Whole blood sample analysis method and sample analyzer

Abstract

The invention provides a whole blood sample analysis method applied to a sample analyzer, which comprises the following steps: performing dissociation treatment on a whole blood sample by adopting a dissociation agent, and then adding a diluent to promote precipitation so as to obtain a supernatant containing free-state small molecules; the concentration of the small molecules in the whole blood sample can be reflected by detecting the concentration of the free-state small molecules in the obtained supernate. According to the analysis method provided by the invention, the participation of a centrifugal machine is not needed in the whole process, the micromolecules combined with blood cells or binding proteins in the whole blood sample can be dissociated under the condition of mild conditions, so that the micromolecules in a free state are released, and the dissociated micromolecules in the free state can also be directly used for subsequent detection. Furthermore, the obtained free-state small molecules can be used for a sandwich detection method based on a compound antibody, and the sensitivity of small molecule detection can be remarkably improved.

Description

Technical Field

[0001] The present invention relates to a fully automatic whole blood sample analysis method and a corresponding sample analyzer. Background Art

[0002] Small molecules generally refer to a class of substances with a molecular weight of less than 5000 Daltons and simple structures. Due to the structural characteristics of small molecules themselves, their molecular size and steric hindrance are limited. Small molecules usually have only one antigenic epitope and cannot provide two or more epitopes for antibody binding. On the other hand, small molecules usually exist in a bound state in the sample. When in the bound state, their epitopes are masked and difficult to be recognized by their specific antibodies. However, both free and bound small molecules in the sample are closely related to the body's physiological state. Therefore, small molecules need to be pre-treated before detection. Conventional technical solutions usually use displacing agents for treatment. However, some small molecules may be more tightly bound to binding proteins in blood cells, such as immunosuppressant drugs. The traditional method is to first treat the blood with an organic solvent, then centrifuge to remove impurities and release the small molecule analyte from the blood cells. The centrifugation process increases the workload of the operator. This step is time-consuming and requires controlled conditions to ensure the quality of the centrifuged sample. It also usually requires the addition of toxic organic solvents such as methanol and whole blood precipitants, which also poses a safety risk to medical staff.

[0003] Therefore, there is an urgent need to develop an analytical method that can dissociate small molecule analytes in blood cells and facilitate full automation on existing analyzers. Summary of the Invention

[0004] In order to solve the above technical problems, a first aspect of the present invention provides a whole blood sample analysis method applied to a sample analyzer.

[0005] According to an embodiment of the present invention, the analysis method includes: controlling the pipette to perform the following operations: Step 1: Controlling a pipette to draw a whole blood sample containing small molecules from a sample storage mechanism and a dissociation agent from a reagent storage mechanism, respectively, and adding them to a first reaction cup to form a first mixture, wherein the working concentration of the dissociation agent in the mixed first mixture is 2-4 mol / L, and the dissociation agent is guanidine thiocyanate; Step 2: After the first mixture in step 1 has been allowed to react for 1-15 minutes, the pipette is controlled to draw a diluent and add it to the first reaction cup to form a second mixture, wherein the diluent is selected from PBS, Tris, MES, HEPES, or Citrate buffer, and the amount of the diluent drawn is at least twice the amount of the first mixture; Step 3: After the second mixture in step 2 is precipitated at 25-37° C. for 5-20 minutes to obtain a supernatant, the pipette is controlled to draw the supernatant from the first reaction cup to the second reaction cup; Step 4: controlling the detection of the concentration of the small molecule in the second reaction cup, wherein the concentration of the small molecule in the second reaction cup reflects the concentration of the small molecule in the whole blood sample; The small molecule is an organic compound with a molecular weight of less than 5000 Daltons.

[0006] In some embodiments, in step 1, the ratio of the volume of the whole blood sample to the volume of the dissociation agent in the first mixture in the first reaction cup is 1:0.5-2.

[0007] In some embodiments of the present invention, in step 2, the volume of the diluent in the first reaction cup is 2-4 times the volume of the first mixture.

[0008] In some embodiments, step 3 also includes controlling the pipette to draw one or more detection reagents from the reagent storage mechanism into the second reaction cup to form a multi-component immune complex, and the control of detecting the concentration of the small molecule in the second reaction cup in step 4 includes controlling the detection of the multi-component immune complex to obtain the concentration of the small molecule in the second reaction cup.

[0009] In some embodiments of the present invention, the detection reagent comprises a small molecule binding partner reagent and a competitive antigen reagent, and the multi-component immune complex is a binary immune complex of small molecule / small molecule binding partner.

[0010] Specifically, the small molecule binding partner reagent and the competitive antigen reagent can be located in the same detection reagent or in different detection reagents.

[0011] In some embodiments of the present invention, the detection reagent comprises a small molecule binding partner reagent and a complex antibody reagent. The multi-component complex is a ternary immune complex having a structure of small molecule / small molecule binding partner complex-complex antibody. The small molecule binding partner is capable of specifically binding to the small molecule to form the small molecule / small molecule binding partner complex, and forming at least one new conformational epitope that is distinct from the free small molecule and the free small molecule binding partner; the complex antibody specifically binds to at least one of the new conformational epitopes.

[0012] In some embodiments, the detection reagent comprises a small molecule binding partner reagent and a complex antibody reagent, and the complex antibody reagent comprises a first complex antibody reagent or a second complex antibody reagent; the multi-component immune complex is a quaternary immune complex with a structure of "first complex antibody-small molecule / small molecule binding partner complex-second complex antibody"; wherein the small molecule binding partner has at least two antigen binding sites, which can bind to at least two of the small molecules to obtain the small molecule / small molecule binding partner complex, and form at least two new conformational epitopes that are different from the free small molecule and the free small molecule binding partner; the first complex antibody and the second complex antibody each specifically bind to at least one new conformational epitope.

