A sample diluent reagent and use thereof

Abstract

The application discloses a sample diluent reagent and application thereof, and belongs to the technical field of biological medicine; the sample diluent reagent provided by the application can effectively reduce the high-dose hook effect of high-concentration sample immunoreaction, improve the accuracy of a detection result, and increase a reportable range by optimizing components in the diluent reagent, the adding amount of the components and the selection of specific components; the sample diluent reagent can maximize the avoidance of matrix effects introduced by sample dilution, improve the precision, accuracy and linear correlation of a detection result; the sample diluent reagent can maximize the degradation, neutralization or inhibition of interferents, improve the specificity of an immunoreaction detection result; the sample diluent reagent can be matched with corresponding immunoreaction reagents, can shorten the immunoreaction time, or can more accurately detect the level of a to-be-detected substance; and the sample diluent reagent can prevent the degradation of the to-be-detected substance in a diluted sample, and improve the stability of the to-be-detected substance in a storage condition and in an experimental reaction process.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a sample diluent reagent and its application. Background Technology

[0002] Sample diluents are substances used to dilute and liquefy test samples to facilitate detection using in vitro diagnostic reagents or instruments. They do not directly participate in the detection process themselves. In immunoassays, when the concentration of the analyte in the sample exceeds the linear range, but a reliable concentration of the analyte in the sample is required, or when non-specific substances interfere with the sample, the sample needs to be diluted appropriately to reduce the interference to an acceptable level before specific detection can be performed.

[0003] The most commonly used sample diluent in the past was physiological saline. However, clinical application has revealed that most commonly used sample diluents still have the following problems: (1) The matrix effect is obvious, which often leads to inaccurate detection and poor repeatability; (2) After the sample is diluted, the color of the sample itself becomes significantly lighter, and it is easy to miss the sample during the process of adding diluted sample, which is not easy to detect; (3) When the sample is diluted with some sample diluents with added pigments, the pigments in some sample diluents have a large interference with the detection when the signal value is detected by optical instruments, resulting in poor detection repeatability; (4) Some analytes for immunoassays degrade quickly after being diluted with sample diluents; (5) The sample diluents have poor versatility, which leads to the need to prepare a variety of sample diluents for immunoassay; (6) When the sample interfering substances are only diluted, it is impossible to reduce the high concentration of interfering substances to the interference limit, and there is still a large interference effect. Therefore, there is a need for an improved sample dilution reagent that has a weak matrix effect, facilitates sample addition with dye color without interfering with signal detection, is stable in diluted environments, is highly versatile, and can eliminate or reduce interference. When used in conjunction with immunoassay reagents for sample detection, this reagent can increase the reportable range of analyte detection results, improve accuracy, specificity, and precision, enhance the stability of diluted samples, facilitate the checking of missed samples, and reduce operational errors. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a sample diluent reagent and its application that is highly versatile and can effectively improve the precision, accuracy, specificity, reportable range, linear correlation, dilute sample stability, versatility and sample loading speed of immunoassay, while reducing sample loading error and signal interference.

[0005] To achieve the above objectives, in a first aspect, the present invention provides a sample diluent reagent comprising the following components at mass concentrations: 0.001‰–0.1‰ pigment, 0.0078%–10% protein stabilizer, 0.05%–5% protein, 0.02%–2% surfactant, 0.0215%–0.344% complexing agent, 0.01%–10.1% blocking agent, 0.85%–3.4% salts, and 0.01%–1% preservative. The sample diluent reagent further comprises a buffer solution with an ionic strength of 10–50 mmol / L and a pH of 7.0–7.9.

[0006] The surfactant is selected from at least two of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, stearic acid, fatty acid glycerides, fatty acid sorbitan, polysorbate, sucrose esters, long-chain fatty acid esters, fatty alcohol esters, and tritons.

[0007] The protein stabilizer is selected from at least two of glycerol, benzylamine hydrochloride, and trehalose;

[0008] The pigment is sodium erythrosine B.

[0009] This invention provides a sample diluent reagent that, through optimized selection of components, their amounts, and specific components, can achieve the following advantages during subsequent sample dilution: First, it effectively reduces the high-dose hook effect in high-concentration sample immunoreactions, improving the accuracy of detection results and expanding the reportable range. Second, it minimizes matrix effects introduced by sample dilution, improving the precision, accuracy, and linear correlation of detection results. Third, it maximizes the degradation, neutralization, or inhibition of interfering substances, improving the specificity of immunoreaction detection results. Fourth, it can be used with corresponding immunoreaction reagents, shortening the immunoreaction time or enabling more accurate detection of analyte levels. Fifth, it prevents the degradation of analytes in diluted samples, improving the stability of analytes in diluted samples under storage conditions and during experimental reactions. Specifically, for example, adding a specially selected concentration of a specific pigment to the sample diluent reagent can not only prevent missed addition of diluted samples but also avoid interference from conventional pigments on optical signals during optical detection, improving sample addition speed and the accuracy and precision of detection results.

[0010] As a preferred embodiment of the sample diluent reagent of the present invention, at least one of the following (a) to (f) is used:

[0011] (a) The protein is selected from at least one of bovine serum albumin, bovine globulin, gelatin, casein, and peptone;

[0012] (b) The complexing agent is selected from at least one of N-(P-isothiocyanobenzyl)-DTTA, N-(P-isothiocyanobenzyl)-DDTA, aminophenyl-EDTA, diethylenetriaminepentaacetic anhydride, benzyl isothiocyanate-DATT, aminophenyl-tetraazacyclododecanetetraacetic acid, 1-(P-phenylbis(2-)EDTA, and ethylenediaminetetraacetic acid;

[0013] (c) The blocking agent is selected from fetal bovine serum, mouse serum, and TRU Block. TM At least one of them;

[0014] (d) The salts are selected from at least one of sodium chloride and potassium chloride;

[0015] (e) The preservative is selected from at least one of sodium azide, sodium thiomercurate salicylate, gentamicin, Germall II, and ProClin 300;

[0016] (f) The buffer solution is selected from at least one of phosphate buffer and tris(hydroxymethyl)aminomethane-hydrochloric acid buffer.

