Sample diluent as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological detection, in particular to a sample diluent as well as a preparation method and application thereof. The sample diluent comprises a dilution component, and the dilution component comprises the following raw materials in percentage by mass: 0.005%-0.02% of thermally denatured immunoglobulin G, 0.5%-2.0% of a protein protective agent, 0.5%-1.5% of a metal ion chelating agent, 0.1%-0.5% of an ampholytic surfactant and 0.5%-2.0% of a sugar alcohol compound. According to the sample diluent, through the interaction of all the components, the ability of the sample diluent to reduce the matrix effect of serum and plasma samples can be remarkably improved, so that the interference of non-specific reaction and matrix effect of serum and plasma on the detection process is reduced, and the accuracy of serum and plasma sample detection can be improved; and a more reliable and accurate experiment result can be obtained.

Description

Technical Field

[0001] The present application relates to the technical field of biological detection, and particularly relates to a sample diluent, a preparation method thereof, and an application thereof. Background Art

[0002] Enzyme-Linked Immunosorbent Assay (ELISA) is an enzyme immunoassay developed after immunofluorescence and radioimmunoassay techniques. It has the characteristics of sensitivity, specificity, economy, simplicity, safety, etc. Currently, ELISA is the most commonly used detection method in immunology laboratories and has been widely used in the fields of biomedical research and the diagnosis and treatment of clinical diseases. At present, the main detection methods of ELISA are two mainstream methodologies, namely the competitive method and the double antibody sandwich method. Among them, the general process of the double antibody sandwich method is: binding a specific antibody to a solid phase carrier to form a solid phase antibody, then binding with the corresponding antigen in the test sample to form an immune complex, subsequently adding a biotinylated antibody, which will bind with the antigen of the immune complex to form a biotinylated antibody-antigen-solid phase antibody complex, then adding HRP enzyme-labeled avidin, and finally adding TMB substrate for color development. The antigen content in the sample can be quantitatively analyzed through color labeling. In the ELISA test, serum or plasma is the most commonly used specimen for various types of ELISA tests and other tests to detect various antigens or antibodies in the blood. Currently, the commonly used diluents for serum or plasma specimens are usually at least one of PBS buffer, TRIS buffer, and HEPES buffer, and then mixed with BSA with a mass concentration of 0.5% - 2%, Tween-20 with a mass concentration of 0.05% - 0.1%, and ProClin300 with a mass concentration of 0.05% - 0.1%. Such diluents have the advantages of easy preparation, low cost, stable batches, and easy storage.

[0003] However, at the present stage, the chemical components of serum and plasma samples are relatively complex. Using such sample diluents cannot effectively reduce the matrix effect of serum and plasma samples, and the accuracy of the results is often affected by the matrix in serum and plasma samples to varying degrees. Summary of the Invention

[0004] The present application provides a sample diluent, a preparation method thereof, and an application thereof to solve the following technical problem: how to improve the ability of the sample diluent to reduce the matrix effect of serum and plasma samples.

[0005] In a first aspect, the present application provides a sample diluent, and the sample diluent includes a dilution component. In terms of mass fraction, the raw materials of the dilution component satisfy: heat-denatured immunoglobulin G: 0.005% - 0.02%, protein protectant: 0.5% - 2.0%, metal ion chelating agent: 0.5% - 1.5%, zwitterionic surfactant: 0.1% - 0.5%, and sugar alcohol compound: 0.5% - 2.0%.

[0006] Optionally, the types of the heat-denatured immunoglobulin G include at least one of the following: mouse IgG, rat IgG, and rabbit IgG; and / or

[0007] The protein protectant includes at least one of the following: bovine serum albumin, horse serum albumin, and goat serum albumin; and / or

[0008] The metal ion chelating agent includes trisodium ethylenediamine disuccinate or tetrasodium glutamate diacetate; and / or

[0009] The zwitterionic surfactant includes a solution of 3-[3-(cholamidopropyl)dimethylammonio]propanesulfonate inner salt; and / or

[0010] The sugar alcohol compound includes at least one of the following: arabinose, allulose, and isomaltulose.

[0011] Optionally, the mass of the heat-denatured immunoglobulin G is 50 mg - 200 mg.

[0012] Optionally, the raw materials of the dilution component further satisfy: the mass fraction of the preservative is 0.05% - 0.1%.

[0013] Optionally, the type of the preservative includes Proclin300.

[0014] Optionally, the sample diluent further includes a basic salt buffer solution, and the molar concentration of the basic salt buffer solution is 0.005 mol / L - 0.015 mol / L.

[0015] Optionally, the pH values of the basic salt buffer solution and the sample diluent are 7 - 8 respectively.

[0016] In a second aspect, the present application provides a method for preparing the sample diluent described in the first aspect, and the method includes:

[0017] Prepare a basic salt buffer solution;

[0018] Mix the protein protectant, metal ion chelating agent, zwitterionic surfactant, sugar alcohol compound, and the basic salt buffer solution to obtain a first diluent;

[0019] Mix the heat - denatured immunoglobulin G, preservative and the first diluent to obtain a sample diluent.

[0020] In a third aspect, the present application provides a kit, and the kit includes the sample diluent described in the first aspect.

[0021] The above - mentioned technical solutions provided by the embodiments of the present application have the following advantages compared with the prior art:

[0022] A sample diluent provided by an embodiment of the present application, the sample diluent includes heat - denatured immunoglobulin G, protein protectant, metal ion chelator, zwitterionic surfactant and sugar alcohol compound. Among them, the zwitterionic surfactant generally has an amphiphilic structure, that is, it contains a hydrophilic group and a hydrophobic group. The zwitterionic surfactant with this structure can effectively dissolve membrane proteins and broken cell fragments, while protecting the hydrophobic region of transmembrane proteins and preventing them from being damaged during the dissolution process. In addition, the hydrophilic group of the zwitterionic surfactant can interact with water molecules, and the hydrophobic group can interact with the hydrophobic part of lipids or proteins. This property can improve the ability of the zwitterionic surfactant to reduce the non - specific adsorption of serum and blood; in addition, heat - denatured immunoglobulin G, as an effective interference substance remover, can effectively reduce the interference of substances such as human anti - mouse antibodies, rheumatoid factors and heterophilic antibodies in serum and blood during the detection process; in addition, the protein protectant can avoid the competitive binding of the target protein in the test sample with serum albumin in serum and blood, so as to reduce the non - specific adsorption of serum and blood and the influence of matrix effect on the detection process; in addition, the sugar alcohol compound has strong thermal stability and acid - base stability. As an osmotic pressure regulator, it can maintain the osmotic pressure balance of the test sample during the dilution process, prevent cells from rupturing or shrinking due to osmotic pressure changes, so as to protect the target protein of the test sample and reduce the influence of matrix effect of serum and blood on the detection process; in addition, the metal ion chelator can bind with metal ions in the test sample, reduce the interference of these metal ions on the detection process, so as to reduce the non - specific adsorption of serum and blood and the influence of matrix effect on the detection process; therefore, through the interaction between heat - denatured immunoglobulin G, protein protectant, metal ion chelator, zwitterionic surfactant and sugar alcohol compound, the ability of the sample diluent to reduce the matrix effect of serum and plasma samples can be significantly improved, so as to reduce the non - specific reaction of serum and plasma and the interference of matrix effect on the detection process, thereby improving the accuracy of the detection of serum and plasma samples and obtaining more reliable and accurate experimental results. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings here are incorporated into the specification and constitute a part of this specification, showing embodiments consistent with the present application, and are used together with the specification to explain the principles of the present application.

