WO2021128065A1

WO2021128065A1 - Human fgf-23 fluorescence immunochromatographic test paper and human fgf-23 fluorescence immunochromatographic test kit

Info

Publication number: WO2021128065A1
Application number: PCT/CN2019/128332
Authority: WO
Inventors: 郭志程
Original assignee: 广州菲康生物技术有限公司
Priority date: 2019-12-25
Filing date: 2019-12-25
Publication date: 2021-07-01

Abstract

Human FGF-23 fluorescence immunochromatographic test paper and a human FGF-23 fluorescence immunochromatographic test kit. The test paper comprises: a substrate (H), and a sample pad (A), a fluorescent microsphere pad (B), a blood filtering pad (C), a reaction film (D), and a water absorbing pad (G) which are provided on the substrate (H) in sequence in a contact manner along a chromatography direction of a sample to be tested. The reaction film (D) is separately provided with a test region (E) and a quality control region (F). The fluorescent microsphere pad (B) is coated with a first antibody (1) of human FGF-23 which is labeled with a fluorescent microsphere and specifically binds to the human FGF-23 of the sample to be tested and a third antibody (6) which is labeled with a fluorescent microsphere and used for quality control. The test region (E) is coated with a second antibody (7) of human FGF-23 which specifically binds to the human FGF-23 of the sample to be tested. The quality control region (F) is coated with a fourth antibody (8) for quality control. The fluorescence immunochromatographic test paper can solve the technical defects of low specificity and poor sensitivity of the existing human FGF-23 protein test kits.

Description

Human FGF-23 fluorescence immunochromatographic test paper and human FGF-23 fluorescence immunochromatographic detection kit

Technical field

The invention relates to the field of biological detection, in particular to a fluorescent immunochromatographic test paper for human FGF-23 and a fluorescent immunochromatographic detection kit for human FGF-23.

Background technique

Human FGF-23 (fibroblast growth factor-23) is synthesized by bone cells and osteoblasts, and can act on a variety of tissues and organs, such as kidney, intestine and bone, and can regulate renal phosphoric acid Salt is reabsorbed and participates in the body's phosphate metabolism. It is a new member of the recently discovered related protein family. It is a functional protein molecule with 248 amino acids and a molecular weight of 32KDa. It is one of the 179th arginine and 180th serine of the protein molecule. It is hydrolyzed by protease into N-terminal and C-terminal fragments. It is mainly secreted by secretory cells to regulate the metabolism of phosphate and 1,25(OH)2D. The N-terminus (aa 1-24) of FGF-23 is hydrophobic and may be used as a signal peptide for entering the blood circulation; its C-terminus (aa 180-248) is limited to other members of the FGF protein family Homology. FGF-23 is the closest to FGF-21 (~24% is a homologous sequence) and FGF-19 (~22% is a homologous sequence).

Loss of renal phosphate can lead to hypophosphatemia, which is one of the reasons for bone mineralization and defects in growth plate development. Patients with autosomal dominant hypophosphatemia rickets (ADHR, a rare genetic disease) carry a FGF-23 mutant that protects the protein from hydrolysis. Moreover, tumor patients with oncogenic osteomalacia (OOM) have been confirmed to have excessive FGF-23mRNA expression, which seems to indicate that the increase in the concentration of FGF-23 in the blood is the cause of the loss of phosphate in these patients. After applying recombinant FGF-23 to rodents, it was found that the excretion of phosphorus in urine increased, which led to hyposquamia and osteomalacia/rickets, which also confirmed this conclusion. In conclusion, the existing literature shows that FGF-23 is directly or indirectly related to the regulation of phosphate homeostasis.

Experiments have shown that FGF-23 is an effective inhibitor of 1,25-dihydroxyvitamin D, and 1,25-dihydroxyvitamin D is the most significant inducer of FGF-23. Studies have found that serum FGF-23 levels are associated with increased cardiovascular risk, hypophosphatemia and hyperparathyroidism. In addition, it has been reported that overexpression of FGF-23 in patients with chronic kidney disease (CKD) is related to left ventricular hypertrophy. Therefore, FGF-23 can be used as a biomarker for early treatment and intervention of CKD.

