CN210665764U

CN210665764U - P1NP test paper and P1NP detection kit

Info

Publication number: CN210665764U
Application number: CN201921203954.7U
Authority: CN
Inventors: 马岚; 吴峰; 岑瑜; 毛茅
Original assignee: Shenzhen Graduate School Tsinghua University
Current assignee: Shenzhen Graduate School Tsinghua University
Priority date: 2019-07-29
Filing date: 2019-07-29
Publication date: 2020-06-02
Anticipated expiration: 2029-07-29

Abstract

The utility model discloses a P1NP test paper, P1NP detect reagent box and detection method with better sensitivity. This P1NP test paper is including the mark pad that connects gradually, envelope membrane and the pad that absorbs water, the peridium has P1NP fluorescence labeling first antibody on the mark pad, the envelope membrane is equipped with detection line and quality control line in proper order along the chromatography direction, the peridium has P1NP second antibody on the detection line, the peridium has the anti-antibody on the quality control line, anti-antibody specificity combines P1NP fluorescence labeling first antibody, detection line and quality control line alternate segregation, P1NP fluorescence labeling first antibody and P1NP second antibody combine together with the different epitopes of P1NP antigen. The utility model provides a P1NP test paper utilizes double antibody sandwich chromatography detection principle, and the specificity that obvious improvement detected shortens and detects the required time. The detection sensitivity can be obviously improved by fluorescently labeling the first antibody.

Description

P1NP test paper and P1NP detection kit

Technical Field

The utility model belongs to the technical field of the immunodetection technique and specifically relates to a P1NP test paper and P1NP detect reagent box.

Background

Osteoporosis (osteoporotis) is an imbalance between bone formation and bone resorption, resulting in a net movement of calcium from the bone to the blood, and is characterized by bone loss and destruction of bone tissue, bone fragility, and susceptibility to fracture. With the increase of the life span of the population and the increase of the aged population, the primary osteoporosis metabolic disease has become one of serious diseases affecting health. In China, 9700 ten thousand osteoporosis patients exist at present, and two thirds of women account for the osteoporosis patients. The prevalence rate of osteoporosis among five major diseases of the elderly at present becomes the social problem of wide attention in China and even the world, and osteoporosis harms the health of people like coronary heart disease and cancer, and has urgent importance for diagnosis and treatment of osteoporosis.

The diagnosis of degenerative osteoporosis needs to be determined by comprehensive analysis such as clinical manifestations, bone density detection, X-ray film and bone transformation biochemical indexes. The bone mineral density detection of the dual-energy X-ray is the gold standard for diagnosing osteoporosis, and if the bone mineral density is lower than the standard value of 2.5 standard variance values of young people, the osteoporosis can be diagnosed. However, bone density-based assays are less sensitive and often require more than half a year or even a year to dynamically change. Therefore, the detection of biochemical indicators of bone turnover, particularly markers in the process of bone turnover, has become an important means for diagnosing osteoporosis. Bone turnover markers can be generally divided into two types, namely bone resorption markers and bone formation markers, and bone formation is an important index in the bone metabolic process and is in a state of relative balance with bone resorption, so that the bone turnover markers have important diagnostic significance for researching the expression of the bone formation indexes of osteoporosis patients. The serum I type procollagen amino terminal peptide (P1NP) is a specific index for reflecting osteoblast activity and bone formation in a certain range, and can be used as an osteoporosis marker for diagnosis.

At present, a common detection method of human bone metabolism markers is a colloidal gold immunochromatography, and colloidal gold detection is faster than ELISA (enzyme-linked immunosorbent assay), but sensitivity needs to be improved, and accurate measurement cannot be carried out. Therefore, it is necessary to provide a P1NP detection product with better sensitivity.

SUMMERY OF THE UTILITY MODEL

The utility model aims to solve the technical problem how to provide a P1NP test paper and P1NP detect reagent box with better sensitivity.