[0013] A second aspect of the present invention provides a sample analyzer for detecting a whole blood sample containing small molecules, comprising: A sample storage mechanism, used for storing sample tubes carrying whole blood samples; A reagent storage mechanism for storing reagents, wherein the reagents include a dissociation agent, a diluent, and one or more detection reagents; The reaction device has at least one placement position, wherein the placement position is used to place a reaction cup and incubate the reaction liquid in the reaction cup; A measuring device, used to perform optical measurement on the reaction solution after incubation to obtain the test result of the sample to be tested; A pipette for transferring the whole blood sample or the reagent into the reaction cup The control device is electrically connected to the sample storage mechanism, the reagent storage mechanism, the reaction device, the measuring device and the pipette, and is configured to: receiving a test instruction, wherein the test instruction includes the type of small molecules to be tested in the whole blood sample; In response to the test instruction: Step 1: Controlling a pipette to draw a whole blood sample containing small molecules from a sample storage mechanism and a dissociation agent from a reagent storage mechanism, respectively, and adding them to a first reaction cup to form a first mixture, wherein the working concentration of the dissociation agent in the mixed first mixture is 2-4 mol / L, and the dissociation agent is guanidine thiocyanate; Step 2: After the first mixture in step 1 is allowed to react for 1-15 minutes, the pipette is controlled to draw a diluent from the reagent storage mechanism and add it to the first reaction cup to form a second mixture, wherein the diluent is selected from PBS, Tris, MES, HEPES, and Citrate buffer, and the amount of the diluent in the second mixture is more than twice the amount of the first mixture; Step 3: After the second mixture is precipitated for 5-20 minutes in step 2 to obtain a supernatant, the pipette is controlled to draw the supernatant from the first reaction cup to the second reaction cup; Step 4: controlling the calculation of the concentration of the small molecule in the second reaction cup according to the signal value in the second reaction cup measured by the measuring device, so as to reflect the concentration of the small molecule in the whole blood sample; The small molecule is an organic compound with a molecular weight of less than 5000 Daltons.

[0014] The present invention provides a method for treating a whole blood sample with a dissociating agent, then adding a diluent to promote precipitation to obtain a supernatant containing free small molecules. The concentration of free small molecules in the supernatant is detected to reflect the concentration of small molecules in the whole blood sample. This analytical method does not require the involvement of a centrifuge throughout the entire process and can dissociate small molecules bound to blood cells or binding proteins in the whole blood sample under mild conditions, thereby releasing free small molecules. The dissociated free small molecules can then be directly used for subsequent detection. Furthermore, the obtained free small molecules can be used in a sandwich detection method based on complex antibodies, significantly improving the sensitivity of small molecule detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 Schematic diagram of a binary immune complex of a small molecule / small molecule binding partner provided in one embodiment of the present invention.

[0017] Figure 2 Schematic diagram of a ternary immune complex of a small molecule / small molecule binding partner complex-complex antibody provided by one embodiment of the present invention.

[0018] Figure 3 This is a schematic diagram of a quaternary immune complex of a first complex antibody-small molecule small molecule binding partner complex-second complex antibody provided by one embodiment of the present invention.

[0019] Figure 4 This is a diagram showing the stratification of the liquid after adding a dissociation agent to a whole blood sample for 10 minutes of dissociation, and then adding a diluent containing a defoaming agent and a diluent without a defoaming agent to precipitate for 5 minutes, as provided by an embodiment of the present invention.

[0020] Figure 5FIG. 1 is a schematic diagram of the composition of a sample analyzer provided in one embodiment of the present invention. DETAILED DESCRIPTION

[0021] A first aspect of the present invention relates to a whole blood sample analysis method applied to a sample analyzer, the method comprising: Step 1: Controlling a pipette to draw a whole blood sample containing small molecules from a sample storage mechanism and a dissociation agent from a reagent storage mechanism, respectively, and adding them to a first reaction cup to form a first mixture, wherein the working concentration of the dissociation agent in the mixed first mixture is 2-4 mol / L, and the dissociation agent is guanidine thiocyanate; Step 2: After the first mixture in step 1 has been allowed to react for 1-15 minutes, the pipette is controlled to draw a diluent and add it to the first reaction cup to form a second mixture, wherein the diluent is selected from PBS, Tris, MES, HEPES, or Citrate buffer, and the amount of the diluent drawn is at least twice the amount of the first mixture; Step 3: After the second mixture in step 2 is precipitated at 25-37° C. for 5-20 minutes to obtain a supernatant, the pipette is controlled to draw the supernatant from the first reaction cup to the second reaction cup; Step 4: controlling the detection of the concentration of the small molecule in the second reaction cup, wherein the concentration of the small molecule in the second reaction cup reflects the concentration of the small molecule in the whole blood sample; The small molecule is an organic compound with a molecular weight of less than 5000 Daltons.

[0022] The inventors of the present application have found that by treating whole blood samples in a high concentration environment of guanidine isothioate, small molecules can be dissociated from whole blood samples, especially suitable for the dissociation of sirolimus from its binding proteins in blood (affinity constant ≥ 10 -13 ), and then by adding a diluent with a volume of more than 2 times the volume of the first mixture, the proteins and other components suspended in the high concentration environment of guanidine isothioate can be caused to settle, thereby achieving the purpose of separation. At the same time, the above-mentioned analytical method will not destroy organic compounds with a molecular weight of less than 5000 Daltons, thereby not affecting the subsequent detection steps.

[0023] It should be noted that the pipette used in the analysis method can be one or more.

[0024] It should also be noted that the sample analyzer further includes one or more reaction cup transfer mechanisms for loading and transferring the first reaction cup and / or the second reaction cup.

[0025] In some embodiments of the present invention, the reaction temperature of the first mixture during the reaction for 1-15 minutes is 25-37°C.

[0026] In some embodiments of the present invention, the reaction temperature for the precipitation treatment of the second mixture for 5-20 min is 25-37°C.

[0027] In some embodiments of the present invention, the small molecule is selected from any one of drugs, biotoxins, environmental pollutants, hormones, and metabolites.

[0028] In some embodiments of the present invention, the small molecule is an immunosuppressant selected from any one of sirolimus, everolimus, tacrolimus, and cyclosporine.

[0029] In some embodiments of the present invention, in step 1, the ratio of the volume of the whole blood sample to the volume of the dissociation agent in the first mixture in the first reaction cup is 1:0.5-2. This is because the dissociation agent will cause a dilution effect when mixed with the whole blood sample, and excessive dilution can affect detection sensitivity.

[0030] In some embodiments of the present invention, in step 2, the volume of the diluent in the first cuvette is 2-4 times the volume of the first mixture. This is because the diluent also dilutes the first mixture after mixing. Excessive dilution can affect detection sensitivity, while excessive dilution can result in a smaller amount of supernatant after precipitation, hindering the sample analyzer from aspirating the supernatant into the second cuvette.