[0024] To more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0025] Figure 1 Schematic diagram of a product of a sample diluent provided by an embodiment of the present application;

[0026] Figure 2 Schematic diagram of the process flow for preparing a sample diluent provided by an embodiment of the present application;

[0027] Figure 3 Schematic diagram of a standard curve of a sample diluent provided by Embodiment 1 of the present application;

[0028] Figure 4 Schematic diagram of a standard curve of a sample diluent provided by Embodiment 2 of the present application;

[0029] Figure 5 Schematic diagram of a standard curve of a sample diluent provided by Embodiment 3 of the present application;

[0030] Figure 6 Schematic diagram of a standard curve of a sample diluent provided by Comparative Example 1 of the present application;

[0031] Figure 7 Schematic diagram of a standard curve of a sample diluent provided by Comparative Example 2 of the present application;

[0032] Figure 8 Schematic diagram of a standard curve of a sample diluent provided by Comparative Example 3 of the present application;

[0033] Figure 9 Schematic diagram of a standard curve of a sample diluent provided by Comparative Example 4 of the present application;

[0034] Figure 10 Schematic diagram of a standard curve of a sample diluent provided by Comparative Example 5 of the present application;

[0035] Figure 11 Schematic diagram of a standard curve of a sample diluent provided by Comparative Example 6 of the present application;

[0036] Figure 12 Schematic diagram of a standard curve of a sample diluent provided by Comparative Example 7 of the present application;

[0037] Figure 13 Schematic diagram of a standard curve of a sample diluent provided by Comparative Example 8 of the present application. Detailed implementation manners

[0038] To make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are some, rather than all, of the embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative efforts shall fall within the scope of protection of the present application.

[0039] The various embodiments of the present application may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be construed as a rigid limitation on the scope of the present application; therefore, it should be considered that the described range description has specifically disclosed all possible sub-ranges and single values within that range; for example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of what the range is; in addition, whenever a numerical range is indicated herein, it means including any cited number (fraction or integer) within the indicated range.

[0040] In this text, terms such as "comprising" mean "including but not limited to". Relative terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. "And / or" describes the associated relationship of associated objects, indicating that three relationships can exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone; where A and B can be singular or plural. "At least one" means one or more, and "multiple" means two or more; "at least one kind", "at least one of the following items" or similar expressions refer to any combination of these items, including any combination of single item or plural items; for example, "at least one of a, b, or c", or "at least one of a, b, and c" can both represent: a, b, c, a-b (that is, a and b), a-c, b-c, or a-b-c, where a, b, and c can be single or multiple respectively. "Parts representation" such as parts by weight, parts by mass, etc. represents the proportional relationship between each component. In the proportional relationships involved in this text, the parameters that need to be described by the ratio should be understood as the antecedents of the ratio formula in the order of description, and the ratio numbers should be understood as the consequents of the ratio formula. For example, if the mass ratio of substance A, substance B, and substance C is 1:2:3, then substance A, substance B, and substance C should correspond one by one with the ratio numbers in the ratio formula in the order of description, that is, the mass of substance A: the mass of substance B: the mass of substance C = 1:2:3.

[0041] Unless otherwise specifically stated, all kinds of raw materials, reagents, instruments, equipment, etc. used in this text can be obtained through market purchase or can be prepared by existing methods.

[0042] It should be noted that the matrix in the serum and plasma samples refers to the components other than the analyte in the sample, and these components may have a significant interfering effect on the analyte during the analysis process, thus affecting the accuracy of the analysis results. Common matrix interferents in serum and plasma are human anti-animal antibodies (such as human anti-mouse antibody, HAMA), rheumatoid factor (RF), heterophilic antibodies, etc., and these matrix interferents will seriously affect the accuracy of the sample determination by the enzyme-linked immunosorbent assay kit.

[0043] Therefore, how to improve the ability of the sample diluent to reduce the matrix effect of serum and plasma samples is a technical problem that urgently needs to be solved at present.

[0044] Figure 1 Exemplarily shows a product schematic diagram of a sample diluent provided by an embodiment of the present application;

[0045] Such as Figure 1As shown, an embodiment of the present application provides a sample diluent, which includes a dilution component. The raw materials of the dilution component meet the following requirements, calculated by mass fraction: heat-denatured immunoglobulin G: 0.005% to 0.02%, protein protective agent: 0.5% to 2.0%, metal ion chelating agent: 0.5% to 1.5%, amphoteric surfactant: 0.1% to 0.5% and sugar alcohol compound: 0.5% to 2.0%.

[0046] It should be noted that the mass fraction of heat-denatured immunoglobulin G can be 0.005% to 0.02%, so that the sample diluent contains a sufficient amount of heat-denatured immunoglobulin G. A sufficient amount of heat-denatured immunoglobulin G can effectively reduce the interference of substances such as human anti-mouse antibodies, rheumatoid factors and heterophilic antibodies in serum and blood during the detection process, thereby reducing the impact of nonspecific adsorption and matrix effects of serum and blood on the detection process.

[0047] The mass fraction of the protein protectant can be 0.5% to 2.0%, so that the sample diluent contains a sufficient amount of protein protectant. A sufficient amount of protein protectant can avoid competitive binding between the target protein of the sample to be tested and the serum albumin of the serum and blood, thereby reducing the impact of nonspecific adsorption and matrix effects of serum and blood on the detection process.

[0048] The mass fraction of the metal ion chelator can be 0.5% to 1.5%, so that the sample diluent has a sufficient amount of metal ion chelator. The sufficient amount of metal ion chelator can combine with the metal ions of the sample to be tested, reducing the interference of these metal ions on the detection process, thereby reducing the impact of nonspecific adsorption and matrix effects of serum and blood on the detection process.

[0049] The mass fraction of the amphoteric surfactant can be 0.1% to 0.5%, so that the sample diluent contains an amphoteric surfactant. A sufficient amount of the amphoteric surfactant can effectively dissolve membrane proteins and broken cell fragments, while protecting the hydrophobic regions of transmembrane proteins to prevent them from being damaged during the dissolution process. In addition, the hydrophilic groups of the sufficient amount of the amphoteric surfactant can interact with water molecules, while the hydrophobic groups can interact with the hydrophobic parts of lipids or proteins. This characteristic can enhance the ability of the amphoteric surfactant to reduce the nonspecific adsorption of serum and blood.

