CN117990902A

CN117990902A - Small molecule fluorescent immunochromatography test strip, detection card and kit

Info

Publication number: CN117990902A
Application number: CN202410284573.5A
Authority: CN
Inventors: 章观雄; 杨家保; 刘宁
Original assignee: Xiamen Baotai Biotechnology Co ltd
Current assignee: Xiamen Baotai Biotechnology Co ltd
Priority date: 2024-03-13
Filing date: 2024-03-13
Publication date: 2024-05-07

Abstract

The invention provides a small molecular fluorescent immunochromatography test strip, a detection card and a kit. The test strip comprises a bottom plate, a blood filtering film sample pad, a glass fiber combination pad, a nitrocellulose film and an absorption pad, wherein the blood filtering film sample pad, the glass fiber combination pad, the nitrocellulose film and the absorption pad are sequentially overlapped and stuck on the bottom plate, fluorescent microsphere markers marked by marked antibodies are adsorbed on the glass fiber combination pad, a detection line and a quality control line are sequentially arranged on the nitrocellulose film from front to back according to the flow direction of the sample, and the detection line is coated with capture antibodies; the fluorescent microsphere marker marked by the marked antibody can be combined with a small molecule to form an immune complex, and the capture antibody can be specifically combined with the immune complex.

Description

Small molecule fluorescent immunochromatography test strip, detection card and kit

Technical Field

The invention belongs to the technical field of medical inspection, and relates to a small molecular fluorescent immunochromatographic test strip, a detection card and a kit.

Background

The small molecule immunoassay method comprises the following steps: radioimmunoassay (radioimmunoassay, RIA), enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA), chemiluminescent immunoassay (chemiluminescenceimmunoassay, CLIA), and analyte analysis are performed mainly by competition methods.

Major drawbacks of the competition method: 1. the reaction signal intensity is inversely related to the content of the small molecules to be detected in the sample, and is in an indirect detection mode, so that the specific quantity of the objects to be detected in the system cannot be directly reflected; 2. the ability of the labeled small molecules to bind to the solid phase antibody should be equivalent to that of the small molecules to be detected, and unfair competition may produce false negative or false positive results; 3. the competition method belongs to reagent limited analysis, the amount of the solid-phase antibody and the labeled antigen is fixed, the binding site of the solid-phase antibody and the labeled antigen is less than the total amount of the labeled antigen and the antigen to be detected, and if the solid-phase antibody or the labeled antigen is not matched, the detection range and the sensitivity are seriously affected; 4. competition methods are susceptible to interference from changes in external conditions and reagent stability is often poor.

The conventional sandwich method belongs to a non-competitive binding assay, and does not have the disadvantages of the competition method, but is only suitable for detecting multivalent antigens having at least two or more immunological binding sites in the molecule, and cannot be used for detecting small molecule haptens, such as 25-hydroxy vitamin D,25- (OH) D.

Disclosure of Invention

The invention provides a small molecular fluorescent immunochromatography test strip, a detection card and a kit, which can effectively solve the problems.

The invention is realized in the following way:

In one aspect, the invention provides a small molecular fluorescent immunochromatography test strip, which comprises a bottom plate, a blood filtering membrane sample pad, a glass fiber combination pad, a nitrocellulose membrane and an absorption pad, wherein the blood filtering membrane sample pad, the glass fiber combination pad, the nitrocellulose membrane and the absorption pad are sequentially overlapped and stuck on the bottom plate, fluorescent microsphere markers marked by marked antibodies are adsorbed on the glass fiber combination pad, a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane from front to back according to the flow direction of the sample, and the detection line is coated with capture antibodies;

The fluorescent microsphere marker marked by the marked antibody can be combined with the small molecule to form an immune complex, and the capture antibody can be specifically combined with the immune complex.

In some embodiments, the small molecule is vitamin D.

In some embodiments, the small molecule is 25- (OH) D.

In some embodiments, the fluorescent microspheres in the labeled antibody-labeled fluorescent microsphere markers are time resolved fluorescent microspheres.

The time-resolved fluorescence microsphere is designed aiming at an immunochromatography detection (lateral flow immunoassay) reagent, is formed by copolymerizing time-resolved fluorescence dye and styrene, and has the characteristics of large stokes displacement, no internal quenching effect during aggregation, strong detection signal, fluorescent dye being wrapped in the microsphere, being not easily influenced by external environment, stable fluorescence and the like.