[0017] The inventors discovered that when the selected components are of the specific types mentioned above, the resulting sample dilution reagent test samples exhibit better stability, accuracy, precision, and specificity.

[0018] Preferably, the buffer solution is a phosphate buffer with an ionic strength of 20 mmol / L and a pH of 7.4 ± 0.1, or a tris(hydroxymethyl)aminomethane-hydrochloric acid buffer with an ionic strength of 50 mmol / L and a pH of 7.8 ± 0.1.

[0019] More preferably, the buffer solution is a tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution with an ionic strength of 50 mmol / L and a pH of 7.8 ± 0.1.

[0020] Preferably, the erythrosine B sodium salt is food grade with a purity of ≥99%.

[0021] More preferably, the pigment is 0.01‰ food-grade sodium erythrosine B.

[0022] As a preferred embodiment of the sample diluent reagent of the present invention, the protein stabilizer is a mixture of glycerol and benzylamine hydrochloride.

[0023] Preferably, the mass concentration ratio of glycerol to benzalkonium hydrochloride is glycerol:benzalkonium hydrochloride = 5:0.016.

[0024] Preferably, the protein is 0.05% to 0.8% sodium caseinate.

[0025] More preferably, the protein is 0.2% sodium caseinate.

[0026] As a preferred embodiment of the sample diluent reagent of the present invention, the surfactant is at least two of sodium dodecyl sulfate, Triton, and Tween-20.

[0027] Preferably, the surfactant is a mixture of sodium dodecyl sulfate, Triton, and Tween-20.

[0028] More preferably, the mass concentration ratio of sodium dodecyl sulfate, Triton, and Tween-20 is sodium dodecyl sulfate: Triton: Tween-20 = 0.04:0.5:1.

[0029] In a preferred embodiment of the sample diluent reagent of the present invention, the complexing agent is sodium ethylenediaminetetraacetate.

[0030] Preferably, the complexing agent is 0.086% sodium ethylenediaminetetraacetate.

[0031] In a preferred embodiment of the sample diluent reagent of the present invention, the preservative is ProClin300.

[0032] Preferably, the preservative is 0.1% ProClin300.

[0033] In a preferred embodiment of the sample diluent reagent of the present invention, the blocking agent is fetal bovine serum or TRUBlock. TM At least one of them.

[0034] Preferably, the blocking agent is calf serum and TRU Block. TM A mixture.

[0035] More preferably, the calf serum and TRU Block TM The mass concentration ratio is calf serum: TRU Block TM =5:0.0005.

[0036] The inventors discovered that when the types and amounts of pigments, protein stabilizers, proteins, surfactants, complexing agents, blocking agents, salts, and preservatives are further selected within the above range, the resulting product exhibits superior sample stability and, in actual testing, better resolution, lower light interference, lower false positive rate, wider reportable range, and lower sample omission rate; that is, a superior overall effect.

[0037] As a preferred embodiment of the sample diluent reagent of the present invention, the sample diluent reagent further includes at least one of anti-human IgG, dithiothreitol, cysteine, β-mercaptoethanol, reduced glutathione, urea, guanidine hydrochloride, guanidine isothiocyanate, hydrogen peroxide, polyethylene glycol, and 8-aniline-1-naphthalenesulfonic acid.

[0038] Preferably, the hydrogen peroxide is an aqueous solution of hydrogen peroxide, and the mass percentage of hydrogen peroxide is 10% to 30%.

[0039] The inventors discovered that adding the above-mentioned substances, depending on the specific substance being tested, can make the test results more reliable and the overall effect better.

[0040] In a second aspect, the present invention provides the application of the sample diluent reagent in immunoassay.

[0041] As a preferred embodiment of the application described in this invention, the specific steps of the application are any one of the following (A) to (H):

[0042] (A) The sample to be tested is diluted with sample diluent reagent and then added to a solid-phase carrier coated with antigen or antibody for reaction, and then immunoassay is performed;

[0043] (B) Add the sample to be tested to a solid-phase carrier coated with antigen or antibody, and simultaneously add sample dilution reagent to react, and then perform immunoassay.

[0044] (C) Add the sample to be tested to a solid-phase carrier coated with antigen or antibody, and simultaneously add the working reagent of the detection antigen or antibody diluted with the sample dilution reagent to react, and then perform immunoassay.

[0045] (D) After adding the sample to be tested to a solid-phase carrier coated with antigen or antibody and reacting, wash the sample, then add the working reagent of the detection antigen or antibody diluted with the sample dilution reagent and react, and then perform immunoassay.

[0046] (E) Add sample dilution reagent to a solid-phase carrier coated with antigen or antibody, and add an appropriate volume of sample to react, and then perform immunoassay.

[0047] (F) The sample to be tested is diluted with sample diluent reagent and added to a solid-phase carrier coated with antigen or antibody. Sample diluent reagent is added at the same time to react and then immunoassay is performed.

[0048] (G) The bridging antigen or antibody diluted with the sample diluent reagent is added to a solid-phase carrier coated with a bridging antigen or antibody, reacted, washed, and then the sample to be tested diluted with the sample diluent reagent is added to the solid-phase carrier bound with the bridging antigen or antibody for reaction, and then immunoassay is performed.

