Colloidal gold immunochromatographic test paper card for detecting the ratio of fPSA to tPSA, detection method, and kit

Abstract

The present invention discloses a colloidal gold immunochromatographic test paper card and a detection method and a kit for detecting the ratio of fPSA to tPSA, and relates to the field of in vitro test paper detection technology. The qualitative detection test paper card provided by the present invention utilizes the colloidal gold immunochromatographic double antibody sandwich method, and the tPSA and fPSA antigens in the sample to be tested can be specifically bound to the colloidal gold-labeled antibody and the capture antibody on the solid phase carrier, and the tPSA and fPSA content in the sample can be qualitatively detected by comparing the colorimetric of the pre-set quality control line and the colorimetric of the tPSA and fPSA detection lines. The present invention is simple to operate, does not require complex equipment and professionals, and is completely interpreted by the user independently, reducing the difficulty and cost of operation. It is fast and simple, has good stability, and the test results can be directly judged by observing the color development with the naked eye, which is suitable for qualitative or semi-quantitative screening or family self-testing by various testing institutions.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro test paper detection, and in particular to a colloidal gold immunochromatographic test paper card for detecting the ratio of fPSA to tPSA, a detection method and a kit. Background Art

[0002] Prostate Specific Antigen (PSA) is a proteolytic enzyme produced by prostate epithelial cells. PSA exists in two forms in the blood circulation. The complex PSA bound to the proteolytic enzyme inhibitor accounts for more than 85%, and the free prostate specific antigen (fPSA) accounts for about 15%. The total prostate specific antigen (tPSA) is the sum of the complex PSA and free PSA. In the diagnosis and monitoring of prostate cancer, PSA determination has irreplaceable advantages. The current routine operation is to first conduct a quantitative test on one of the indicators, tPSA. Under normal circumstances, tPSA is ≤4ng / mL; when the tPSA content is greater than 10ng / mL, it indicates a high risk of prostate cancer, and the biopsy positive rate is 50%~75%; and when the tPSA concentration value is in the gray area (4ng / mL~10ng / mL), another indicator fPSA needs to be measured, and the ratio of free prostate-specific antigen (fPSA) to total prostate-specific antigen (tPSA) (fPSA / tPSA) is calculated to distinguish between prostate cancer and benign prostatic hyperplasia. When fPSA / tPSA is less than 0.16, the probability of prostate cancer is 17.4%. Generally, the combined determination of tPSA and fPSA can increase the detection rate of prostate malignant diseases to more than 90%.

[0003] In the related technology, chemiluminescence, immunoturbidimetry, enzyme-linked immunosorbent assay (ELISA) and other detection methods are usually used to quantify tPSA and fPSA, respectively, for prostate cancer diagnosis and monitoring evaluation. Although the above methods have high sensitivity and specificity, they usually require professional equipment and technicians to operate, which is time-consuming and not convenient for rapid screening and on-site testing. For example, Chinese patent CN202311032099.9, "A kit for detecting tPSA and fPSA using flow cytometry", discloses a detection mode of "microspheres + capture antibody A + tPSA + capture antibody B + fluorescein 1" and "microspheres + capture antibody A + fPSA + capture antibody C + fluorescein 2" formed by a double antibody sandwich method; the tPSA and fPSA contents are obtained by a single detection by flow cytometry, and the fPSA / tPSA ratio is calculated. This detection method detects the fluorescence value of the sandwich structure through flow cytometry, realizes quantitative detection of tPSA and fPSA in the sample, and then completes the calculation of the fPSA / tPSA ratio through the built-in calculation system. Three indicators are obtained in one test, which greatly facilitates the detection and analysis. However, this method requires professional equipment and technicians to operate, which is time-consuming and expensive; it also requires professional personnel to interpret the gray area and test results, which is not convenient for rapid screening and on-site testing; and fluorescein is used as a signal indicator, and fluorescein may undergo fluorescence quenching under certain conditions, which will affect the signal intensity of the test and thus affect the accuracy of the experimental results.

[0004] Therefore, it is of great significance to develop a qualitative detection system that is simple to operate, low in cost, can provide test results in a short time, does not require professional personnel, and can be completely determined by the test subject independently. The system is suitable for grassroots units and on-site rapid testing, and can perform tPSA and fPSA testing at the same time. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a colloidal gold immunochromatographic test paper card and a detection method and a kit for detecting the ratio of fPSA to tPSA, aiming to solve the problem that most of the current methods for detecting prostate cancer markers are quantitative detection, which requires professional equipment, professional personnel to operate and interpret the test results, is time-consuming and costly, and is not convenient for rapid screening and on-site detection.

[0006] An embodiment of the first aspect of the present invention provides a colloidal gold immunochromatographic test paper card for detecting the ratio of fPSA to tPSA, comprising a base and an fPSA test strip and a tPSA test strip disposed on the base; The tPSA test strip comprises a first sample pad, a first immune colloidal gold pad, a control area colloidal gold pad, a first nitrocellulose membrane, and a first absorbent pad in sequence; the first immune colloidal gold pad has tPSA antibody 1 labeled with colloidal gold adsorbed therein, and the control area colloidal gold pad has independent antigen labeled with colloidal gold adsorbed therein; the first nitrocellulose membrane is provided with a detection area T1 and a control area C1 at intervals in sequence, the detection area T1 is fixed with tPSA antibody 2, and the control area C1 is fixed with an independent antibody that can specifically bind to the independent antigen; The fPSA test paper includes a second sample pad, a second immunocolloidal gold pad, a second nitrocellulose membrane, and a second absorbent pad in sequence; the second immunocolloidal gold pad is adsorbed with fPSA antibody 1 labeled with colloidal gold; the second nitrocellulose membrane is provided with detection area T2 and quality control area C in sequence at intervals, the detection area T2 is fixed with fPSA antibody 2, and the quality control area C is fixed with a secondary antibody that can bind to fPSA antibody 1.

[0007] The colloidal gold immunochromatographic test paper card according to the first embodiment of the present invention has at least the following beneficial effects: The present invention provides a qualitative test paper card for detecting total prostate-specific antigen (tPSA) and free prostate-specific antigen (fPSA) in a sample, which includes an fPSA test strip and a tPSA test strip. The qualitative test paper card provided by the present invention utilizes a colloidal gold immunochromatographic double antibody sandwich method, and the tPSA and fPSA antigens in the sample to be tested can specifically bind to the colloidal gold-labeled antibody and the capture antibody on the solid phase carrier, and the tPSA and fPSA content in the sample can be qualitatively detected by comparing the colorimetric values ​​of the pre-set quality control line and the colorimetric values ​​of the tPSA and fPSA detection lines. Specifically: first confirm whether it is in the gray area (4ng / mL~10ng / mL) through the tPSA test strip colorimetry. When tPSA is in the gray area, the fPSA / tPSA value can be directly evaluated by comparing the colorimetry of the tPSA test strip and the fPSA test strip: when the colorimetry of the fPSA test strip T2 line is darker than that of the tPSA test strip T1, it means that the fPSA / tPSA ratio is greater than 0.16, indicating a negative result and a low risk of prostate cancer; when the colorimetry of the fPSA test strip T2 line is lighter than that of the tPSA test strip T1, it means that the fPSA / tPSA ratio is less than 0.16, indicating a high risk of prostate cancer.