Principle of time-resolved fluorescence immunochromatography (TRFILF): when a sample containing an antigen (antibody) to be detected is dripped in a sample adding area, the antigen (antibody) in the sample to be detected is combined with the fluorescent nanoparticle labeled antibody (antigen) in a combining pad and is subjected to forward chromatography through capillary action, and after the sample reaches a detection area, the antigen (antigen) is combined with an antibody (antigen) fixed on a detection line to form a particle-antibody-antigen-antibody sandwich compound and is fixed on the detection line, and redundant fluorescent microsphere markers are subjected to forward chromatography and are combined with a secondary antibody fixed on a quality control line. After the reaction is finished, the ultraviolet light source (340 nm) is used for scanning and detecting the detection area, and the fluorescent nano-microspheres on the detection line and the quality control line emit high-intensity fluorescence (615 nm) with longer decay time. By delaying the measurement time, after the naturally occurring short-life fluorescence (1-10 ns) in the sample matrix decays completely, the specific fluorescence of the rare earth element is measured, so that the interference of the specific background fluorescence can be completely eliminated. The concentration of the object to be detected in the sample can be analyzed through the intensity and the ratio of the fluorescence intensity of the detection line and the quality control line.

In some embodiments, the mass ratio of the amount of the fluorescent microsphere to the amount of the labeled antibody in the labeled antibody labeled fluorescent microsphere label is 1:0.030 to 0.135.

In some embodiments, the method of making a fluorescent microsphere marker labeled with the 25- (OH) D-labeled antibody comprises:

S11, taking time-resolved fluorescent microspheres, adding MES solution with pH of 4.5-6.5, centrifugally washing, removing supernatant to obtain washed fluorescent microspheres, and adding MES solution with pH of 4.5-6.5 into the washed fluorescent microspheres for ultrasonic dispersion to prepare microsphere suspension I;

In some embodiments, the suspension of fluorescent microspheres at a concentration of 10mg/mL is centrifuged at 14000rpm for 10-20 min to yield time-resolved fluorescent microspheres.

In some embodiments, in step S11, the volume of MES solution added each time is twice that of the fluorescent microsphere suspension.

In some embodiments, the MES solution is added and centrifuged at 14000rpm for 10-20 minutes.

S12, adding EDC and NHS into the microsphere suspension I, uniformly mixing, oscillating at a shaking table of 37 ℃, centrifuging after the reaction is finished, removing supernatant, and adding HEPES solution with pH of 7.0-8.0 for ultrasonic dispersion to prepare microsphere suspension II;

In some embodiments, EDC and NHS are both dissolved to 10mg/mL with MES solution at pH 4.5-6.5.

In some embodiments, the time-resolved fluorescent microsphere, EDC, NHS are used in a mass ratio of 1:0.01 to 0.2:0.01 to 0.2.

In some embodiments, shaking is performed at 37℃for 0.3 to 1 hour.

In some embodiments, the ratio of time-resolved fluorescent microsphere to HEPES solution is 1mg: 100. Mu.L.

S13, adding a 25- (OH) D labeled antibody into the microsphere suspension II, uniformly mixing, vibrating at a temperature of 37 ℃, adding bovine serum albumin after the reaction is finished, uniformly mixing, sealing at the temperature of 37 ℃, centrifuging to remove supernatant, adding a cleaning buffer solution, centrifuging, washing, and removing the supernatant to obtain the fluorescent microsphere marker labeled by the 25- (OH) D labeled antibody;

In some embodiments, 25- (OH) D-labeled antibody is added to the microsphere suspension ii and mixed well, supplemented with HEPES solution at ph 7.0-8.0, and the ratio of time-resolved fluorescent microspheres to supplemented liquid is 1mg: 200. Mu.L.

In some embodiments, shaking is performed at 37 ℃ for 3.5 hours. After the reaction is finished, adding bovine serum albumin, uniformly mixing, sealing for 30min on a shaking table at 37 ℃, and centrifuging for 10-20 min at 14000rpm to remove supernatant.

In some embodiments, the time-resolved fluorogenic microsphere is used in a mass ratio to bovine serum albumin of 1:0.5 to 2.0.

Preferably, the cleaning buffer is a tween 20 solution with a mass fraction of 3%.

Preferably, the fluorescent microsphere marker marked by the 25- (OH) D-labeled antibody is stored in a storage buffer. Further preferably, the ratio of the amount of time-resolved fluorescent microsphere to the amount of preservation buffer is 1mg: 100. Mu.L.