According to the utility model discloses a first aspect, the utility model provides a P1NP test paper, according to the utility model discloses an embodiment, this P1NP test paper is including the mark pad that connects gradually, envelope membrane and the pad that absorbs water, the envelope is gone up the peridium and is had the first antibody of P1NP fluorescence labeling, the envelope membrane is equipped with detection line and quality control line along chromatography direction in proper order, the peridium has the P1NP second antibody on the detection line, the peridium has the anti-antibody on the quality control line, anti-antibody specificity combines the first antibody of P1NP fluorescence labeling, detection line and quality control line alternate segregation, the first antibody of P1NP fluorescence labeling and the different epitopes of P1NP second antibody and P1NP antigen combine together.

The utility model has the advantages that:

the utility model provides a P1NP test paper utilizes double antibody sandwich chromatography detection principle, the P1NP fluorescence labeling first antibody of parcel on the mark pad, the P1NP second antibody of envelope district parcel is after adding the sample that awaits measuring and carrying out the membrane chromatography, can form the double antibody sandwich complex of P1NP fluorescence labeling first antibody-P1 NP antigen-P1 NP second antibody on the envelope, unnecessary P1NP fluorescence labeling first antibody combines to form fluorescence labeling immune complex at the quality control line with anti-antibody, the concentration of the antigen that contains in the sample is surveyed based on the fluorescence intensity of the fluorescence labeling of the first antibody upper band in the detection line and the fluorescence intensity and the standard concentration curve of the fluorescence labeling of the P1NP fluorescence labeling first antibody on the quality control line. The utility model discloses a specificity that this test paper can be obvious improvement detects shortens and detects the required time. The detection sensitivity can be obviously improved by fluorescently labeling the first antibody.

According to the utility model discloses an embodiment, the fluorescence label of the first antibody of P1NP fluorescence label is fluorescence microsphere, forms the first antibody of P1NP fluorescence microsphere label, and the first antibody of P1NP passes through peptide bond covalent bonding with fluorescence microsphere to improve the stability of marker, avoided the influence of antibody steric hindrance, be favorable to improving sensitivity and specificity.

In one embodiment of the present invention, the diameter of the fluorescent microsphere is in the nanometer range, and the particle size is in the range of 10nm to 600nm, preferably 40nm to 300 nm. The fluorescent substance is loaded on the fluorescent powder and is solid particles which can excite fluorescence under the stimulation of external energy.

According to an embodiment of the present invention, a fluorescent microsphere includes a fluorescent substance and a polymer layer coating the fluorescent substance. The fluorescent material can be quantum dots or rare earth complexes, and quantum dot fluorescent microspheres or rare earth complex fluorescent microspheres are formed correspondingly. The fluorescence emitted by the two fluorescent substances under the excitation of the ultraviolet light source has long service life and is not easy to bleach, and the intensity of the fluorescent substances can be effectively applied to quantitative detection. The quantum dots are preferably CdSe/CdS and the rare earth complexes are preferably Eu (TTA)₃Phen。

According to an embodiment of the invention, the polymer layer is an activated polymer layer.

According to the embodiment of the present invention, the polymer layer is modified with active functional groups, so that the P1NP antibody can be directionally connected to the surface of the fluorescent microsphere in a covalent coupling manner. The active functional group can be carboxyl, amino, hydroxyl or sulfhydryl.

According to an embodiment of the invention, the polymer layer is a styrene-carboxy polyvinyl alcohol copolymer layer. The single styrene polymer has the advantages of difficult control of synthesis conditions, low fluorescence efficiency and nonuniform particle size, and the fluorescent microspheres prepared from the styrene-carboxyl polyvinyl alcohol copolymer have higher fluorescence efficiency and more uniform particle size.

According to an embodiment of the invention, the marking mat, the envelope membrane and the absorbent mat are fixed to the bottom plate of the same solid matrix. The bottom plate of the solid phase matrix mainly plays a bearing role, and is convenient to operate in the detection process. The type of the solid phase matrix is not particularly limited, and may be an inert material that does not react with the sample to be measured or affect the binding of antigen and antibody, such as a cardboard, a plastic plate, or the like.