[0008] The present invention has the following beneficial effects: 1. It is suitable for qualitative or semi-quantitative screening by various testing institutions or self-testing at home, effectively avoiding over-testing when the sample PSA is in the gray area. A single test can provide a more accurate risk assessment of prostate cancer.

[0009] 2. Use chromatography to simplify sample addition operations: No complex instruments or professional operators are required. Simple sample addition operations and reagent design reduce the difficulty of operation. Users can directly interpret the results after one sample injection, making the test more portable and user-friendly.

[0010] 3. Use colloidal gold colorimetry to interpret the results: The test results can usually be judged directly by observing the color with the naked eye, which reduces the dependence on professional analysis equipment. There is no need for complex equipment or professional medical staff to interpret the results. The results can be completely determined by the subject independently through visual colorimetry of the test area.

[0011] 4. Fast and easy: Colloidal gold immunochromatography testing can be completed in a relatively short time (the test results can be interpreted within 10 minutes at the minimum), which is suitable for primary medical institutions and on-site rapid screening.

[0012] 4. Cost-effectiveness: Since it does not require expensive equipment and professional personnel to operate, the overall cost of colloidal gold immunochromatographic testing is relatively low.

[0013] 5. Good stability: Colloidal gold labeling has high stability, and the labeled samples can be stored for a long time at room temperature without signal attenuation.

[0014] In some embodiments of the present invention, the tPSA antibody 1 and tPSA antibody 2 are different antibodies, and both can specifically bind to the tPSA antigen. Specifically, the tPSA antibody 1 can include mouse antibodies, rabbit antibodies, etc., preferably mouse anti-tPSA antibodies; the tPSA antibody 2 can include mouse antibodies, rabbit antibodies, etc., preferably mouse anti-tPSA antibodies.

[0015] In some embodiments of the present invention, the fPSA antibody 1 and fPSA antibody 2 are different antibodies, and both can specifically bind to the fPSA antigen. Specifically, the fPSA antibody 1 can include mouse antibodies, rabbit antibodies, etc., preferably mouse anti-fPSA antibodies; the fPSA antibody 2 can include mouse antibodies, rabbit antibodies, etc., preferably mouse anti-fPSA antibodies.

[0016] In some embodiments of the present invention, the independent antigen includes IgG antigen, chicken IgY antigen, DNP antigen, etc., preferably IgG antigen, and more preferably rabbit IgG antigen.

[0017] In some embodiments of the present invention, the independent antibody that can specifically bind to the independent antigen includes IgG antibody, chicken IgY antibody, DNP antibody, etc., preferably IgG antibody, and more preferably rabbit IgG antibody.

[0018] The present invention uses an independent quality control line, because the quality control line C1 of tPSA needs to be used for colorimetry with the detection line T1, and its color development needs to be stable at a chromaticity, and setting up an independent quality control will increase stability. The quality control line can use an IgG quality control system, or a chicken IgY quality control system, a small molecule DNP quality control system, or other available colloidal gold immunochromatography independent quality control system.

[0019] In some embodiments of the present invention, the final concentration of the tPSA antibody II coated in the detection area T1 is 0.5-2.0 mg / mL, preferably about 0.8 mg / mL.

[0020] In some embodiments of the present invention, the final concentration of the independent antibody coated in the control area C1 is 0.5-1.0 mg / mL, preferably about 0.5 mg / mL.

[0021] In some embodiments of the present invention, the final concentration of the fPSA antibody II coated in the detection area T2 is 0.5-2.0 mg / mL, preferably about 1 mg / mL.

[0022] In some embodiments of the present invention, the final concentration of the secondary antibody that binds to the fPSA antibody is 0.5-2.0 mg / mL, preferably about 1 mg / mL, when coated in the quality control area C.

[0023] In the present invention, the color depth of the detection area and the quality control area is related to the fixed antibody concentration and the antibody concentration labeled with the colloidal gold pad, and may also be affected by other conditions. Based on this, the present invention provides a preferred coating concentration of the antibody, but is not limited thereto, and can be appropriately adjusted according to changes in antigens, antibodies or other conditions, all of which are considered within the scope of the present invention.

[0024] In some embodiments of the present invention, in the fPSA test strip and the tPSA test strip, the sample pad overlaps with the immunocolloidal gold pad by 1 to 3 mm; the immunocolloidal gold pad overlaps with the nitrocellulose membrane by 1 to 3 mm; the nitrocellulose membrane overlaps with the absorbent pad by 2 to 4 mm; the detection area T1 and the control area C1 are spaced 3 to 6 mm apart, and the detection area T2 and the quality control area C are spaced 3 to 6 mm apart; the first immunocolloidal gold pad and the control area colloidal gold pad are placed overlapping each other by 1 to 3 mm, the sample pad overlaps with the lower gold pad by 1 to 3 mm, and the upper gold pad overlaps with the nitrocellulose membrane by 1 to 3 mm.

[0025] In some embodiments of the present invention, the width of the test strip is 2.5-3.5 mm.

[0026] Specifically, the width of the tPSA test strip is 2.5~3.5mm. In the test strip, the first sample pad overlaps with the first immune colloidal gold pad by 1~3mm; the first immune colloidal gold pad overlaps with the first nitrocellulose membrane by 1~3mm; the first nitrocellulose membrane overlaps with the first absorbent pad by 2~4mm; the detection area T1 and the control area C1 are spaced 3~6mm apart; the first immune colloidal gold pad and the control area colloidal gold pad are placed overlapping 1~3mm, the sample pad presses the lower gold pad by 1~3mm, and the upper gold pad presses the nitrocellulose membrane by 1~3mm.

[0027] Specifically, the width of the fPSA test strip is 2.5-3.5 mm. In the test strip, the second sample pad overlaps with the second immunocolloidal gold pad by 1-3 mm; the second immunocolloidal gold pad overlaps with the second nitrocellulose membrane by 1-3 mm; the second nitrocellulose membrane overlaps with the second absorbent pad by 2-4 mm; and the detection area T2 and the quality control area C are spaced 3-6 mm apart.

[0028] In some embodiments of the present invention, the absorbent pad comprises absorbent paper, but is not limited thereto.

[0029] In some embodiments of the present invention, the sample pad includes a glass cellulose membrane, but is not limited thereto.

[0030] In some embodiments of the present invention, the colloidal gold immunochromatographic test paper card further includes a card cover, the card cover covers the fPSA test strip and the tPSA test strip, and the card cover is provided with a first sample addition port, a first observation port, a second sample addition port, and a second observation port; The first sample addition port is disposed above the first sample pad to expose part or all of the first sample pad area; the first observation port is disposed above the first nitrocellulose membrane to expose the detection area T1 and the control area C1 of the first nitrocellulose membrane; The second sample addition port is disposed above the second sample pad to expose part or all of the second sample pad area; the second observation port is disposed above the second nitrocellulose membrane to expose the detection area T2 and the quality control area C of the second nitrocellulose membrane.