In some embodiments, the ratio of the amount of time-resolved fluorescent microsphere to the amount of 25- (OH) D-labeled antibody is 1:0.030 to 0.135. In some embodiments, the ratio of the amount of time-resolved fluorescent microsphere to the amount of 25- (OH) D-labeled antibody is 1:0.030 to 0.10, 1:0.10 to 0.135. In some embodiments, the ratio of the amount of time-resolved fluorescent microsphere to the amount of 25- (OH) D-labeled antibody is 1:0.030, 1:0.10, 1:0.135.

In some embodiments, the adsorption method of the fluorescent microsphere marker with the labeled antibody adsorbed on the glass fiber bonding pad is as follows: uniformly spraying a line of fluorescent microsphere marker marked by the marked antibody with the concentration of 0.3-1.25 mg/mL on the glass fiber bonding pad, wherein the dosage is 2-4 mu L/cm of the bonding pad.

In some embodiments, the preservation buffer storing the fluorescent microsphere marker marked by the 25- (OH) D-labeled antibody is diluted by 8-30 times by microsphere dilution liquid, a line is uniformly sprayed on the glass fiber bonding pad, the usage amount is 2-4 mu L/cm of the bonding pad, and the concentration of the fluorescent microsphere marker marked by the labeled antibody in the preservation buffer storing the fluorescent microsphere marker marked by the labeled antibody is 10mg/mL.

Preferably, the microsphere dilution is a Tris-HCl solution with mass fractions of 0.5% sodium chloride, 0.5% S9, 2% trehalose and 0.1% bovine serum albumin, wherein the Tris-HCl solution has a pH of 8.0.

In some embodiments, the glass fiber-binding pad sprayed with fluorescent microsphere markers labeled with 25- (OH) D-labeled antibodies is dried overnight at 37 ℃.

In some embodiments, the coating method of the detection line coated with the capture antibody is as follows: and marking the capture antibody with the concentration of 0.5-1.5 mg/mL on the nitrocellulose membrane as the detection line for coating, wherein the coating dosage is 0.5-1.5 mu L/cm membrane.

Preferably, the concentration of the capture antibody is adjusted to 0.5-1.5 mg/mL by using a coating buffer solution, and the capture antibody is marked on the nitrocellulose membrane as the detection line for coating, wherein the coating dosage is 0.5-1.5 mu L/cm membrane.

Preferably, the coating buffer is 10mM PBS buffer containing 3.0% trehalose by mass.

In some embodiments, the quality control line is a rabbit anti-DNP antibody coated quality control line.

In some embodiments, the coating method of the quality control line coated with the rabbit anti-DNP antibody comprises the following steps: and (3) marking the rabbit anti-DNP antibody with the concentration of 0.3-1.0 mg/mL on the nitrocellulose membrane as the quality control line for coating, wherein the coating dosage is 0.5-1.5 mu L/cm membrane.

Preferably, the rabbit anti-DNP antibody is coated by drawing the quality control line on the nitrocellulose membrane by adjusting the concentration to 0.3-1.0 mg/mL with the coating buffer, and the coating dosage is 0.5-1.5 mu L/cm membrane.

Preferably, the detection line and the quality control line are parallel to each other, and the interval distance is 6-8 mm.

In some embodiments, the nitrocellulose membrane coated with the capture antibody and rabbit anti-DNP antibody is dried at 55 ℃ overnight.

In some embodiments, the blood filtering membrane sample pad is a pretreated blood filtering membrane, the pretreatment is to spray sample pad treatment liquid on the blood filtering membrane in parallel and uniformly, the dosage is 2-5 mu L/cm sample pad, and the sample pad is dried at 37 ℃;

preferably, the sample pad treatment solution is a Tris-HCl solution with mass fractions of 20mM and pH8.0 containing 0.5% sodium chloride, 0.5% S9 and 0.1% bovine serum albumin respectively.

In some embodiments, the base plate is a PVC base plate.

In some embodiments, the absorbent pad is made of paper material with high water absorption capacity.

In another aspect, the present invention provides a test card, where the test card includes the test strip and a card case described above;

Preferably, the clamping shell comprises an upper clamping shell and a lower clamping shell which are mutually clamped, a clamping groove for placing the test strip is formed in the inner surface of the lower clamping shell, a sample adding port is formed in the position, corresponding to the sample pad of the blood filtering membrane, of the upper clamping shell, an observation port is formed in the position, corresponding to the nitrocellulose membrane, of the upper clamping shell, and the detection line and the quality control line are both exposed at the position of the observation port.