However, the current kits for detecting human FGF-23 protein are basically enzyme-linked immunosorbent kits (ELISA), and enzyme-linked immunosorbent kits can only detect serum or plasma, not whole blood. Moreover, the operation is cumbersome, there are many pre-processing steps for the sample, and the detection requires a long time, which cannot meet the requirements of rapid detection at the bedside. At the same time, the capture antibody and detection antibody used in the existing human FGF-23 protein enzyme-linked immunosorbent kit have low specificity and poor sensitivity, and cannot quickly and accurately detect the content of human FGF-23 protein.

Summary of the invention

The present invention provides a fluorescent immunochromatographic test paper for human FGF-23 and a fluorescent immunochromatographic detection kit for human FGF-23, which can effectively solve the low specificity and poor sensitivity of the existing human FGF-23 protein detection kit , Can not quickly and accurately detect the technical defects of human FGF-23 protein.

The technical scheme of human FGF-23 fluorescence immunochromatographic test paper disclosed in the present invention includes:

A substrate; a sample pad, a fluorescent microsphere pad, a blood filter pad, a reaction membrane, and a water-absorbing pad arranged in contact on the substrate along the chromatographic direction of the sample to be tested;

A detection zone and a quality control zone are respectively provided on the reaction membrane along the sample chromatographic direction; the sample pad is used for absorbing the sample to be tested;

The fluorescent microsphere pad is coated with a first antibody of human FGF-23 labeled with fluorescent microspheres, and the first antibody of human FGF-23 specifically binds to the human FGF-23 of the sample to be tested, and The fluorescent microsphere pad is also coated with a third antibody labeled with fluorescent microspheres for quality control;

The detection area is coated with a second antibody of human FGF-23, the second antibody of human FGF-23 specifically binds to the human FGF-23 of the sample to be tested, and the quality control area is coated with a second antibody for quality The fourth antibody for quality control, the fourth antibody for quality control specifically binds to the third antibody for quality control;

Wherein, the method for preparing the first antibody of human FGF-23 is: immunizing mice with a prokaryotic expression recombinant protein with an amino acid sequence of SEQ. No. 2 as an antigen, and obtaining the first antibody of human FGF-23 by using monoclonal technology;

The preparation method of the second antibody of the human FGF-23 is as follows: the amino acid sequence of SEQ. No. 3 and SEQ. No. 4 are respectively coupled to the carrier protein to obtain the carrier protein-based SEQ. No. 3 polypeptide antigen and No. 4 polypeptide antigen coupled with a carrier protein; then, the carrier protein-coupled SEQ. No. 3 polypeptide antigen and the carrier protein-coupled SEQ. No. 4 polypeptide antigen are used as antigens to immunize mice, Monoclonal technology was used to obtain the secondary antibody of human FGF-23.

Among them, the second antibody of human FGF-23 includes an antibody against the SEQ. No. 3 polypeptide antigen coupled with a carrier protein and an antibody against the SEQ. No. 4 polypeptide antigen coupled with a carrier protein.

Preferably,

The diameter of the fluorescent microspheres is 160-400 nm.

Preferably,

Stable in the ground state, the fluorescent microspheres emit fluorescence with a wavelength range of 550-650 nm under the action of an excitation light source of 300-400 nm.

Preferably,

The fluorescent substance of the fluorescent microspheres is fluorescein isothiocyanate, tetraethyl rhodamine, tetramethyl rhodamine isothiocyanate or X-rhodamine.

Preferably,

The carrier protein is selected from one or more of KLH carrier protein, BSA carrier protein and OVA carrier protein.

Preferably,

The third antibody used for quality control labeled with fluorescent microspheres is a chicken IgY antibody labeled with microspheres; the fourth antibody used for quality control is a rabbit anti-chicken IgY antibody.

Preferably,

The substrate is a PVC board.

Preferably,

The reaction membrane is a nitrocellulose membrane.

