 4.835, respectively; 5.175; 5.448, respectively; 5.551, respectively; 5.414, the average of the T/C values was calculated to be 5.201, and this value was substituted into the standard curve to calculate a concentration of 2.692ng/mL and a recovery of 134.62% (recovery ═ concentration of test sample/concentration of added sample × 100%).
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A time-resolved fluorescence immunochromatographic reagent strip is characterized by comprising a sample pad, a combination pad, a coating film, an absorption pad and a bottom plate;
the sample pad, the combination pad and the coating film are sequentially arranged on the bottom plate, and the absorption pad is arranged on the coating film and is in contact with the coating film;
the binding pad is provided with a hemp monoclonal antibody marked by a time-resolved fluorescent microsphere, the surface of the time-resolved fluorescent microsphere is modified with a functional group, and the time-resolved fluorescent microsphere is filled with a substance containing lanthanide;
the coating film is provided with a detection line and a quality control line, the detection line is closer to the binding pad than the quality control line, the detection line is coated with a hemp-BSA antigen, and the quality control line is coated with an antibody capable of binding with the hemp-BSA antigen.
2. The time-resolved fluoroimmunochromatography reagent strip according to claim 1, wherein the functional group includes a carboxyl group.
3. The time-resolved fluorescence immunochromatography reagent strip according to claim 1, wherein the particle size of the time-resolved fluorescence microsphere is 100-500nm, the excitation wavelength is 300-400nm, and the emission wavelength is 600-700 nm.
4. The time-resolved fluoroimmunochromatographic reagent strip according to any one of claims 1 to 3, wherein the distance between the quality control line and the detection line is 3 to 8 mm.
5. A preparation method of a time-resolved fluorescence immunochromatography reagent strip is characterized by comprising the following steps:
preparing a hemp monoclonal antibody marked by the time-resolved fluorescent microspheres: adding the time-resolved fluorescent microspheres into a coupling buffer solution, adding a hemp monoclonal antibody to be marked, coupling, sealing, centrifuging and storing at 4 ℃ after heavy suspension, wherein the hemp monoclonal antibody is added in the following content: 10-100 μ g of cannabis monoclonal antibody/100 uL of cannabis monoclonal antibody;
sample pad preparation: dropwise adding the sample pretreatment solution on a sample pad, and drying;
preparing a bonding pad: diluting the obtained hemp monoclonal antibody marked by the time-resolved fluorescent microspheres by using a microsphere diluent to obtain an antibody diluent, wherein the content of the time-resolved fluorescent microspheres is as follows: 0.5-1.5uL of hemp monoclonal antibody/1 mL of microsphere diluent, dripping the antibody diluent on a combination pad, and drying;
preparation of coating film: respectively marking hemp-BSA antigen and goat anti-mouse IgG antibody diluted by PBS solution on a coating film to serve as a detection line and a quality control line, wherein the detection line is closer to a binding pad than the quality control line, and storing at 37 ℃;
and sequentially adhering the prepared sample pad, the prepared combination pad and the prepared coating film on a bottom plate, adhering an absorption pad on the bottom plate, and enabling the quality control line to be close to the absorption pad.
6. The method for preparing the time-resolved fluoroimmunoassay reagent strip according to claim 5, wherein the coupling time is 5-60min, the blocking solution is added for 5-60min, and the strip is washed and resuspended by the preservation solution and preserved at 4 ℃.
7. The method for preparing a time-resolved fluoroimmunoassay reagent strip according to claim 5 or 6, wherein the drying temperature in the step of preparing the sample pad and/or the step of preparing the conjugate pad is 45 ℃ and the drying time is 3 to 4 hours.
8. The method for preparing a time-resolved fluoroimmunochromatographic reagent strip according to claim 5 or 6, wherein in the step of preparing a coating film, the coating amount of the detection line and/or the quality control line is 0.5 to 1 μ L/cm.
9. A method for quantitatively detecting the concentration of hemp is characterized by comprising the following steps:
placing the time-resolved fluoroimmunoassay reagent strip according to any one of claims 1 to 4 or the preparation method according to any one of claims 5 to 8 and a sample to be tested at room temperature;
and dropwise adding a sample to be detected to the time-resolved fluorescence immunochromatography reagent strip, detecting the fluorescence intensity of a detection line and a quality control line on the time-resolved fluorescence immunochromatography reagent strip after reaction, obtaining a T value, a C value and a T/C value, and obtaining the concentration of cannabis in the sample to be detected through a standard curve.
10. A kit comprising the time-resolved fluoroimmunoassay reagent strip according to any one of claims 1 to 4 or produced by the production method according to any one of claims 5 to 8.
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