In yet another aspect, the present invention provides a kit comprising the test strip or the test card described above;

preferably, the kit further comprises a sample diluent, the sample buffer comprising the following components in mass fraction: HEPES 0.5-1.5%, perfluorooctanoic acid 0.5-2.0%, sodium chloride 1.0-2.0%, S9.1-1.0%, and the pH value of the sample buffer is 7.0-9.0.

The beneficial effects of the invention are as follows:

The invention provides a small molecule fluorescent immunochromatography test strip, which adopts a double-antibody sandwich form to detect small molecules, wherein a fluorescent microsphere marker is formed after a labeled antibody and a fluorescent microsphere are labeled, then when a sample is detected, the fluorescent microsphere marker is combined with the small molecules in the sample to form a fluorescent microsphere-labeled antibody-small molecule immune complex, and the immune complex provides a novel immune binding site, so that a capture antibody can be combined with the immune complex to form a final double-antibody sandwich complex, thereby meeting the requirements of a sandwich method and solving the defects of methodologies brought by the detection of a conventional immune competition method.

Furthermore, the invention adopts a time-resolved fluorescence immunochromatography to detect small molecules, so that the interference of nonspecific fluorescence can be effectively eliminated, and the analysis sensitivity is greatly improved.

Furthermore, the reagent is prepared in the form of a solid-phase test strip, so that the reagent has a wider preservation condition range, can be preserved at the normal temperature of 2-30 ℃, can be transported and preserved at the normal temperature, solves the problems that the conventional liquid reagent needs to be preserved in a refrigerating way and has larger cold chain transportation cost, and improves the use convenience.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some examples of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of a test strip and a test card according to an embodiment of the present invention.

FIG. 2 is a schematic diagram of a double antibody sandwich method for detecting 25- (OH) D provided by the embodiment of the invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention. Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention.

Example 1

Abbreviations description:

MES:2-Morpholinoethanesulfonic Acid, 2-morpholinoethanesulfonic acid.

EDC:1-Ethyl-3- [3-dimethylaminopropyl ] carbodiimide Hydrochloride, 1-Ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride.

NHS: N-Hydroxysuccinimide, N-hydroxysuccinimide.

HEPES:4- (2-hydroxyerhyl) piperazine-1-erhanesulfonic Acid, N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid.

PBS: phosphate Buffer Saline phosphate buffer salts.

Tris-HCl: tri (Hydroxymethyl) Amino Methane Hydrochloride tris (hydroxymethyl) aminomethane hydrochloride.

NC film: nitrocellulose membrane.

The time-resolved fluorescence microsphere used in the embodiment of the invention is purchased from the Boyue organism, and the product number is EU0100C; the 25- (OH) D-labeled antibody was purchased from the European Kaplan organism under the designation R484j4; capture antibodies were purchased from the organisms euroki under the accession number R483k3; rabbit anti-DNP antibodies were purchased from south kyo under the trade designation T112501A.

Vitamin D (VD) is an important regulator in the human body, and is widely accepted to have beneficial effects on the human body, and besides participating in calcium and phosphorus metabolism in the human body, recent researches have found that Vitamin D plays an important role in diseases such as hypertension, cardiovascular diseases, diabetes, tumor and the like. Low levels of VD concentrations can negatively affect these diseases.

25 Hydroxy vitamin D is the main transport form of VD in blood, its nature is stable (half-life 2 weeks), its level can reflect the total amount of VD after human body intake and self-synthesis. The detection of 25- (OH) D levels in patients with hand-foot disease, pre-eclampsia, vitiligo, hepatitis B, gestational diabetes, lung cancer, chronic heart failure and the like shows that the content of the D is obviously reduced compared with that of normal people and the reduction degree is positively correlated with the disease severity, so that the detection of the 25- (OH) D level can reflect the light and heavy degree of the disease, and thus, the clinical attention of the measurement of the 25- (OH) D level is growing.

And 25- (OH) D is a small molecule, and two or more immune binding sites are not present, so that the requirement of the traditional sandwich method cannot be met, and the detection can be carried out only through an indirect competition mode.

The embodiment of the invention provides a vitamin D time-resolved fluorescence immunochromatography test strip.