[0031] In some embodiments of the present invention, the diluent further comprises a defoaming agent, wherein the concentration of the defoaming agent in the diluent is in the range of 0.005% to 0.02%. The addition of the defoaming agent can prevent the formation of bubbles in the supernatant, which can prevent some proteins from settling quickly and affect subsequent sampling from the supernatant due to the inhalation of bubbles, resulting in inaccurate sampling.

[0032] In some preferred embodiments of the present invention, the main component of the defoaming agent is an organosilicon substance, selected from any one of polydimethylsiloxane, polyether-modified organosilicon, and fluorosilicone-modified polyether.

[0033] In some embodiments of the present invention, step 3 also includes controlling the pipette to draw one or more detection reagents from the reagent storage mechanism into the second reaction cup to form a multi-component immune complex, and step 4 includes controlling the detection of the concentration of the small molecule in the second reaction cup, including controlling the detection of the multi-component immune complex to obtain the concentration of the small molecule in the second reaction cup.

[0034] In some embodiments of the present invention, the detection reagent comprises a small molecule binding partner reagent and a competitive antigen reagent, and the multi-component immune complex is a binary immune complex of small molecule / small molecule binding partner, such as Figure 1 shown.

[0035] In some embodiments of the present invention, the detection reagent comprises a small molecule binding partner reagent and a complex antibody reagent. The multi-component complex is a ternary immune complex with a structure of small molecule / small molecule binding partner complex-complex antibody, such as Figure 2 The small molecule binding partner can specifically bind to the small molecule to form the small molecule / small molecule binding partner complex, and form at least one new conformational epitope that is different from the free small molecule and the free small molecule binding partner; the complex antibody specifically binds to at least one of the new conformational epitopes.

[0036] It should be noted that the complex antibody is not an antibody against the small molecule antigen or a specific antibody against the small molecule binding partner.

[0037] Due to the simple structure and small molecular weight of small molecules, at least one new conformational epitope is obtained by forming a small molecule / antigen binding fragment small molecule binding partner complex. This allows the construction of a detection system based on a complex-antibody sandwich, which can significantly improve the sensitivity of the detection results. Experiments have demonstrated that the small molecules obtained by dissociation with a high concentration of guanidine isothiocyanate followed by dilution and precipitation can be detected using the sandwich detection principle based on the aforementioned ternary immune complex.

[0038] In some embodiments, the detection reagent comprises a small molecule binding partner reagent and a complex antibody reagent, wherein the complex antibody reagent comprises a first complex antibody reagent or a second complex antibody reagent; the multicomponent immune complex is a quaternary immune complex having a structure of "first complex antibody-small molecule / small molecule binding partner complex-second complex antibody"; wherein the small molecule binding partner has at least two antigen-binding sites capable of binding to at least two small molecules to form the small molecule / small molecule binding partner complex, and forming at least two new conformational epitopes distinct from the free small molecule and the free small molecule binding partner; and the first complex antibody and the second complex antibody each specifically bind to at least one new conformational epitope. The use of a double complex antibody sandwich detection system further enhances the sensitivity of the detection results. Experiments have demonstrated that small molecules obtained by dissociation with a high-concentration guanidine isothiocyanate dissociator followed by dilution and precipitation can be detected using the aforementioned sandwich detection principle based on the quaternary immune complex.

[0039] In some embodiments of the present invention, the affinity constant between the complex antibody and the bound novel conformational epitope is K D1 , the affinity constant between the complex antibody and the small molecule binding partner is K D2 , then K D2 / K D1 ≥10 2 :1, or ≥10 3 :1, or ≥10 4 : 1. The above-mentioned difference in affinity is conducive to the first complex antibody and the second complex antibody to bind more specifically to the new conformational epitope, thereby improving the detection sensitivity.

[0040] It is easy to understand that the K of the first complex antibody D1、 K D2 and the K of the second complex antibody D1、 K D2 can be the same or different values, but their K D2 / K D1 All satisfy the above proportional relationship.

[0041] In some embodiments, the affinity constant K between the small molecule binding partner and the small molecule is D3 ≤9×10 -9 .

[0042] In some embodiments, K D1 ≤9×10 -9 .

[0043] In some embodiments, K D2 ≥1×10 -6 .

[0044] In some embodiments, 1×10 -6 ≤K D2 ≤9×10 -4 , or 1×10 -6 ≤K D2 ≤9×10 -5 .

[0045] In some embodiments, the complexed antibody does not bind or binds non-specifically to free small molecules.

[0046] The present invention uses the SPR method to detect affinity constants. The detection principle is based on optical principles: when biomolecules bind or dissociate on the sensor chip surface, they cause changes in the chip surface's refractive index, which in turn leads to changes in the SPR angle. By monitoring these changes, intermolecular interactions can be analyzed in real time.

[0047] The exemplary process mainly includes the following steps: first, the anti-small molecule antibody / complex antibody to be tested is fixed on the surface of the sensor chip; then, different concentration gradients of the test standard / anti-small molecule antibody are flowed through the chip in sequence as the mobile phase, and the dynamic changes of the SPR angle during the molecular binding and dissociation process are monitored in real time to generate the binding / dissociation curve; finally, the affinity constant (K) is calculated by fitting the curve data through the software. D ), thereby evaluating antibody affinity.

[0048] In some embodiments, in the aforementioned ternary immune complex or quaternary immune complex sandwich detection system, the small molecule binding partner is an anti-small molecule antibody.

[0049] In some embodiments, the anti-small molecule antibody and the complex antibody are complete immunoglobulins or antibody fragments. More advantageously, the small molecule antibody and the complex antibody are both monoclonal antibodies.

[0050] In some embodiments, the complex antibody comprises one or more antibody variable regions, which may further be Fv, Fab, Fab' or F(ab')2 fragments.

[0051] In some embodiments, in the aforementioned four-component immune complex sandwich detection principle, the first complex antibody and the second complex antibody have the same or different CDR sequences. In some preferred embodiments, the first complex antibody and the second complex antibody are derived from the same monoclonal antibody. Advantageously, both the first complex antibody and the second complex antibody are monoclonal antibodies.

[0052] In some embodiments, the anti-small molecule antibody is any one of IgG, IgA, IgM, IgD, and IgE.

[0053] In some embodiments, the anti-small molecule antibody is a whole immunoglobulin or an antibody fragment.

