阅读说明：本技术 一种检测降钙素原的荧光免疫层析试纸及其制备方法 (Fluorescence immunochromatographic test paper for detecting procalcitonin and preparation method thereof ) 是由 戴瞻 于 2019-11-04 设计创作，主要内容包括：一种检测降钙素原的荧光免疫层析试纸,包括PVC底板；PVC底板上从左到右依次设有样品垫、结合垫、硝酸纤维素膜及吸水垫；样品垫的一端固定在PVC底板上,样品垫的另一端搭设在结合垫上,样品垫的中心处设有样品滴入孔；结合垫的一端固定在PVC底板上,结合垫的另一端搭设在硝酸纤维素膜上；硝酸纤维素膜上从左到右依次设有检测线和质控线,吸水垫的左端搭设在硝酸纤维素膜上。本发明检测试纸通过时间分辨免疫荧光微球对抗体蛋白进行标记,并且多抗进行捕获,从而使得检测灵敏度更高。大大缩短了检测时间,效率高,具有良好的稳定性和重复性。(A fluorescence immunochromatographic test paper for detecting procalcitonin comprises a PVC base plate; a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the PVC bottom plate from left to right; one end of the sample pad is fixed on the PVC bottom plate, the other end of the sample pad is overlapped on the combination pad, and a sample dropping hole is formed in the center of the sample pad; one end of the combination pad is fixed on the PVC bottom plate, and the other end of the combination pad is erected on the nitrocellulose membrane; the nitrocellulose membrane is sequentially provided with a detection line and a quality control line from left to right, and the left end of the water absorption pad is erected on the nitrocellulose membrane. The detection test paper marks the antibody protein through the time-resolved immunofluorescence microspheres, and captures the polyclonal antibody, so that the detection sensitivity is higher. The detection time is greatly shortened, the efficiency is high, and the stability and the repeatability are good.)

1. A fluorescence immunochromatographic test paper for detecting procalcitonin comprises a PVC base plate; the method is characterized in that: a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the PVC bottom plate from left to right; the sample pad is arranged at the leftmost end of the PVC base plate, one end of the sample pad is fixed on the PVC base plate, the other end of the sample pad is overlapped on the combination pad, and a sample dropping hole is formed in the center of the sample pad; one end of the combination pad is fixed on the PVC bottom plate and is positioned below the other end of the sample pad, and the other end of the combination pad is erected on the nitrocellulose membrane; the combination pad contains a plurality of time-resolved immunofluorescent microspheres; the surface of the time-resolved immunofluorescence microsphere is modified with carboxyl groups which are covalently coupled with protein or antibody; a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane from left to right, and the detection line and the quality control line are arranged on the nitrocellulose membrane in parallel at a distance; the detection line is a scribing line of PCT multiple antibody, and the concentration of the detection line is 1.5 mg/ml; the quality control line is drawn by goat anti-mouse IgG antibody, and the concentration of the quality control line is 1 mg/ml; the water absorption pad is arranged at the rightmost end of the PVC bottom plate, and the left end of the water absorption pad is erected on the nitrocellulose membrane.

2. The fluorescence immunochromatographic test strip for detecting procalcitonin according to claim 1, which is characterized in that: each time-resolved immunofluorescence microsphere is wrapped with a fluorescent substance, the fluorescent substance is a rare earth europium ion complex, the excitation wavelength of the fluorescent substance is 365nm, the reflection wavelength of the fluorescent substance is 610nm, and the diameter of the time-resolved immunofluorescence microsphere is 200 nm.

3. The fluorescence immunochromatographic test strip for detecting procalcitonin according to claim 1, which is characterized in that: the nitrocellulose membrane is a nitrocellulose membrane, the nitrocellulose membrane is a white or opalescent membrane with a smooth surface, the water content of the nitrocellulose membrane is 25-50%, and the membrane aperture of the nitrocellulose membrane is 8 μm.