Referring to fig. 1, the test strip includes a base plate, a blood filtering membrane sample pad, a glass fiber bonding pad, a nitrocellulose membrane and an absorption pad, wherein the blood filtering membrane sample pad, the glass fiber bonding pad, the nitrocellulose membrane and the absorption pad are sequentially lapped and stuck on the base plate, fluorescent microsphere markers marked by 25- (OH) D marked antibodies are adsorbed on the glass fiber bonding pad, a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane from front to back in the direction of sample flow (chromatography), and the detection line is coated with capture antibodies;

The fluorescent microsphere marker labeled by the 25- (OH) D labeled antibody can be combined with the 25- (OH) D to form an immune complex, and the capture antibody can be specifically combined with the immune complex.

The reagent strip can be prepared by the following preparation method:

(1) The preparation of the fluorescent microsphere marker marked by the 25- (OH) D marked antibody comprises the following steps:

S1, placing 100 mu L of fluorescent microsphere suspension with the concentration of 10mg/mL into a test tube, centrifuging for 20min at the rotation speed of 14000rpm, removing supernatant, adding 200 mu L of MES solution with the pH of 6.5, washing twice, centrifuging for 20min at the rotation speed of 14000rpm, removing supernatant, adding 200 mu L of MES solution with the pH of 6.5, and performing ultrasonic dispersion to prepare microsphere suspension I.

S2, 0.2mg of EDC is dissolved to 10mg/mL by using MES solution with pH of 6.5, 0.2mg of NHS is dissolved to 10mg/mL by using MES solution with pH of 6.5, the mixture is added into microsphere suspension I prepared in the step S1 respectively, the mixture is uniformly mixed, shaking is carried out for 1h at 37 ℃, supernatant is removed by centrifugation after the reaction is finished, 100 mu L of HEPES solution with pH of 8.0 is added, and the mixture is subjected to ultrasonic dispersion to prepare microsphere suspension II.

S3, sequentially adding 100 mug of 25- (OH) D labeled antibody into the microsphere suspension II prepared in the step S2, supplementing HEPES solution with pH of 8.0 to a total volume of 200 mug, uniformly mixing, shaking for 3.5 hours under a shaking table at 37 ℃, adding 2.0mg of bovine serum albumin after the reaction is finished, uniformly mixing, sealing for 30 minutes on the shaking table at 37 ℃, centrifuging for 20 minutes at 14000rpm, removing the supernatant, adding 200 mug of Tween 20 solution (washing buffer solution) with the mass fraction of 3 percent, washing twice to obtain fluorescent microsphere marker labeled by the 25- (OH) D labeled antibody, and adding 100 mug of preservation buffer solution for storage.

(2) Preparation of a glass fiber bonding pad:

The preservation buffer solution of the fluorescent microsphere marker prepared in the step (1) and stored with the 25- (OH) D-labeled antibody is diluted 8 times by a Tris-HCl solution (microsphere dilution) containing 0.5% of sodium chloride, 0.5% of S9.5% of trehalose and 0.1% of bovine serum albumin by mass percent and having the pH of 8.0, a line is uniformly sprayed on the binding pad at the dosage of 4 mu L/cm, and the binding pad is placed in an oven and dried at 37 ℃ overnight.

(3) Preparation of nitrocellulose membrane:

and a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane. The detection line and the quality control line are parallel to each other, and the interval distance is 6-8 mm.

The detection line is coated with a capture antibody, and the quality control line is coated with a rabbit anti-DNP antibody.

The capture antibody and the rabbit anti-DNP antibody were adjusted to 1.5mg/mL and 1.0mg/mL, respectively, using 10mM PBS buffer (coating buffer) containing 3.0% trehalose. I.e., the capture antibody coating concentration in the detection line was 1.5mg/mL. The coating concentration of the rabbit anti-DNP antibody in the quality control line is 1.0mg/mL.

Coating buffer solution containing capture antibody and coating buffer solution containing rabbit anti-DNP antibody as detection line and quality control line respectively on nitrocellulose membrane in parallel, wherein the coating amount is 1.5 μl/cm membrane, and drying overnight at 55deg.C.

(4) Preparation of a hemofilter sample pad:

A sample pad of 5. Mu.L/cm was sprayed uniformly in parallel on a hemofilter with a 20mM, pH8.0 Tris-HCl solution containing 0.5% by mass of sodium chloride, 0.5% by mass of S9.5% by mass of bovine serum albumin and 0.1% by mass of bovine serum albumin, and dried overnight at 37 ℃.