[0054] In some embodiments, in the aforementioned sandwich detection method of the quaternary immune complex, the anti-small molecule antibody comprises two or more antibody variable regions, which may further be F(ab')2. In some preferred embodiments, the anti-small molecule antibody comprises two or more antibody variable regions, and the sequences of the two or more antibody variable regions are identical or inconsistent.

[0055] In some embodiments, in the aforementioned sandwich detection principle of the ternary immune complex, in the detection reagent, one of the small molecule binding partner and the complex antibody exists in the form of being conjugated to the solid phase, and the other of the complex antibody and the small molecule binding partner exists in the form of being coupled to a signal generator.

[0056] In some embodiments, in the aforementioned sandwich detection principle of the quaternary immune complex, the small molecule binding partner exists in the small molecule binding partner reagent in a free state, the first complex antibody exists in the first complex antibody reagent in the form of being conjugated to the solid phase, and the second complex antibody exists in the second complex antibody reagent in the form of being coupled to a signal generator.

[0057] It should be noted that, in the present invention, a signal generator refers to any substance capable of generating a signal for direct or indirect detection. Therefore, the signal generator can be detected directly or indirectly. For direct detection, signal generators suitable for the present invention can be selected from any known chemiluminescent compounds, electrochemiluminescent compounds, enzymes, and fluorescent dyes (e.g., fluorescein, coumarin, rhodamine, oxazine, resorufin, cyanine, and their derivatives). Furthermore, the signal generator is selected from any one of luminol, isoluminol and its derivatives, horseradish peroxidase, alkaline phosphatase, acridinium esters and their derivatives, and terpyridine ruthenium. Common isoluminol derivatives include one or more of ABEI, AHEI, ABEN, isoluminol NHSester, isoluminol-derivatized acridinium esters, ALP / HRP-Linked Isoluminol Conjugates, and Poly-ABEI-Dendrimer / Polymer. Common ABEI derivatives include one or more of ABEI-NHS ester, ABEI-COOH, ABEI-SS-COOH, ABEI-BSA covalent complex, and ABEI-Dendrimer.

[0058] It should also be noted that, as used herein, the solid phase refers to a solid-phase material that interacts with a liquid-phase reagent via a heterogeneous reaction. The uses of solid phases are well known in the fields of chemistry, biochemistry, pharmacy, and molecular biology. Solid phases can be classified into various types based on their properties, including organic polymers such as polystyrene, polypropylene, glass, and silicone rubber; inorganic materials such as silicon-based materials, metal materials, and aluminum oxide; nanomaterials such as nanoparticles and nanofibers; and composite materials such as magnetic microspheres and nanocomposites. Immobilization on a solid phase can be achieved using the following methods: the solid phase is modified or activated to contain functional groups capable of covalently coupling to the antibody or antibody complex. The functional groups are selected from one or more of carboxyl, amino, sulfhydryl, and tosyl groups. The antibody or antibody complex can also be non-covalently attached to the solid phase, for example, via ionic or hydrophobic mechanisms. Furthermore, the solid phase can be in the form of a sheet, a preformed disk, a cylinder, fibers, or particles. The size of the solid phase suitable for use in the methods of the present invention can vary depending on the method chosen. The antibody may be bound to only one solid phase (e.g., a multiwell plate), or may be bound to many solid phases (e.g., beads). In one embodiment, the solid phase may be fibrous or granular to allow for optimal contact. The size of the solid phase may vary and may be selected based on the method to be performed.

[0059] In the present invention, the shape of the solid phase can generally vary. For example, in a particular embodiment, the solid phase is spherical. However, it should be understood that the present invention also contemplates other shapes, such as plates, rods, disks, strips, tubes, irregular shapes, and the like. In addition, the size of the solid phase can also vary. For example, the average size (e.g., diameter) of the solid phase can preferably range from 0.1 nm to 1 mm, such as 1 nm, 10 nm, 100 nm, 500 nm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 500 μm. Preferably, the solid phase is a magnetic sphere, such as ferrosoferric oxide nanomagnetic microspheres.

[0060] In some preferred embodiments, controlling the pipette to draw one or more detection reagents from the reagent storage mechanism into the second reaction cup to form a multiplexed immune complex comprises: controlling the pipette to draw the small molecule binding partner reagent from the reagent storage mechanism into the second reaction cup to form a small molecule / small molecule binding partner complex; The pipette is controlled to draw the first complex antibody reagent and the second complex antibody reagent from the reagent storage mechanism into the second reaction cup to obtain the multi-component immune complex containing the "first complex antibody-small molecule / small molecule binding partner complex-second complex antibody" structure.

[0061] In some more preferred embodiments, the method further comprises controlling the pipette to draw the first complex antibody reagent and the second complex antibody reagent from the reagent storage mechanism into the second reaction cup to obtain the multi-component immune complex containing the structure of "first complex antibody-small molecule / small molecule binding partner complex-second complex antibody", and further comprises at least one washing step to separate unbound substances from the solid phase.

[0062] It should be noted that the sample analyzer also includes a cleaning mechanism, which controls the reaction cup transfer mechanism to transfer the second reaction cup to the cleaning mechanism to perform the cleaning step, and can also control the pipette to add reagents to the second reaction cup by removing the reaction cup from the cleaning mechanism.

[0063] It should also be noted that the unbound substances mainly include impurities in the reaction system of the second reaction cup that are not bound to the solid phase, free small molecule binding partners that are not bound to small molecules in the system, and second complex antibodies coupled with signal substances that do not participate in the formation of a multi-complex; in this process, the washing step can be used to remove free small molecule binding partners that are non-specifically bound to the complex antibody.

[0064] Specifically, it can be a two-step washing process. The first washing step is performed after the small molecule / small molecule binding partner complex and the first complex antibody reagent are contacted and incubated until the two are fully reacted, so that the first unbound substance is separated from the solid phase. The first unbound substance mainly includes impurities in the sample that are not bound to the solid phase and free small molecule binding partners in the system that are not bound to small molecules. In this process, the washing step can be used to remove free small molecule binding partners that are non-specifically bound to the complex antibody. The second washing step is performed after the second complex antibody reagent is contacted with the solid phase obtained in the first washing step and incubated until the two are fully bound to form a multi-component immune complex, so that the second unbound substance is separated from the multi-component complex. The second unbound substance mainly includes the second complex antibody coupled with the signal substance that does not participate in the formation of the multi-component complex.