Preferably,

The material of the blood filter pad is filter paper or absorbent cotton.

The application also provides a fluorescence immunochromatographic detection kit for human FGF-23, including the above-mentioned fluorescence immunochromatographic test paper for human FGF-23.

The purpose of the present invention: using fluorescence immunochromatography (FIA), it can directly detect whole blood, serum, and plasma samples, which can be detected by simple processing, and the results can be obtained within 15 minutes. The detection instrument is small and portable, and the bedside detection is realized. Demand. The reagent can be stored at 2～30℃. At the same time, the sensitivity and linear range are equivalent to similar products, and the precision and accuracy are within the range of industry requirements.

The beneficial effects of using the above technical solution are:

The human FGF-23 (fibroblast growth factor 23, FGF23) fluorescence immunochromatographic test paper disclosed in the present application includes a substrate, and sample pads and fluorescent fluorescent lamps are arranged in contact with each other along the sample chromatography direction on the substrate. Microsphere pad, blood filter pad, reaction membrane and absorbent pad. The sample chromatographic direction on the reaction membrane is provided with a detection area and a quality control area; the sample pad is used to absorb the detection sample and diluent, and the fluorescent microsphere pad is coated with fluorescence The primary antibody of FGF-23 labeled with microspheres, the fluorescent microsphere pad is coated with the third antibody labeled with fluorescent microspheres for quality control, and the detection area is coated with the secondary antibody of FGF-23 (FGF-23 The second antibody specifically binds to the antigen in the test sample), and the quality control area is coated with a fourth antibody for quality control (the fourth antibody for quality control specifically binds to the third antibody for quality control) ), this application uses the double-antibody sandwich method to detect human FGF-23 protein. When in use, drop the test sample on the sample pad, and the test sample moves toward the fluorescent microsphere pad, blood filter pad, reaction membrane, and absorbent pad along the chromatographic direction of the sample. Moving in the direction, the first antibody of human FGF-23 labeled with fluorescent microspheres on the fluorescent microsphere pad can specifically bind to the antigen (FGF-23) in the test sample to form the first conjugate. Then, the first conjugate and the fluorescent microsphere The ball-labeled third antibody for quality control moves together to the detection area. The second antibody of FGF-23 in the detection area specifically binds to the antigen in the first conjugate to form a second conjugate, and the second conjugate stays in the On the detection area, the third antibody for quality control labeled with fluorescent microspheres continues to move to the quality control area, and the third antibody for quality control labeled with fluorescent microspheres specifically binds to the fourth antibody for quality control. The third conjugate, the third conjugate stays on the quality control area, and the rest of the test sample continues to move to the absorbent pad and is absorbed by the absorbent pad.

The immunofluorescence chromatography method of the present application makes the performance of the first antibody of human FGF-23 and the second antibody of FGF-23 more stable, and has the following advantages:

1. This application can directly detect whole blood, serum, and plasma samples with a small amount of sample. Only a blood collector, a straw, and a small amount of blood samples are needed to complete the sample collection, and the detection can be performed after simple processing;

2. The testing equipment used in this application is small, simple and portable, and meets the needs of point-of-care testing (POCT);

3. The storage conditions of this application are 2-30°C, and low temperature storage is not required.

Description of the drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only It is an embodiment of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on the provided drawings without creative work.

FIG. 1 is a diagram of the assembly structure and reaction principle of a fluorescent immunochromatographic test paper of human FGF-23 provided by an embodiment of the present invention;

Figure 2 is a crude extract of human FGF-23 recombinant protein prepared in an embodiment of the application and an SDS-PAGE identification gel image after preliminary purification;

Fig. 3 is an SDS-PAGE gel image of the recombinant human FGF-23 protein prepared in an embodiment of the application after repurification;

Fig. 4 shows the correlation analysis between the fluorescence immunochromatography test paper using human FGF-23 and the existing enzyme-linked immunosorbent technique in the embodiment of the application.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