[0050] The mass fraction of the sugar alcohol compound can be 0.5% to 2.0%, so that the sample diluent contains a sufficient amount of sugar alcohol compounds. The sufficient amount of sugar alcohol compounds can act as osmotic pressure regulators to maintain the osmotic pressure balance of the sample to be tested during the dilution process, prevent cells from rupturing or shrinking due to osmotic pressure changes, and thus protect the target protein of the sample to be tested, thereby reducing the influence of the matrix effect of serum and blood on the detection process during the detection process.

[0051] The mass fraction of the heat-denatured immunoglobulin G can be 0.005%, 0.01%, 0.015% or 0.02%.

[0052] The mass fraction of the protein protectant can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2.0%.

[0053] The mass fraction of the metal ion chelating agent can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4% or 1.5%.

[0054] The mass fraction of the amphoteric surfactant can be 0.1%, 0.2%, 0.3%, 0.4% or 0.5%.

[0055] The mass fraction of the sugar alcohol compound can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2.0%.

[0056] It should be noted that the non-specific adsorption means that some substances in serum and blood will cross-react with the antibodies used in the experiment, which will affect the accuracy of the detection of the sample to be tested.

[0057] It should be noted that through the interaction between the heat-denatured immunoglobulin G, the protein protectant, the metal ion chelating agent, the amphoteric surfactant and the sugar alcohol compound, the sample diluent can significantly improve the ability to reduce the matrix effect of serum and plasma samples, so as to reduce the interference of non-specific reactions and matrix effects of serum and plasma on the detection process, thereby improving the accuracy of the detection of serum and plasma samples to obtain more reliable and accurate experimental results.

[0058] It should be noted that the heat-denatured immunoglobulin G refers to the one obtained by subjecting normal immunoglobulin G to heat treatment for 20 min to 30 min under the condition of water bath heating at 60°C to 65°C.

[0059] In some optional embodiments, the types of the heat-denatured immunoglobulin G include at least one of the following: mouse IgG, rat IgG and rabbit IgG; and / or

[0060] The protein protectant includes at least one of the following: bovine serum albumin, horse serum albumin and goat serum albumin; and / or

[0061] The metal ion chelating agent includes trisodium ethylenediamine disuccinate or tetrasodium glutamate diacetate;

[0062] The amphoteric surfactant includes a solution of 3-[3-(cholamidopropyl)dimethylammonio]propanesulfonate inner salt;

[0063] The sugar alcohol compounds include at least one of the following: arabinose, allulose, and isomaltulose;

[0064] In these embodiments, the types of heat-denatured immunoglobulin G can include at least one of mouse IgG, rat IgG, and rabbit IgG, which can cover most of the heat-denatured immunoglobulin G. The heat-denatured immunoglobulin G can effectively reduce the interference of substances such as human anti-mouse antibodies, rheumatoid factors, and heterophilic antibodies in serum and blood during the detection process, thereby reducing the non-specific adsorption of serum and blood and the matrix effect on the detection process; in addition, the protein protectant can include at least one of bovine serum albumin, horse serum albumin, and goat serum albumin, which can cover most of the types of protein protectants. The protein protectant can prevent the target protein of the sample to be tested from competitively binding with the serum albumin in serum and blood, so as to reduce the non-specific adsorption of serum and blood and the matrix effect on the detection process; in addition, the metal ion chelating agent includes trisodium ethylenediamine disuccinate or tetrasodium glutamate diacetate, which can bind to the metal ions in the sample to be tested and reduce the interference of these metal ions on the detection process, thereby reducing the non-specific adsorption of serum and blood and the matrix effect on the detection process; in addition, the amphoteric surfactant can include a solution of 3-[3-(cholamidopropyl)dimethylammonio]propanesulfonate inner salt, which can effectively dissolve membrane proteins and broken cell fragments, while protecting the hydrophobic regions of transmembrane proteins and preventing them from being damaged during the dissolution process. In addition, the hydrophilic groups of a sufficient amount of amphoteric surfactants can interact with water molecules, while the hydrophobic groups can interact with the hydrophobic parts of lipids or proteins. This property can improve the ability of amphoteric surfactants to reduce the non-specific adsorption of serum and blood; in addition, the sugar alcohol compounds can include at least one of arabinose, allulose, and isomaltulose, which can be used as an osmotic pressure regulator to maintain the osmotic pressure balance during the dilution of the sample to be tested, prevent cells from rupturing or shrinking due to osmotic pressure changes, and thus protect the target protein of the sample to be detected, so as to reduce the matrix effect of serum and blood on the detection process during the detection process.

[0065] It should be noted that 3-[3-(cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS) is a non-denaturing zwitterionic detergent that can dissolve the membrane proteins of blood and serum and disrupt protein-protein interactions. CHAPS is generally used to stabilize various protein-DNA complexes and can also retain the biochemical activity of proteins in solution. CHAPS has a low micelle molecular weight (6150) and a high critical micelle concentration (6 mM to 10 mM), and can be removed from the sample by dialysis later.

[0066] It should be noted that CHAPS can also protect the structure of proteins in serum or blood, preventing them from denaturing during processing. Additionally, it can enhance the stability of the detection signal during the detection process. Based on this property, CHAPS is particularly important for experiments such as immunoassays that require high sensitivity and high specificity.

[0067] In some optional embodiments, the weight of the heat-denatured immunoglobulin G is 50 mg to 200 mg;

[0068] In these embodiments, the weight of the heat-denatured immunoglobulin G can be 50 mg to 200 mg, ensuring that there is a sufficient amount of heat-denatured immunoglobulin G in the sample diluent. A sufficient amount of heat-denatured immunoglobulin G can effectively reduce the interference of substances such as human anti-mouse antibodies, rheumatoid factors, and heterophilic antibodies in serum and blood during the detection process, thereby reducing the non-specific adsorption of serum and blood and the impact of matrix effects on the detection process.

[0069] The weight of the heat-denatured immunoglobulin G can be 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, or 200 mg.

[0070] In some optional embodiments, the raw materials of the dilution component further satisfy: the mass fraction of the preservative is 0.05% to 0.1%;

[0071] In these embodiments, the raw materials of the dilution component can further satisfy: the mass fraction of the preservative can be 0.05% to 0.1%, ensuring that there is a sufficient amount of preservative in the sample diluent. A sufficient amount of preservative can prevent the heat-denatured immunoglobulin G, protein protectant, metal ion chelator, zwitterionic surfactant, and sugar alcohol compounds from being inactivated during the detection process, improving the stability of the dilution component, and thereby reducing the non-specific adsorption of serum and blood and the impact of matrix effects on the detection process.