[0049] (H) Add the sample to a solid-phase carrier coated with antigen or antibody, along with sample diluent reagent. After washing for an appropriate reaction time, add an appropriate amount of bridging antigen or antibody diluted with sample diluent reagent to react, and then perform immunoassay.

[0050] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0051] The sample diluent reagent provided by this invention has strong versatility and can effectively improve the precision, accuracy, specificity, reportable range, linear correlation, dilute sample stability, versatility and sample addition speed of immunoassay, while reducing sample addition error and signal interference. Furthermore, the sample diluent reagent provided by this invention can be widely used in immunoassay and can be applied in different ways, thus having a wide range of applications. Detailed Implementation

[0052] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0053] Unless otherwise specified, the reagents, methods and equipment used in this invention are all conventional reagents, methods and equipment in the field.

[0054] Buffer 1: Phosphate buffer with an ionic strength of 20 mmol / L and a pH of 7.4 ± 0.1;

[0055] Buffer 2: Tris(hydroxymethyl)aminomethane-hydrochloric acid buffer with an ionic strength of 50 mmol / L and a pH of 7.8 ± 0.1;

[0056] Pigment 1: Sodium erythrosine B, food grade, purity ≥99%;

[0057] Pigment 2: Brilliant Blue, food grade, purity ≥99%;

[0058] Pigment 3: Sodium eosin;

[0059] Pigment 4: Sunset Yellow;

[0060] Protein stabilizer 1: a mixture of glycerol and benzylamine hydrochloride, with a mass concentration ratio of glycerol to benzylamine hydrochloride of 5:0.016;

[0061] Protein stabilizer 2: benzalkonium hydrochloride;

[0062] Protein stabilizer 3: Glycerol;

[0063] Protein stabilizer 4: Trehalose;

[0064] Protein stabilizer 5: a mixture of glycerol and trehalose, with a mass concentration ratio of glycerol to trehalose of 5:0.016;

[0065] Surfactant 1: A mixture of Tween-20, sodium dodecyl sulfate, and X-100 Triton, wherein the mass concentration ratio of Tween-20, sodium dodecyl sulfate, and X-100 Triton is Tween-20:sodium dodecyl sulfate:X-100 Triton = 1:0.04:0.5;

[0066] Surfactant 2: A mixture of sodium dodecyl sulfate and X-100 Triton, with a mass concentration ratio of sodium dodecyl sulfate to X-100 Triton of 0.04:0.5.

[0067] Surfactant 3: A mixture of Tween-20 and X-100 Trilaton, with a mass concentration ratio of Tween-20:X-100 Trilaton = 1:0.5;

[0068] Surfactant 4: A mixture of Tween-20 and sodium dodecyl sulfate, with a mass concentration ratio of Tween-20 to sodium dodecyl sulfate of 1:0.04.

[0069] Surfactant 5: Tween-20;

[0070] Surfactant 6: Sodium dodecyl sulfate;

[0071] Surfactant 7: X-100 Triton;

[0072] Surfactant 8: Sodium dodecylbenzenesulfonate;

[0073] Surfactant 9: Stearic acid;

[0074] Surfactant 10: Fatty acid glycerides;

[0075] Surfactant 11: Sorbitan fatty acid;

[0076] Surfactant 12: Sucrose ester;

[0077] Surfactant 13: Long-chain fatty acid ester (Myrij);

[0078] Surfactant 14: Fatty alcohol ester (Brij);

[0079] Surfactant 15: A mixture of Tween-20, sodium lauryl sulfate, and stearic acid, wherein the mass concentration ratio of Tween-20, sodium lauryl sulfate, and stearic acid is Tween-20:sodium lauryl sulfate:stearic acid = 1:0.04:0.5;

[0080] Protein 1: Sodium caseinate;

[0081] Salt 1: Sodium chloride;

[0082] Complexing agent 1: Disodium ethylenediaminetetraacetate;

[0083] Complexing agent 2: N-(P-isothiocyanobenzyl)-DTTA;

[0084] Complexing agent 3: N-(P-isothiocyanobenzyl)-DDTA;

[0085] Complexing agent 4: aminophenyl-EDTA;

[0086] Complexing agent 5: Diethylenetriaminepentaacetic anhydride;

[0087] Complexing agent 6: Benzyl isothiocyanate-DATT;

[0088] Complexing agent 7: aminophenyl-tetraazacyclododecanetetraacetic acid;

[0089] Complexing agent 8: 1-(P-phenylbisazone)-EDTA;

[0090] Preservative 1: Procline 300;

[0091] Preservative 2: Germall II;

[0092] Blocker 1: Fetal bovine serum and TRU Block TM A mixture of calf serum and TRU Block TM The mass concentration ratio is calf serum: TRU Block TM = 5:0.0005, of which the protein content in calf serum is 6%;

[0093] Blocker 2: Fetal bovine serum and TRU Block TM A mixture of calf serum and TRU Block TM The mass concentration ratio is calf serum: TRU Block TM = 10:0.0005, of which the protein content in calf serum is 3%;

[0094] Blocker 3: Fetal bovine serum and TRU Block TM A mixture of calf serum and TRU Block TMThe mass concentration ratio is calf serum: TRU Block TM = 3:0.0005, of which the protein content in calf serum is 10%;

[0095] Blocker 4: Fetal bovine serum and TRU Block TM A mixture of calf serum and TRU Block TM The mass concentration ratio is calf serum: TRU Block TM = 5:0.0001, of which the protein content in calf serum is 6%;

[0096] Blocker 5: Fetal bovine serum and TRU Block TM A mixture of calf serum and TRU Block TM The mass concentration ratio is calf serum: TRU Block TM = 5:0.001, of which the protein content in calf serum is 6%;