As shown in Fig. 1, Fig. 1 is a structural diagram of a fluorescence immunochromatographic test paper for human FGF-23 provided by an embodiment of the present application. The fluorescence immunochromatographic test paper for human FGF-23 in the embodiment of the present application includes a substrate H. The sample pad A, the fluorescent microsphere pad B, the blood filter pad C, the reaction membrane D and the absorbent pad G are arranged in contact along the sample chromatographic direction in sequence on the H. The reaction membrane is provided with a detection area E along the sample chromatographic direction. And quality control area F; sample pad A is used to receive test sample 4 and diluent 5, fluorescent microsphere pad B is adsorbed with fluorescent microsphere labeled FGF-23 primary antibody 1, and fluorescent microsphere pad B is also adsorbed The third antibody 6 labeled with fluorescent microspheres for quality control, the second antibody 7 of FGF-23 is adsorbed on the detection area E (the second antibody 7 of FGF-23 is used to specifically bind to antigen 2, and antigen 2 is FGF-23 in the sample to be tested), the fourth antibody 8 for quality control is adsorbed on the quality control area F (the fourth antibody 8 for quality control specifically binds to the third antibody 6 for quality control), and the antigen It is 2 and the antibody is 3. Among them, the fluorescent microspheres used in this embodiment have a diameter of 160-400 nm, which are stable in the ground state, and emit fluorescence with a wavelength range of 550-650 nm under the action of an excitation light source of 300-400 nm.

Specifically, the blood filter pad C is filter paper or absorbent cotton, the substrate H is a PVC board, and the reaction membrane D is a nitrocellulose membrane (NC membrane).

The preparation method of the fluorescence immunochromatographic test paper of human FGF-23 in Figure 1 is:

1) There is a test paper card. The test paper card is sequentially set up from bottom to top: PVC board, sample pad A, fluorescent microsphere pad B, blood filter pad C, nitrocellulose membrane (NC membrane) and absorbent pad G.

2) Fluorescent microsphere pad B is prepared by the following steps: Pretreatment solution containing 0.1～0.4mg/ml mouse anti-RBC antibody (containing 0.6% Tris, 0.5% sodium caseinate, 3% trehalose, pH 7.4) Pre-treat the microsphere pad, then put it in an oven at 50°C to dry for 5 hours, for later use.

3) The microsphere pad is made by the following steps:

Activation of fluorescent microspheres: Take appropriate amount of fluorescent microspheres, wash once with activation buffer by centrifugation, remove the supernatant, and resuspend with the above buffer; the volume ratio of microspheres: EDC (20mg/mL) = 50:1 ratio Add EDC, mix immediately, and rotate for 30 minutes; after the reaction, centrifuge and wash twice, remove the supernatant, resuspend in coupling buffer, and sonicate;

Preparation of FGF-23 primary antibody 1 labeled with fluorescent microspheres: Divide the activated microspheres into the required volume, and then press FGF-23 primary antibody: microsphere mass ratio = 0.02:1 ～0.12:1 Add the dialyzed protein and mix it upside down immediately, then place it on a rotary mixer and rotate for 2h; after the reaction, add 5% BSA, immediately mix upside down, and react on the rotary mixer for 1h ; After the reaction is completed, centrifuge and wash twice, remove the supernatant, resuspend in the preservation solution, and sonicate; add the preservation solution to make the final concentration of the first antibody 1 of FGF-23 labeled with fluorescent microspheres 1 mg/mL, and place it in 2 Store at ~8℃ in the dark, and the shelf life is 6 months.

Dilute the prepared fluorescent microsphere-labeled FGF-23 primary antibody 1 and chicken IgY microsphere-labeled antibody to the target concentration with microsphere labeling diluent to obtain fluorescent microsphere-labeled FGF-23 primary antibody 1 and The chicken IgY labeled with fluorescent microspheres was sprayed onto the pretreated fluorescent microsphere pad B under the condition that the humidity was lower than 35%, and the spray volume was set to 4 μL/cm, and dried at 50° C. for 5 hours before use.