[0072] In some optional embodiments, the types of the preservative include Proclin300;

[0073] In these embodiments, the types of preservatives may include Proclin 300, which can prevent the inactivation of heat-denatured immunoglobulin G, protein protectants, metal ion chelating agents, amphoteric surfactants, and sugar alcohol compounds during the detection process, so as to improve the stability of the dilution component, thereby reducing the influence of non-specific adsorption of serum and blood and matrix effects on the detection process.

[0074] It should be noted that ProClin 300 is a liquid biopreservative, which is mainly obtained through biological culture, extraction, and separation techniques. It has the functions of inhibiting and killing microorganisms, antibacterial, and antiseptic. Therefore, ProClin 300 is applicable to the in vitro medical diagnostic product market, can effectively extend the shelf life of detection reagents, and improve the safety of detection reagents.

[0075] In some optional embodiments, the sample diluent further includes a basic salt buffer solution, and the molar concentration of the basic salt buffer solution is 0.05 mol / L to 0.15 mol / L;

[0076] In these embodiments, the sample diluent may further include a basic salt buffer solution, and the molar concentration of the basic salt buffer solution may be 0.05 mol / L to 0.15 mol / L, which can make the sample diluent have a sufficient concentration of the basic salt buffer solution. The sufficient concentration of the basic salt buffer solution can promote the stable binding of heat-denatured immunoglobulin G, protein protectants, metal ion chelating agents, amphoteric surfactants, and sugar alcohol compounds with the test sample, thereby further reducing the risk of non-specific adsorption and matrix effects of the sample diluent during the detection of serum and blood samples.

[0077] The molar concentration of the basic salt buffer solution may be 0.05 mol / L, 0.06 mol / L, 0.07 mol / L, 0.08 mol / L, 0.09 mol / L, 0.10 mol / L, 0.11 mol / L, 0.12 mol / L, 0.13 mol / L, 0.14 mol / L, or 0.15 mol / L.

[0078] It should be noted that the basic salt buffer solution may also be a phosphate diluent, and the components of the phosphate diluent may be sodium chloride, potassium chloride, disodium hydrogen phosphate anhydrous, and potassium dihydrogen phosphate anhydrous.

[0079] In some optional embodiments, the pH values of the basic salt buffer solution and the sample diluent are 7 to 8 respectively;

[0080] In these embodiments, the pH values of the basic salt buffer solution and the sample diluent can be 7-8 respectively, which can maintain the pH values of the basic salt buffer solution and the sample diluent at a neutral level, avoid the phenomenon of over-acidity or over-alkalinity of the basic salt buffer solution and the sample diluent, and thus reduce the risk of non-specific adsorption and matrix effect during the detection of serum and blood samples.

[0081] The pH values of the basic salt buffer solution and the sample diluent can be 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0 respectively.

[0082] Figure 2 Exemplarily shown is a schematic flowchart of a method for preparing a sample diluent provided by an embodiment of the present application;

[0083] Based on a general inventive concept, as Figure 2 shown, an embodiment of the present application provides a method for preparing the sample diluent, and the method includes:

[0084] S1. Prepare a basic salt buffer solution;

[0085] S2. Mix a protein protectant, a metal ion chelator, an amphoteric surfactant, a sugar alcohol compound and the basic salt buffer solution to obtain a first diluent;

[0086] S3. Mix heat-denatured immunoglobulin G, a preservative and the first diluent to obtain a sample diluent.

[0087] This method is for the preparation method of the above sample diluent, and the specific composition of the sample diluent can refer to the above embodiments. Since this method adopts some or all of the technical solutions of the above embodiments, it at least has all the beneficial effects brought by the technical solutions of the above embodiments, and will not be elaborated here one by one.

[0088] It should be noted that the mixing process of the heat-denatured immunoglobulin G, the preservative and the first diluent also needs to be adjusted in terms of acid and alkali to maintain the pH value of the sample diluent at a neutral level.

[0089] Based on a general inventive concept, an embodiment of the present application provides a kit, and the kit includes the sample diluent.

[0090] This kit is realized based on the above sample diluent, and the specific composition of the sample diluent can refer to the above embodiments. Since this kit adopts some or all of the technical solutions of the above embodiments, it at least has all the beneficial effects brought by the technical solutions of the above embodiments, and will not be elaborated here one by one.

[0091] In summary, the sample diluent provided by the embodiments of the present application has the following advantages compared with traditional sample diluents:

[0092] 1. High sample stability. Through the interaction between heat-denatured immunoglobulin G, protein protectant, metal ion chelator, zwitterionic surfactant, and sugar alcohol compounds, this sample diluent can significantly improve the ability of the sample diluent to reduce the matrix effect of serum and plasma samples, thereby improving the stability of the detection process for serum and plasma samples.

[0093] 2. High accuracy and reliability. Through the interaction between the dilution components, this sample diluent can effectively reduce the matrix effect of serum and plasma samples, thereby improving the accuracy of the detection of serum and plasma samples to obtain more reliable and accurate experimental results.

[0094] The following further elaborates the present application with specific embodiments. For the experimental methods without specific conditions indicated in the following embodiments, they are usually determined according to national standards / industry standards; if there are no corresponding national standards / industry standards, they are carried out according to general international standards, conventional conditions, or the conditions recommended by the manufacturer.

[0095] Example 1

[0096] A sample diluent, the sample diluent includes dilution components. By mass fraction, the raw materials of the dilution components satisfy: heat-denatured immunoglobulin G: 0.01%, protein protectant: 1%, metal ion chelator: 0.6%, zwitterionic surfactant: 0.2%, and sugar alcohol compound: 1.8%.

[0097] The type of heat-denatured immunoglobulin G is mouse IgG;

[0098] The protein protectant includes bovine serum albumin;

[0099] The metal ion chelator is trisodium ethylenediamine disuccinate;

[0100] The zwitterionic surfactant is a solution of 3-[3-(cholamidopropyl)dimethylammonio]propanesulfonate inner salt;

[0101] The sugar alcohol compound is arabinose.

[0102] The mass of heat-denatured immunoglobulin G is 100 mg.

[0103] The raw materials of the dilution components also satisfy: the mass fraction of the preservative is 0.1%.

[0104] The type of the preservative is Proclin300.

[0105] The sample diluent further includes a basic salt buffer solution, and the molar concentration of the basic salt buffer solution is 0.01 mol / L.

[0106] The basic salt buffer solution is a phosphate diluent.

[0107] The pH values of the basic salt buffer solution and the sample diluent are 7.4 respectively.

[0108] As Figure 2 shown, a method for preparing a sample diluent includes:

[0109] S1. Prepare the basic salt buffer solution:

[0110] Weigh 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate anhydrous, and 0.24 g of potassium dihydrogen phosphate anhydrous, dissolve them together in 800 mL of purified water, stir until completely dissolved, and then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid to obtain a phosphate diluent;

[0111] S2. Mix 10 g of protein protectant, 6 g of metal ion chelator, 2 g of zwitterionic surfactant, 18 g of sugar alcohol compound and the basic salt buffer solution, and stir until completely dissolved to obtain a first diluent;

[0112] S3. Mix 100 mg of heat-inactivated immunoglobulin G, 1 mL of preservative and the first diluent, then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid, and make up the volume to 1 L to obtain the sample diluent.

