Combination pad soak solution, combination pad working solution and application of combination pad soak solution and combination pad working solution

Abstract

The invention discloses a conjugate pad soaking solution, a conjugate pad working solution and application thereof, and relates to the technical field of immunodetection. The invention provides the conjugate pad soak solution, the conjugate pad soak solution contains the nonionic fluorine-containing surfactant with extremely high surface activity, the surface tension of a conjugate pad soak solution system is greatly reduced by adding the fluorine-containing surfactant, and the precision of a test result is improved. The Good's buffer solution, the protein protective agent, the saccharides and the polyol are beneficial to improving the stability of the antibody. The preservative inhibits growth and reproduction of microorganisms such as bacteria in the solution and prevents the solution from going bad. A small amount of monohydric alcohol is added to promote rapid volatilization of liquid in the drying process of the conjugate pad, so that the antibody activity can be preserved. Experiments prove that after the conjugate pad prepared from the conjugate pad soak solution provided by the invention is prepared into a test strip, the test strip is good in detection repeatability, has a relatively strong improvement effect on reagent performance, and has a very good commercial value.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to a conjugate pad soaking solution, a conjugate pad working solution and applications thereof. Background Art

[0002] Point-of-care testing (POCT) reagents have the advantages of rapid detection and convenient use. They play an increasingly important role in various medical fields, especially in scenarios that require rapid decision-making and emergency treatment, such as emergency departments, ICUs, CCUs, etc. Precision is crucial for POCT reagents and reflects the repeatability of test results. In the prior art, in order to improve the precision of reagents, the test strip structure, particle labeling conditions, chromatographic membrane processing conditions, etc. are usually optimized, but the results are often unsatisfactory.

[0003] In view of this, the present invention is proposed. Summary of the invention

[0004] The object of the present invention is to provide a bonding pad soaking liquid, a bonding pad working liquid and applications thereof to solve the above technical problems.

[0005] The present invention is achieved in that:

[0006] In a first aspect, the present invention provides a conjugate pad soaking solution, which includes the following raw materials with final concentrations: 10-100 mM buffer, 0.1 wt%-1 wt% nonionic surfactant, 0.5 wt%-5 wt% protein protectant, 2 wt%-10 wt% sugar, 0.5 v / v%-5 v / v% monohydric alcohol, 0.1 wt / v%-1 wt / v% polyol and 0.05 wt%-1 wt% preservative, the buffer is selected from at least one of Tris, MOPS, MES, HEPES and Bis-tris propane, the nonionic surfactant includes a first nonionic surfactant and a second nonionic surfactant, the first nonionic surfactant is selected from at least one of Tween-20, Tween-80 and Triton X-100, the second nonionic surfactant is selected from fluorinated surfactant, the final concentration of the first nonionic surfactant is 0.1 wt%-0.9 wt%, and the final concentration of the second nonionic surfactant is 0.005 wt%-0.05 wt%;

[0007] The protein protective agent comprises a first protein protective agent and a second protein protective agent, the first protein protective agent is selected from at least one of BSA, glycerol and gelatin, and the second protein protective agent is selected from at least one of caseinate sodium, SC01, SC02 and SG01; the final concentration of the first protein protective agent is 0.25wt% to 2.5wt%, and the final concentration of the second protein protective agent is 0.25wt% to 2.5wt%;

[0008] The sugar is selected from at least one of sucrose and trehalose, the monohydric alcohol is selected from at least one of methanol, ethanol, propanol and butanol, and the polyhydric alcohol is selected from at least one of ethylene glycol, propylene glycol, glycerol, mannitol, xylitol and sorbitol.

[0009] In a second aspect, the present invention provides a conjugate pad working solution or a kit, which comprises the conjugate pad soaking solution and a marker with an antibody.

[0010] In a third aspect, the present invention provides use of a conjugate pad soaking solution in preparing a conjugate pad.

[0011] In a fourth aspect, the present invention provides a method for preparing a conjugate pad, comprising the following steps (1) or (2):

[0012] (1) soaking the above-mentioned conjugate pad soaking liquid in a conjugate pad substrate, and after drying, spraying a marker with an antibody onto the dried conjugate pad substrate;

[0013] (2) Soak the bonding pad substrate in the bonding pad working solution and dry it.