4) The nitrocellulose membrane (NC membrane) coated with the detection area E (T line) and the quality control area F (C line) is prepared by the following steps: the second antibody 7 of FGF-23 is used, and the coating protein is used. The diluent dilutes the FGF-23 secondary antibody 7 (T-line antibody) to 0.5-2mg/ml, and the spray volume is set to 1μL/cm under the condition of 50%-65% humidity, and the diluted FGF-23 Mark the second antibody 7 with a T line on the detection area E, and dry at 50°C for 2 hours after the marking; use the coating protein diluent to use the fourth antibody 8 for quality control (this application uses rabbit anti-chicken IgY antibody, namely Line C antibody) is diluted to 0.05～1mg/ml, the spray volume is set to 1μL/cm under the condition of humidity of 50%～65%, and the diluted fourth antibody for quality control 8 is placed on the quality control area F Draw C line, dry at 50°C for 2 hours after scribing, and put it in a sealed bag under the condition that the wet bottom is less than 35%.

5) Sample pad A is prepared by the following steps: use a sample pad pretreatment solution containing 0.01～0.4mg/ml mouse anti-RBC antibody and 0.1-1.5mg/ml blocking agent (containing 0.1M Tris, 1% S17, 1 %Tween 20, pH 7.4) pre-treated the sample pad, and then placed it in an oven to dry.

6) The steps of using the fluorescent immunochromatographic test paper of human FGF-23 in the examples of this application:

Equilibrate the test reagents and samples to room temperature, remove the test paper card, and lay it flat;

Adding whole blood sample: Take 50μL of whole blood sample and add sample diluent;

Serum/plasma sample addition: Take 40μL of serum/plasma sample and add it to the sample diluent, shake and mix, and then drop 100μL vertically to the sample pad A of the human FGF-23 fluorescence immunochromatography test paper; be careful not to inhale air bubbles when sampling ；

Card reading: Turn on the instrument, select the project file corresponding to the kit batch number; select the sample type, "whole blood" or "serum/plasma"; read the results on the instrument 15 minutes after the sample is added to the test card.

Example 1

Example 1 of the present application provides the preparation steps of the first antibody 1 of human FGF-23:

Get the information of the human FGF-23 gene (AF263537) or protein peptide sequence (AAG09917.1FGF23[Homo sapiens]) through the gene database, remove the N-terminal signal peptide sequence, reconstruct the two restriction sites NdeI and XhoI, and add Purify the tag 6×His sequence, and finally obtain the complete reconstructed target gene sequence (SEQ. No. 1,744 bases), which encodes a polypeptide with a length of 248 amino acids (SEQ. No. 2,248 amino acids). Insert the reconstructed gene sequence into the pET-28a(+) vector, transfect BL21(DE3)plysS expression strain, and finally shake the flask to culture the bacteria to obtain the human FGF-23 recombinant protein. The target protein expression is identified by SDS-PAGE The purification effect is shown in Figures 2 to 3. Figure 2 is a crude extract of human FGF-23 recombinant protein prepared in an example of the application and the SDS-PAGE identification gel image after preliminary purification, and Figure 3 is an example of the application SDS-PAGE gel image of human FGF-23 recombinant protein after repurification, where A and B in Fig. 2 are the preliminarily purified protein, M: protein molecule Marker; S1-10 in Fig. 3 is the A protein in Fig. 2 The protein after repurification, the loading volume is 10 microliters, S1-5 is the protein after the repurification of protein A in Figure 2, the loading volume is 5 microliters, M: protein molecule Marker.

SEQ.No 1 is:

SEQ.No 2 is:

The human FGF-23 reconstituted protein obtained in this example was made into a 1 mg/mL solution, and the monoclonal antibody was prepared by immunizing mice with the single-cloning technique, which was labeled as the first antibody 1 of human FGF-23.