[0097] Blocker 6: Fetal bovine serum and TRU Block TM A mixture of calf serum and TRU Block TM The mass concentration ratio is calf serum: TRU Block TM = 5:0.0005, of which the protein content in calf serum is 2%;

[0098] Blocker 7: Fetal bovine serum and TRU Block TM A mixture of calf serum and TRU Block TM The mass concentration ratio is calf serum: TRU Block TM = 5:0.0005, of which the protein content in calf serum is 15%;

[0099] Blocker 8: Fetal bovine serum and TRU Block TM A mixture of calf serum and TRU Block TM The mass concentration ratio is calf serum: TRU Block TM = 5:0.00005, of which the protein content in calf serum is 6%;

[0100] Blocker 9: Fetal bovine serum and TRU Block TM A mixture of calf serum and TRU Block TM The mass concentration ratio is calf serum: TRU Block TM = 5:0.0015, of which the protein content in calf serum is 6%;

[0101] Example 1

[0102] This invention provides a sample diluent reagent comprising the following components at the following mass concentrations: 0.01‰ pigment 1, 5.016% protein stabilizer 1, 1.54% surfactant 1, 0.2% protein 1, 0.85% sodium chloride, 0.086% complexing agent 1, 0.1% preservative 1, and 5.0005% blocking agent. The sample diluent reagent also includes a buffer solution 1.

[0103] Example 2

[0104] This invention provides a sample diluent reagent, the only difference between the sample diluent reagent and that of Example 1 is that buffer 2 is used instead of buffer 1.

[0105] Comparative Example 1

[0106] The present invention provides a sample diluent reagent in a comparative example, the only difference between the sample diluent reagent and Example 2 is that no pigment is added.

[0107] Comparative Example 2

[0108] The present invention provides a sample diluent reagent in a comparative example, the only difference between the sample diluent reagent and Example 2 is that no protein stabilizer is added.

[0109] Comparative Example 3

[0110] The present invention provides a sample diluent reagent in a comparative example, the only difference between the sample diluent reagent and that in Example 2 is that no surfactant is added.

[0111] Comparative Example 4

[0112] The present invention provides a sample diluent reagent in a comparative example, the only difference between the sample diluent reagent and that in Example 2 is that no protein is added.

[0113] Comparative Example 5

[0114] The present invention provides a sample diluent reagent in a comparative example, the only difference between the sample diluent reagent and Example 2 is that no salts are added.

[0115] Comparative Example 6

[0116] The present invention provides a sample diluent reagent in a comparative example, the only difference between the sample diluent reagent and that in Example 2 is that no complexing agent is added.

[0117] Comparative Example 7

[0118] The present invention provides a sample diluent reagent in a comparative example, the only difference between the sample diluent reagent and Example 2 is that no preservative is added.

[0119] Comparative Example 8

[0120] The present invention provides a sample diluent reagent in a comparative example, the only difference between the sample diluent reagent and that in Example 2 is that no blocking agent is added.

[0121] Examples 3-35 and Comparative Examples 9-32

[0122] Examples 3-35 and Comparative Examples 9-32 of this invention provide a sample diluent reagent, the only difference between the sample diluent reagent and Example 2 is the difference in the component types and / or mass concentrations listed in Table 1;

[0123] Table 1

[0124]

[0125]

[0126]

[0127]

[0128] Examples 36-42

[0129] Examples 36-42 of this invention provide a sample diluent reagent, wherein the sample diluent reagent is based on Example 2 with the addition of substances of the concentrations shown in Table 2;

[0130] Table 2

[0131] Example 36 5 μg / mL anti-human IgG Example 40 17% hydrogen peroxide Example 37 1 mol / L dithiothreitol Example 41 1.75% polyethylene glycol 6000 Example 38 1‰ β-mercaptoethanol Example 42 1 mol / L 8-anilino-1-naphthalenesulfonic acid Example 39 8mol / L urea

[0132] Example 1

[0133] The effectiveness of this invention is verified by examples 1-35 and comparative examples 1-32 in immunoassay. The hepatitis C virus antibody detection kit (time-resolved fluorescence immunoassay) was used for qualitative detection of hepatitis C virus antibodies (anti-HCV) in human serum samples, specifically including the following steps:

[0134] Add 50 μL of anti-HCV calibrator and the test sample sequentially to the wells of a microplate, and add 50 μL of sample dilution reagent to each well. Cover with a sealing film. Incubate the microplate at room temperature with gentle shaking for 45 min. After the first incubation, carefully remove and discard the sealing film, wash 5 times with a plate washer, and pat dry. Add 100 μL of biotin-labeled antigen working solution diluted with the sample dilution reagent to each well, and cover with a sealing film. Incubate the microplate at room temperature with gentle shaking for 45 min. After the second incubation, carefully remove and discard the sealing film, wash 5 times with a plate washer, and pat dry. Add 100 μL of prepared europium-labeled working solution to each well, and cover with a sealing film. Incubate the microplate at room temperature with gentle shaking for 15 min. After the third incubation, carefully remove and discard the sealing film, wash 5 times with a plate washer, and pat dry. Add 100 μL of enhancement solution to each well. After slowly shaking the microwell reaction strip at room temperature for 5 min, perform the analysis. The analysis should be completed within 30 min.

[0135] The performance indicators are as follows:

[0136] (1) Background: The fluorescence value of calibrator A should be less than 1,000. The lower the background, the better the detection background.

[0137] (2) Resolution: The ratio of the fluorescence value of calibrator B to the fluorescence value of calibrator A. The larger the ratio, the better the resolution and the better the sensitivity.

[0138] (3) Light interference: The adsorption of certain substances can easily cause non-specific infection when using optical principles for detection. The lower the coefficient of variation (CV) of the repeatability test results, the less light interference and the stronger the specificity.