[0031] Figure 1 The figure shows one embodiment of the test paper card provided by the present invention, as shown in the figure, which includes two detection modules, the two detection modules are left and right sample addition holes and windows, and the main body of the detection module is a test paper strip. One is a tPSA test paper strip, including a sample pad, an immune colloidal gold pad, a control area colloidal gold pad, a nitrocellulose membrane where the detection area T1 and the control area C1 are located, and a water absorbent pad; the other is an fPSA test paper strip, including a sample pad, an immune colloidal gold pad, a nitrocellulose membrane where the detection area T2 and the quality control area C are located, and a water absorbent pad.

[0032] One of the schematic diagrams of tPSA test strips and fPSA test strips is as follows Figure 2 As shown, the test strip includes a base plate and a sample pad, a colloidal gold pad, a nitrocellulose membrane, and a water-absorbing pad sequentially adhered to the base plate along the length direction of the base plate. The width of the test strip is 2.5~3.5mm, the sample pad and the colloidal gold pad overlap by 1~3mm, the colloidal gold pad and the nitrocellulose membrane overlap by 1~3mm, the nitrocellulose membrane and the water-absorbing pad overlap by 2~4mm, and the interval between the test line and the quality control is 3~6mm. The difference between the tPSA test strip and the fPSA test strip is that the gold pad of the tPSA test strip is stacked in two layers, one for the first immune colloidal gold pad and the other for the control area colloidal gold pad.

[0033] The present invention fixes specific antigens or antibodies on a membrane in the form of strips, and a colloidal gold labeling reagent is adsorbed on a binding pad. When the sample to be tested is added to the sample pad at one end of the test strip, it moves forward by capillary action, dissolves the colloidal gold labeling reagent on the binding pad, and reacts with each other. When it moves to the region of the fixed antibody, the conjugate of the sample to be tested and the colloidal gold labeling reagent is specifically bound to it and is intercepted, and is gathered on the detection band, and the color development result can be observed by the naked eye. This solution is quick to detect and simple to operate.

[0034] The embodiment of the second aspect of the present invention provides a method for preparing the colloidal gold immunochromatographic test paper card for detecting the ratio of fPSA to tPSA, comprising the steps of: S101, preparing a colloidal gold solution; S102, reacting the colloidal gold solution with the tPSA antibody, and then adding the tPSA gold-labeled solution to prepare a tPSA gold-labeled solution; S103, adsorbing the tPSA gold-labeled solution onto a glass cellulose membrane to prepare a first immune colloidal gold pad adsorbed with colloidal gold-labeled tPSA antibody 1; S104, reacting the colloidal gold solution with an independent antigen to prepare an independent antigen gold labeled solution; S105, adsorbing the independent antigen gold-labeled solution onto a glass cellulose membrane to prepare a control area colloidal gold pad adsorbed with the colloidal gold-labeled independent antigen; S106, using a membrane streaking instrument to fix the C1 line antibody and the T1 line antibody to separate regions on the same nitrocellulose membrane, to prepare a first nitrocellulose membrane, wherein the C1 line antibody is an independent antibody that can specifically bind to the independent antigen, and the T1 line antibody is tPSA antibody II; S107, sequentially sticking the sample pad, the first immune colloidal gold pad, the control area colloidal gold pad, the first nitrocellulose membrane, and the absorbent pad on the PVC bottom plate to obtain a tPSA test strip; S108, reacting the colloidal gold solution with the fPSA antibody, and then adding the fPSA gold labeling solution to prepare an fPSA gold labeling solution; S109, adsorbing the fPSA gold-labeled solution onto a glass cellulose membrane to prepare a second immunocolloidal gold pad adsorbed with colloidal gold-labeled fPSA antibody 1; S110, using a membrane scribing instrument to fix the C-line antibody and the T2-line antibody to separate regions on the same nitrocellulose membrane to prepare a second nitrocellulose membrane, wherein the C-line antibody is a secondary antibody that can bind to the fPSA antibody one, and the T2-line antibody is the fPSA antibody two; S111, sequentially sticking the sample pad, the second immunocolloidal gold pad, the second nitrocellulose membrane, and the absorbent pad on the PVC bottom plate to obtain an fPSA test strip; S112, after assembling the tPSA test strip and the fPSA test strip, obtaining the colloidal gold immunochromatographic test card for detecting the ratio of fPSA to tPSA.

[0035] In the present invention, the above steps are not strictly in order, and can be reasonably adjusted according to actual needs. For example, steps S102 to S107 can be after steps S108 to S111, and will not have any impact on the results of the present invention. The above preparation method is simple to operate, has low equipment requirements, and is suitable for large-scale industrial production.

[0036] In some embodiments of the present invention, the method for preparing the tPSA gold-labeled complex solution comprises: 2-10 wt.% of sucrose, 0.5-3 wt.% of Casein-Na, 0.5-2 wt.% of BSA, 0.1-0.5 wt.% of S9, 0.2-0.5 wt.% of glycine, 0.05-0.5 wt.% of Tween-20, and 0.01-0.2 wt.% of PC300 were added to the PBS buffer, and the pH was adjusted to 8.0±0.2 to obtain the tPSA gold-labeled complex solution.

[0037] Preferably, 5wt.% sucrose, 2.5wt.% Casein-Na, 1wt.% BSA, 0.2wt.% S9, 0.5wt.% glycine, 0.2wt.% Tween-20, and 0.03wt.% PC300 are added to 0.02mol PBS buffer, and the pH is adjusted to 8.0±0.2 to obtain the tPSA gold-labeled complex solution.

[0038] In some embodiments of the present invention, the method for preparing the fPSA gold-labeled solution comprises: 0.5~2wt.% sodium chloride, 5~15wt.% sucrose, 0.5~3wt.% Casein-Na, 0.5~2wt.% BSA, 0.2~2wt.% S9, 0.2~1.5wt.% Tween-20, and 0.01~0.2wt.% PC300 were added to the PBS buffer, and the pH was adjusted to 8.0±0.2 to obtain the fPSA gold-labeled complex solution.

[0039] Preferably, 1wt.% sodium chloride, 10wt.% sucrose, 2wt.% Casein-Na, 1wt.% BSA, 0.5wt.% S9, 0.5wt.% Tween-20, and 0.03wt.% PC300 are added to 0.02mol PBS buffer, and the pH is adjusted to 8.0±0.2 to obtain the fPSA gold-labeled complex solution.

[0040] In some embodiments of the present invention, the method for preparing the sample pad comprises: 0.2-1 wt.% Casein-Na, 0.05-0.5 wt.% Tween-20, 0.02-0.2 mg / mL RBC, and 0.01-0.2 wt.% PC300 were added to the PB buffer to obtain a sample pad buffer. The sample pad buffer was evenly spread on a glass cellulose membrane and dried for 12-36 h to obtain the sample pad.

[0041] Preferably, 0.5wt.% Casein-Na, 0.2wt.% Tween-20, 0.05mg / mL RBC, and 0.03wt.% PC300 are added to 0.02mol PB buffer, and 45mL / sheet (25.4*30cm) is evenly spread on a glass cellulose membrane, and dried at 37°C for 12-36h to obtain the sample pad.