[0065] Specifically, it can also be a one-step washing step, which is only performed after the small molecule / small molecule binding partner complex contacts with the first complex antibody reagent and the second complex antibody reagent and the system is incubated until it is fully bound to form a multi-component immune complex, so that the above-mentioned unbound substances are separated from the system at one time in this washing step, wherein the washing step in this process can remove the free small molecule binding partner that non-specifically binds to the complex antibody.

[0066] In some embodiments, the sample analyzer includes a sample storage mechanism 10 , a reagent storage mechanism 20 , a reaction device 30 , a measuring device 40 , a pipette 50 , and a control device 60 .

[0067] Wherein, the sample storage mechanism 10 is used to store sample tubes carrying whole blood samples; A reagent storage mechanism 20 is used to store reagents, including a dissociation agent, a diluent, and one or more detection reagents; The reaction device 30 has at least one placement position, wherein the placement position is used to place a reaction cup and incubate the reaction liquid in the reaction cup; The measuring device 40 is used to perform optical measurement on the reaction solution after incubation to obtain the test result of the sample to be tested; Pipette 50, used to transfer the whole blood sample or the reagent to the reaction cup The control device 60 is electrically connected to the sample storage mechanism 10, the reagent storage mechanism 20, the reaction device 30, the measuring device 40 and the pipette 50, and is configured to: receiving a test instruction, wherein the test instruction includes the type of small molecules to be tested in the whole blood sample; In response to the test instruction: Step 1: Controlling a pipette to draw a whole blood sample containing small molecules from a sample storage mechanism and a dissociation agent from a reagent storage mechanism, respectively, and adding them to a first reaction cup to form a first mixture, wherein the working concentration of the dissociation agent in the mixed first mixture is 2-4 mol / L, and the dissociation agent is guanidine thiocyanate; Step 2: After the first mixture in step 1 is allowed to react for 1-15 minutes, the pipette is controlled to draw a diluent from the reagent storage mechanism and add it to the first reaction cup to form a second mixture, wherein the diluent is selected from PBS, Tris, MES, HEPES, and Citrate buffer, and the amount of the diluent in the second mixture is more than twice the amount of the first mixture; Step 3: After the second mixture is precipitated for 5-20 minutes in step 2 to obtain a supernatant, the pipette is controlled to draw the supernatant from the first reaction cup to the second reaction cup; Step 4: controlling the calculation of the concentration of the small molecule in the second reaction cup according to the signal value in the second reaction cup measured by the measuring device, so as to reflect the concentration of the small molecule in the whole blood sample; The small molecule is an organic compound with a molecular weight of less than 5000 Daltons.

[0068] As an embodiment, the sample storage mechanism is used to store sample tubes containing whole blood samples.

[0069] The sample tube is used to load samples collected from patients, which are whole blood samples in this solution. The sample tube can be stored in a sample rack for storing batch sample tubes or in a sample container for storing single sample tubes.

[0070] The sample storage mechanism includes a sample storage assembly and a sample loading assembly. The sample storage assembly is used to store sample tubes. Specifically, the sample storage assembly is used to store multiple sample racks or sample containers. The sample loading assembly is used to transport the sample tubes to the sample aspiration position for the pipette to aspirate samples at the sample aspiration position. Specifically, the sample loading assembly removes the sample rack or sample container loaded with sample tubes from the sample storage assembly and transports it to the sample aspiration position. Of course, in specific applications, the sample storage mechanism may also be provided without the sample loading assembly. For example, as an alternative embodiment, the sample tubes loaded with samples are placed by an operator at the sample aspiration position for the pipette to aspirate samples.

[0071] As an embodiment, the reagent storage mechanism is used to store reagents, and the reagents include a dissociation agent, a diluent, and one or more detection reagents.

[0072] The reagent storage mechanism is used to store a test kit, which includes a dissociation solution, a diluent, and one or more detection reagents, wherein the one or more detection reagents include an antigen or antibody reagent coupled to a solid phase and an antigen or antibody reagent conjugated to a signal generator. In some embodiments, the reagent storage mechanism can be a reagent bin provided with a plurality of reagent positions, the reagent positions being used to carry the reagents required for detection by a sample analyzer, and the pipette moves to the corresponding reagent position to absorb the required reagents. In some embodiments, the reagent storage mechanism can also be a reagent disc, which is arranged in a disc-shaped structure and has a plurality of reagent positions for carrying the reagents required for detection by a sample analyzer. The reagent storage mechanism 20 can rotate and drive the reagents it carries to rotate, and is used to rotate the reagent position to a specific position, such as the position where the pipette 50 absorbs the reagent. The number of reagent storage mechanisms can be one or more.

[0073] In one embodiment, the reaction device has at least one placement position for placing a reaction cup and incubating the reaction liquid in the reaction cup. For example, the reaction device can be a reaction disk, which is arranged in a disc-shaped structure and has one or more placement positions for placing reaction cups. The reaction disk can rotate and drive the reaction cups in its placement positions to rotate, which is used to arrange the reaction cups in the reaction disk and incubate the reaction liquid in the reaction cups. The incubation temperature can be 25-37°C. In a sample analysis, there can be one or more reaction devices.

[0074] In one embodiment, the measuring device is configured to perform optical measurement on the reaction solution after incubation to obtain a test result of the sample to be tested. For example, based on the optical signal information obtained from the optical measurement of the reaction solution, the concentration of the test component in the sample can be calculated using a calibration curve. The test component can be a target antigen or a target antibody in the sample.

[0075] In one embodiment, a pipette is used to transfer the whole blood sample or the reagent into the reaction cup. The pipette utilizes a two-dimensional or three-dimensional drive mechanism to perform two-dimensional or three-dimensional motion in space, thereby enabling the pipette to move to a sample or reagent aspiration position to aspirate the sample or reagent. It should be noted that one or more pipettes may be used during this analytical method.

[0076] In one embodiment, the sample analyzer further includes a cuvette transfer mechanism for transferring the cuvette. The cuvette transfer mechanism utilizes a two-dimensional or three-dimensional drive mechanism to perform two-dimensional or three-dimensional motion in space, and can transfer the cuvette to a designated position.