(5) Assembly of test strips

The embodiment of the invention also provides a vitamin D time-resolved fluorescence immunochromatography detection card.

The detection card comprises the test strip and a card shell. The card shell includes the last card shell and the lower card shell of mutual joint, the internal surface of lower card shell is provided with and is used for placing the draw-in groove of test strip, go up the card shell correspond to straining blood membrane sample pad department is equipped with the application of sample mouth, correspond to nitrocellulose membrane department is equipped with the observation mouth, the detection line with the matter control line all expose in observation mouth department. Wherein, the detection line is close to the application of sample mouth, and the matter control line is kept away from the application of sample mouth.

The clamping shell not only protects the test strip and prevents the test strip from being polluted by damage, but also plays a role in fixing, so that the test strip is not easy to slide and the measurement is affected. The upper clamping shell and the lower clamping shell can fix and compress the test strip, the labeled antibody on the combining pad and the sample diluent can synchronously run, and the uniform flow speed of the liquid is ensured, so that the CV coefficient is further reduced, and the precision and the accuracy are improved.

The embodiment of the invention also provides a vitamin D time-resolved fluorescence immunochromatography detection kit, which comprises the test strip or the detection card.

The kit further comprises a sample diluent, wherein the sample buffer comprises the following components in percentage by mass: HEPES1.5%, perfluorooctanoic acid 2.0%, sodium chloride 2.0%, S9.1.0%, the pH of the sample buffer was 7.0.

Principle of time-resolved fluorescence immunochromatography (TRFILF)

When a sample containing an antigen (antibody) to be detected is dripped in a sample adding area, the antigen (antibody) in the sample to be detected is combined with the fluorescent nanoparticle labeled antibody (antigen) in a combining pad and is subjected to forward chromatography through capillary action, and after the sample reaches a detection area, the antigen (antigen) is combined with an antibody (antigen) fixed on a detection line to form a particle-antibody-antigen-antibody sandwich compound and is fixed on the detection line, and redundant fluorescent microsphere markers are subjected to forward chromatography and are combined with a secondary antibody fixed on a quality control line. After the reaction is finished, the ultraviolet light source (340 nm) is used for scanning and detecting the detection area, and the fluorescent nano-microspheres on the detection line and the quality control line emit high-intensity fluorescence (615 nm) with longer decay time. By delaying the measurement time, after the naturally occurring short-life fluorescence (1-10 ns) in the sample matrix decays completely, the specific fluorescence of the rare earth element is measured, so that the interference of the specific background fluorescence can be completely eliminated. The concentration of the object to be detected in the sample can be analyzed through the intensity and the ratio of the fluorescence intensity of the detection line and the quality control line.

The test strip, the test card and the kit of the embodiment of the invention perform the detection operations as follows: collecting clinical plasma samples, adding sample diluent to a blood filtering film sample pad of a detection card, wherein the dosage is 130 mu L, then dripping the samples to the blood filtering film sample pad of a test strip, wherein the dosage is 15 mu L, inserting the detection card with the added samples into an immunofluorescence card slot, synchronously inserting an SD card loaded with reagent calibration curve information, reading the detection card at 15min, and converting fluorescent value signals read by an instrument into concentration information in a corresponding calibration curve.

The test strip, the test card and the kit provided by the embodiment of the invention are simple and quick to operate, are convenient for clinically and rapidly detecting the 25- (OH) D level in a sample, and have important application values.

The invention adopts the double-antibody sandwich method to detect the antigen, which belongs to the direct detection of the object to be detected, can effectively avoid the methodological defects caused by the competition method, can effectively reduce the storage and transportation costs when the test paper is stored at normal temperature, and improves the use convenience.

Example 2

The difference from example 1 is that:

in step S3, the amount of the 25- (OH) D-labeled antibody was 30. Mu.g.

(2) The preservation buffer solution in which the fluorescent microsphere marker marked by the 25- (OH) D-labeled antibody is stored is diluted by 30 times by microsphere dilution solution, and a line is uniformly sprayed on the bonding pad, wherein the dosage is 2 mu L/cm of the bonding pad.

(3) The concentration of the capture antibody coating in the medium detection line is 0.5mg/mL. The coating concentration of the rabbit anti-DNP antibody in the quality control line is 0.3mg/mL. The coating amount was 0.5. Mu.L/cm film.