According to an embodiment of the present invention, the marking pad is a glass cellulose membrane and the coating membrane is a nitrocellulose membrane (NC membrane). The glass fiber membrane is chemically inert, does not contain a binder, is made of 100% borosilicate glass fiber, and is beneficial to the specific combination of the P1NP fluorescence labeled first antibody coated on the glass fiber membrane and a target antigen in a sample to be detected. The NC membrane is added with a surfactant to improve the hydrophilic capability, has a certain buffer system, has a capillary fiber structure, can adsorb more water than the same cellulose filter paper, has high flow rate and high temperature resistance, and is beneficial to the specific binding reaction between the P1NP secondary antibody coated on the NC membrane and the P1NP fluorescence labeled primary antibody-antigen to excite fluorescence.

According to embodiments of the present invention, the P1NP primary/secondary antibodies can specifically bind to different surface determinants of the P1NP antigen. The P1NP primary/secondary antibody may be any one of P1NP polyclonal and monoclonal antibodies. The utility model discloses a this test paper is based on the research to fluorescence labeling, antigen and antibody characteristic, carries out directional covalent chemical coupling through selecting suitable fluorescence labeling and the antibody of specificity, obtains the first antibody analysis of fluorescence labeling to through optimizing the various conditions of two antibody sandwich immunoreactions, prepare the test paper who obtains above-mentioned P1 NP.

According to an embodiment of the invention, the anti-antibody is a goat anti-mouse IgG antibody. The fluorescent probe can be specifically combined with the P1NP fluorescent-labeled primary antibody, namely, the fluorescent probe is combined with the redundant P1NP primary antibody to form an immune complex, and fluorescence is excited, so that the immune complex can be detected qualitatively and/or quantitatively.

According to the utility model discloses an embodiment, the width of NTx test paper is 3mm ~ 5 mm.

According to the embodiment of the present invention, the P1NP test paper can be prepared by the following steps:

s1: preparation of P1NP fluorescent microsphere labeled antibody

After the carboxyl on the surface of the fluorescent microsphere is activated by adopting a proper carboxyl modified fluorescent microsphere, the P1NP first antibody is directionally connected to the surface of the carboxyl modified fluorescent microsphere by adopting a covalent coupling mode.

S2: coating of antigen antibody at T line and C line of test region

And (3) spraying a P1NP second antibody at the detection line of the coating film and spraying a goat anti-mouse IgG antibody at the quality control line by using a film spraying instrument.

S3: coating of P1NP fluorescent microsphere labeled antibody

And (3) spraying P1NP fluorescent microspheres to mark the first antibody at the position of the marking pad by using a spraying instrument, wherein the specific position is used as a subsequent 'sample adding end'.

S4: assembling and forming of detection test paper

A coating film serving as a test area is adhered to the middle of the plastic support back plate, a marking pad is adhered to one end, close to the test line, of the coating film, and a water absorption pad is adhered to one end, close to the quality control line, of the coating film. The paper is cut into paper strips with a certain width, such as 4mm, by a test paper cutting machine, and the paper strips are loaded into a clamping piece and packaged by an aluminum foil bag filled with a drying agent.

According to the second aspect of the present invention, the present invention further provides a P1NP detection kit, according to the present invention, the P1NP detection kit includes the above-mentioned P1NP detection test paper.

According to a third aspect of the present invention, the present invention further provides a method for detecting P1NP for non-diagnostic purposes, according to an embodiment of the present invention, the method for detecting P1NP for non-diagnostic purposes comprises the following steps:

adding a sample to be detected to the P1NP detection test paper or a label pad in the detection kit;

and (3) measuring the fluorescence intensity of the detection line and the quality control line area, and determining the concentration of P1NP in the sample to be detected.

According to the utility model discloses an embodiment, this detection method includes following step:

adding a sample to be detected to a marking pad area in any test paper/kit;

and (3) detecting the fluorescence intensity of the detection line area and the quality control line area on the envelope, obtaining specific values of the fluorescence intensity of the detection line area and the quality control line area, and then quantitatively determining the concentration of P1NP in the sample to be detected based on a fluorescence intensity ratio (the fluorescence intensity of the detection line/the fluorescence intensity of the quality control line) and a concentration standard curve formula.