[0042] The third aspect of the present invention provides a method for detecting the ratio of fPSA to tPSA based on the colloidal gold immunochromatographic test paper card, comprising the steps of: S1. Add the sample to be tested to the sample pads of the fPSA test strip and the tPSA test strip respectively and simultaneously. The sample to be tested migrates to different areas of the test strip and reacts with the corresponding antibody; S2. Determine the range of the ratio of fPSA to tPSA based on the number of bands that appear in the detection area of ​​the test strip and the depth of color.

[0043] In some embodiments of the present invention, step S2, the specific steps of determining the range of the ratio of fPSA to tPSA according to the number of bands and the depth of coloration in the detection area of ​​the test strip include: First, observe the bands of the tPSA test strip: when a band appears in the control area C1 and no band appears in the detection area T1, it means that the tPSA content in the sample to be tested is less than 4 ng / mL; Alternatively, when bands appear in both the control area C1 and the detection area T1, and the color of the band in the detection area T1 is greater than that of the band in the control area C1, it means that the tPSA content in the sample to be tested is greater than 10 ng / mL; Alternatively, when bands appear in both the control area C1 and the detection area T1, and the color of the band in the detection area T1 is less than that of the band in the control area C1, it means that the tPSA content in the sample to be tested is between 4 and 10 ng / mL; at this time, further observe the bands of the fPSA test strip: when the color of the band in the detection area T1 is greater than that of the band in the detection area T2, it means that the fPSA / tPSA ratio is less than 0.16; when the color of the band in the detection area T1 is less than that of the band in the detection area T2, it means that the fPSA / tPSA ratio is greater than 0.16.

[0044] The method provided by the present invention, during the detection, 1) the mixed sample is added to the detection wells of the left and right modules at the same time, the gold-labeled antibodies pre-coated on the surface of the colloidal gold particles in the tPSA and fPSA reagent strips in the sample are combined, and the conjugate migrates upward to the detection area with the capillary action of the membrane, and combines with the capture antibody fixed on the nitrocellulose membrane, and a red strip appears in the detection area; regardless of whether the analyte is present in the sample, when the liquid level continues to move to the control area C1 and the quality control area C, a red strip will definitely appear; 2) When no band appears in the tPSA detection area T1, it means that the total prostate-specific antigen (tPSA) content in the sample is less than 4ng / mL, indicating that there is no obvious pathological change in the prostate tissue and the risk of prostate cancer is low; when a band appears and the color is greater than that of the control area C1, it means that the total prostate-specific antigen (tPSA) content in the sample is greater than 10ng / mL, indicating a higher risk of prostate cancer, and further diagnostic examination is recommended; when a band appears in the tPSA detection area T1 but the color is lighter than that of the control area C1, it means that the total prostate-specific antigen (tPSA) content in the sample is between 4 and 10ng / mL, indicating a possible risk of prostate cancer. At this time, it is further compared with the free prostate-specific antigen T2 color of another detection module; 3) When the T1 color is greater than T2, it means that the fPSA / tPSA ratio is less than 0.16, indicating a higher risk of prostate cancer; when the T1 color is less than T2, it means that the fPSA / tPSA ratio is greater than 0.16, indicating a lower risk of prostate cancer.

[0045] In a specific embodiment, the method for detecting the ratio of fPSA to tPSA based on the above-mentioned colloidal gold immunochromatographic test paper card comprises the steps of: a. Take the sample and add 60 μL to each of the tPSA and fPSA wells; b. On the tPSA test paper, the PSA antigen in the sample first combines with the tPSA antibody 1 coated on the surface of the colloidal gold particles on the gold pad under capillary action to form a conjugate, which then moves forward to the detection area and combines with the capture antibody (tPSA antibody 2) fixed on the nitrocellulose membrane. The liquid continues to move forward, and the independent antigen (gold-labeled rabbit IgG) combines with the independent antibody (sheep anti-rabbit IgG) fixed in the control area to form a red strip. Regardless of whether there is PSA antigen in the sample, a red strip will appear in the control area C1. c. When a red stripe appears in the test area T1, it indicates that the tPSA content of the sample is greater than 4ng / mL. When the colorimetric value of the test area T1 is greater than that of the control area C1, it indicates that the tPSA content of the sample is greater than 10ng / mL. The subject has a higher risk of prostate cancer and further diagnostic examination is recommended. When there is no obvious red stripe in the test area T1, it indicates a negative result, indicating that the subject has no obvious pathological changes in the prostate tissue and has a low risk of prostate cancer. d. On the fPSA test paper, the free PSA antigen in the sample first combines with the fPSA antibody 1 coated on the surface of the colloidal gold particles on the gold pad under capillary action to form a conjugate. The conjugate continues to move forward to the detection area and combines with the fPSA antibody 2 fixed on the detection area T2 on the nitrocellulose membrane. The liquid continues to move forward and combines with the secondary antibody (sheep anti-mouse IgG) fixed in the quality control area C to form a red strip. Regardless of whether there is free PSA antigen in the specimen, a red strip will appear in the quality control area C. e. When a band appears in the test area T1 and the colorimetry is lower than that in the control area C1, it indicates that the PSA antigen in the sample is between 4 and 10 ng / mL. This is the gray area of ​​clinical prostate-specific antigen detection and needs to be compared with the fPSA concentration. At this time, compare the colorimetry of T1 and T2. When the color of T1 is greater than that of T2, it indicates a positive result, indicating that the fPSA / tPSA ratio is less than 0.16, indicating a higher risk of prostate cancer. When the color of T1 is less than that of T2, it indicates a negative result, indicating that the fPSA / tPSA ratio is greater than 0.16, indicating a lower risk of prostate cancer.

[0046] The use of the above reagent card is simple and reliable, and the entire detection process only takes 10 minutes.

[0047] The fourth aspect of the present invention provides an embodiment of the colloidal gold immunochromatographic test paper card for detecting the ratio of fPSA to tPSA or the colloidal gold immunochromatographic test paper card for detecting the ratio of fPSA to tPSA prepared by the above preparation method, and uses it in at least one of the following (a1) to (a3): (a1) detecting the ratio of fPSA to tPSA in a sample, wherein the sample comprises serum, plasma or whole blood; (a2) preparing products for monitoring and / or detecting prostate diseases; (a3) preparing a product for assessing the risk of prostate cancer; The use described is not for diagnostic or therapeutic purposes.

[0048] An embodiment of the fifth aspect of the present invention provides a kit for detecting the ratio of fPSA to tPSA, comprising the above-mentioned colloidal gold immunochromatographic test paper card for detecting the ratio of fPSA to tPSA or the colloidal gold immunochromatographic test paper card for detecting the ratio of fPSA to tPSA prepared by the above-mentioned preparation method. Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures particularly pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 A schematic diagram of a test paper card provided in an embodiment of the present invention; Figure 2 Schematic diagram of a tPSA test strip and an fPSA test strip provided in an embodiment of the present invention; Figure 3 A schematic diagram of the negative sample test result of the test paper card provided in an embodiment of the present invention; Figure 4 A schematic diagram of the test results of a positive sample detected by a test paper card provided in an embodiment of the present invention; Figure 5 A schematic diagram of the results of a negative sample in the gray area detected by a test paper card provided in an embodiment of the present invention; Figure 6 This is a schematic diagram of the results of gray area positive sample detection using the test paper card provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0050] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0051] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0052] In the description of the present invention, unless otherwise specified, the numerical range "a to b" represents an abbreviation of any real number combination between a and b, wherein a and b are real numbers. Unless otherwise specified, each reaction or operation step may be performed in sequence or not. Preferably, the reaction method of the present invention is performed in sequence.