[0139] (4) Sample stability: The sample diluted with the sample dilution reagent was frozen at -20℃, or frozen at -20℃ and then repeatedly frozen and thawed, or stored at room temperature (25℃). The change rate was within ±10% compared with the control (freshly diluted sample), indicating that it was stable. If the stability time of freezing at -20℃ was longer, the number of repeated freeze-thaw cycles under stable results was more, and the stability time of storage at room temperature was longer, the sample degradation was slower, indicating that the sample dilution reagent had a better effect on sample stability.

[0140] (5) False positive rate: The number of false positives generated when testing negative samples (1000 cases). The lower the false positive rate, the better the specificity.

[0141] (6) Linear range: The linear range refers to the range of changes in the concentration of the test substance that meets the requirements of precision and accuracy when using a method. The wider the linear range, the wider the range that can be accurately quantified.

[0142] (7) Reportable range: If the concentration of the analyte in the sample exceeds the linear range, the sample can be pretreated by dilution, concentration, etc., to bring the concentration of the analyte within the analytical measurement range, and then multiplied by the dilution or concentration factor. The wider the reportable range, the wider the range of analyte concentrations that are clinically and diagnostically significant.

[0143] The results are shown in Table 3.

[0144] Table 3

[0145]

[0146]

[0147]

[0148] As can be seen from Table 3, when the technical solution of the present invention is adopted, the overall test indicators are excellent, the background is below 865, the light interference is below 6.64%, it can be stable for more than 18 months after freezing at -20℃, or can be repeatedly frozen and thawed more than 4 times after freezing at -20℃, or can be stored at room temperature (25℃) for more than 18 hours, the false positive rate is below 2%, the sample omission rate is below 1‰, the linear range is between 0.05 and 4 NCU / mL, and the reportable range is between 0.032 and 320 NCU / mL.

[0149] As can be seen from Table 3:

[0150] Tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (buffer 2) with an ionic strength of 50 mmol / L and a pH of 7.8 ± 0.1 significantly reduced background, improved resolution and precision, and reduced false positive rate compared to phosphate buffer (buffer 1) with an ionic strength of 20 mmol / L and a pH of 7.4 ± 0.1.

[0151] For pigments: 0.01‰ of food-grade erythrosine B sodium salt (purity ≥99%) can prevent missed addition of diluted samples and avoid interference from conventional pigments on optical signals when using optical principles for detection, thus improving the sample addition speed and the accuracy and precision of the detection results. When the content of food-grade erythrosine B sodium salt in Example 3 is reduced to 0.001‰, the color of the sample diluent reagent becomes lighter compared to Example 2, so missed addition of samples will begin to occur when used for sample dilution. When the content of food-grade erythrosine B sodium salt in Comparative Example 9 is increased to 0.15‰, the background will increase significantly, reaching 1084 CPs, and the resolution and precision will decrease accordingly. When 0.01‰ of food-grade brilliant blue, 0.01‰ of eosin, and 0.01‰ of sunset yellow were used in Comparative Examples 10–12, the light interference was very large (the coefficient of variation was between 12.34% and 15.66%), and false positives occurred. When no pigment was added in Comparative Example 1, the omission rate increased significantly, reaching 5‰.

[0152] Regarding protein stabilizers: Protein stabilizer 1 is a mixture of glycerol and benzalkonium hydrochloride, with a mass concentration ratio of 5:0.016. At a mass concentration of 5.016%, protein stabilizer 1 significantly improves sample stability and reduces the false positive rate, indicating a synergistic effect between glycerol and benzalkonium hydrochloride. In Example 6, when the mass concentration of protein stabilizer 1 was reduced to 0.009%, resolution and sample stability decreased, and precision slightly decreased. In Example 7, when protein stabilizer 1 was increased to 9.8%, resolution and precision slightly decreased. However, in Comparative Example 13, when protein stabilizer 1 was increased to 15%, resolution significantly decreased, precision slightly decreased, and the reportable range narrowed. In Comparative Example 2, without protein stabilizer, sample stability was significantly reduced; it remained stable for only 6 months after freezing at -20°C, or only twice after repeated freeze-thaw cycles at -20°C, or only for 8 hours at room temperature (25°C).

[0153] Regarding the surfactant: Surfactant 1 is a mixture of Tween-20, sodium dodecyl sulfate, and X-100 Triton, with a mass concentration ratio of Tween-20:sodium dodecyl sulfate:X-100 Triton = 1:0.04:0.5. At a mass concentration of 1.54% surfactant 1, resolution and precision are significantly improved, particularly the false positive rate is reduced, indicating a synergistic effect among the three surfactants. In Example 12, when the mass concentration of surfactant 1 is reduced to 0.04%, false positives occur. However, in Comparative Example 14, when the mass concentration of surfactant 1 is increased to 4%, resolution significantly decreases, and the reportable range significantly narrows. In Comparative Example 3, without surfactant, the background is significantly increased, resolution and precision are significantly decreased, the false positive rate is significantly increased, and the reportable range is significantly narrowed.

[0154] For proteins, protein 1: sodium caseinate at a mass concentration of 0.05%–5% showed better resolution, precision, and a lower false positive rate compared to 8% concentration. In comparative example 4, without added protein, the background was significantly increased, resolution and precision were significantly decreased, the false positive rate was significantly increased, and the reportable range was significantly narrowed.

[0155] For salts, salt 1: sodium chloride at a mass concentration of 0.85%–3.4% showed better resolution and precision than at 4.25%, but the reporting range was significantly narrower. In Comparative Example 5, without salts, the background was significantly higher, the precision slightly lower, and the false positive rate significantly higher.