The fluorescence intensity of the detection line area and the quality control line area refers to a numerical value obtained after the bound fluorescent microspheres respectively retained at the detection line and the quality control line are measured by a fluorescence detector. And (3) drawing a standard concentration curve by measuring a large number of samples with different standard concentrations under the condition of double-antibody sandwich immunoreaction, obtaining a standard concentration curve equation, and calculating the concentration of the detected sample according to the standard concentration curve equation. And the quality control line measurement result is used as the quality control internal standard of the measurement method.

Utilize the utility model discloses a this detection method with the utility model discloses an above-mentioned test paper can realize the quick and quantitative determination to P1NP, has that sensitivity is high, the specificity is strong, quick, simple and convenient, can realize the advantage of objectification survey.

Drawings

Fig. 1 is a schematic structural diagram of a P1NP test strip according to an embodiment of the present invention.

Fig. 2 is a graph showing a detection value and a concentration standard curve of the P1NP test strip according to an embodiment of the present invention.

Detailed Description

The conception, specific structure, and technical effects of the present invention will be described clearly and completely with reference to the following embodiments, so that the objects, features, and effects of the present invention can be fully understood. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and other embodiments obtained by those skilled in the art without inventive labor based on the embodiments of the present invention all belong to the protection scope of the present invention. In addition, the term "coated" in the present invention is an immunological term, including the meaning of immobilization and/or adsorption. Materials, reagents and the like used in the following examples are conventionally available from commercial sources unless otherwise specified.

Example 1

Fig. 1 is a schematic structural diagram of a P1NP test strip according to an embodiment of the present invention. As shown in FIG. 1, the P1NP test strip comprises a back plate (not shown in FIG. 1), and a label pad 1, a coating film 2 and a water absorption pad 3 formed on the back plate and connected in sequence, wherein a detection line 21 and a quality control line 22 are arranged on the coating film 2 in sequence along the chromatography direction. The label pad 1 is a glass cellulose membrane coated with a P1NP fluorescently labeled primary antibody. The coating film 2 is a nitrocellulose film, the detection line 21 is coated with a P1NP second antibody, and the quality control line 22 is coated with a goat anti-mouse IgG antibody.

The preparation method of the P1NP test paper specifically comprises the following steps:

preparation of P1NP fluorescent microsphere labeled antibody

After styrene is washed by 10 percent sodium hydroxide solution to remove a protective agent, 0.1 percent CdSe/CdS quantum dots (the preparation method refers to Hanifi et al, Science 363, 1199-containing 1202(2019)) are added and ultrasonically mixed to obtain a solution a. Dissolve 1% carboxylated polyvinyl alcohol and 0.05% sodium bicarbonate in water to give solution b. Adding the solution a into the solution b, performing ultrasonic treatment for 15min, introducing nitrogen for 30min, stirring to remove oxygen, and heating to 80 ℃. Adding 0.1% potassium persulfate to react for 24h to obtain the polymer fluorescent microsphere, and filtering, centrifuging and washing with deionized water to obtain the purified carboxyl modified fluorescent microsphere.

10mg of the carboxyl modified fluorescent microspheres are washed by MES buffer solution (0.1M, pH4.7) and centrifuged, then resuspended by 1mL of MES buffer solution (0.1M, pH4.7), 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) is added to a final concentration of 5mM, NHS (N-hydroxysuccinimide) is added to a final concentration of 10mM, and the mixture is protected from light at room temperature and reacted for half an hour to obtain activated carboxyl modified fluorescent microspheres.

Washing the activated carboxyl modified fluorescent microspheres with 50mM borax buffer solution with pH8.5, and respectively mixing 0.2mg of the P1NP first antibody to be marked and the activated carboxyl modified fluorescent microspheres into 50mM borax buffer solution with pH8.5 to be fully mixed. Reacting for 2h at room temperature in a dark place, and enabling the antibody and the fluorescent microsphere to form stable peptide bond covalent bonding to obtain the conjugate of the fluorescent microsphere and the first antibody. After the reaction is finished, adding BSA solution with the final concentration of 1% (mass percentage content) to seal the residual active carboxyl sites on the conjugate of the fluorescent microsphere and the antibody/antigen, and reacting at room temperature in a dark place for 0.5 h. After completion, the mixture was washed with 0.02MPBS buffer (pH 7.4) and resuspended to obtain P1NP fluorescent microspheres labeled with the primary antibody, and the mixture was stored at 4 ℃ until use.