[0014] The present invention has the following beneficial effects:

[0015] The present invention provides a conjugate pad soaking solution, which contains a non-ionic fluorinated surfactant with extremely high surface activity. The addition of the fluorinated surfactant greatly reduces the surface tension of the conjugate pad soaking solution system. On the one hand, according to the Hofmeister effect, the fluorinated surfactant increases the solubility of proteins such as antibodies, promotes salt dissolution, and avoids the aggregation of antibodies and labeled microspheres in the conjugate pad soaking solution; on the other hand, due to the wetting effect of the surfactant, during the conjugate pad manufacturing process, the marker (particles) are quickly and evenly dispersed in the fiber membrane, improving the uniformity of laying; after the test strip is prepared, during the detection process, due to the presence of the fluorinated surfactant, the particles are quickly redissolved and fully released from the conjugate pad, improving the precision of the test results.

[0016] Good's buffer, protein protectant, sugar and polyol are helpful to improve the stability of antibodies. Preservatives inhibit the growth and reproduction of bacteria and other microorganisms in the solution, preventing the solution from deteriorating. The addition of a small amount of monohydric alcohol promotes rapid evaporation of the liquid during the drying process of the conjugate pad, which is beneficial to preserve the activity of the antibody.

[0017] Experimental verification shows that after the conjugate pad prepared by the conjugate pad soaking solution provided by the present invention is prepared into a test strip, the test strip has good detection repeatability, has a strong improvement effect on the reagent performance, and has good commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 This is a graph showing the results of the plasma clinical correlation experiment in Example 1;

[0020] Figure 2 This is a graph showing the results of a plasma clinical correlation experiment in Comparative Example 8;

[0021] Figure 3 A standard curve diagram of the conjugate pad soaking solution provided in Example 1;

[0022] Figure 4 A standard curve diagram of the conjugate pad soaking solution provided in Comparative Example 1;

[0023] Figure 5 A standard curve diagram of the conjugate pad soaking solution provided for Comparative Example 2;

[0024] Figure 6 The standard curve of the conjugate pad soaking solution provided in Comparative Example 3;

[0025] Figure 7 A standard curve diagram of the conjugate pad soaking solution provided for Comparative Example 4;

[0026] Figure 8 A standard curve diagram of the conjugate pad soaking solution provided for Comparative Example 5;

[0027] Fig. 9 A standard curve diagram of the conjugate pad soaking solution provided for Comparative Example 6;

[0028] Fig.10 A standard curve diagram of the conjugate pad soaking solution provided for Comparative Example 7;

[0029] Fig.11 This is a standard curve diagram of the conjugate pad soaking solution provided in Comparative Example 8. DETAILED DESCRIPTION

[0030] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0031] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0032] The invention found that for lateral flow chromatography reagents, the uniformity of the particles in the conjugate pad and the release of the particles during the re-dissolution of the conjugate pad directly determine the precision performance of the reagent. Based on this, the present invention provides a conjugate pad soaking solution, which contains a non-ionic fluorinated surfactant with extremely high surface activity. The addition of the fluorinated surfactant greatly reduces the surface tension of the conjugate pad soaking solution system. On the one hand, according to the Hofmeister effect, the fluorinated surfactant increases the solubility of proteins such as antibodies, promotes salt dissolution, and avoids the aggregation of antibodies and labeled microspheres in the conjugate pad soaking solution; on the other hand, due to the wetting effect of the surfactant, during the conjugate pad manufacturing process, the marker (particles) are quickly and evenly dispersed in the fiber membrane, which improves the uniformity of the laying; after the test strip is prepared. During the detection process, due to the presence of the fluorinated surfactant, the particles are quickly re-dissolved and fully released from the conjugate pad, which improves the precision of the test results.

[0033] In addition, in order to further improve the precision of the reagent, Good's buffer, protein protectant, sugar and polyol are set in the conjugate pad soaking solution. Buffer, protein protectant, sugar and polyol are beneficial to improve the stability of the antibody. The preservative inhibits the growth and reproduction of microorganisms such as bacteria in the solution and prevents the solution from deteriorating. The addition of a small amount of monohydric alcohol promotes the rapid volatilization of the liquid during the drying process of the conjugate pad, which is beneficial to preserve the activity of the antibody.