[0156] For the complexing agents, when the mass concentration of complexing agent 1 (disodium EDTA) is 0.0215%–0.344%, it exhibits better precision and a lower false positive rate compared to complexing agents 2, 3, 4, 5, 6, 7, and 8. In comparative example 6, without the addition of a complexing agent, the background is significantly increased, the resolution is significantly decreased, the precision is significantly reduced, and the false positive rate is increased.

[0157] Regarding preservatives, preservative 1: Procline 300 had a lower false positive rate than preservative 2. When no preservative was added in comparative example 7, the sample stability was significantly reduced.

[0158] The blocking agents are fetal bovine serum and TRU Block. TM The mixture must contain at least 3% calf serum albumin, and TRU Block must be present. TM The concentration should not be lower than 0.0001%; ​​if it is lower, the false positive rate increases. When the concentration of the blocking agent is 12%, the resolution decreases significantly, and the reportable range narrows significantly. In Comparative Example 8, without the addition of the blocking agent, the background is significantly increased, the resolution is significantly decreased, the precision is significantly decreased, and the false positive rate increases.

[0159] In other words, regardless of the absence of any component in this invention or the fact that the component type or mass percentage of the component is outside the scope of this invention, the overall effect of the obtained sample dilution is significantly reduced, such as decreased stability, increased false positive rate, increased background, and reduced reportable range.

[0160] Example 2

[0161] The effectiveness examples of this invention verify the application of the sample diluent reagents of Examples 1 and 2 in immunoassay, specifically as follows:

[0162] The Pregnancy-Related Plasma Protein A Assay Kit (Time-Resolved Fluorescence Immunoassay) is used to quantitatively detect the level of pregnancy-related plasma protein A in the serum samples of pregnant women. Clinically, it is mainly used to assess the risk of Down syndrome in the first trimester of pregnancy. Before testing, the serum sample should be diluted with sample diluent at a ratio of 1:5, for example, 50 μL of sample should be added to 200 μL of sample diluent. Add 100 μL of the antibody working solution diluted with sample diluent to the microwells of the solid-phase carrier. Cover the reaction plate with a sealing film and incubate at room temperature for 30 min (±10 min), shaking gently with a low-speed shaker. Remove and discard the sealing film, wash the reaction plate twice, and blot dry. Add 100 μL of the prepared label working solution and 50 μL of calibrator or serum sample diluted with sample diluent to each well. Cover the reaction plate with a sealing film and shake at room temperature for 2 h (±10 min). Remove and discard the sealing film, wash the reaction plate 6 times, and blot dry. Add 200 μL of enhancement solution to each well. Apply a sealing film to the microplate and shake it at room temperature for 5 minutes. Remove and discard the sealing film, and measure the results on a suitable instrument. The test should be completed within 30 minutes. Dilute serum samples 1:5; the final concentration result needs to be multiplied by the dilution factor when back-calculating.

[0163] The results are shown in Table 4.

[0164] Table 4

[0165]

[0166]

[0167] The effects of Examples 1 and 2 were compared, and the results of undiluted samples were also compared. Table 4 shows that when the samples were diluted with hormone-free serum matrix, 20 mmol / L phosphate buffer (pH 7.4 ± 0.1), and 50 mmol / L tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (pH 7.8 ± 0.1), the bias between sample test results did not exceed ±10%, the correlation was greater than 0.9900, and there was no statistically significant difference after paired t-test (P > 0.05). However, undiluted sample testing showed a significant high-dose hook effect, with high-concentration samples showing significantly lower test results. Therefore, 20 mmol / L phosphate buffer (pH 7.4 ± 0.1) and 50 mmol / L tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (pH 7.8 ± 0.1) are equivalent sample diluents. The prepared sample diluent reagents are matrix interchangeable with the hormone serum matrix. Diluting the samples before testing can reduce the high-dose hook effect, resulting in more accurate test results and expanding the reportable range.

[0168] Example 3

[0169] The effectiveness examples of this invention verify the application of the sample diluent reagents prepared in Example 2, Comparative Example 1, and Comparative Examples 10-12 in immunoassay, specifically as follows:

[0170] The Free β-human chorionic gonadotropin (FCG) Assay Kit (Time-Resolved Fluorescence Immunoassay) is used to quantitatively detect the content of free β-human chorionic gonadotropin in human serum. Pipette 25 μL of Fβ-hCG calibrator or test sample, and simultaneously add 100 μL of the labeled working solution diluted with sample diluent to each well, then seal with a sealing film. Incubate the microplate strips at room temperature with gentle shaking for 1.5 h. After incubation, wash 6 times with a plate washer and blot dry. After washing, add 100 μL of enhancement solution to each well. Incubate the microplate strips at room temperature with gentle shaking for 5 min before testing; the detection should be completed within 30 min.

[0171] The results are shown in Table 5.

[0172] Table 5

[0173]

[0174] As shown in Table 5, Brilliant Blue, Eosin Sodium, and Sunset Yellow are easily affected by light, and their coefficients of variation (CV) under light are significantly higher than those of Erysrubin B Sodium. Adding Erysrubin B Sodium to the sample diluent allows for color-coded identification, preventing missed samples and improving sample addition efficiency.

[0175] Example of effect 4

[0176] The application of the sample diluent reagents prepared in Example 2, Comparative Example 8, and Comparative Example 19 in immunoassay is verified by the following examples:

[0177] The Alpha-Fetoprotein (AFP) Assay Kit (Time-Resolved Fluorescence Immunoassay) is used to quantitatively determine the AFP content in human serum. Pipettes 25 μL of AFP calibrator or test sample, and simultaneously adds 100 μL of the labeled working solution diluted with sample diluent to each well, then seals the plate. Incubate the microplate at room temperature with gentle shaking for 1.5 h. After incubation, wash 6 times with a plate washer and blot dry. After washing, add 100 μL of enhancement buffer to each well. Incubate the microplate at room temperature with gentle shaking for 5 min before testing; the assay should be completed within 30 min.