Example 2

Example 1 of the present application provides the preparation steps of the second antibody 7 of FGF-23:

Artificial synthesis of the first epitope protein, the sequence of the first epitope protein is SEQ. No 3, SEQ. No 3: N'-CTPIPRRHTRSAEDDSERDPL-C', and the second epitope protein, the second epitope protein The sequence of SEQ. No 4, SEQ. No 4: N'-CSQELPSAEDNSPMASDPL-C', the first epitope protein and the second epitope protein are respectively combined with KLH (Keyhole Limpet Hemocyanin, hemocyanin) carrier protein Coupling to obtain SEQ. No. 3 polypeptide antigen coupled with carrier protein KLH and SEQ. No. 4 polypeptide antigen coupled with carrier protein KLH; the first epitope protein and the second epitope protein are respectively combined with BSA (Bovine SerumAlbumin, bovine serum albumin) carrier protein is coupled to obtain the SEQ. No. 3 polypeptide antigen coupled based on the carrier protein BSA and SEQ. No. 4 polypeptide antigen coupled based on the carrier protein BSA, which will be based on the carrier protein KLH No 3 polypeptide antigen coupled, SEQ. No 4 polypeptide antigen coupled based on carrier protein KLH, SEQ. No 3 polypeptide antigen coupled based on carrier protein BSA, and SEQ. No 4 polypeptide antigen coupled based on carrier protein BSA The same amount was combined to prepare a 1 mg/mL solution to immunize mice to prepare monoclonal antibodies. The second antibody 7 of FGF-23 includes antibodies directed against the first epitope protein and antibodies directed against the second epitope protein, that is, against SEQ The antibody of No. 3 and the antibody against SEQ. No. 4.

Among them, SEQ. No. 3 and SEQ. No. 4 are respectively conjugated to KLH, BSA, or OVA through a sulfhydryl group (side chain of Cys) to couple. The coupling in this embodiment uses a commercial kit for coupling.

Example 3

Example 1 of the present application provides a specific preparation method of human FGF-23 fluorescence immunochromatographic test paper, and the steps are as follows:

1. Preparation of sample pad A: use a sample pad pretreatment solution containing 0.08mg/ml mouse anti-RBC antibody and 0.4mg/ml blocking agent (containing 0.1M Tris, 1% S17, 1% Tween 20, pH 7.4 ) The sample pad is pretreated and then dried in an oven to obtain sample pad A.

2. Preparation of fluorescent microsphere pad B with primary antibody 1 of FGF-23 labeled with fluorescent microspheres: use a pretreatment solution containing 0.05 mg/ml mouse anti-RBC antibody (containing 0.6% Tris, 0.5% casein) Sodium, 3% trehalose, pH 7.4) pretreated the microsphere pad, and then placed it in an oven at 50°C for drying for 5 hours, and then set aside.

Activation of fluorescent microspheres: take appropriate amount of fluorescent microspheres, wash once with activation buffer by centrifugation, remove the supernatant, and resuspend with the above buffer; the volume ratio of microspheres: EDC (20mg/mL) = 50:1 ratio At the same time, add EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), mix immediately, rotate and react for 30 minutes; after the reaction, centrifuge and wash twice, remove the supernatant, and use Resuspend in coupling buffer and sonicate;

Preparation of the first antibody 1 of FGF-23 labeled with fluorescent microspheres: Divide the activated microspheres into the required volume, and then press the mass ratio of the first antibody of FGF-23 1: fluorescent microspheres = 0.08 :1 Add the dialyzed protein, turn it upside down and mix it immediately, then place it on a rotary mixer and rotate for 2h; after the reaction, add 5% BSA, immediately turn it upside down to mix, and react on the rotary mixer for 1h; After the end, centrifuge and wash twice, remove the supernatant, resuspend in the preservation solution, and sonicate; add the preservation solution to make the final concentration of the primary antibody 1 of FGF-23 labeled with fluorescent microspheres 1 mg/mL, and place it at 2-8 Store at ℃ protected from light, the shelf life is 6 months.

Dilute the FGF-23 primary antibody 1 and chicken IgY microsphere labeled antibody to the target concentration with the microsphere labeling diluent to obtain the fluorescent microsphere labeled FGF-23 primary antibody 1 and fluorescent microsphere labeled chicken IgY. Spray on the pre-treated fluorescent microsphere pad B under the condition of humidity lower than 35%, set the spray volume to 4 μL/cm, and dry at 50° C. for 5 hours for use.