[0113] Example 2

[0114] On the basis of the content disclosed in Example 1, the following further modifications are made:

[0115] In terms of mass fraction, the raw materials of the dilution component satisfy: heat-inactivated immunoglobulin G: 0.006%, protein protectant: 0.6%, metal ion chelator: 1.4%, zwitterionic surfactant: 0.4% and sugar alcohol compound: 1%.

[0116] The type of heat-inactivated immunoglobulin G is mouse IgG;

[0117] The protein protectant is goat serum albumin;

[0118] The metal ion chelator is tetrasodium glutamate diacetate;

[0119] The zwitterionic surfactant is a solution of 3-[3-(cholamidopropyl)dimethylammonio]propanesulfonate inner salt;

[0120] The sugar alcohol compound is allulose.

[0121] The weight of heat - denatured immunoglobulin G is 60 mg.

[0122] The raw materials of the dilution component also meet the requirement that the mass fraction of the preservative is 0.08%.

[0123] A method for preparing a sample diluent, comprising:

[0124] S1. Prepare a basic salt buffer solution:

[0125] Weigh 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate anhydrous, and 0.24 g of potassium dihydrogen phosphate anhydrous, dissolve them together in 800 mL of purified water, stir until completely dissolved, and then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid to obtain a phosphate diluent;

[0126] S2. Mix 6 g of protein protectant, 14 g of metal ion chelator, 4 g of zwitterionic surfactant, 10 g of sugar alcohol compound and the basic salt buffer solution, and stir until completely dissolved to obtain a first diluent;

[0127] S3. Mix 60 mg of heat - denatured immunoglobulin G, 0.8 mL of preservative and the first diluent, then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid, and make up the volume to 1 L to obtain the sample diluent.

[0128] Example 3

[0129] Based on the content disclosed in Example 1, the following further modifications are made:

[0130] In terms of mass fraction, the raw materials of the dilution component meet the requirements: heat - denatured immunoglobulin G: 0.018%, protein protectant: 1.9%, metal ion chelator: 1%, zwitterionic surfactant: 0.3%, and sugar alcohol compound: 0.6%.

[0131] The type of heat - denatured immunoglobulin G is: rat IgG;

[0132] The protein protectant is horse serum albumin;

[0133] The metal ion chelator is trisodium ethylenediaminedisuccinate;

[0134] The zwitterionic surfactant is a solution of 3 - [3 - (cholamidopropyl) dimethylammonio] propanesulfonate inner salt;

[0135] The sugar alcohol compound is isomaltulose.

[0136] The weight of heat - denatured immunoglobulin G is 180 mg.

[0137] The raw materials of the dilution component also meet the requirement that the mass fraction of the preservative is 0.06%.

[0138] A method for preparing a sample diluent, comprising:

[0139] S1. Prepare a basic salt buffer solution:

[0140] Weigh 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate anhydrous, and 0.24 g of potassium dihydrogen phosphate anhydrous, dissolve them together in 800 mL of purified water, stir until completely dissolved, and then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid to obtain a phosphate diluent;

[0141] S2. Mix 19 g of a protein protectant, 10 g of a metal ion chelator, 3 g of an amphoteric surfactant, 6 g of a sugar alcohol compound, and the basic salt buffer solution, and stir until completely dissolved to obtain a first diluent;

[0142] S3. Mix 180 mg of heat - denatured immunoglobulin G, 0.6 mL of a preservative, and the first diluent, then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid, and make up the volume to 1 L to obtain a sample diluent.

[0143] Comparative Example 1

[0144] On the basis of the content disclosed in Example 1, further modifications are made as follows:

[0145] Do not add heat - denatured immunoglobulin G, metal ion chelator, amphoteric surfactant, and sugar alcohol compound.

[0146] A method for preparing a sample diluent, comprising:

[0147] S1. Prepare a basic salt buffer solution:

[0148] Weigh 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate anhydrous, and 0.24 g of potassium dihydrogen phosphate anhydrous, dissolve them together in 800 mL of purified water, stir until completely dissolved, and then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid to obtain a phosphate diluent;

[0149] S2. Mix 10 g of a protein protectant and the basic salt buffer solution, and stir until completely dissolved to obtain a first diluent;

[0150] S3. Mix 1 mL of a preservative and the first diluent, then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid, and make up the volume to 1 L to obtain a sample diluent.

[0151] Comparative Example 2

[0152] On the basis of the content disclosed in Example 1, further modifications are made as follows:

[0153] Do not add heat - denatured immunoglobulin G, metal ion chelating agents, and sugar alcohols. A method for preparing a sample diluent includes:

[0154] S1. Prepare a basic salt buffer solution:

[0155] Weigh 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of anhydrous disodium hydrogen phosphate, and 0.24 g of anhydrous potassium dihydrogen phosphate, dissolve them together in 800 mL of purified water, stir until completely dissolved, and then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid to obtain a phosphate diluent;

[0156] S2. Mix 8 g of protein protectant, 3 g of amphoteric surfactant, and the basic salt buffer solution, stir until completely dissolved to obtain a first diluent;

[0157] S3. Mix 1 mL of preservative and the first diluent, then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid, and make up the volume to 1 L to obtain the sample diluent.

[0158] Comparative Example 3

[0159] Based on the content disclosed in Example 1, further modifications are made as follows:

[0160] Do not add heat - denatured immunoglobulin G and sugar alcohols. A method for preparing a sample diluent includes:

[0161] S1. Prepare a basic salt buffer solution:

[0162] Weigh 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of anhydrous disodium hydrogen phosphate, and 0.24 g of anhydrous potassium dihydrogen phosphate, dissolve them together in 800 mL of purified water, stir until completely dissolved, and then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid to obtain a phosphate diluent;

[0163] S2. Mix 14 g of protein protectant, 6 g of metal ion chelating agent, 2 g of amphoteric surfactant, and the basic salt buffer solution, stir until completely dissolved to obtain a first diluent;

[0164] S3. Mix 1 mL of preservative and the first diluent, then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid, and make up the volume to 1 L to obtain the sample diluent.

[0165] Comparative Example 4

[0166] Based on the content disclosed in Example 1, further modifications are made as follows:

[0167] Do not add heat - denatured immunoglobulin G. A method for preparing a sample diluent includes:

[0168] S1. Prepare a basic salt buffer solution:

[0169] Weigh 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of anhydrous disodium hydrogen phosphate, and 0.24 g of anhydrous potassium dihydrogen phosphate, dissolve them together in 800 mL of purified water, stir until completely dissolved, and then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid to obtain a phosphate diluent;

[0170] S2. Mix 10 g of a protein protectant, 10 g of a metal ion chelator, 4 g of an amphoteric surfactant, 10 g of a sugar alcohol compound, and the basic salt buffer solution, and stir until completely dissolved to obtain a first diluent;

[0171] S3. Mix 1 mL of a preservative and the first diluent, then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid, and make up the volume to 1 L to obtain a sample diluent.