[0178] The results are shown in Table 6.

[0179] Table 6

[0180] Background 1014 1056 1027 False positive 0 0.5‰ 0 Precision 2.56％ 2.68％ 8.64％

[0181] As can be seen from Table 6, calf serum and TRU Block™ significantly improved the specificity of the test results and reduced false positives.

[0182] Example 5

[0183] The application of the sample diluent reagents prepared in Examples 2 and 36 in immunoassay is verified by the following examples:

[0184] The Rubella Virus IgM Antibody Assay Kit (Time-Resolved Fluorescence Immunoassay) is used for the qualitative detection of rubella virus IgM antibodies in serum samples. Dilute the blood sample and sample diluent at a volume ratio of 1:100 (e.g., add 10 μL of the test sample to 1 mL of sample diluent). Add 100 μL of rubella virus IgM antibody calibrator, control, and the diluted test sample sequentially to the wells of a microplate, and attach a sealing film. Incubate the microplate at room temperature with gentle shaking for 45 min. After the first incubation, carefully remove and discard the sealing film, wash the plate 5 times with a plate washer, and pat dry. Add 100 μL of the prepared biotin-labeled antigen and europium-labeled working solution to each well, and attach a sealing film. Incubate the microplate at room temperature with gentle shaking for 45 min. After the second incubation, carefully remove and discard the sealing film, wash the plate 5 times with a plate washer, and pat dry. Add 100 μL of enhancement solution to each well. After slowly shaking the microwell reaction strip at room temperature for 5 min, perform the analysis. The analysis should be completed within 30 min.

[0185] The results are shown in Table 7.

[0186] Table 7

[0187]

[0188]

[0189] As can be seen from Table 7, adding anti-human IgG to the test based on Example 2 can effectively improve the sensitivity, specificity and precision of rubella virus IgM antibody detection.

[0190] Example 6

[0191] The application of the sample diluent reagents prepared in Examples 2 and 37-38 of this invention in immunoassay is verified by the following examples:

[0192] The Hepatitis B Virus Core Antibody Assay Kit (Time-Resolved Fluorescence Immunoassay) is used to quantitatively detect the content of Hepatitis B Virus Core Antibody (anti-HBc) in human serum samples. Add 25 μL of sample diluent to each well of the reaction plate containing the sample to be tested. Add 50 μL of anti-HBc calibrator or the sample to be tested to each well, along with 50 μL of neutralizing antigen and a sealing film. Incubate the reaction plate at room temperature with gentle shaking for 45 min. After the first incubation, carefully remove and discard the sealing film, wash the plate four times with a plate washer, and pat dry. Add 100 μL of prepared label working solution to each well and a sealing film. Incubate the reaction plate at room temperature with gentle shaking for 45 min. After the second incubation, carefully remove and discard the sealing film, wash the plate six times with a plate washer, and pat dry. Add 100 μL of enhancement solution to each well. The microporous reaction strips were slowly shaken at room temperature for 5 minutes before being tested on the instrument, and the test was completed within 30 minutes.

[0193] The results are shown in Table 8.

[0194] Table 8

[0195] Negative concordance rate (550 samples) 98％ 100％ 100％ Positive concordance rate (550 samples) 100％ 100％ 100％

[0196] As can be seen from Table 8, adding dithiothreitol or β-mercaptoethanol to the assay for hepatitis B virus core antibody based on Example 2 can effectively improve the specificity.

[0197] Example 7

[0198] The application of the sample diluent reagents prepared in Examples 2 and 39 of this invention in immunoassay is verified by the following examples:

[0199] The Rubella Virus IgG Antibody Assay Kit (Time-Resolved Fluorescence Immunoassay) is used for the qualitative or quantitative detection of rubella virus IgG antibodies in serum samples. Dilute the blood sample and sample diluent at a volume ratio of 1:100 (e.g., add 10 μL of the test sample to 1 mL of sample diluent). Add 100 μL of rubella virus IgG antibody calibrator, control, and the diluted test sample sequentially to the wells of a microplate, and attach a sealing film. Incubate the microplate at room temperature with gentle shaking for 45 min. After the first incubation, carefully remove and discard the sealing film, wash the plate 5 times with a plate washer, and blot dry. Add 100 μL of prepared europium-labeled working solution to each well, and attach a sealing film. Incubate the microplate at room temperature with gentle shaking for 45 min. After the second incubation, carefully remove and discard the sealing film, wash the plate 5 times with a plate washer, and blot dry. Add 100 μL of enhancement solution to each well. The microporous reaction strips were slowly shaken at room temperature for 5 minutes before being tested on the instrument, and the test was completed within 30 minutes.

[0200] The results are shown in Table 9.

[0201] Table 9

[0202] RV IgM- / IgG+(120 cases) 0 6％ 94％ RV IgM+ / IgG+(102 cases) 23％ 6％ 71％

[0203] As shown in Table 9, by combining the detection results of Example 39 and Example 2, the affinity index (AI) was calculated as (result with protein denaturant / result without protein denaturant) × 100%. AI < 30% indicates low affinity antibody, interpreted as primary infection; AI between 30% and 50% indicates intermediate affinity antibody, interpreted as suspected primary infection; and AI > 50% indicates high affinity antibody, interpreted as past infection (including viral activation or reinfection). Adding urea to the antibody detection results of rubella virus IgG in Example 2, combined with the results of Example 2, effectively differentiates between primary and past rubella virus infections.