4. The fluorescence immunochromatographic test strip for detecting procalcitonin according to claim 1, which is characterized in that: the plastic shell comprises a plastic upper shell and a plastic lower shell; the fluorescence immunochromatographic test paper is arranged in a plastic shell formed by buckling a plastic upper shell and a plastic lower shell, the plastic upper shell is provided with a sample adding hole and an observation window, the sample adding hole is arranged corresponding to a sample pad on the test paper strip, and the observation window is arranged corresponding to a detection line and a quality control line on a nitrocellulose membrane.

5. The method for preparing the fluorescence immunochromatographic test strip for detecting procalcitonin according to claim 1, which is characterized in that: comprises the following steps:

(1) pretreatment of time-resolved immunofluorescent microspheres

Dispersing the time-resolved immunofluorescence microsphere with ultrasound for 5min, centrifuging at 14000rpm for 15min with 50ul, removing supernatant, washing the precipitate with 190ul10mmol/L MES buffer solution with pH 6.0; respectively preparing 20mg/ml carbodiimide and succinimide, adding 5ul of the carbodiimide and the succinimide respectively, reacting at room temperature for 30min, centrifuging at 14000rpm for 15min at a high speed, washing the precipitate with MES solution with pH of 6.0, and resuspending to 500ul to obtain time-resolved immunofluorescence microsphere solution;

(2) preparation of time-resolved immunofluorescence microsphere combined with PCT monoclonal antibody

Adding 50ug of PCT monoclonal antibody into the time-resolved immunofluorescence microsphere solution processed in the step (1), mixing uniformly, reacting for 2 hours at room temperature, blocking for 2 hours at room temperature by 4% BSA, centrifuging at 14000rpm for 15min at high speed, and centrifuging by using a solution containing 0.1% BSA, 0.2% Tween20 and 0.1% NaN ₃Washing with Tris-HCl preservation solution (50 mmol/L, pH is 8.0), resuspending to 100ul, and storing at 4 deg.C in dark place to obtain binding PCT monoclonal antibodyThe time-resolved immunofluorescence microsphere solution of (1);

(3) gold spraying and film scratching treatment

Diluting goat anti-chicken IgG and mouse anti-human PCT monoclonal antibody with 10mmol/LPBS buffer solution containing 1% sucrose to 1mg/ml, respectively, collecting 100ul of the time-resolved immunofluorescence microsphere solution combined with the PCT monoclonal antibody obtained in step (2), and centrifuging at 14000rpm for 15 min; resuspend with 250ul of a 50mmol/L, pH Tris-HCl microsphere resuspension of 7.5 Tris-HCl microsphere suspension containing 0.5BSA, 0.3% Tween20, 10% sucrose; spraying a control line and a detection line on the nitrocellulose membrane in parallel by using a gold spraying membrane scribing instrument in an amount of 1ul/cm, wherein the interval between the control line and the detection line is 4mm, and spraying a time-resolved immunofluorescence microsphere line on the nitrocellulose membrane in parallel by using the gold spraying membrane scribing instrument in an amount of 2ul/cm, wherein the interval between the time-resolved immunofluorescence microsphere line and the detection line is 4 mm; drying in a 37 ℃ oven for 10 hours in a dark place, adding a drying agent, and sealing for later use;

(4) assembly of test strips

Sequentially and mutually staggered and adhered with a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad by 2mm on a PVC (polyvinyl chloride) base plate, wherein a detection line is close to the combination pad, and a quality control line is close to the water absorption pad, so that a test paper plate is obtained, and then the test paper plate is cut into test paper strips with the thickness of 4 mm; and then assembling the test strip in a plastic shell formed by buckling a plastic upper shell and a plastic lower shell, wherein the plastic upper shell is provided with a sample adding hole and an observation window, the sample adding hole corresponds to a sample pad on the test strip, and the observation window is arranged corresponding to a detection line and a quality control line on the test strip.