[0077] As an embodiment, the control device is electrically connected to the sample storage mechanism, the reagent storage mechanism, the reaction device, the measuring device, the reaction cup transfer mechanism and the pipette.

[0078] Wherein, the control device is configured to: receive a test instruction, wherein the test instruction includes the type of small molecules to be tested in the whole blood sample; In response to the test instruction: Step 1: controlling the pipette 50 to draw a whole blood sample containing small molecules from the sample storage mechanism and a dissociation agent from the reagent storage mechanism and adding them to the first reaction cup to form a first mixture, wherein the working concentration of the dissociation agent in the mixed first mixture is 2-4 mol / L, and the dissociation agent is guanidine thiocyanate; comprising: The cuvette transfer mechanism is controlled to load the first cuvette into the reaction apparatus, and the pipette is controlled to draw a corresponding volume of the whole blood sample from the sample storage mechanism and a corresponding volume of the dissociation agent from the reagent storage mechanism into the first cuvette in the reaction apparatus. Without limitation, the cuvette transfer mechanism can be used to transfer the cuvette to a location near the sample storage mechanism or the reagent storage mechanism in order to reduce the pipette's travel distance during the transfer of the whole blood sample and the dissociation agent.

[0079] In some specific embodiments, in step 1, the ratio of the volume of the whole blood sample to the volume of the dissociation agent in the first mixture in the first reaction cup is 1:0.5-2.

[0080] As an embodiment, the controller is further configured to: execute step 2 after step 1.

[0081] In some specific embodiments, step 2 includes: after the first mixture in step 1 is allowed to react for 1-15 minutes, controlling the pipette to draw a diluent from the reagent storage mechanism and adding it to the first reaction cup to form a second mixture, wherein the diluent comprises a buffer solution such as PBS, tris, MES, HEPES, etc., and the amount of the diluent in the second mixture is more than twice the amount of the first mixture.

[0082] More specifically, the first reaction cup containing the first mixed solution is allowed to react in the reaction device for 1-15 minutes at an incubation temperature of 25-37° C., and then the pipette is controlled to draw a corresponding volume of diluent from the reagent storage mechanism and add it to the first reaction cup.

[0083] It should be noted that, during the static reaction process, it is permitted for the reaction device or the reaction cup transfer mechanism to control the first reaction cup to move smoothly on the horizontal plane without affecting the reaction effect.

[0084] In some specific embodiments, in step 2, the volume of the diluent in the second mixture is 2-4 times the volume of the first mixture.

[0085] In some embodiments, the small molecule is selected from any one of drugs, biotoxins, environmental pollutants, hormones, and metabolites.

[0086] In some embodiments, the small molecule is an immunosuppressant selected from any one of sirolimus, everolimus, tacrolimus, and cyclosporine.

[0087] In some specific embodiments, the diluent further comprises a defoaming agent, and the concentration of the defoaming agent in the diluent is in the range of 0.005%-0.02%.

[0088] In some more specific embodiments, the main component of the defoaming agent is an organosilicon substance, and the organosilicon substance is selected from any one of polydimethylsiloxane, polyether-modified organosilicon, and fluorosilicone-modified polyether.

[0089] As an embodiment, the controller is further configured to execute step 3 after step 2.

[0090] In some specific embodiments, step 3 includes: after the second mixture is precipitated for 5-20 minutes in step 2 to obtain a supernatant, controlling the pipette to draw the supernatant from the first reaction cup to the second reaction cup.

[0091] Specifically, step 3 includes: Allow the first reaction cup containing the second mixture to settle in the reaction device for 5-20 minutes, The reaction cup transfer mechanism is controlled to load the second reaction cup into the reaction device, wherein the first reaction cup and the second reaction cup can be located in the same reaction device or in different reaction devices. After the precipitation treatment is completed, the pipette is controlled to absorb the supernatant from the liquid surface of the first reaction cup into the second reaction cup to avoid inhaling sediment. At the same time, the pipette's suction port is controlled to always be below the liquid surface during the absorption process to avoid inhaling air.

[0092] In some specific embodiments, step 3 further includes controlling the pipette to draw one or more detection reagents from the reagent storage mechanism into the second reaction cup to form a multiplexed immune complex.

[0093] In some more specific embodiments, the detection reagent comprises a small molecule binding partner reagent and a complex antibody reagent, and the multi-component immune complex is a ternary immune complex with a structure of small molecule / small molecule binding partner complex-complex antibody, and the small molecule binding partner can specifically bind to the small molecule to obtain the small molecule / small molecule binding partner complex, and form a new conformational epitope that is different from the free small molecule and the free small molecule binding partner, and the complex antibody specifically binds to at least one of the new conformational epitopes.

[0094] In some more specific embodiments, the detection reagent comprises a small molecule binding partner reagent and a complex antibody reagent, and the complex antibody reagent comprises a first complex antibody reagent and a second complex antibody reagent; the multi-component immune complex is a quaternary immune complex with a structure of "first complex antibody-small molecule / small molecule binding partner complex-second complex antibody"; wherein the small molecule binding partner has at least two antigen binding sites, which can bind to at least two of the small molecules to obtain the small molecule / small molecule binding partner complex comprising at least two new conformational epitopes; the first complex antibody and the second complex antibody each specifically bind to at least one new conformational epitope.

[0095] In some more specific embodiments, in the aforementioned sandwich detection principle of the ternary immune complex, in the detection reagent, one of the small molecule binding partner and the complex antibody exists in the form of being conjugated to the solid phase, and the other of the complex antibody and the small molecule binding partner exists in the form of being coupled to a signal generator.

[0096] In some more specific embodiments, in the aforementioned sandwich detection principle of the quaternary immune complex, the small molecule binding partner exists in the small molecule binding partner reagent in a free state, the first complex antibody exists in the first complex antibody reagent in the form of being conjugated to the solid phase, and the second complex antibody exists in the second complex antibody reagent in the form of being coupled to a signal generator.

[0097] In some more specific embodiments, controlling the pipette to draw one or more detection reagents from the reagent storage mechanism into the second reaction cup to form a multiplexed immune complex comprises: controlling the pipette to draw the small molecule binding partner reagent from the reagent storage mechanism into the second reaction cup to form a small molecule / small molecule binding partner complex; The pipette is controlled to draw the first complex antibody reagent and the second complex antibody reagent from the reagent storage mechanism into the second reaction cup to obtain the multi-component immune complex containing the "first complex antibody-small molecule / small molecule binding partner complex-second complex antibody" structure.