[0034] In a first aspect, the present invention provides a conjugate pad soaking solution, which includes the following raw materials with final concentrations: 10-100 mM buffer, 0.1 wt%-1 wt% nonionic surfactant, 0.5 wt%-5 wt% protein protectant, 2 wt%-10 wt% sugar, 0.5 v / v%-5 v / v% monohydric alcohol, 0.1 wt / v%-1 wt / v% polyol and 0.05 wt%-1 wt% preservative, the buffer is selected from at least one of Tris, MOPS, MES, HEPES and Bis-tris propane, the nonionic surfactant includes a first nonionic surfactant and a second nonionic surfactant, the first nonionic surfactant is selected from at least one of Tween-20, Tween-80 and Triton X-100, the second nonionic surfactant is selected from fluorinated surfactant, the final concentration of the first nonionic surfactant is 0.1 wt%-0.9 wt%, and the final concentration of the second nonionic surfactant is 0.005 wt%-0.05 wt%;

[0035] The protein protective agent comprises a first protein protective agent and a second protein protective agent, the first protein protective agent is selected from at least one of BSA, glycerol and gelatin, and the second protein protective agent is selected from at least one of caseinate sodium, SC01, SC02 and SG01; the final concentration of the first protein protective agent is 0.25wt% to 2.5wt%, and the final concentration of the second protein protective agent is 0.25wt% to 2.5wt%;

[0036] The sugar is selected from at least one of sucrose and trehalose, the monohydric alcohol is selected from at least one of methanol, ethanol, propanol and butanol, and the polyhydric alcohol is selected from at least one of ethylene glycol, propylene glycol, glycerol, mannitol, xylitol and sorbitol.

[0037] The inventors have found that if the protein protective agent is set only as the first protein protective agent and does not contain the second protein protective agent, the precision of the reagent will be reduced. The absence of monohydric alcohol or polyhydric alcohol will also cause the precision of the reagent to be significantly reduced. If a fluorinated surfactant is not provided in the conjugate pad soaking solution, the precision of the reagent will be extremely low and the repeatability will be extremely poor. In addition, a carbohydrate content of less than 2wt% will also cause the precision of the reagent to be significantly reduced when detecting low-concentration samples. Therefore, selecting a conjugate pad soaking solution with the above-mentioned ratio will help improve the precision of the reagent.

[0038] In one embodiment, the conjugate pad soak solution contains a buffer at a final concentration of 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM.

[0039] In one embodiment, the conjugate pad soaking solution contains a nonionic surfactant at a final concentration of 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt% or 1 wt%.

[0040] In one embodiment, the conjugate pad soaking solution contains a protein protectant at a final concentration of 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt% or 5wt%.

[0041] In one embodiment, the conjugate pad soaking solution contains sugar at a final concentration of 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt% or 10 wt%.

[0042] In one embodiment, the conjugate pad soaking solution contains a monohydric alcohol at a final concentration of 0.5 v / v%, 1 v / v%, 1.5 v / v%, 2 v / v%, 2.5 v / v%, 3 v / v%, 3.5 v / v%, 4 v / v%, 4.5 v / v% or 5 v / v%.

[0043] In one embodiment, the conjugate pad soaking solution contains a polyol at a final concentration of 0.1 wt / v%, 0.2 wt / v%, 0.3 wt / v%, 0.4 wt / v%, 0.5 wt / v%, 0.6 wt / v%, 0.7 wt / v%, 0.8 wt / v%, 0.9 wt / v% or 1 wt / v%.

[0044] In one embodiment, the conjugate pad soaking solution contains a preservative at a final concentration of 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.1wt%, 0.15wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt% or 1wt%.

[0045] In a preferred embodiment of the present invention, it includes the following raw materials with final concentrations: 50-100 mM buffer, 0.1wt%-1wt% non-ionic surfactant, 0.5wt%-5wt% protein protective agent, 5wt%-10wt% sugar, 1v / v%-5v / v% monohydric alcohol, 0.25wt / v%-1wt / v% polyol and 0.1wt%-1wt% preservative.

[0046] In a preferred embodiment of the present invention, the nonionic surfactant includes Tween-20 with a final concentration of 0.5wt% to 0.8wt% and a fluorine-containing surfactant with a final concentration of 0.01wt% to 0.05wt%.