6. The method for preparing the fluorescence immunochromatographic test strip for detecting procalcitonin according to claim 5, which is characterized in that: the size of the PVC bottom plate is 80 x 300 mm; the sample pad is a whole blood filter pad, the size of the sample pad is 30 x 300mm, and the sample pad is made of glass fiber cotton; the size of the nitrocellulose membrane is 25 x 300mm, and the nitrocellulose membrane is made of nitrocellulose; the absorbent pad size was 28 x 300 mm.

Technical Field

The invention belongs to the field of biotechnology application, and relates to a fluorescence immunochromatographic test paper for detecting procalcitonin and a preparation method thereof.

Background

Procalcitonin (PCT) is a calcitonin precursor with no hormonal activity, and is a glycoprotein consisting of 116 amino acids and having a molecular weight of 13 KD. PCT is secreted by cells within the nerve (including thyroid, lung and pancreatic tissue cells), and cleaved enzymatically into Calcitonin (CT)), carboxy-terminal peptides and amino-terminal peptides. The half-life period of PCT in human body is 25-30h, the stability is good, and the content of PCT in the serum of normal human is extremely low. In many cases leading to Systemic Inflammatory Response Syndrome (SIRS), such as bacterial infections, pancreatitis, burns, multiple wounds, the level of PCT in the blood is abnormally elevated without significant changes in CT. PCT levels in blood are positively correlated with the severity of infection and injury, and have become effective indicators for diagnosis and monitoring of therapeutic efficacy of sepsis, severe sepsis, septic shock.

PCT (procalcitonin) is a protein whose levels in plasma are elevated when severe bacterial, fungal, parasitic infections and sepsis and multi-organ failure. PCT does not rise upon autoimmunity, allergy and viral infection. Localized limited bacterial infection, mild infection and chronic inflammation did not result in elevation. Bacterial endotoxins play a crucial role in the induction process.

PCT reflects the activity of the systemic inflammatory response. Factors affecting PCT levels include the size and type of the organ infected, the type of bacteria, the degree of inflammation and the status of the immune response. At present, there are many methods for detecting PCT, and besides the past gel layer analysis and high performance liquid chromatography which are time-consuming and difficult to automate, the current methods for detecting PCT are more specific and sensitive: enzyme-linked immunosorbent assay, radioimmunoassay and immunofluorescence assay.

The enzyme-linked immunosorbent assay is used for detecting the PCT in serum by using a double-antibody sandwich method principle of two mouse monoclonal antibodies, and the method is specific and has no cross reaction. However, the enzyme-linked immunosorbent assay has low detection sensitivity, the lowest limit is only 10ug/L, and the PCT concentration in the serum of normal human cannot be detected.

The radioimmunoassay can detect both free PCT and bound PCT, and has high detection sensitivity, with the lowest limit of 4 pg/L. However, the radioimmunoassay takes a long time, usually several hours, and contamination with radioactive elements limits the application of this method.

The immunofluorescence method is a quantitative immunofluorescence detection method, two kinds of mouse monoclonal antibodies are combined with two different binding sites of PCT antigen, one kind of antibody is fluorescently labeled (tracer), the other kind of antibody is fixed on the wall of a test tube, the antibody reacts with PCT molecules in serum to form a sandwich compound, the fluorescently labeled antibody is bound on the wall of the test tube, the content of a fluorescent marker is counted through a luminescent reagent, the intensity of a fluorescent signal is in direct proportion to the concentration of the PCT in a sample, a standard substance is simultaneously measured, a standard curve is made according to the PCT with known antigen concentration, the concentration of the PCT in the sample is obtained quantitatively, and the sensitivity of the method is similar to that of the radioimmunoassay. However, immunofluorescence requires the provision of expensive immunofluorescence detectors.

Therefore, how to solve the above problems is an important research content for those skilled in the art.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide the fluorescence immunochromatographic test paper for detecting procalcitonin and the preparation method thereof.