[0172] Comparative Example 5

[0173] On the basis of the content disclosed in Example 1, the following further modifications are made:

[0174] The mass fraction of heat - denatured immunoglobulin G > 0.02%, and the mass fraction of this heat - denatured immunoglobulin G is 0.05%. A method for preparing a sample diluent includes:

[0175] S1. Prepare a basic salt buffer solution:

[0176] Weigh 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of anhydrous disodium hydrogen phosphate, and 0.24 g of anhydrous potassium dihydrogen phosphate, dissolve them together in 800 mL of purified water, stir until completely dissolved, and then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid to obtain a phosphate diluent;

[0177] S2. Mix 8 g of a protein protectant, 8 g of a metal ion chelator, 4 g of an amphoteric surfactant, 12 g of a sugar alcohol compound, and the basic salt buffer solution, and stir until completely dissolved to obtain a first diluent;

[0178] S3. Mix 500 mg of heat - denatured immunoglobulin G, 1 mL of a preservative, and the first diluent, then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid, and make up the volume to 1 L to obtain a sample diluent.

[0179] Comparative Example 6

[0180] On the basis of the content disclosed in Example 1, the following further modifications are made:

[0181] The mass fraction of the metal ion chelating agent is 2.0%. A method for preparing a sample diluent includes:

[0182] S1. Prepare a basic salt buffer solution:

[0183] Weigh 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate anhydrous, and 0.24 g of potassium dihydrogen phosphate anhydrous, dissolve them together in 800 mL of purified water, stir until completely dissolved, and then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid to obtain a phosphate diluent;

[0184] S2. Mix 10 g of protein protectant, 20 g of metal ion chelating agent, 3 g of amphoteric surfactant, 8 g of sugar alcohol compound, and the basic salt buffer solution, stir until completely dissolved to obtain a first diluent;

[0185] S3. Mix 100 mg of heat - denatured immunoglobulin G, 1 mL of preservative, and the first diluent, then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid, and make up the volume to 1 L to obtain the sample diluent.

[0186] Comparative Example 7

[0187] On the basis of the content disclosed in Example 1, the following further modifications are made:

[0188] The mass fraction of the amphoteric surfactant is 1.0%.

[0189] A method for preparing a sample diluent includes:

[0190] S1. Prepare a basic salt buffer solution:

[0191] Weigh 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate anhydrous, and 0.24 g of potassium dihydrogen phosphate anhydrous, dissolve them together in 800 mL of purified water, stir until completely dissolved, and then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid to obtain a phosphate diluent;

[0192] S2. Mix 10 g of protein protectant, 8 g of metal ion chelating agent, 10 g of amphoteric surfactant, 10 g of sugar alcohol compound, and the basic salt buffer solution, stir until completely dissolved to obtain a first diluent;

[0193] S3. Mix 120 mg of heat - denatured immunoglobulin G, 1 mL of preservative, and the first diluent, then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid, and make up the volume to 1 L to obtain the sample diluent.

[0194] Comparative Example 8

[0195] On the basis of the content disclosed in Example 1, the following further modifications are made:

[0196] The mass fraction of the sugar alcohol compound is 3.0%. A method for preparing a sample diluent includes:

[0197] S1. Prepare a basic salt buffer solution:

[0198] Weigh 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of anhydrous disodium hydrogen phosphate, and 0.24 g of anhydrous potassium dihydrogen phosphate, dissolve them together in 800 mL of purified water, stir until completely dissolved, and then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid to obtain a phosphate diluent;

[0199] S2. Mix 10 g of a protein protectant, 8 g of a metal ion chelator, 10 g of an amphoteric surfactant, 30 g of the sugar alcohol compound, and the basic salt buffer solution, and stir until completely dissolved to obtain a first diluent;

[0200] S3. Mix 120 mg of heat-denatured immunoglobulin G, 1 mL of a preservative, and the first diluent, then adjust the pH value to about 7.4 using sodium hydroxide or hydrochloric acid, and make up the volume to 1 L to obtain a sample diluent.

[0201] Related experiments and effect data:

[0202] I. Tests on standards:

[0203] 1. Preparation of standards:

[0204] (1) Take out the freeze-dried standard, centrifuge it at a speed of 6000 rpm to 10000 rpm for 30 s. Dissolve it separately with 1 mL of the above sample diluent, and repeatedly pipette 5 times against the bottom of the cryotube to assist dissolution, and mix well to obtain standard S7, and set it aside for use.

[0205] (2) Take out the above several groups of 7 1.5 mL centrifuge tubes (S0 - S6) and arrange them in sequence. Add 250 μL of the above sample diluent to each group. Pipette 250 μL of standard S7 into the first centrifuge tube (S6), and gently pipette and mix well. Pipette 250 μL from S6 into the second EP tube (S5), and gently pipette and mix well. And so on for serial dilution of the standard. S0 is the sample diluent.

[0206] 2. Wash working solution

[0207] Dilute the concentrated detergent solution with deionized water at a volume ratio of 1:25. For example, use a graduated cylinder to measure 240 mL of deionized water, pour it into a beaker or other clean container, then measure 10 mL of concentrated detergent solution, add it evenly, and stir to mix thoroughly. Prepare the solution immediately before use. Since concentrated detergent solution may precipitate salts when stored at low temperatures, warm it in a water bath to aid dissolution during dilution.

[0208] 3. Biotinylated Antibody Working Solution

[0209] Dilute the biotinylated antibody solution according to the usage ratio. For example, if the dilution is 1:100 by volume with sample diluent, add 10 μL of biotinylated antibody to 990 μL of sample diluent, mix gently, and prepare within 10 minutes before use.

[0210] 4. Horseradish peroxidase labeled avidin working solution

[0211] Dilute the 1:40 volume ratio of horseradish peroxidase-labeled avidin intermediate solution with sample diluent at a 1:100 volume ratio. For example, add 10 μL of horseradish peroxidase-labeled avidin to 990 μL of sample diluent, mix gently, and prepare within 10 minutes before use.

[0212] 2. Sample Processing

[0213] (1) Serum: Whole blood specimens should be placed at room temperature for 2 hours or at 4°C overnight, then centrifuged at 2°C to 8°C at 1000 x g for 15 minutes. The supernatant can be immediately tested. Alternatively, the specimens can be aliquoted and stored at -20°C or -80°C, but repeated freezing and thawing should be avoided. Thawed samples should be centrifuged again before testing.

[0214] (2) Plasma: EDTA or heparin can be used as an anticoagulant. Within 30 minutes after specimen collection, centrifuge the sample at 2°C to 8°C and 1000 x g for 15 minutes. The supernatant can be immediately tested. Alternatively, the supernatant can be aliquoted and the sample can be stored at -20°C or -80°C, but repeated freezing and thawing should be avoided. Thawed samples should be centrifuged again before testing.