[0053] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The materials and reagents used in the examples are commercially available unless otherwise specified.

[0054] Example 1 In this embodiment, a colloidal gold immunochromatographic test paper card for detecting the ratio of fPSA to tPSA is prepared, and the steps are as follows: (I) Preparation of colloidal gold solution: 1. Weigh an appropriate amount of chloroauric acid and dissolve it in pure water to prepare a 1% (w / v) chloroauric acid solution; 2. Weigh an appropriate amount of sodium citrate and dissolve it in pure water to prepare a 2% (w / v) sodium citrate solution; 3. Add 1L of pure water to the flask, heat to boiling, then add 60mL of the 1% chloroauric acid solution prepared in step 1, continue heating to boiling, then add 45mL of the 2% sodium citrate solution prepared in step 2, continue heating to boiling and react for 10 minutes, and cool the prepared colloidal gold solution to room temperature for use.

[0055] (II) Preparation of tPSA colloidal gold pad: 4. Take 50 mL of the prepared colloidal gold solution, add 0.3 mL of 0.2 mol / L potassium carbonate solution to adjust the pH, mix well with a vortex instrument, then add 1 mg of mouse tPSA monoclonal antibody to a final concentration of 20 μg / mL, mix well and let it react for 15 minutes; 5. Take 1 mL of 20% BSA blocking solution and add it to the reaction system in step 4, mix well and let it react for 5 minutes; 6. Place the liquid from step 5 into a centrifuge and centrifuge at 12,000 rpm for 20 min. Discard the supernatant and add 50 mL of tPSA gold labeling solution to resuspend it to 100-200% of the original volume for later use as tPSA gold labeling solution.

[0056] Preparation of tPSA gold-labeled complex solution: Add 5% sucrose, 2.5% Casein-Na, 1% BSA, 0.2% S9, 0.5% glycine, 0.2% Tween-20, and 0.03% PC300 to 0.02 mol PBS buffer and adjust the pH to 8.0±0.2.

[0057] 7. Take 45 mL of the tPSA gold-labeled solution from step 6 and spread it evenly on a (25.4*30 cm) glass cellulose membrane. Place it at 37°C and dry it for 24 hours to make a tPSA colloidal gold pad for use.

[0058] (III) Preparation of rabbit IgG colloidal gold pad: 8. Take 50 mL of the prepared colloidal gold solution, add 0.6 mL of 0.2 mol / L potassium carbonate, mix well with a vortex instrument, then add 1 mg of rabbit IgG, the final concentration is 20 μg / mL, mix well and let it react for 15 minutes; 9. Take 1 mL of 20% BSA blocking solution and add it to the reaction system in step 8, mix well and let it react for 5 minutes; 10. Place the liquid from step 9 into a centrifuge and centrifuge at 12000 rpm for 20 min. Discard the supernatant and add 150 mL of tPSA gold-labeled solution to resuspend the solution as rabbit IgG gold-labeled solution for later use.

[0059] 11. Take 45 mL of the rabbit IgG gold-labeled solution from step 10 and spread it evenly on a (25.4*30 cm) glass cellulose membrane. Place it at 37°C and dry it for 24 hours to make a rabbit IgG colloidal gold pad for use.

[0060] (IV) Preparation of tPSA test strips: 12. Stick the nitrocellulose membrane on the PVC base plate, take goat anti-rabbit IgG antibody as C1 line antibody and mouse tPSA monoclonal antibody as T1 line antibody, dilute the goat anti-rabbit IgG antibody to a final concentration of 0.5 mg / ml with coating diluent, and dilute the mouse tPSA monoclonal antibody to a final concentration of 0.8 mg / ml, use a film scribing instrument to coat the diluted antibodies on the nitrocellulose membrane at 1 μL / cm, and dry at 37°C for 24 hours.

[0061] Preparation of coating diluent: Add 2% sucrose to 0.02 mol PBS buffer and adjust the pH to 7.4-8.0.

[0062] 13. Sample pad preparation: Add 0.5% Casein-Na, 0.2% Tween-20, 0.05mg / mL RBC, and 0.03% PC300 to 0.02mol PBS buffer. Spread evenly on the glass cellulose membrane at 45mL / sheet (25.4*30cm), and dry at 37℃ for 24 hours for use.

[0063] 14. Cut the dried tPSA colloidal gold pad and rabbit IgG colloidal gold pad into 3mm and stick them on the above PVC base plate, and paste a 19*30cm sample pad and 20.5*30cm absorbent paper, and cut them into 3mm tPSA test strips.

[0064] (V) Preparation of fPSA colloidal gold pad: 15. Take 50 mL of the prepared colloidal gold solution, add 0.5 mL of 0.2 mol / L potassium carbonate, mix well with a vortex instrument, then add 1 mg of mouse fPSA monoclonal antibody to a final concentration of 20 μg / mL, mix well and let react for 15 minutes; 16. Add 1 mL of 20% BSA blocking solution to the reaction system in step 15, mix well, and let react for 5 minutes; 17. Place the liquid from step 16 in a centrifuge and centrifuge at 12000rpm for 20min. Discard the supernatant and add 50mL of fPSA gold-labeled solution to resuspend as fPSA gold-labeled solution for later use. Pour the above fPSA gold-labeled solution into a glass fiber at a rate of 45ml / sheet (25.4*30cm) and dry at 37℃ for 24 hours for later use.

[0065] Preparation of fPSA gold-labeled complex solution: Add 1% sodium chloride, 10% sucrose, 2% Casein-Na, 1% BSA, 0.5% S9, 0.5% Tween-20, and 0.03% PC300 to 0.02 mol PBS buffer, and adjust the pH to 8.0±0.2.

[0066] (VI) Preparation of fPSA test strips: 18. Stick the nitrocellulose membrane on the PVC base plate, take goat anti-mouse IgG antibody as the C-line antibody, and mouse fPSA monoclonal antibody as the T2-line antibody, dilute the goat anti-mouse IgG antibody to a final concentration of 1 mg / ml with coating diluent, and dilute the mouse fPSA monoclonal antibody to a final concentration of 1 mg / ml, use a film scribing instrument to coat the diluted antibodies on the nitrocellulose membrane at 1 μL / cm, and dry at 37°C for 24 hours for use.

[0067] 19. Cut the dried fPSA colloidal gold pad into 3mm and stick it on the above PVC base plate, and paste a 19*30cm sample pad and 20.5*30cm absorbent paper, and cut it into 3mm fPSA test strips.

[0068] (VII) Preparation of test paper cards: 20. Assemble the PVC base plate and card cover with the tPSA test strip and fPSA test strip attached to form a test strip card for use.