In order to achieve the above objects and other related objects, the present invention provides a fluorescence immunochromatographic test strip for detecting procalcitonin, comprising a PVC base plate; a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the PVC bottom plate from left to right; the sample pad is arranged at the leftmost end of the PVC base plate, one end of the sample pad is fixed on the PVC base plate, the other end of the sample pad is overlapped on the combination pad, and a sample dropping hole is formed in the center of the sample pad; one end of the combination pad is fixed on the PVC bottom plate and is positioned below the other end of the sample pad, and the other end of the combination pad is erected on the nitrocellulose membrane; the combination pad contains a plurality of time-resolved immunofluorescent microspheres; the surface of the time-resolved immunofluorescence microsphere is modified with carboxyl groups which are covalently coupled with protein or antibody; a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane from left to right, and the detection line and the quality control line are arranged on the nitrocellulose membrane in parallel at a distance; the detection line is a scribing line of PCT multiple antibody, and the concentration of the detection line is 1.5 mg/ml; the quality control line is drawn by goat anti-mouse IgG antibody, and the concentration of the quality control line is 1 mg/ml; the water absorption pad is arranged at the rightmost end of the PVC bottom plate, and the left end of the water absorption pad is erected on the nitrocellulose membrane.

In the above scheme, the following is explained:

1. in the scheme, tens of thousands of fluorescent substances are wrapped in each time-resolved immunofluorescent microsphere, so that leakage is avoided, the marking efficiency of fluorescence is greatly improved, and the analysis sensitivity is effectively improved. The fluorescent substance is a rare earth europium ion complex, the excitation wavelength of the fluorescent substance is 365nm, the reflection wavelength of the fluorescent substance is 610nm, and the diameter of the time-resolved immunofluorescence microsphere is 200 nm. The time-resolved immunofluorescence microsphere is a microsphere with special functions, carboxyl or other functional groups with certain density are modified on the surface of the microsphere, and the microsphere is covalently coupled with protein or an antibody, so that the stability of a marker is greatly improved.

2. In the scheme, the nitrocellulose membrane is a nitrocellulose membrane, the nitrocellulose membrane is a white or opalescent membrane with a smooth surface, the water content of the nitrocellulose membrane is 25-50%, and the membrane aperture of the nitrocellulose membrane is 8 μm.

3. In the above scheme, the plastic shell is further included, and the plastic shell includes a plastic upper shell and a plastic lower shell; the fluorescence immunochromatographic test paper is arranged in a plastic shell formed by buckling a plastic upper shell and a plastic lower shell, the plastic upper shell is provided with a sample adding hole and an observation window, the sample adding hole is arranged corresponding to a sample pad on the test paper strip, and the observation window is arranged corresponding to a detection line and a quality control line on a nitrocellulose membrane.

The invention also provides a preparation method of the fluorescence immunochromatographic test paper for detecting procalcitonin, which comprises the following steps:

(1) pretreatment of time-resolved immunofluorescent microspheres

Dispersing the time-resolved immunofluorescence microsphere with ultrasound for 5min, centrifuging at 14000rpm for 15min with 50ul, removing supernatant, washing the precipitate with 190ul10mmol/L MES buffer solution with pH 6.0; respectively preparing 20mg/ml carbodiimide and succinimide, adding 5ul of the carbodiimide and the succinimide respectively, reacting at room temperature for 30min, centrifuging at 14000rpm for 15min at a high speed, washing the precipitate with MES solution with pH of 6.0, and resuspending to 500ul to obtain time-resolved immunofluorescence microsphere solution;

(2) preparation of time-resolved immunofluorescence microsphere combined with PCT monoclonal antibody

Adding 50ug of PCT monoclonal antibody into the time-resolved immunofluorescence microsphere solution treated in the step (1), mixing uniformly, reacting for 2 hours at room temperature, blocking for 2 hours at room temperature by 4% BSA, centrifuging at 14000rpm for 15min at high speed, and centrifuging by using a solution containing 0.1% BSA, 0.2% Tween20 and 0.1% NaN ₃Washing with a Tris-HCl preservation solution of which the concentration is 50mmol/L, pH is 8.0, resuspending to 100ul, and storing at 4 ℃ in a dark place to obtain a time-resolved immunofluorescence microsphere solution combined with the PCT monoclonal antibody;