[0098] As an embodiment, the control device is further configured to execute step 4 after the reaction cup is fully filled in step 3.

[0099] In some specific embodiments, step 4 includes: after step 3, controlling the calculation of the concentration of the small molecule in the second reaction cup based on the signal value in the second reaction cup measured by the measuring device, reflecting the concentration of the small molecule in the whole blood sample.

[0100] In some more specific embodiments, after the second reaction cup is fully reacted to obtain a multi-component immune complex, step 4 includes controlling the second reaction cup to be transferred to a measuring device, detecting the signal value of the multi-component immune complex in the second reaction cup to obtain the concentration of the small molecule in the second reaction cup.

[0101] In one embodiment, the sample analyzer further comprises a washing mechanism for performing at least one washing step to separate unbound substances from the solid phase, specifically by repeatedly adding and withdrawing a washing solution to wash the unbound substances in the second reaction cup.

[0102] In some specific embodiments, step 3 of controlling the pipette to draw the first complex antibody reagent and the second complex antibody reagent from the reagent storage mechanism into the second reaction cup to obtain the multi-component immune complex containing the "first complex antibody-small molecule / small molecule binding partner complex-second complex antibody" structure also includes at least one washing step to separate unbound substances from the solid phase.

[0103] Specifically, the second reaction cup is transferred from the reaction device to the cleaning mechanism by controlling the reaction cup transfer mechanism to perform the cleaning step.

[0104] More specifically, a two-step wash process can be employed. In the first wash, after the small molecule / small molecule binding partner complex and the first complex antibody reagent are brought into contact and incubated until they fully react, the cuvette transfer mechanism is controlled to transfer the second cuvette to a wash mechanism for a wash operation, thereby separating the first unbound material from the solid phase. The first unbound material primarily includes impurities in the sample that are not bound to the solid phase and free small molecule binding partner in the system that is not bound to the small molecule. This wash process removes free small molecule binding partner that nonspecifically binds to the complex antibody. The cuvette transfer mechanism is then controlled to transfer the second cuvette to a reaction apparatus, and a pipette is then controlled to draw the second complex antibody reagent from the actual storage mechanism into the second cuvette. After sufficient binding to form a multicomponent immune complex, the cuvette transfer mechanism is again controlled to transfer the second cuvette to a wash mechanism for a wash operation, thereby separating the second unbound material from the multicomponent complex. The second unbound material primarily includes the second complex antibody conjugated to the signaling substance that did not participate in the formation of the multicomponent complex.

[0105] Specifically, it can also be a one-step cleaning. After the small molecule / small molecule binding partner complex contacts the first complex antibody reagent and the second complex antibody reagent and the system is incubated until it is fully combined to form a multi-component immune complex, the reaction cup transfer mechanism is controlled to transfer the second reaction cup to the cleaning mechanism to perform a cleaning operation, so that the above-mentioned unbound substances are separated from the system at one time in this cleaning. In this process, the free small molecule binding partner that non-specifically binds to the complex antibody can be removed by using the cleaning step.

[0106] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. For experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention, and may also be made to experimental manuals or conventional conditions in the art, other experimental methods known in the art, or conditions recommended by the manufacturer.

[0107] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0108] Example 1 Antibody Preparation

[0109] 1. Source of anti-small molecule antibodies: small molecule antibodies in the Snibe Sirolimus Detection Kit (Product No. 130207008M).

[0110] 2. Source of complex antibody: The complex antibody in the Snibe Sirolimus Detection Kit (Product No. 130207008M).

[0111] 3. Binding characteristics of complex antibodies to complexes, anti-small molecule antibodies, and free antigens, as well as binding affinity assessment.

[0112] The binding affinity assessment assay for the sirolimus complex antibody is performed as follows.

[0113] a. Affinity detection of complex antibodies with small molecules or anti-small molecule antibodies.

[0114] (1) Immobilization of complex antibodies: The ligand was immobilized by direct amino coupling. First, EDC / NHS (prepared and used immediately, 1:1 mixture) was injected for 5-10 minutes to activate the carboxyl group. Then, the complex antibody was diluted to 10 μg / ml with sodium acetate buffer at pH 4.5 and injected into the detection channel to the target coupling level (small molecule analytes, high coupling, 20,000 RU; large analytes, low coupling, 100-500 RU). Then, ethanolamine (pH 8.5) was injected for 5-10 minutes to block the unreacted sites. Finally, the equilibration buffer was used to rinse until the baseline was stable.

[0115] (2) Analyte detection: Small molecules were diluted to 100 μM, or anti-small molecule antibodies were diluted to 10 μM, and then diluted 2-fold to 6 gradients, plus 0 concentration for a total of 8. A multi-cycle kinetic detection method was used, with injection from low to high concentrations at a flow rate of 10 μL / min for 180 s, a dissociation time of 600 s, and regeneration with 10 mM Gly-HCl (pH 2.0) for 120 s.

[0116] b. Affinity detection of complex antibody and small molecule / anti-small molecule antibody complex.

[0117] (1) Immobilization of anti-small molecule antibodies: Ligand immobilization is performed using the amino direct coupling method. First, EDC / NHS (freshly prepared and used, 1:1 mixture) is injected for 5-10 minutes to activate the carboxyl group; then the anti-small molecule antibody is diluted to 10 μg / ml with pH 4.5 sodium acetate buffer and injected into the detection channel to the target coupling level (large analyte, low coupling, 100-500 RU); then ethanolamine (pH 8.5) is injected for 5-10 minutes to block unreacted sites; (4) Rinse with equilibrium buffer until the baseline is stable.

[0118] (2) Small molecule injection: The small molecule solution was diluted to 100 μM and injected until the curve stabilized.

[0119] (3) Co-injection of small molecule and complex antibody: The complex antibody was diluted to 10 μM (containing 100 μM of the small molecule), and then diluted 2-fold to 6 concentrations (the dilution solution contained 100 μM of the small molecule). A multi-cycle kinetic detection method was used, and the samples were injected in the order from low concentration to high concentration at a flow rate of 10 μL / min for 120 s. The dissociation phase was still carried out with a 100 μM small molecule solution, followed by regeneration and baseline equilibration.

[0120] c. Use analysis software to fit and analyze data and calculate affinity.