[0047] In a preferred embodiment of the present invention, the fluorinated surfactant is selected from at least one of FS-10, FS-30 and FS-31. FS-10 may also be referred to as Capstone FS-10.

[0048] In a preferred embodiment of the present invention, the protein protective agent includes BSA with a final concentration of 1 wt% to 1.5 wt% and sodium caseinate with a final concentration of 1 wt% to 1.5 wt%. Sodium caseinate is also known as sodium caseinate, CAS number: 9005-46-3.

[0049] In a preferred embodiment of the present invention, the preservative is selected from at least one of Proclin 300, thimerosal, sodium benzoate, nitrate, nitrite and sodium azide.

[0050] In a second aspect, the present invention provides a conjugate pad working solution or a kit, which comprises the conjugate pad soaking solution and a marker with an antibody.

[0051] The above-mentioned marker refers to a class of substances that have characteristics such as luminescence, color development, radioactivity, etc. that can be directly observed by the naked eye or detected or detected by an instrument, and the qualitative or quantitative detection of the corresponding target can be achieved through the characteristics. In the actual use process, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter what kind of marker is used, it belongs to the protection scope of the present invention.

[0052] In a preferred embodiment of the present invention, the marker is selected from at least one of nanoparticle markers, fluorescent dyes, radioactive isotopes, and chemiluminescent agents.

[0053] In a preferred embodiment of the present invention, the nanoparticle marker is selected from nanoparticles or colloidal metals; the nanoparticles are selected from organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles, such as latex microspheres.

[0054] Fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5 .5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), prephycocyanin-chlorophyll protein (preCP), etc.).

[0055] In an alternative embodiment, the radioactive isotopes include but are not limited to 212 Bi, 131 I. 111 In, 90 Y. 186 Re, 211 At 125 I. 188 Re, 153 Sm, 213 Bi, 32 P. 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn、 97 Such as 62 Cu, 64 Cu, 86 Y. 88 Y. 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu and 18 F.

[0056] In an optional embodiment, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0057] In alternative embodiments, the colloidal metal includes, but is not limited to, colloidal gold or colloidal silver.

[0058] In a third aspect, the present invention provides use of a conjugate pad soaking solution in preparing a conjugate pad.

[0059] The conjugate pad substrate is immersed in a conjugate pad working solution, mixed with a marker having an antibody, and dried to prepare a conjugate pad;

[0060] Alternatively, the conjugate pad substrate is soaked in a conjugate pad soaking solution, dried, and then the labeling substance with the antibody is sprayed onto the dried conjugate pad substrate.

[0061] In a fourth aspect, the present invention provides a method for preparing a conjugate pad, comprising the following steps (1) or (2):

[0062] (1) soaking the above-mentioned conjugate pad soaking liquid in a conjugate pad substrate, and after drying, spraying a marker with an antibody onto the dried conjugate pad substrate;

[0063] (2) soaking the bonding pad substrate in the bonding pad working solution and drying;

[0064] In a preferred embodiment of the present invention, the binding pad substrate is a glass fiber membrane, a cotton fiber membrane or a nitrocellulose membrane;

[0065] In a preferred embodiment of the present invention, the drying is vacuum drying, and the vacuum drying condition is to maintain the pressure below 100 Pa for 0.5 to 1 hour.

[0066] In other embodiments, the drying method may also be direct drying at room temperature or drying under ventilation conditions.

[0067] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0068] Example 1

[0069] The present embodiment provides a conjugate pad soaking solution, which includes Tris buffer with a final concentration of 0.05M, 5wt% trehalose, 1wt% BSA, 1wt% casein Na, 1v / v% ethanol, 0.25wt / v% mannitol, 0.5wt% Tween-20, 0.01wt% FS-31 (manufacturer: Capstone, CAS: 52550-44-4) and 0.1wt% NaN3, and the final pH is 9.20±0.05.

[0070] Example 2

[0071] The present embodiment provides a conjugate pad soaking solution, which includes MES buffer with a final concentration of 0.06 M, 5wt% sucrose, 1wt% glycerol, 1wt% SC01, 1v / v% methanol, 0.25wt / v% xylitol, 0.8wt% Tween-80, 0.05wt% FS-10 and 0.1wt% NaN3, and the final pH is 6.50±0.05.