[0204] Example 8

[0205] The application of the sample diluent reagents prepared in Examples 2 and 40 of this invention in immunoassay is verified by the following examples:

[0206] The Inhibin A Assay Kit (Time-Resolved Fluorescence Immunoassay) is used for the in vitro quantitative detection of inhibin A levels in human serum samples. The sample and sample diluent are mixed at a 4:1 ratio. 100 μL of INHIBIN-A calibrator and the treated sample are added, along with 100 μL of prepared europium-labeled working solution to each well. The mixture is then mixed thoroughly and sealed. The microplate is incubated at room temperature with gentle shaking for 4 hours. After incubation, the microplate is washed 6 times and patted dry. After washing, 100 μL of enhancement solution is added to each well. The microplate is then incubated at room temperature with gentle shaking for 5 minutes before being analyzed. The analysis is completed within 30 minutes.

[0207] The results are shown in Table 10.

[0208] Table 10

[0209] Reactivity (mean fluorescence value) 3456 648732 3548 Correlation 0.3178 0.9856 0.3621

[0210] As shown in Table 10, serum samples from the second trimester, treated with the sample diluent of Example 2 and without sample diluent, exhibited low reactivity (fluorescence value) and low correlation with commercially accepted methods. Serum samples from the second trimester, treated with the sample diluent of Example 40, showed high reactivity and good correlation with commercially accepted methods. This indicates that adding hydrogen peroxide to the results from Example 2 can improve the correlation between inhibin A assay results and commercially accepted methods.

[0211] Example 9

[0212] The application of the sample diluent reagents prepared in Examples 2 and 41 of this invention in immunoassay is verified by the following examples:

[0213] The Neonatal Thyroid-Stimulating Hormone Assay Kit (Time-Resolved Fluorescence Immunoassay) is used to quantitatively determine the thyroid-stimulating hormone (TSH) content in dried blood samples taken from neonatal filter paper. Using a punch, 3 mm (1 / 8 inch) diameter pieces of the calibrator, control, and test sample are punched from the circular spots on the filter paper of the Neo TSH blood smear calibrator, control, and test sample (the punched paper pieces must be soaked in blood). These pieces are then placed sequentially into a microplate, one piece per well. 150 μL of the labeled working solution prepared with sample dilution is added to each well, and a sealing film is attached. The microplate strips are incubated at room temperature by rapid shaking for 15 min, then allowed to stand overnight at 2–8°C, followed by slow shaking for 60 min at room temperature the next day (overnight incubation method); or by rapid shaking for 15 min, followed by slow shaking for 4 h at room temperature (rapid incubation method). After incubation, the filter paper pieces are removed, washed 6 times with a plate washer, and then patted dry. After washing, add 150 μL of enhancement solution to each well. The microwell reaction strips are then slowly shaken at room temperature for 10 min before being tested. The test is completed within 30 min.

[0214] The results are shown in Table 11.

[0215] Table 11

[0216] After standing overnight at 2–8°C, the reaction proceeds to room temperature. 3h 1h room temperature reaction 8h 4h Coefficient of variation (CV) 10.98％ 5.98％

[0217] As can be seen from Table 11, adding polyethylene glycol 6000 to the neonatal thyroid-stimulating hormone (TSH) assay based on step 2 significantly shortened the reaction saturation time and improved the precision.

[0218] Example 10

[0219] The application of the sample diluent reagents prepared in Examples 2 and 42 of this invention in immunoassay is verified by the following examples:

[0220] The Thyroxine Assay Kit (Time-Resolved Fluorescence Immunoassay) is used for the in vitro quantitative detection of thyroxine levels in human serum samples. Add 25 μL of T4 calibrator and the sample to be tested sequentially to the wells of a microplate. Add 150 μL of europium-labeled working solution diluted with sample diluent to each well, and seal the plate. Incubate at room temperature with gentle shaking for 60 min. After incubation, wash the plate 6 times with a plate washer and blot dry. Add 200 μL of enhancement solution to each well and gently shake at room temperature for 5 min. Perform the assay on the microplate; the detection should be completed within 30 min.

[0221] The results are shown in Table 12;

[0222] Table 12

[0223] Accuracy -55％～-26％ 0.56％～7.65％ Linear correlation 0.5364 0.9876

[0224] As can be seen from Table 12, the addition of 8-aniline-1-naphthalenesulfonic acid to the assay for thyroxine based on step 2 resulted in high accuracy of the test samples and better linear correlation with the results of commercially recognized methods.

[0225] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A sample diluent reagent, characterized in that, The sample diluent reagent comprises the following components at mass concentrations: 0.001‰~0.1‰ pigment, 0.0078%~10% protein stabilizer, 0.05%~5% protein, 0.02%~2% surfactant, 0.0215%~0.344% complexing agent, 0.01%~10.1% blocking agent, 0.85%~3.4% salts, and 0.01%~1% preservative. The sample diluent reagent also includes a buffer solution with an ionic strength of 10~50 mmol / L and a pH of 7.0~7.

9. The surfactant is a mixture of sodium dodecyl sulfate, X-100 Triton, and Tween-20; The protein stabilizer is a mixture of glycerol and benzylamine hydrochloride; The protein is sodium caseinate; The complexing agent is disodium ethylenediaminetetraacetate; The pigment is sodium erythrosine B salt; The blocking agent is fetal bovine serum and TRU Block. TM A mixture; The salt is sodium chloride; The preservative is Procline 300; The buffer solution is a phosphate buffer.

2. The sample diluent reagent according to claim 1, characterized in that, The sample diluent reagent also includes at least one of the following: anti-human IgG, dithiothreitol, cysteine, β-mercaptoethanol, reduced glutathione, urea, guanidine hydrochloride, guanidine isothiocyanate, hydrogen peroxide, polyethylene glycol, and 8-aniline-1-naphthalenesulfonic acid.

3. The use of the sample diluent reagent as described in any one of claims 1-2 in immunoassay.

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