(3) gold spraying and film scratching treatment

Diluting goat anti-chicken IgG and mouse anti-human PCT monoclonal antibody with 10mmol/LPBS buffer solution containing 1% sucrose to 1mg/ml, respectively, collecting 100ul of the time-resolved immunofluorescence microsphere solution combined with the PCT monoclonal antibody obtained in step (2), and centrifuging at 14000rpm for 15 min; resuspend with 250ul of a 50mmol/L, pH Tris-HCl microsphere resuspension of 7.5 Tris-HCl microsphere suspension containing 0.5BSA, 0.3% Tween20, 10% sucrose; spraying a control line and a detection line on the nitrocellulose membrane in parallel by using a gold spraying membrane scribing instrument in an amount of 1ul/cm, wherein the interval between the control line and the detection line is 4mm, and spraying a time-resolved immunofluorescence microsphere line on the nitrocellulose membrane in parallel by using the gold spraying membrane scribing instrument in an amount of 2ul/cm, wherein the interval between the time-resolved immunofluorescence microsphere line and the detection line is 4 mm; drying in a 37 ℃ oven for 10 hours in a dark place, adding a drying agent, and sealing for later use;

(4) assembly of test strips

Sequentially and mutually staggered and adhered with a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad by 2mm on a PVC (polyvinyl chloride) base plate, wherein a detection line is close to the combination pad, and a quality control line is close to the water absorption pad, so that a test paper plate is obtained, and then the test paper plate is cut into test paper strips with the thickness of 4 mm; and then assembling the test strip in a plastic shell formed by buckling a plastic upper shell and a plastic lower shell, wherein the plastic upper shell is provided with a sample adding hole and an observation window, the sample adding hole corresponds to a sample pad on the test strip, and the observation window is arranged corresponding to a detection line and a quality control line on the test strip.

Further, the size of the PVC bottom plate is 80 x 300 mm; the sample pad is a whole blood filter pad, the size of the sample pad is 30 x 300mm, and the sample pad is made of glass fiber cotton; the size of the nitrocellulose membrane is 25 x 300mm, and the nitrocellulose membrane is made of nitrocellulose; the absorbent pad size was 28 x 300 mm.

Due to the application of the technical scheme, compared with the prior art, the invention has the following beneficial effects:

the conventional test strip detects the PCT antigen and is used for capturing monoclonal antibody. The PCT polyclonal antibody is captured, so that the sensitivity is higher than that of the traditional test strip; the detection test paper marks the antibody protein through the time-resolved immunofluorescence microspheres, and captures the polyclonal antibody, so that the detection sensitivity is higher. The detection time is greatly shortened, the efficiency is high, and the stability and the repeatability are good. Simple operation and no need of special instruments and equipment. The invention has outstanding substantive characteristics and remarkable progress.

Drawings

FIG. 1 is a schematic structural diagram of the fluorescence immunochromatographic test strip of the present invention;

FIG. 2 is a schematic diagram of a standard curve for detecting PCT concentration by time-resolved fluorescence immunochromatography according to the present invention.

In the figure: 1. a PVC base plate; 2. a sample pad; 3. a sample dropping hole; 4. a bonding pad; 5. a nitrocellulose membrane; 6. detecting lines; 7. a quality control line; 8. an absorbent pad.

Detailed Description

Other advantages and capabilities of the present invention will be readily apparent to those skilled in the art from the disclosure of the present specification by describing the embodiments of the present invention with reference to the specific embodiments thereof.