[0215] 3. Operation steps

[0216] (1) Move all reagents to room temperature (18℃~25℃) and equilibrate for at least 30 minutes. Prepare the reagents according to the above method and set aside.

[0217] (2) Sample addition: Set up wells for standards and wells for samples to be tested. Add 100 μL of standard or sample to each well, cover with a plate sticker, and incubate at 37°C for 120 min.

[0218] (3) Discard the liquid in the wells, centrifuge to dry, and wash the plate twice. Immerse each time for 1 minute, 200 μL per well, and centrifuge to dry.

[0219] (4) Add biotinylated detection antibody: Add 100 μL of the biotinylated antibody working solution to each well, cover with a plate sticker, and incubate at 37 °C for 60 minutes.

[0220] (5) Discard the liquid in the wells, centrifuge to dry, and wash the plate three times. Immerse each time for 1 minute, 200 μL per well, and centrifuge to dry.

[0221] (6) Add 100 μL of the horseradish peroxidase-labeled avidin working solution to each well, cover with a plate sticker, and incubate at 37 °C for 60 minutes.

[0222] (7) Discard the liquid in the wells, centrifuge to dry, and wash the plate five times. Immerse each time for 1 minute, 200 μL per well, and centrifuge to dry.

[0223] (8) Sequentially add 90 μL of the substrate solution to each well, and incubate at 37 °C for 20 minutes for color development.

[0224] (9) Sequentially add 50 μL of the termination solution to each well to terminate the reaction.

[0225] (10) Within 5 minutes after the reaction is terminated, measure the optical density (OD value) of each well in sequence using an ELISA reader at a wavelength of 450 nm.

[0226] IV. Experimental Parameter Verification

[0227] 1. Comparative experiment:

[0228] Conduct a comparative test on the parameters (standard curve test, sample linearity test, sample recovery rate test) of the Human Epididymis Protein 4 (Human HE4 / WFDC2) ELISA kit. The sample diluent is the sample diluent prepared in Examples 1-3 and Comparative Examples 1-8. The detailed data is as follows:

[0229] (1) Compare the linearity of the standard curves. The results are shown in Table 1.

[0230] Table 1 Standard Curves of the Sample Diluents in Examples 1-3 and Comparative Examples 1-8

[0231]

[0232]

[0233] Figure 3 Exemplarily shows a schematic diagram of the standard curve of a sample diluent provided in Example 1 of the present application;

[0234] Figure 4Exemplarily shown is a schematic diagram of the standard curve of a sample diluent provided in Embodiment 2 of the present application;

[0235] Figure 5 Exemplarily shown is a schematic diagram of the standard curve of a sample diluent provided in Embodiment 3 of the present application;

[0236] Figure 6 Exemplarily shown is a schematic diagram of the standard curve of a sample diluent provided in Comparative Example 1 of the present application;

[0237] Figure 7 Exemplarily shown is a schematic diagram of the standard curve of a sample diluent provided in Comparative Example 2 of the present application;

[0238] Figure 8 Exemplarily shown is a schematic diagram of the standard curve of a sample diluent provided in Comparative Example 3 of the present application;

[0239] Figure 9 Exemplarily shown is a schematic diagram of the standard curve of a sample diluent provided in Comparative Example 4 of the present application;

[0240] Figure 10 Exemplarily shown is a schematic diagram of the standard curve of a sample diluent provided in Comparative Example 5 of the present application;

[0241] Figure 11 Exemplarily shown is a schematic diagram of the standard curve of a sample diluent provided in Comparative Example 6 of the present application;

[0242] Figure 12 Exemplarily shown is a schematic diagram of the standard curve of a sample diluent provided in Comparative Example 7 of the present application;

[0243] Figure 13 Exemplarily shown is a schematic diagram of the standard curve of a sample diluent provided in Comparative Example 8 of the present application;

[0244] According to the data in Table 1, the standard curves of Embodiments 1 to 3 and Comparative Examples 1 to 8 are respectively plotted, and the results are as Figure 3-13 shown, wherein, the standard curve formula of the sample diluent in Embodiment 1 is: y = (a + bx) / (1 + cx + dx 2 ), where a = -137.458, b = 1187.666, c = -0.185, d = -0.007; the standard curve formula of the sample diluent in Embodiment 2 is: y = (a + bx) / (1 + cx + dx 2 ), where a = -309.623, b = 2160.423, c = 0.621, d = -0.242; the standard curve formula of the sample diluent in Embodiment 3 is: y = (a + bx) / (1 + cx + dx 2), where a = -187.47, b = 1461.36, c = 0.005, d = -0.059; The standard curve formula of the sample diluent of Comparative Example 1 is y = (a + bx) / (1 + cx + dx 2 ), where a = -247.022, b = 1839.532, c = 0.271, d = -0.137; The standard curve formula of the sample diluent of Comparative Example 2 is y = (a + bx) / (1 + cx + dx 2 ), where a = -60.881, b = 998.195, c = -0.269, d = 0.015;

[0245] The standard curve formula of the sample diluent of Comparative Example 3 is y = (a + bx) / (1 + cx + dx 2 ), where a = -158.641, b = 1429.863, c = 0.116, d = -0.099; The standard curve formula of the sample diluent of Comparative Example 4 is y = (a + bx) / (1 + cx + dx 2 ), where a = -232.342, b = 1796.008, c = 0.354, d = -0.170; The standard curve formula of the sample diluent of Comparative Example 5 is y = (a + bx) / (1 + cx + dx 2 ), where a = -363.75, b = 1652.80, c = 0.38, d = -0.18; The standard curve formula of the sample diluent of Comparative Example 6 is y = (a + bx) / (1 + cx + dx 2 ), where a = -325.11, b = 1333.97, c = -0.16, d = -0.03; The standard curve formula of the sample diluent of Comparative Example 7 is y = (a + bx) / (1 + cx + dx 2 ), where a = -277.70, b = 1014.15, c = -0.35, d = 0.04; The standard curve formula of the sample diluent of Comparative Example 8 is y = (a + bx) / (1 + cx + dx 2 ), where a = -1160.66, b = 5418.10, c = 3.12, d = -1.05.

[0246] As can be seen from the above formula, the relevant parameters of the standard curves of the sample diluents in Example 1, Example 2, and Example 3 are: R > 0.999, P / N > 22, which indicates that the three standard curves in Examples 1-3 all meet the industry requirements standards; the relevant parameters of the standard curves of the sample diluents in Comparative Examples 1-4 are: R > 0.999, P / N > 21, and the three standard curves in Comparative Examples 1-4 all meet the industry requirements standards; the relevant parameters of the standard curves of the sample diluents in Comparative Examples 5-8 are: R < 0.999, P / N < 18, and the standard curves of the sample diluents in Comparative Examples 5-8 do not meet the requirements and no subsequent relevant verification needs to be done; therefore, through the sample diluent provided in the embodiments of the present application, the relevant linearity of the standard curve of the kit prepared therefrom is not affected.