[0072] Example 3

[0073] The present embodiment provides a conjugate pad soaking solution, which includes HEPES buffer with a final concentration of 0.06 M, 10 wt% sucrose, 5 wt% glycerol, 2 wt% SC01, 5 v / v% methanol, 1 wt / v% sorbitol, 0.8 wt% Triton X-100, 0.05 wt% FS-10 and 0.1 wt% NaN3, and the final pH is 8.00±0.05.

[0074] Comparative Example 1

[0075] This comparative example provides a conjugate pad soaking solution, which is different from Example 1 only in the buffer solution. This comparative example includes a PBS buffer solution with a final concentration of 0.05 M (pH 8.00±0.05).

[0076] Comparative Example 2

[0077] This comparative example provides a conjugate pad soaking solution, which is different from Example 1 only in the sugar content. This comparative example includes trehalose with a final concentration of 1%.

[0078] Comparative Example 3

[0079] This comparative example provides a conjugate pad soaking solution, which is different from Example 1 only in that the protein protective agent is different. This comparative example includes BSA with a final concentration of 1%.

[0080] Comparative Example 4

[0081] This comparative example provides a conjugate pad soaking solution, which is different from Example 1 only in that it does not contain monohydric alcohol, that is, does not contain 1% ethanol.

[0082] Comparative Example 5

[0083] This comparative example provides a conjugate pad soaking solution, which is different from Example 1 only in that it does not contain polyol, that is, does not contain 0.25% mannitol.

[0084] Comparative Example 6

[0085] This comparative example provides a conjugate pad soaking solution, which is different from Example 1 only in that the surfactant is different. This comparative example includes Tween-20 with a final concentration of 0.5 wt %.

[0086] Comparative Example 7

[0087] This comparative example provides a conjugate pad soaking solution, which is different from Example 1 only in that the active agent is different. This comparative example includes Tween-20 and FS-31 at a final concentration of 0.05 wt %.

[0088] Comparative Example 8

[0089] This embodiment provides a conjugate pad soaking solution, which includes PBS buffer (pH 8.00±0.05) with a final concentration of 0.05M, 1wt% trehalose, 1wt% BSA, 0.5wt% Tween-20 and 0.1wt% NaN3.

[0090] Experimental Example 1

[0091] Fluorescent latex microspheres with a particle size of 300 nm were coupled with anti-human IL-6 mouse monoclonal antibody (manufacturer: GenScript, catalog number: 55E9) by the EDC / NHS method to form fluorescent particle-labeled anti-IL-6 antibody (concentration of 1 mg / mL).

[0092] The fluorescent particle-labeled anti-IL-6 antibody was added to the binding pad soaking solution prepared in the above Example 1 and Comparative Examples 1-7, mixed, laid on a glass fiber membrane, evenly coated with a coating rod, and placed in a vacuum drying oven for drying (maintaining pressure below 100 Pa for 0.5 h).

[0093] Anti-human IL-6 mouse monoclonal antibody (manufacturer: Nearshore Protein, catalog number: 19D6) was used as the capture antibody (concentration of 1 mg / mL) to streak on the nitrocellulose chromatography membrane as the detection line, and goat anti-mouse IgG was used as the quality control line antibody (concentration of 1 mg / mL). After streaking, the membrane was placed in a vacuum drying oven for drying, and the drying condition was maintained at a pressure below 100 Pa for 0.5 h.

[0094] The dried conjugate pad semi-finished product and the nitrocellulose membrane semi-finished product, the sample pad, the buffer pad and the water absorbent pad are assembled into a reagent sheet.

[0095] The kit uses a double antibody sandwich method to quantitatively detect the concentration of IL-6. Under chromatography, IL-6 in the sample combines with fluorescent particles labeled with anti-human IL-6 monoclonal antibodies in the conjugate pad to form a particle-antigen complex. As the chromatography continues, the complex reaches the detection line coated with anti-human IL-6 monoclonal antibodies to form a sandwich complex. After excitation with 520nm wavelength light, the fluorescence intensity of the reaction product is measured by the matching instrument. The fluorescence intensity is closely related to the IL-6 concentration in the sample. The content of the analyte is determined by a known multi-point calibration curve.