[0069] Comparative Example 1 The only difference between Comparative Example 1 and Example 1 is that the concentration of Casein-Na in the tPSA gold complex solution is 1%. Preparation of tPSA gold-labeled complex solution: 5% sucrose, 1% Casein-Na, 1% BSA, 0.2% S9, 0.5% Gly, 0.2% Tween-20, 0.03% PC300 were added to 0.02 mol PBS buffer, and the pH was adjusted to 8.0±0.2.

[0070] The rest are the same as in Example 1 and will not be described again.

[0071] Comparative Example 2 The only difference between Comparative Example 2 and Example 1 is that the final concentration of the tPSA antibody 2 coated on the nitrocellulose is diluted to 1 mg / ml.

[0072] The rest are the same as in Example 1 and will not be described again.

[0073] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that 0.5% S9 is removed from the fPSA gold-labeled complex solution.

[0074] The rest are the same as in Example 1 and will not be described again.

[0075] Comparative Example 4 The only difference between Comparative Example 4 and Example 1 is that the concentration of Casein-Na in the sample pad pretreatment solution is 0.2%.

[0076] The rest are the same as in Example 1 and will not be described again.

[0077] Test Example 1 This test example uses the test paper card prepared in Example 1 to detect negative samples.

[0078] Take the sample and add 60 μL to each of the tPSA and fPSA sample wells. After the reaction is completed, judge based on the color development results of the strips.

[0079] The results are as follows Figure 3 As shown, the detection area T1 does not show color, indicating that the total prostate-specific antigen is less than 4 ng / mL, a low risk of prostate cancer, and the result is negative.

[0080] Test Example 2 This test example uses the test paper card prepared in Example 1 to detect positive samples.

[0081] Take the sample and add 60 μL to each of the tPSA and fPSA sample wells. After the reaction is completed, judge based on the color development results of the strips.

[0082] The results are as follows Figure 4 As shown, the color development of the test area T1 is greater than that of the control area C1, indicating that the total prostate-specific antigen is greater than 10 ng / mL, which indicates a higher risk of prostate cancer and the result is positive.

[0083] Test Example 3 This test example uses the test paper card prepared in Example 1 to detect negative samples in the gray area.

[0084] Take the sample and add 60 μL to each of the tPSA and fPSA sample wells. After the reaction is completed, judge based on the color development results of the strips.

[0085] The results are as follows Figure 5 As shown, when a band appears in T1 and the color development is lower than that of the control area C1, it indicates that the total prostate-specific antigen is between 4 and 10 ng / mL. At this time, comparing the color development of T1 and T2, the color development of T1 is less than that of T2, indicating that the fPSA / tPSA ratio is greater than 0.16, indicating that the risk of prostate cancer is low, and the result is negative in the gray area.

[0086] Test Example 4 This test example uses the test paper card prepared in Example 1 to detect gray area positive samples.

[0087] Take the sample and add 60 μL to each of the tPSA and fPSA sample wells. After the reaction is completed, judge based on the color development results of the strips.

[0088] The results are as follows Figure 6 As shown, when a band appears on T1 and the color is lower than that of the control area C1, it indicates that the total prostate-specific antigen is between 4 and 10 ng / mL. At this time, comparing the color of T1 and T2, the color of T1 is greater than that of T2, indicating that the fPSA / tPSA ratio is less than 0.16, indicating a higher risk of prostate cancer, and the result is positive in the gray area.

[0089] Test Example 5 This test example uses the test paper card prepared in Example 1 to detect the quality control product. The tPSA quality control product tests the colorimetric display of the tPSA test paper; the fPSA quality control product tests the colorimetric display of the fPSA test paper.

[0090] Six groups of quality control products were tested. The total prostate-specific antigen (tPSA) quality control products tC1, tC2, tC3, tC4, tC5, and tC6 had concentrations of 2ng / mL, 4ng / mL, 6ng / mL, 8ng / mL, 10ng / mL, and 15ng / mL, respectively; the free prostate-specific antigen (fPSA) quality control products fC1, fC2, fC3, fC4, fC5, and fC6 had concentrations of 0.32ng / mL, 0.64ng / mL, 1.00ng / mL, 1.20ng / mL, 1.7ng / mL, and 2.0ng / mL, respectively.

[0091] Take the sample and add 60 μL to each of the tPSA and fPSA sample wells. After the reaction is completed, judge based on the color development results of the strips.

[0092] The results are shown in Table 1: Table 1

[0093] The test results in Table 1 show that the content of the analyte is positively correlated with the colorimetric index of the detection area. When tPSA is less than 4 ng / mL, tPSA in the T1 area does not show color, the test result is negative, and the test results of the quality control products tC1 and fC1 are negative; when tPSA is greater than 10 ng / mL, the colorimetric index of T1 is greater than the colorimetric index of the control area C1, the test result is positive, and the test results of the quality control products tC6 and fC6 are positive. When the colorimetric degree of T1 ≤ that of the control area C1, it indicates that the tPSA concentration is between 4 and 10 ng / mL. At this time, compare the colorimetric degrees of the detection areas T1 and T2. When the colorimetric degree of tPSA in area T1 ≥ that of fPSA in area T2, it indicates that fPSA / tPSA ≤ 0.16, the test result is positive, and the test results of the quality control products tC2, fC2, tC4, and fC4 are positive; when the colorimetric degree of tPSA in area T1 < that of fPSA in area T2, it indicates that fPSA / tPSA > 0.16, the test result is negative, and the test results of the quality control products tC3, fC3, tC5, and fC5 are negative.

[0094] The test results of the quality control products in this test case meet the requirements, indicating that the detection accuracy and sensitivity of the reagent card meet the test needs.

[0095] Test Example 6 The reagent card prepared in Comparative Example 1 was used to test the quality control product. The tPSA quality control product tested the colorimetric display of the tPSA test paper; and the fPSA quality control product tested the colorimetric display of the fPSA test paper.

[0096] Six groups of quality control products were tested. The total prostate-specific antigen (tPSA) quality control products tC1, tC2, tC3, tC4, tC5, and tC6 had concentrations of 2ng / mL, 4ng / mL, 6ng / mL, 8ng / mL, 10ng / mL, and 15ng / mL, respectively; the free prostate-specific antigen (fPSA) quality control products fC1, fC2, fC3, fC4, fC5, and fC6 had concentrations of 0.32ng / mL, 0.64ng / mL, 1.00ng / mL, 1.20ng / mL, 1.7ng / mL, and 2.0ng / mL, respectively.

[0097] Take the sample and add 60 μL to each of the tPSA and fPSA sample wells. After the reaction is completed, judge based on the color development results of the strips.

[0098] The results are shown in Table 2: Table 2

[0099] In this test case, the test results of the quality control products tC2 and fC2 (tPSA concentration between 4 and 10 ng / mL, fPSA / tPSA≤0.16) were negative (should be positive); the test results of tC4 and fC4 (tPSA concentration between 4 and 10 ng / mL, fPSA / tPSA≤0.16) were negative (should be positive). This indicates that there are false negative results in the test, and the detection accuracy and sensitivity of the reagent card are poor.