[0247] 2. Sample linearity test

[0248] To evaluate the linearity of the measured samples, the above-mentioned several sample diluents were used to continuously dilute the samples containing high-concentration human HE4 to produce samples with values within the dynamic range of the measurement. The samples were diluted before this measurement to determine the influence of matrix effect interference on the sample measurement.

[0249] The samples were diluted using the sample diluent of Example 1, and the measured stability results are shown in Table 2.

[0250] Table 2 Stability of samples diluted with the sample diluent of Example 1

[0251]

[0252]

[0253] The samples were diluted using the sample diluent of Example 2, and the measured stability results are shown in Table 3.

[0254] Table 3 Stability of samples diluted with the sample diluent of Example 2

[0255]

[0256] The samples were diluted using the sample diluent of Example 3, and the measured stability results are shown in Table 4.

[0257] Table 4 Stability of samples diluted with the sample diluent of Example 3

[0258]

[0259] The samples were diluted using the sample diluent of Comparative Example 1, and the measured stability results are shown in Table 5.

[0260] Table 5 Stability of samples diluted with the sample diluent of Comparative Example 1

[0261]

[0262] Dilute the sample with the sample diluent of Comparative Example 2, and the measured stability results are shown in Table 6.

[0263] Table 6 Stability of the sample diluted with the sample diluent of Comparative Example 2

[0264]

[0265] Dilute the sample with the sample diluent of Comparative Example 3, and the measured stability results are shown in Table 7.

[0266] Table 7 Table of stability results of the sample diluted with the sample diluent of Comparative Example 3

[0267]

[0268]

[0269] Dilute the sample with the sample diluent of Comparative Example 4, and the measured stability results are shown in Table 8.

[0270] Table 8 Table of stability results of the sample diluted with the sample diluent of Comparative Example 4

[0271]

[0272] As can be seen from Tables 2 to 8, the sample diluents provided in Examples 1, 2, and 3 of the present application have a measured linear range between 80% and 120%, meeting the quality control standards of the kit industry. For the sample diluents provided in Comparative Examples 1 to 4, the measured partial linear average recovery rates are lower than 80%, and there are obvious matrix interferences in the samples with a recovery rate range exceeding 80% to 120%, not meeting the quality control standards of the kit industry.

[0273] 3. Sample recovery rate test:

[0274] Statistically analyze the recovery rates of human HE4 incorporated into the entire measurement range in various matrices in the above experiments to verify the accuracy of sample detection. The results are shown in Table 5.

[0275] Table 9 Table of recovery rate results of human HE4 in Examples 1 - 3 and Comparative Examples 1 - 4

[0276]

[0277]

[0278] As can be seen from Table 5, the recovery rates of serum and plasma tested with the sample diluents of Example 1, Example 2 and Example 3 are all within the range of 80% - 120%, meeting the quality control standards of the kit industry; while the serum recovery rates and plasma recovery rates tested in Comparative Examples 1 - 4 exceed the industry quality control standard range of 80% - 120%, indicating that the sample diluents of Comparative Examples 1 - 4 have obvious matrix interference.

[0279] V. Actual detection of the kit product:

[0280] Select an existing enzyme - linked immunosorbent assay kit for detecting the content of human epididymis protein 4 (Human HE4 / WFDC2) (from Wuhan Huamei Biological Engineering Co., Ltd.). The performance of this kit, such as the linearity of the standard curve, sample linearity test, sample recovery rate test, etc., meets the requirements. The sample diluents obtained in each example and comparative example are respectively combined with this kit for parameter (linearity of the standard curve, sample linearity test, sample recovery rate test) comparison tests, and the results are shown in Table 10.

[0281] Table 10 Comparison test of actual detection of the kit product

[0282]

[0283] As can be seen from Table 10, a sample diluent provided by an embodiment of the present application can accurately and reliably detect the target protein during the actual detection process.

[0284] In summary, a sample diluent provided by an embodiment of the present application can significantly improve the ability of the sample diluent to reduce the matrix effect of serum and plasma samples through the interaction between heat - denatured immunoglobulin G, protein protectant, metal ion chelating agent, amphoteric surfactant and sugar alcohol compounds, so as to reduce the interference of non - specific reactions and matrix effects of serum and plasma on the detection process, thereby improving the accuracy of serum and plasma sample detection and obtaining more reliable and accurate experimental results.

[0285] In addition, a preparation method of a sample diluent provided by an embodiment of the present application only requires multiple materials to be mixed in batches, and the operation steps are simple and the production difficulty is small, which can be used for the scale production of the sample diluent of the kit.

[0286] The above are only specific embodiments of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined in the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown in the present application, but rather to the broadest scope consistent with the principles and novel features claimed in the present application.

Claims

1. A sample diluent, comprising a dilution component, wherein the raw materials of the dilution component meet the following requirements, calculated by mass fraction: heat-denatured immunoglobulin G: 0.005% to 0.02%, protein protective agent: 0.5% to 2.0%, metal ion chelating agent: 0.5% to 1.5%, amphoteric surfactant: 0.1% to 0.5% and sugar alcohol compound: 0.5% to 2.0%.

2. The sample diluent according to claim 1, wherein the type of heat-denatured immunoglobulin G comprises at least one of the following: mouse IgG, rat IgG and rabbit IgG; and / or The protein protective agent comprises at least one of the following: bovine serum albumin, horse serum albumin and goat serum albumin; and / or The metal ion chelating agent includes trisodium ethylenediamine disuccinate or tetrasodium glutamate diacetate; and / or The amphoteric surfactant includes a solution of 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt; and / or The sugar alcohol compound includes at least one of the following: arabinose, psicose and isomalt. The sample diluent according to claim 1 , wherein the mass of the heat-denatured immunoglobulin G is 50 mg to 200 mg.

4. The sample diluent according to claim 1, wherein the raw materials of the dilution component further satisfy the following requirement: the mass fraction of the preservative is 0.05% to 0.1%. The sample diluent according to claim 4 , wherein the preservative comprises Proclin 300. The sample diluent according to claim 1 , further comprising a basal salt buffer solution, wherein the molar concentration of the basal salt buffer solution is 0.005 mol / L to 0.015 mol / L. 7 . The sample diluent according to claim 6 , wherein the pH values of the basic salt buffer and the sample diluent are respectively 7-8.

8. A method for preparing the sample diluent according to any one of claims 1 to 7, comprising: Prepare basal salt buffer; Mixing a protein protective agent, a metal ion chelating agent, an amphoteric surfactant, a sugar alcohol compound, and the basic salt buffer to obtain a first dilution solution; The heat-denatured immunoglobulin G, the preservative and the first diluent are mixed to obtain a sample diluent.

9. A kit comprising the sample diluent according to any one of claims 1 to 7.

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