3. Preparation of nitrocellulose membrane (NC membrane) 4 coated with a detection line (T line) in the detection area E and a quality control line (C line) in the quality control area F:

Using FGF-23 secondary antibody 7, dilute the FGF-23 secondary antibody 7 (T-line antibody) with the coating protein diluent to 1mg/ml, and set the spray volume under the condition of 50%-65% humidity At 1μL/cm, mark the diluted FGF-23 secondary antibody 7 on the detection area E and dry it at 50°C for 2 hours;

Dilute the rabbit anti-chicken IgY antibody (C-line antibody) to 0.8 mg/ml with the coating protein diluent, and set the spray volume to 1 μL/cm under the condition of 50% to 65% humidity, and use the diluted solution for quality The controlled fourth antibody 8 is marked with a line C on the quality control area F, dried at 50°C for 2 hours after marking, and put into a sealed bag under the condition that the wet bottom is lower than 35%.

The assembly of the test paper card: sample pad A, fluorescent microsphere pad B, blood filter pad C, and reaction membrane D (coated with nitric acid with detection line and quality control line) arranged in contact in sequence along the sample chromatography direction on the PVC plate Cellulose membrane) and absorbent pad G. After assembling, a large plate of human FGF-23 fluorescence immunochromatographic test paper is obtained, which is cut into 4mm width as required, and the test paper is put into a plastic card to form a human FGF-23 fluorescence immunochromatographic test paper .

Example 4

Example 3 of this application provides a specific experiment using human FGF-23 fluorescent immunochromatographic test paper for detection, and the steps are as follows:

1. Use the fluorescence immunochromatographic test paper of human FGF-23 prepared in Example 3 and the applicable fluorescence immunoassay instrument.

2. Fluorescence immunoassay analyzer parameter setting: After setting the test paper card process parameters on the fluorescence immunoassay analyzer, take the assembled test paper card above and use 0, 50, 100, 200, 400, 800, 1600pg/ The standard human FGF-23 calibrator of mL is measured with a test paper card to obtain the fluorescence intensity value of each calibrator, and the result is input into the parameters of the analyzer to complete the parameter setting of the analyzer.

3. Main testing materials: clinical samples obtained from relevant hospitals, a total of 80 ELISA samples, including serum samples, plasma samples and whole blood samples, including 30 serum samples and 30 plasma samples , 20 whole blood samples, the distribution range of human FGF-23 content is between 10 and 1600 pg/mL.

4. Detection method:

Step 1: Equilibrate the detection reagents and samples to room temperature, take out the fluorescence immunochromatographic test paper of human FGF-23, and lay it flat;

Step 2: Add sample: whole blood sample: take 50μL of whole blood sample and add sample diluent, serum/plasma sample: take 40μL of serum/plasma sample into sample diluent, shake and mix well, take 100μL and drop it vertically into human FGF-23 Fluorescence immunochromatography test paper on the sample pad; be careful not to inhale air bubbles when sampling;

Step 3: Read the card: turn on the instrument, select the project file corresponding to the kit batch number; select the sample type, "whole blood" or "serum/plasma"; read the results on the instrument 15 minutes after the sample is added to the test card.

5. Analysis of test results:

After the preparation of the clinical sample testing reagents, all clinical samples are tested according to the testing method, and the testing results are analyzed. The results are shown in Figure 4. As shown in FIG. 4, FIG. 4 is a detection correlation analysis between the fluorescent immunochromatographic test paper using human FGF-23 and the existing enzyme-linked immunosorbent technology in the embodiment of the application. Take the test value of the experimental system on the Y-axis and the test value of the control system on the X-axis, draw a scatter plot, and perform correlation analysis. Clinical sample detection: 80 clinical fixed-value samples were tested. The average deviation of the samples was less than 10%, the maximum deviation was less than 20%, and R ² >0.95. The test results show that the detection kit of the human FGF-23 fluorescence immunochromatographic test paper provided in this application has good performance, is suitable for clinical testing, and meets the differentiated needs of different customers in different testing occasions.