Example 3

The difference from example 1 is that:

in step S3, the amount of the 25- (OH) D-labeled antibody was 135. Mu.g.

(2) The preservation buffer solution in which the fluorescent microsphere marker marked by the 25- (OH) D-labeled antibody is stored is diluted by 20 times by microsphere dilution solution, and a line is uniformly sprayed on the bonding pad, wherein the usage amount of the line is 3 mu L/cm of the bonding pad.

(3) The concentration of the capture antibody coating in the medium detection line is 1.0mg/mL. The coating concentration of the rabbit anti-DNP antibody in the quality control line is 0.7mg/mL. The coating amount was 1.0. Mu.L/cm film.

Example 4

The difference from example 1 is that:

in step S3, (3), the concentration of the capture antibody coating was 0.5mg/mL.

Detection example 1

And (3) precision testing:

Two sets of clinical plasma samples were tested 10 times each using the kit prepared in example 1 of the present invention. Test experiments 1-10 are a first set of clinical plasma samples. Test experiments 11-20 are a second set of clinical plasma samples.

The experimental results are shown in table 1, and the CV (coefficient of variation ) of the two sets of samples were within 10%. The test paper strip, the detection card and the kit have good precision.

TABLE 1 plasma sample precision test results

Detection example 2

Correlation test:

23 sets of clinical plasma samples were tested using the kit prepared in example 1 of the present invention and compared to clinical outcome values.

Experimental results are shown in table 2, correlation with clinical outcome values, R ² = 0.9727. The test paper strip, the detection card and the kit have good correlation.

Table 2 comparison of test results with clinical results for the kit

Test sequence	Clinical outcome value ng/mL	Detection result value ng/mL of kit
			1	3	2.75
2	9.34	7.63
			3	10	11.17
4	12.23	14.24
			5	16.73	15.31
6	17.2	16.78
			7	18	18.90
8	19.23	20.88
			9	19.55	18.99
10	23.83	19.82
			11	22.46	25.42
12	26.84	30.57
			13	28.99	29.88
14	29.58	31.86
			15	31.21	32.46
16	33.2	34.28
			17	34.04	32.47
18	34.40	37.49
			19	34.47	35.01
20	36.56	36.62
			21	38.58	36.69
22	44.15	43.17
			23	49.49	47.02
	Correlation R ²	0.9727

Detection example 3

And (3) precision testing:

Two sets of clinical plasma samples were tested 10 times each using the kit prepared in example 2 of the present invention. Test experiments 1-10 are a first set of clinical plasma samples. Test experiments 11-20 are a second set of clinical plasma samples.

The experimental results are shown in Table 3, and the CV (coefficient of variation ) of the two groups of samples is within 10%. The test paper strip, the detection card and the kit have good precision.

TABLE 3 plasma sample precision test results

Detection example 4

Correlation test:

23 sets of clinical plasma samples were tested using the kit prepared in example 2 of the present invention and compared to clinical outcome values.

Experimental results are shown in table 4, correlation with clinical outcome values, R ² = 0.9734. The test paper strip, the detection card and the kit have good correlation.

TABLE 4 comparison of test results with clinical results for the kit

Detection example 5

And (3) precision testing:

Two sets of clinical plasma samples were tested 10 times each using the kit prepared in example 3 of the present invention. Test experiments 1-10 are a first set of clinical plasma samples. Test experiments 11-20 are a second set of clinical plasma samples.

The experimental results are shown in Table 5, and the CV (coefficient of variation ) of the two sets of samples was within 10%. The test paper strip, the detection card and the kit have good precision.

TABLE 5 plasma sample precision test results

Detection example 6

Correlation test:

23 sets of clinical plasma samples were tested using the kit prepared in example 3 of the present invention and compared to clinical outcome values.

Experimental results are shown in table 6, correlation with clinical outcome values, R ² = 0.9735. The test paper strip, the detection card and the kit have good correlation.

Table 6 comparison of test results with clinical results for the kit

Detection example 7

And (3) precision testing:

Two sets of clinical plasma samples were tested 10 times each using the kit prepared in example 4 of the present invention. Test experiments 1-10 are a first set of clinical plasma samples. Test experiments 11-20 are a second set of clinical plasma samples.