[0100] Test Example 7 The reagent card prepared in Comparative Example 2 was used to test the quality control product in the same manner as in Test Example 6.

[0101] The results are shown in Table 3: Table 3

[0102] In this test case, the test results of the quality control products tC1 and fC1 (tPSA < 4ng / mL) were positive (should be negative); the test results of tC3, fC3, tC5, fC5 (tPSA concentration between 4-10ng / mL, fPSA / tPSA > 0.16) were positive (should be negative). This shows that there are false positive results in the test, and the detection accuracy and sensitivity of the reagent card are poor.

[0103] Test Case 8 The reagent card prepared in Comparative Example 3 was used to test the quality control product in the same manner as in Test Example 6.

[0104] The results are shown in Table 4: Table 4

[0105] In this test case, the test results of the quality control products tC3 and fC3 (tPSA concentration between 4 and 10 ng / mL, fPSA / tPSA>0.16) were positive (should be negative); the test results of tC5 and fC5 (tPSA concentration between 4 and 10 ng / mL, fPSA / tPSA>0.16) were positive (should be negative). This indicates that there are false positive results in the test, and the detection accuracy and sensitivity of the reagent card are poor.

[0106] Test Example 9 The reagent card prepared in Comparative Example 4 was used to test the quality control product in the same manner as in Test Example 6.

[0107] The results are shown in Table 5: Table 5

[0108] In this test case, the test results of the quality control products tC2 and fC2 (tPSA concentration between 4 and 10 ng / mL, fPSA / tPSA≤0.16) were negative (should be positive); the test results of tC3 and fC3 (tPSA concentration between 4 and 10 ng / mL, fPSA / tPSA>0.16) were positive (should be negative); and the test results of tC4 and fC4 (tPSA concentration between 4 and 10 ng / mL, fPSA / tPSA≤0.16) were negative (should be positive). This shows that the detection accuracy and sensitivity of the reagent card are poor.

[0109] Test Example 10 This test example uses the test paper cards prepared in Example 1 and Comparative Examples 1-4 to detect clinical samples.

[0110] 30 positive clinical samples and 30 negative clinical samples (tested by chemiluminescence) were selected, and the judgment criteria were: tPSA greater than 10 ng / mL was positive, and tPSA between 4 and 10 ng / mL and fPSA / tPSA less than or equal to 0.16 were also positive. The test strips prepared in Example 1 and Comparative Examples 1-4 were used for testing.

[0111] The results are shown in Table 6: Table 6

[0112] According to the comparison with the above-mentioned total prostate specific antigen (tPSA) and free prostate specific antigen (fPSA) chemiluminescence detection results, the positive coincidence rate of Example 1 is (30 / 30) 100%, and the negative coincidence rate is (30 / 30) 100%.

[0113] The negative coincidence rate (29 / 30) of comparative example 1 was 96.7%, and the positive coincidence rate (25 / 30) was 83.3%. The results show that Example 1 of the present invention is significantly better than comparative example 1 in terms of the positive and negative coincidence rate, proving that increasing the Casein-Na of the tPSA gold complex solution to 2.5% can effectively improve the positive and negative coincidence rate of the detection.

[0114] The negative coincidence rate (27 / 30) of comparative example 2 was 90%, and the positive coincidence rate (30 / 30) was 100%. The results showed that Example 1 of the present invention was significantly better than comparative example 2 in terms of the negative and positive coincidence rate, proving that reducing the final concentration of tPSA antibody 2 dilution from 1 mg / ml to 0.8 mg / ml can effectively improve the negative coincidence rate of the detection.

[0115] The negative coincidence rate (26 / 30) of comparative example 3 was 86.7%, and the positive coincidence rate (30 / 30) was 100%. The results show that Example 1 of the present invention is significantly better than comparative example 3 in terms of the negative and positive coincidence rate, proving that adding 0.5% S9 to the fPSA gold-labeled complex solution can effectively improve the negative coincidence rate of the detection.

[0116] The negative coincidence rate (28 / 30) of comparative example 4 was 93.3%, and the positive coincidence rate (25 / 30) was 83.3%. The results show that Example 1 of the present invention is significantly better than comparative example 4 in terms of the positive and negative coincidence rate, proving that increasing the Casein-Na from 0.2% to 0.5% in the sample pad pretreatment solution can effectively improve the positive and negative coincidence rate of detection.

[0117] The above is a specific description of the preferred implementation of the present application, but the present application is not limited to the above-mentioned implementation mode. Technical personnel familiar with the field can also make various equivalent deformations or substitutions without violating the spirit of the present application. These equivalent deformations or substitutions are all included in the scope defined by the claims of the present application.

Claims

1. A colloidal gold immunochromatographic test paper card for detecting the ratio of fPSA to tPSA, characterized in that: It includes a base and an fPSA test strip and a tPSA test strip arranged on the base; The tPSA test strip comprises a first sample pad, a first immune colloidal gold pad, a control area colloidal gold pad, a first nitrocellulose membrane, and a first absorbent pad in sequence; the first immune colloidal gold pad has tPSA antibody 1 labeled with colloidal gold adsorbed therein, and the control area colloidal gold pad has independent antigen labeled with colloidal gold adsorbed therein; the first nitrocellulose membrane is provided with a detection area T1 and a control area C1 at intervals in sequence, the detection area T1 is fixed with tPSA antibody 2, and the control area C1 is fixed with an independent antibody that can specifically bind to the independent antigen; The fPSA test paper includes a second sample pad, a second immunocolloidal gold pad, a second nitrocellulose membrane, and a second absorbent pad in sequence; the second immunocolloidal gold pad is adsorbed with fPSA antibody 1 labeled with colloidal gold; the second nitrocellulose membrane is provided with detection area T2 and quality control area C in sequence at intervals, the detection area T2 is fixed with fPSA antibody 2, and the quality control area C is fixed with a secondary antibody that can bind to fPSA antibody 1.

2. The colloidal gold immunochromatographic test paper card according to claim 1, characterized in that: The tPSA antibody 1 and tPSA antibody 2 are different antibodies, and both can bind to the tPSA antigen; And / or, the fPSA antibody 1 and fPSA antibody 2 are different antibodies, and both can bind to the fPSA antigen; and / or, the final concentration of the tPSA antibody II coated in the detection area T1 is 0.5-2.0 mg / mL; and / or, the final concentration of the independent antibody coated in the control area C1 is 0.5-1.0 mg / mL; and / or, the final concentration of the fPSA antibody II coated in the detection area T2 is 0.5-2.0 mg / mL; And / or, the final concentration of the secondary antibody that binds to the fPSA antibody in the quality control area C is 0.5-2.0 mg / mL.

3. The colloidal gold immunochromatographic test paper card according to claim 1, characterized in that: In the fPSA test strip and the tPSA test strip, the sample pad overlaps with the immunocolloidal gold pad by 1-3 mm; the immunocolloidal gold pad overlaps with the nitrocellulose membrane by 1-3 mm; the nitrocellulose membrane overlaps with the absorbent pad by 2-4 mm; the detection area T1 and the control area C1 are spaced 3-6 mm apart, and the detection area T2 and the quality control area C are spaced 3-6 mm apart; The first immune colloidal gold pad and the control area colloidal gold pad are placed with an overlap of 1-3 mm, the sample pad overlaps the lower gold pad by 1-3 mm, and the upper gold pad overlaps the nitrocellulose membrane by 1-3 mm.