Preparation of P1NP test paper

The coating film is prepared by using a P1NP secondary antibody and a goat anti-mouse IgG antibody, and the specific method is as follows:

preparing a goat anti-mouse IgG antibody into a solution with the concentration of 0.8mg/mL by using 0.02M PBS buffer solution with the pH value of 7.4, preparing a solution with the concentration of 2mg/mL by using a P1NP secondary antibody, spraying the goat anti-mouse IgG antibody to a quality control line (C line) position of a coating film by using an XYZ3050 film spraying system of Biodot, spraying the P1NP secondary antibody to a detection line (T line) position, dehumidifying for 4 hours in a drying workshop with the relative humidity of less than 10%, and drying for later use to obtain the coating film with the detection line and the quality control line.

Soaking the glass cellulose membrane for half an hour by using a membrane treatment buffer solution, wherein the soaking temperature is 37 ℃, after dehumidifying for 4 hours under the same dehumidifying condition, diluting the mixed solution with the P1NP fluorescent microsphere marked with the primary antibody obtained in the step (I) to obtain the content of 1 mu g/mL by using the membrane treatment buffer solution, spraying the mixed solution onto the treated glass cellulose membrane by using an XYZ3050 membrane spraying system of BioDot to prepare a marking pad, and drying under the same dehumidifying condition. After dried envelope film, marking pad, water absorption pad and back plate with detection line and quality control line are assembled in a 10 ten thousand clean and dry workshop according to the figure 1, the attached paper board is cut into 4 mm/strip width by adopting a CM4000 cutting system of Biodot, and is loaded into a clamping piece for detection for standby.

Wherein:

the primary antibody to be labeled P1NP is the anti-P1 NP anti-PN-002 monoclonal antibody purchased from Shenzhen Shenkang science and technology Limited.

The P1NP second antibody is the anti-P1 NP monoclonal antibody numbered PN-001 purchased from Shenzhen Shenkang science and technology Limited.

The goat anti-mouse IgG antibody is purchased from the center of the Luoyang Baiotong experimental material and is numbered as C020201.

Glass fiber membranes were purchased from Millipore under catalog number GF-DX 20300.

Absorbent paper used to make absorbent pads is available from Millipore under catalog number CF-SP 22300.

Nitrocellulose membrane was purchased from Millipore under catalog number Hi-Flow Plus HF 135.

The PBS buffer was prepared as follows: 2.3g of Na2HPO are weighed₄、0.524gNaH₂PO₄.H₂O and 8.77g NaCl were dissolved in purified water, and the volume was adjusted to 1L with purified water, and the pH was adjusted to 7.4 to obtain 0.02M PBS buffer solution having a pH of 7.4.

The membrane treatment buffer was prepared as follows: dissolving Tween-20, BSA and sucrose in the 0.02MPBS buffer solution with the pH of 7.4 to ensure that the mass percent of the Tween-20 is 0.2%, the mass percent of the BSA is 1% and the mass percent of the sucrose is 2%, and adjusting the pH to 7.4 to obtain the membrane processing buffer solution.

The borax buffer solution is prepared according to the following method: weighing 1.9g of Na₂B4O₇.10H₂O was dissolved in 100mL of purified water and the pH was adjusted to 8.5 to obtain 50mM borax buffer solution having a pH of 8.5.

Example 2

Test paper performance detection

(I) sensitivity detection

The sensitivity of the test strip P1NP of example 1 was determined using P1NP antigen as the test sample.

P1NP antigen was prepared in a series of concentrations (1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 1.95, 0.98, 0.49, 0.24ng/mL) using 0.02M PBS buffer at pH7.4 containing 1% BSA, added to the loading end of the P1NP test strip obtained in example 1, and detected using a fluorescence detector. A detection step: before detection, the sample to be detected is recovered to room temperature (25 ℃), 60 mu L of the sample to be detected is taken by a precise pipettor and vertically and slowly dripped into the sample adding end, and after 10min, the sample to be detected is detected by a fluorescence detector.