As shown in figure 1, the fluorescence immunochromatographic test paper for detecting procalcitonin comprises a PVC (polyvinyl chloride) base plate 1; the PVC base plate 1 is sequentially provided with a sample pad 2, a combination pad 4, a nitrocellulose membrane 5 and a water absorption pad 8 from left to right; the sample pad 2 is arranged at the leftmost end of the PVC base plate 1, one end of the sample pad 2 is fixed on the PVC base plate 1, the other end of the sample pad 2 is arranged on the combination pad 4 in an overlapping mode, and a sample dropping hole 3 is formed in the center of the sample pad 2; one end of the combination pad 4 is fixed on the PVC base plate 1 and is positioned below the other end of the sample pad 2, and the other end of the combination pad 4 is arranged on the nitrocellulose membrane 5 in a lapping way; the binding pad 4 contains a plurality of time-resolved immunofluorescent microspheres (not shown in the figure); the surface of the time-resolved immunofluorescence microsphere is modified with carboxyl groups which are covalently coupled with protein or antibody; a detection line 6 and a quality control line 7 are sequentially arranged on the nitrocellulose membrane 5 from left to right, and the detection line 6 and the quality control line 7 are arranged on the nitrocellulose membrane 5 in parallel at a distance; the detection line 6 is a scribing line of PCT multiple antibody, and the concentration of the detection line is 1.5 mg/ml; the quality control line 7 is drawn by goat anti-mouse IgG antibody, and the concentration of the quality control line is 1 mg/ml; the water absorption pad 8 is arranged at the rightmost end of the PVC bottom plate 1, and the left end of the water absorption pad 8 is erected on the nitrocellulose membrane 5.

The kit for detecting the PCT by the time-resolved fluorescence immunochromatography method adopts the immunochromatography principle of a double-antibody sandwich method to detect the PCT content in human whole blood. The kit consists of a test strip for detecting PCT by a time-resolved fluorescence immunochromatography method and an ID card containing a PCT standard curve.

Tens of thousands of fluorescent substances are wrapped in each time-resolved immunofluorescent microsphere, so that leakage is avoided, the marking efficiency of fluorescence is greatly improved, and the analysis sensitivity is effectively improved. The fluorescent substance is a rare earth europium ion complex, the excitation wavelength of the fluorescent substance is 365nm, the reflection wavelength of the fluorescent substance is 610nm, and the diameter of the time-resolved immunofluorescence microsphere is 200 nm. The time-resolved immunofluorescence microsphere is a microsphere with special functions, carboxyl or other functional groups with certain density are modified on the surface of the microsphere, and the microsphere is covalently coupled with protein or an antibody, so that the stability of a marker is greatly improved.

The nitrocellulose membrane 5 is a nitrocellulose membrane, the nitrocellulose membrane 5 is a white or opalescent membrane with a smooth surface, the water content of the nitrocellulose membrane 5 is 25% -50%, and the membrane aperture of the nitrocellulose membrane 5 is 8 μm.

The detection line 6 is a scribing line of PCT multiple antibody, and the concentration of the detection line is 1.5 mg/ml; the quality control line 7 is drawn by goat anti-mouse IgG antibody, and the concentration of the quality control line is 1 mg/ml.

The plastic shell comprises a plastic upper shell and a plastic lower shell; the fluorescence immunochromatographic test paper is arranged in a plastic shell formed by buckling a plastic upper shell and a plastic lower shell, the plastic upper shell is provided with a sample adding hole and an observation window, the sample adding hole is arranged corresponding to a sample pad on the test paper strip, and the observation window is arranged corresponding to a detection line and a quality control line on a nitrocellulose membrane.