The results of the experiment are shown in Table 7, and the CV (coefficient of variation ) of the measured values of the two groups of samples are within 10%. The test paper strip, the detection card and the kit have good precision.

TABLE 7 plasma sample precision test results

Detection example 8

Correlation test:

23 sets of clinical plasma samples were tested using the kit prepared in example 4 of the present invention and compared to clinical outcome values.

Experimental results are shown in table 8, correlation with clinical outcome values, R ² = 0.9758. The test paper strip, the detection card and the kit have good correlation.

Table 8 comparison of test results with clinical results for the kit

Test sequence	Clinical outcome value ng/mL	Detection result value ng/mL of kit
			1	3	3.16
2	9.34	8.24
			3	10	11.78
4	12.23	13.63
			5	16.73	15.72
6	17.2	17.39
			7	18	19.51
8	19.23	20.27
			9	19.55	19.40
10	23.83	20.43
			11	22.46	26.03
12	26.84	29.96
			13	28.99	30.29
14	29.58	32.47
			15	31.21	33.07
16	33.2	33.67
			17	34.04	32.88
18	34.40	38.10
			19	34.47	35.62
20	36.56	36.01
			21	38.58	37.10
22	44.15	43.78
			23	49.49	47.63
	Correlation R ²	0.9758

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, and various modifications and variations may be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The small molecular fluorescent immunochromatography test strip is characterized by comprising a bottom plate, a blood filtering membrane sample pad, a glass fiber combination pad, a nitrocellulose membrane and an absorption pad, wherein the blood filtering membrane sample pad, the glass fiber combination pad, the nitrocellulose membrane and the absorption pad are sequentially lapped and stuck on the bottom plate, fluorescent microsphere markers marked by labeled antibodies are adsorbed on the glass fiber combination pad, a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane from front to back according to the flow direction of the sample, and the detection line is coated with capture antibodies;

2. The test strip of claim 1, wherein the small molecule is vitamin D.

3. The test strip of claim 1, wherein the fluorescent microspheres in the fluorescent microsphere markers labeled with the labeled antibodies are time resolved fluorescent microspheres.

4. The test strip of claim 1, wherein the mass ratio of the amount of the fluorescent microsphere to the amount of the labeled antibody in the fluorescent microsphere label labeled by the labeled antibody is 1:0.030 to 0.135.

5. The test strip of claim 1, wherein the glass fiber binding pad is adsorbed with fluorescent microsphere markers marked by labeled antibodies by the following adsorption method: uniformly spraying a line of fluorescent microsphere marker marked by the marked antibody with the concentration of 0.3-1.25 mg/mL on the glass fiber bonding pad, wherein the dosage is 2-4 mu L/cm of the bonding pad.

6. The test strip of claim 1, wherein the detection line is coated with capture antibodies by a coating method comprising: and marking the capture antibody with the concentration of 0.5-1.5 mg/mL on the nitrocellulose membrane as the detection line for coating, wherein the coating dosage is 0.5-1.5 mu L/cm membrane.

7. The test strip of claim 1, wherein the quality control line is a rabbit anti-DNP antibody coated quality control line;

The coating method for coating the quality control line with the rabbit anti-DNP antibody comprises the following steps: and (3) marking the rabbit anti-DNP antibody with the concentration of 0.3-1.0 mg/mL on the nitrocellulose membrane as the quality control line for coating, wherein the coating dosage is 0.5-1.5 mu L/cm membrane.

8. The test strip of claim 1, wherein the method for preparing the fluorescent microsphere marker labeled by the labeled antibody comprises the following steps:

S11, taking fluorescent microspheres, adding MES solution with pH of 4.5-6.5, centrifugally washing, removing supernatant to obtain washed fluorescent microspheres, and adding MES solution with pH of 4.5-6.5 into the washed fluorescent microspheres for ultrasonic dispersion to prepare microsphere suspension I;

S13, adding a small molecular labeled antibody into the microsphere suspension II, uniformly mixing, vibrating at a temperature of 37 ℃, adding bovine serum albumin after the reaction is finished, uniformly mixing, sealing at the temperature of 37 ℃ and vibrating, centrifuging to remove the supernatant, adding a cleaning buffer solution, centrifuging and washing to remove the supernatant, and thus obtaining the fluorescent microsphere marker labeled by the labeled antibody.

9. A test card comprising the test strip of any one of claims 1-8 and a card housing.

10. A kit comprising the test strip of any one of claims 1 to 8 or the test card of claim 9.