The results are shown in Table 1 below. From the detection results, it can be found that the sensitivity of the test paper for detecting P1NP is 0.49ng/mL. Calibration curve is made according to the ratio of the detected values and the concentration, and the correlation coefficient R²The detection range is 0.9997, and the detection range is 0.049-1000 ng/mL.

Fig. 2 is a graph showing a detection value and a concentration standard curve of the P1NP test strip according to an embodiment of the present invention. As shown in FIG. 2, the horizontal axis represents the concentration of P1NP, and the vertical axis represents the ratio of fluorescence intensity (T/C).

TABLE 1 test paper detection values of P1NP for different sample concentrations

Concentration (ng/mL)	0	0.24	0.49	0.98	1.95	3.9	7.8
								T/C detection value	0.0033	0.0041	0.0053	0.0095	0.0240	0.0469	0.0911
Concentration (ng/mL)	15.6	31.3	62.5	125	250	500	1000
								T/C detection value	0.1818	0.3472	0.6513	1.0469	1.8773	3.0573	5.4938

(II) precision detection

Choose the P1NP antigen sample of 3 different concentrations respectively, according to the utility model method repeated measurement 10 times, calculate the interior average deviation CV% value of wholesale according to the result of 10 times. According to 3 batch P1NP test paper that method prepared, select the sample of 3 different concentrations respectively, repeated measurement 10 times respectively, calculate the average deviation CV% value between criticizing according to the result. The results are shown in Table 2 below.

TABLE 2P 1NP test paper batch-to-batch Difference determination

Example 3

A P1NP test paper, which is different from the test paper in example 1 in that the P1NP fluorescence labeling primary antibody is Eu (TTA)₃Phen fluorescent microsphere labeled P1NP antibody.

Example 4

A P1NP test strip, which is different from the test strip of example 1 in that a speed-adjusting pad is arranged between the label pad and the envelope, and the speed-adjusting pad can be made of non-woven fabric, glass fiber or polyester film. The speed regulation pad is arranged to enable the P1NP fluorescence labeled first antibody to be temporarily gathered at the speed regulation pad and then continuously flow to the envelope membrane, so that the P1NP fluorescence labeled first antibody is contacted with the detection line and the quality control line at more uniform concentration and speed.

Example 5

A P1NP detection kit, comprising the detection test paper of P1NP in any one of

embodiments

1, 3 and 4.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the foregoing and various other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention.

Claims

1. The P1NP test paper is characterized by comprising a marking pad, a coating film and a water absorption pad which are sequentially connected, wherein a P1NP fluorescence-labeled first antibody is coated on the marking pad, the coating film is sequentially provided with a detection line and a quality control line along a chromatography direction, a P1NP second antibody is coated on the detection line, an anti-antibody is coated on the quality control line, the anti-antibody is specifically combined with the P1NP fluorescence-labeled first antibody, the detection line and the quality control line are separated from each other, and the P1NP fluorescence-labeled first antibody and the P1NP second antibody are combined with different epitopes of a P1NP antigen.

2. The P1NP test strip of claim 1, wherein the P1NP fluorescently labeled primary antibody is a P1NP fluorescently labeled primary antibody.

3. The P1NP test paper of claim 2, wherein the fluorescent microsphere labeled with the P1NP fluorescent microsphere is 10nm to 600nm in particle size.

4. The P1NP test paper of claim 2, wherein the fluorescent microsphere labeled with the first antibody by the P1NP fluorescent microsphere is any one of a quantum dot fluorescent microsphere and a rare earth complex fluorescent microsphere.

5. The P1NP test paper of claim 4, wherein the quantum dot fluorescent microspheres are CdSe/CdS quantum dot fluorescent microspheres.

6. The P1NP test strip according to any one of claims 1 to 5, wherein the width of the P1NP test strip is 3mm to 5 mm.

7. The P1NP test strip according to any one of claims 1 to 5, wherein the labeling pad is a glass cellulose membrane.

8. The test strip of P1NP according to any one of claims 1 to 5, wherein the coating film is a nitrocellulose film.

9. A P1NP test kit, comprising the P1NP test strip of any one of claims 1 to 8.