The invention also provides a preparation method of the fluorescence immunochromatographic test paper for detecting procalcitonin, which comprises the following steps:

(1) pretreatment of time-resolved immunofluorescent microspheres

Dispersing the time-resolved immunofluorescence microsphere with ultrasound for 5min, centrifuging at 14000rpm for 15min with 50ul, removing supernatant, washing the precipitate with 190ul10mmol/L MES buffer solution with pH 6.0; respectively preparing 20mg/ml carbodiimide and succinimide, adding 5ul of the carbodiimide and the succinimide respectively, reacting at room temperature for 30min, centrifuging at 14000rpm for 15min at a high speed, washing the precipitate with MES solution with pH of 6.0, and resuspending to 500ul to obtain time-resolved immunofluorescence microsphere solution;

(2) preparation of time-resolved immunofluorescence microsphere combined with PCT monoclonal antibody

Adding 50ug of PCT monoclonal antibody into the time-resolved immunofluorescence microsphere solution treated in the step (1), mixing uniformly, reacting for 2 hours at room temperature, blocking for 2 hours at room temperature by 4% BSA, centrifuging at 14000rpm for 15min at high speed, and centrifuging by using a solution containing 0.1% BSA, 0.2% Tween20 and 0.1% NaN ₃Washing with a Tris-HCl preservation solution of which the concentration is 50mmol/L, pH is 8.0, resuspending to 100ul, and storing at 4 ℃ in a dark place to obtain a time-resolved immunofluorescence microsphere solution combined with the PCT monoclonal antibody;

(3) gold spraying and film scratching treatment

Diluting goat anti-chicken IgG and mouse anti-human PCT monoclonal antibody with 10mmol/LPBS buffer solution containing 1% sucrose to 1mg/ml, respectively, collecting 100ul of the time-resolved immunofluorescence microsphere solution combined with the PCT monoclonal antibody obtained in step (2), and centrifuging at 14000rpm for 15 min; resuspend with 250ul of a 50mmol/L, pH Tris-HCl microsphere resuspension of 7.5 Tris-HCl microsphere suspension containing 0.5BSA, 0.3% Tween20, 10% sucrose; spraying a control line and a detection line on the nitrocellulose membrane in parallel by using a gold spraying membrane scribing instrument in an amount of 1ul/cm, wherein the interval between the control line and the detection line is 4mm, and spraying a time-resolved immunofluorescence microsphere line on the nitrocellulose membrane in parallel by using the gold spraying membrane scribing instrument in an amount of 2ul/cm, wherein the interval between the time-resolved immunofluorescence microsphere line and the detection line is 4 mm; drying in a 37 ℃ oven for 10 hours in a dark place, adding a drying agent, and sealing for later use;

(4) assembly of test strips

Sequentially and mutually staggered and adhered with a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad by 2mm on a PVC (polyvinyl chloride) base plate, wherein a detection line is close to the combination pad, and a quality control line is close to the water absorption pad, so that a test paper plate is obtained, and then the test paper plate is cut into test paper strips with the thickness of 4 mm; and then assembling the test strip in a plastic shell formed by buckling a plastic upper shell and a plastic lower shell, wherein the plastic upper shell is provided with a sample adding hole and an observation window, the sample adding hole corresponds to a sample pad on the test strip, and the observation window is arranged corresponding to a detection line and a quality control line on the test strip.

Further, the size of the PVC bottom plate is 80 x 300 mm; the sample pad is a whole blood filter pad, the size of the sample pad is 30 x 300mm, and the sample pad is made of glass fiber cotton; the size of the nitrocellulose membrane is 25 x 300mm, and the nitrocellulose membrane is made of nitrocellulose; the absorbent pad size was 28 x 300 mm.

In the embodiment, the test strip is assembled in the plastic shell to form the kit for detecting the PCT by the medium-time-resolved fluorescence immunochromatography, each reagent box is matched with an ID card containing a PCT standard curve, the standard curves of products in the same batch are the same, calibrators with different concentrations are detected by the PCT test strip, the concentration of the calibrator is taken as an X axis, the ratio of the fluorescence intensity of a detection line to the fluorescence intensity of a quality control line is taken as a Y axis, the standard curve is drawn, corresponding bar code information is written and generated and stored in the ID card, and the matched bar code is printed and pasted on the test strip shell. When the concentration is detected, the ID card is inserted into the ID card socket of the instrument, and the dry type fluorescence immunoassay analyzer reads the corresponding bar code information on the test strip shell to obtain the corresponding standard curve.