[0096] Table 1 Conjugate pad soaking solution

[0097]

[0098]

[0099] The kits of Example 1 and Comparative Examples 1-7 were calibrated and standard curves were drawn using the calibrators. The calibrators used were recombinant human IL-6 antigens with traceable concentrations of 0.00, 2.26, 7.08, 9.25, 75.56, 146.35, 370.49, 720.56, 1293.77, 3078.68, 3350.45, and 4400.00 pg / mL. The standard curves drawn with the conjugate pad soaking solutions provided in Example 1 and Comparative Examples 1-7 were respectively referred to Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Fig. 9 and Fig.10 shown. Figure 3-Figure 10 The left figure in the figure is a standard curve diagram of antigen at low concentration (x-axis (pg / mL)) and fluorescence intensity (y-axis), and the right figure is a standard curve diagram of antigen at high concentration (x-axis (pg / mL)) and fluorescence intensity (y-axis).

[0100] Calibrators with concentrations of 7.08, 238.96, and 1286.95 pg / mL were used for testing to compare the repeatability of the conjugate pad. The concentration values ​​of the calibrators at the three levels of Example 1 and Comparative Examples 1-7 were obtained by back-calculation of the standard curve. The results are shown in Table 2.

[0101] Table 2 Conjugate pad repeatability

[0102]

[0103]

[0104]

[0105] No Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 Comparative Example 7 CV mean 7.19％ 9.82％ 7.41％ 9.50％ 11.30％ 12.12％ 12.66％ 10.13％

[0106] Conclusion: By comparing the mean CV values ​​of each group, it can be seen that the reagent prepared by the conjugate pad soaking solution formula in Example 1 has a repeatability CV that is smaller than that of the comparative example at three calibration concentrations of low, medium and high, and Example 1 has better precision. If the protein protective agent is set only as the first protein protective agent (BSA) and does not contain the second protein protective agent (casein Na), the precision of the reagent will be reduced (Comparative Example 3). The absence of monohydric alcohol or polyhydric alcohol will also cause the precision of the reagent to be significantly reduced (Comparative Examples 4 and 5). If no fluorinated surfactant is provided in the conjugate pad soaking solution, the precision of the reagent will be extremely low and the repeatability will be extremely poor (Comparative Example 6). In addition, the content of carbohydrates is less than 2wt%, which will also cause the precision of the reagent to be significantly reduced when detecting low-concentration samples (Comparative Example 2). Exchanging the buffer for PBS will affect the detection precision of the reagent (Comparative Example 1).

[0107] Therefore, selecting a conjugate pad soaking solution with the above ratio helps to improve the precision of the reagent.

[0108] Experimental Example 2

[0109] Clinically relevant testing.

[0110] Prepare different conjugate pad soaking solutions according to the table below.

[0111]

[0112] The two conjugate pad soaking solutions of Example 1 and Comparative Example 8 were used to prepare conjugate pads and assemble corresponding reagent sheets. The preparation method of the test paper was the same as that of Experimental Example 1.

[0113] A total of 51 EDTA anticoagulated plasma samples were tested using the two kits, and correlation analysis was performed with the target values ​​of the samples detected by the Interleukin-6 Detection Kit (Electrochemiluminescence Method) (Manufacturer: Roche, Catalog Number: 5109442190).

[0114] The specific experimental steps are as follows:

[0115] Comparative Example 8 The kit was calibrated with the 12-level calibrator in Experimental Example 1. The standard curve results are shown in Fig.11 The levels of IL-6 in 51 EDTA anticoagulated plasma samples were detected according to the standard curves and detection methods of Example 1 and Comparative Example 8, respectively.

[0116] The test results were correlated with the sample target value (the results of the Roche kit test). Figure 1 and Figure 2shown.

[0117] Conclusion: The correlation coefficient R between the test results of the reagent prepared by the pad soaking solution formula in Example 1 and the sample target value is 2 is 0.9897, and the R 2 It is 0.9578, and the clinical correlation of Example 1 is better than that of Comparative Example 8.

[0118] In summary, the conjugate pad soaking solution of the present invention has a good effect on improving the precision of POCT reagents, has a strong improvement effect on the performance of the reagents, and has good commercial value.