In summary, this application provides a rapid quantitative fluorescence immunochromatography test paper for human FGF-23 prepared by fluorescence immunochromatography technology, which is suitable for serum/plasma and whole blood samples, and is suitable for clinical single-person testing. The accurate and quantitative detection of the content of human FGF-23 in the sample to be tested has clearer clinical guiding significance, and has the advantages of simple operation, fast response, high sensitivity, strong specificity, suitable for on-site detection, and economical and practical.

The above embodiments are only used to illustrate the technical solutions of the present invention, not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, a person of ordinary skill in the art should understand that: The recorded technical solutions are modified, or some of the technical features are equivalently replaced; these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

The fluorescence immunochromatographic test paper of human FGF-23 is characterized by comprising: a substrate; a sample pad, a fluorescent microsphere pad, a blood filter pad, and a sample pad, a fluorescent microsphere pad, and a blood filter pad that are sequentially arranged in contact on the substrate along the chromatographic direction of the sample to be tested. Reaction membrane and absorbent pad;

A detection zone and a quality control zone are respectively provided on the reaction membrane along the sample chromatography direction;

The fluorescent microsphere pad is coated with a first antibody of human FGF-23 labeled with fluorescent microspheres, and the first antibody of human FGF-23 specifically binds to the human FGF-23 of the sample to be tested, and The fluorescent microsphere pad is also coated with a third antibody labeled with fluorescent microspheres for quality control;

The detection area is coated with a second antibody of human FGF-23, the second antibody of human FGF-23 specifically binds to the human FGF-23 of the sample to be tested, and the quality control area is coated with a second antibody for quality The fourth antibody for quality control, the fourth antibody for quality control specifically binds to the third antibody for quality control;

Wherein, the method for preparing the first antibody of human FGF-23 is: immunizing mice with the protein of SEQ. No. 2 as an antigen, and obtaining the first antibody of human FGF-23 by using monoclonal technology;

The method for preparing the second antibody of human FGF-23 is: coupling SEQ. No. 3 and SEQ. No. 4 to the carrier protein, respectively, to obtain the carrier protein-based SEQ. No. 3 polypeptide antigen and the carrier protein-based coupling. No. 4 polypeptide antigen; then, the carrier protein-based SEQ. No. 3 polypeptide antigen and the carrier protein-based SEQ. No. 4 polypeptide antigen are used as antigens to immunize mice, using a monoclonal Technology to obtain the second antibody of human FGF-23.
The fluorescence immunochromatographic test paper according to claim 1, wherein the diameter of the fluorescent microspheres is 160-400 nm.
The fluorescence immunochromatographic test paper according to claim 1, characterized in that, stable in the ground state, the fluorescent microspheres emit fluorescence with a wavelength range of 550-650 nm under the action of an excitation light source of 300-400 nm.
The fluorescence immunochromatographic test paper according to claim 1, wherein the fluorescent substance of the fluorescent microspheres is fluorescein isothiocyanate, tetraethyl rhodamine, tetramethyl rhodamine isothiocyanate or X -Rhodamine.
The fluorescence immunochromatographic test paper according to claim 1, wherein the carrier protein is selected from one or more of KLH carrier protein, BSA carrier protein and OVA carrier protein.
The fluorescence immunochromatographic test paper according to claim 1, wherein the third antibody for quality control labeled with fluorescent microspheres is a chicken IgY antibody labeled with microspheres; and the fourth antibody for quality control is labeled with microspheres. The antibody is a rabbit anti-chicken IgY antibody.
The fluorescence immunochromatographic test paper according to claim 1, wherein the substrate is a PVC board.
The fluorescence immunochromatographic test paper according to claim 1, wherein the reaction membrane is a nitrocellulose membrane.
The fluorescence immunochromatographic test paper according to claim 1, wherein the material of the blood filter pad is filter paper or absorbent cotton.
The fluorescence immunochromatographic detection kit for human FGF-23 is characterized by comprising the fluorescence immunochromatographic test paper for human FGF-23 according to any one of claims 1-9.