4. The colloidal gold immunochromatographic test paper card according to claim 1, characterized in that: The colloidal gold immunochromatographic test paper card also includes a card cover, which covers the fPSA test strip and the tPSA test strip, and is provided with a first sample addition port, a first observation port, a second sample addition port, and a second observation port; The first sample addition port is disposed above the first sample pad to expose part or all of the first sample pad area; the first observation port is disposed above the first nitrocellulose membrane to expose the detection area T1 and the control area C1 of the first nitrocellulose membrane; The second sample addition port is disposed above the second sample pad to expose part or all of the second sample pad area; the second observation port is disposed above the second nitrocellulose membrane to expose the detection area T2 and the quality control area C of the second nitrocellulose membrane.

5. A method for preparing a colloidal gold immunochromatographic test paper card for detecting the ratio of fPSA to tPSA according to any one of claims 1 to 4, characterized in that: Includes steps: preparing colloidal gold solution; The colloidal gold solution is reacted with the tPSA antibody, and then the tPSA gold-labeled solution is added to prepare a tPSA gold-labeled solution; Adsorbing the tPSA gold-labeled solution onto a glass cellulose membrane to prepare a first immunocolloidal gold pad adsorbed with colloidal gold-labeled tPSA antibody; reacting the colloidal gold solution with an independent antigen to prepare an independent antigen gold labeled solution; Adsorbing the independent antigen gold-labeled solution onto a glass cellulose membrane to prepare a control area colloidal gold pad adsorbed with the colloidal gold-labeled independent antigen; Using a membrane streaking instrument, the C1 line antibody and the T1 line antibody are fixed to the separated areas on the same nitrocellulose membrane to prepare a first nitrocellulose membrane, wherein the C1 line antibody is an independent antibody that can specifically bind to the independent antigen, and the T1 line antibody is tPSA antibody II; Sequentially sticking the sample pad, the first immune colloidal gold pad, the control area colloidal gold pad, the first nitrocellulose membrane, and the absorbent pad on a PVC bottom plate to obtain a tPSA test strip; The colloidal gold solution is reacted with the fPSA antibody, and then the fPSA gold-labeled solution is added to prepare the fPSA gold-labeled solution; Adsorbing the fPSA gold-labeled solution onto a glass cellulose membrane to prepare a second immunocolloidal gold pad adsorbed with colloidal gold-labeled fPSA antibody-1; Using a membrane stripper, the C-line antibody and the T2-line antibody are fixed to separate regions on the same nitrocellulose membrane to prepare a second nitrocellulose membrane, wherein the C-line antibody is a secondary antibody that can bind to the fPSA antibody one, and the T2-line antibody is the fPSA antibody two; Sequentially sticking the sample pad, the second immunocolloidal gold pad, the second nitrocellulose membrane, and the absorbent pad on a PVC bottom plate to obtain an fPSA test strip; After assembling the tPSA test strip and the fPSA test strip, the colloidal gold immunochromatographic test card for detecting the ratio of fPSA to tPSA is obtained.

6. The preparation method according to claim 5, characterized in that: The preparation method of the tPSA gold-labeled complex solution comprises: Add 2-10wt.% sucrose, 0.5-3wt.% Casein-Na, 0.5-2wt.% BSA, 0.1-0.5wt.% S9, 0.2-0.5wt.% glycine, 0.05-0.5wt.% Tween-20, and 0.01-0.2wt.% PC300 to PBS buffer, adjust the pH to 8.0±0.2, and obtain the tPSA gold-labeled complex solution; And / or, the method for preparing the fPSA gold-labeled solution comprises: Add 0.5-2wt.% sodium chloride, 5-15wt.% sucrose, 0.5-3wt.% Casein-Na, 0.5-2wt.% BSA, 0.2-2wt.% S9, 0.2-1.5wt.% Tween-20, and 0.01-0.2wt.% PC300 to the PBS buffer, and adjust the pH to 8.0±0.2 to obtain the fPSA gold-labeled complex solution; And / or, the method for preparing the sample pad comprises: 0.2-1 wt.% Casein-Na, 0.05-0.5 wt.% Tween-20, 0.02-0.2 mg / mL RBC, and 0.01-0.2 wt.% PC300 were added to the PB buffer to obtain a sample pad buffer. The sample pad buffer was evenly spread on a glass cellulose membrane and dried for 12-36 h to obtain the sample pad.

7. A method for detecting the ratio of fPSA to tPSA based on the colloidal gold immunochromatographic test paper card according to any one of claims 1 to 4, characterized in that: Includes steps: Adding the sample to be tested to the sample pads of the fPSA test strip and the tPSA test strip respectively and simultaneously, the sample to be tested migrates to different areas of the test strip and reacts with the corresponding antibody; The range of the ratio of fPSA to tPSA is determined based on the number of bands that appear in the detection area of ​​the test strip and the depth of color.

8. The method according to claim 7, characterized in that The specific steps for determining the range of the ratio of fPSA to tPSA based on the number of bands and the depth of color in the test area of ​​the test strip include: First, observe the bands of the tPSA test strip: when a band appears in the control area C1 and no band appears in the detection area T1, it means that the tPSA content in the sample to be tested is less than 4 ng / mL; Alternatively, when bands appear in both the control area C1 and the detection area T1, and the color of the band in the detection area T1 is greater than that of the band in the control area C1, it means that the tPSA content in the sample to be tested is greater than 10 ng / mL; Alternatively, when bands appear in both the control area C1 and the detection area T1, and the color of the band in the detection area T1 is less than that of the band in the control area C1, it means that the tPSA content in the sample to be tested is between 4 and 10 ng / mL; at this time, further observe the bands of the fPSA test strip: when the color of the band in the detection area T1 is greater than that of the band in the detection area T2, it means that the fPSA / tPSA ratio is less than 0.16; when the color of the band in the detection area T1 is less than that of the band in the detection area T2, it means that the fPSA / tPSA ratio is greater than 0.

16.

9. Use of a colloidal gold immunochromatographic test paper card for detecting the ratio of fPSA to tPSA as claimed in any one of claims 1 to 4 or a colloidal gold immunochromatographic test paper card for detecting the ratio of fPSA to tPSA prepared by the preparation method according to any one of claims 5 to 6 in at least one of the following (a1) to (a3): (a1) detecting the ratio of fPSA to tPSA in a sample, wherein the sample comprises serum, plasma or whole blood; (a2) preparing products for monitoring and / or detecting prostate diseases; (a3) preparing a product for assessing the risk of prostate cancer; The use described is not for diagnostic or therapeutic purposes.

10. A kit for detecting the ratio of fPSA to tPSA, characterized in that: It comprises a colloidal gold immunochromatographic test paper card for detecting the ratio of fPSA to tPSA as described in any one of claims 1 to 4, or a colloidal gold immunochromatographic test paper card for detecting the ratio of fPSA to tPSA prepared by the preparation method according to any one of claims 5 to 6.

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