[0119] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A bonding pad soaking solution, characterized in that: The invention comprises the following raw materials with final concentrations: 10-100 mM buffer, 0.1 wt%-1 wt% nonionic surfactant, 0.5 wt%-5 wt% protein protective agent, 2 wt%-10 wt% sugar, 0.5 v / v%-5 v / v% monohydric alcohol, 0.1 wt / v%-1 wt / v% polyhydric alcohol and 0.05 wt%-1 wt% preservative, wherein the buffer is selected from at least one of Tris, MOPS, MES, HEPES and Bis-tris propane, the nonionic surfactant comprises a first nonionic surfactant and a second nonionic surfactant, the first nonionic surfactant is selected from at least one of Tween-20, Tween-80 and Triton X-100, the second nonionic surfactant is selected from fluorinated surfactant, the final concentration of the first nonionic surfactant is 0.1 wt%-0.9 wt%, and the final concentration of the second nonionic surfactant is 0.005 wt%-0.05 wt%; The protein protective agent comprises a first protein protective agent and a second protein protective agent, wherein the first protein protective agent is selected from at least one of BSA, glycerol and gelatin, and the second protein protective agent is selected from at least one of caseinate sodium, SC01, SC02 and SG01; the final concentration of the first protein protective agent is 0.25wt% to 2.5wt%, and the final concentration of the second protein protective agent is 0.25wt% to 2.5wt%; The sugar is selected from at least one of sucrose and trehalose, the monohydric alcohol is selected from at least one of methanol, ethanol, propanol and butanol, and the polyhydric alcohol is selected from at least one of ethylene glycol, propylene glycol, glycerol, mannitol, xylitol and sorbitol.

2. The conjugate pad soaking solution according to claim 1, characterized in that: The method comprises the following raw materials with final concentrations: 50-100 mM buffer, 0.1 wt%-1 wt% nonionic surfactant, 0.5 wt%-5 wt% protein protective agent, 5 wt%-10 wt% sugar, 1 v / v%-5 v / v% monohydric alcohol, 0.25 wt / v%-1 wt / v% polyhydric alcohol and 0.1 wt%-1 wt% preservative.

3. The conjugate pad soaking solution according to claim 2, characterized in that: The nonionic surfactant comprises Tween-20 with a final concentration of 0.5wt% to 0.8wt% and a fluorine-containing surfactant with a final concentration of 0.01wt% to 0.05wt%.

4. The conjugate pad soaking solution according to claim 3, characterized in that: The fluorine-containing surfactant is selected from at least one of FS-10, FS-30 and FS-31.

5. The conjugate pad soaking solution according to claim 2, characterized in that: The protein protective agent comprises BSA with a final concentration of 1wt% to 1.5wt% and casein sodium with a final concentration of 1wt% to 1.5wt%.

6. The conjugate pad soaking solution according to claim 1, characterized in that: The preservative is selected from at least one of Proclin 300, thimerosal, sodium benzoate, nitrate, nitrite and sodium azide.

7. A conjugate pad working solution or a reagent kit, characterized in that: The invention comprises the conjugate pad soaking solution according to any one of claims 1 to 6 and a marker with an antibody.

8. The conjugate pad working solution or kit according to claim 7, characterized in that: The marker is selected from at least one of nanoparticle markers, fluorescent dyes, radioactive isotopes, and chemiluminescent reagents; Preferably, the nanoparticle marker is selected from nanoparticles or colloidal metals; the nanoparticles are selected from organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

9. Use of the conjugate pad soaking solution according to any one of claims 1 to 6 in preparing a conjugate pad.

10. A method for preparing a conjugate pad, characterized in that: It includes the following steps (1) or (2): (1) soaking a conjugate pad substrate with the conjugate pad soaking solution according to any one of claims 1 to 6, and after drying, spraying a marker with an antibody onto the dried conjugate pad substrate; (2) soaking the binding pad substrate with the binding pad working solution described in any one of claims 7 to 8, and drying; Preferably, the binding pad substrate is a glass fiber membrane, a cotton fiber membrane or a nitrocellulose membrane; Preferably, the drying is vacuum drying, and the vacuum drying condition is to maintain the pressure below 100 Pa for 0.5 to 1 hour.

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