[0121] The test results are shown in Table 1, indicating that the complex antibody in the kit has a high affinity for the small molecule / small molecule antibody complex and has a weak binding to the free anti-small molecule antibody.

[0122] Table 1 Composite antibody Anti-small molecule antibodies Small molecule / anti-small molecule antibody complex Sirolimus complex antibody <![CDATA[3.26*10 -5 ]]> <![CDATA[1.54*10 -10 ]]>

[0123] Example 2 Preparation and performance verification of immunosuppressant kit.

[0124] 1. Components of the immunosuppressant kit.

[0125] Dissociation solution: Guanidine isothiocyanate aqueous solution, 6 mol / L.

[0126] Diluent: 50 mM Tris-HCl, pH 8.0, containing 5 g / L BSA, 5.85 g / L NaCl, 0.1% (v / v) ProClin 300, and 0.01% (v / v) defoaming agent (dimethylsiloxane).

[0127] Immunodetection reagents: 1) Reagent components based on the sandwich detection principle of ternary immune complex: Buffer: Tris buffer containing 0.1% ProClin300; Solid phase reagents: magnetic microspheres coupled to antibodies against small molecule analytes to be detected; Labeling reagent: a complex antibody linked to a signal substance.

[0128] 2) Reagent components based on the sandwich detection principle of quaternary immune complexes: Pre-reaction solution 1: Tris buffer containing 0.1% ProClin300 and anti-small molecule antibody; Solid phase reagents: Tris buffer, magnetic microspheres coupled to complex antibodies; Labeling reagents: Tris buffer, complex antibody linked to signal substance.

[0129] 2. Detection process of immunosuppressant kit

[0130] Method 1: Sandwich assay based on ternary immune complex.

[0131] Pretreatment step: Take 50 μL of sample and transfer it to the first reaction cup. Then add the corresponding volume of dissociation agent according to Table 1 and mix thoroughly to obtain the first mixed solution. Incubate at 37°C for 10 minutes. Then add the corresponding volume and component dilution according to Table 1 and mix thoroughly. At this time, there is a precipitate of denatured protein in the system. Let it settle for 20 minutes.

[0132] Testing steps:

[0133] ① Take 100 μL of the supernatant obtained in the pretreatment step and transfer it to the second reaction cup. Add 80 μL of buffer and 20 μL of solid phase reagent. Mix well and incubate at 37°C for 10 min. Wash and remove the supernatant. ②Add 150 μL of labeling reagent to step ①, mix well, incubate at 37°C for 10 min, and remove the supernatant after washing; ③Add substrate solution and measure the light signal.

[0134] Method 2: Sandwich detection principle based on quaternary immune complex.

[0135] Pretreatment step: Take 50 μL of sample and transfer it to the first reaction cup. Then add the corresponding volume of dissociation agent according to Table 1 and mix thoroughly to obtain the first mixed solution. Incubate at 37°C for 10 minutes. Then add the corresponding volume and component dilution according to Table 1 and mix thoroughly. At this time, there is a precipitate of denatured protein in the system. Let it settle for 20 minutes.

[0136] Testing steps:

[0137] ① Take 100 μL of the supernatant obtained in the pretreatment step into the second reaction cup, add 100 μL of pre-reaction solution 1, mix well, and incubate at 37°C for 4 minutes; ②Add 20 μL of solid phase reagent to step ①, mix well, incubate at 37°C for 4 minutes, and remove the supernatant after washing; ③Add 150 μL of labeling reagent to step ②, mix well, incubate at 37°C for 4 minutes, and remove the supernatant after washing; ④Add substrate solution and measure the light signal.

[0138] The experimental conditions of Examples 2.1-2.10 are shown in Table 1. Example 2.11 is based on the pretreatment steps of Example 2.2, except that "standing for 20 minutes" is changed to "centrifugation at 10,000×g for 5 minutes". Table 2: Dissociation agent working concentration Dissociation agent and sample addition volume ratio Components of the diluent Volume ratio of diluent: first mixed solution Defoamer concentration in dilution solution Example 2.1 2 0.5:1 Tris + dimethylsiloxane 3：1 0.010% Example 2.2 3 1:1 Tris + dimethylsiloxane 3：1 0.010% Example 2.3 3 1:1 Tris + dimethylsiloxane 2：1 0.010% Example 2.4 3 1:1 Tris + dimethylsiloxane 4：1 0.010% Example 2.5 3 1:1 Tris + dimethylsiloxane 3：1 0.005% Example 2.6 3 1:1 Tris + dimethylsiloxane 3：1 0.020% Example 2.7 3 1:1 Tris+polyether modified silicone 3：1 0.010% Example 2.8 3 1:1 Tris+fluorosilicone modified polyether 3：1 0.010% Example 2.9 4 2:1 Tris + dimethylsiloxane 3：1 0.010% Example 2.10 3 1:1 Tris 3：1 /

[0139] 3. Results evaluation.

[0140] 1) Verification of precipitation: Take photos after 5 minutes of precipitation. Take photos of the stratification of Example 2.2 and Example 2.10. Figure 4 The left cup is Example 2.2 with defoaming agent added, and the right cup is Example 2.10 without defoaming agent.

[0141] The experimental results show that obvious stratification can be formed regardless of whether a defoaming agent is added. Furthermore, it can be seen that the liquid surface of Example 2.10 without a defoaming agent contains more bubbles, which will adsorb precipitated particles after oscillation and affect the absorption of the supernatant, thereby affecting the repeatability of the test.

[0142] 2) Sensitivity assessment.

[0143] Testing instrument: MAGLUMI X3.

[0144] Sample preparation: Mixed whole blood (sirolimus 10.92 ng / mL) assigned by mass spectrometry was used as the positive sample; mixed whole blood from drug-free subjects was used as the negative sample.

[0145] Indicator verification method: Whole blood samples were pretreated according to different examples / comparative examples, and the test was repeated multiple times on the instrument to obtain relative light intensity (RLU).

[0146] Indicator interpretation method: [Average RLU of positive samples / Average RLU of negative samples] (hereinafter referred to as RLU ratio) reflects the detection sensitivity of the pretreatment solution. The larger the RLU ratio, the better the dissociation effect and the better the detection sensitivity.

[0147] 3) Repeatability assessment.

[0148] Testing instrument: MAGLUMI X3.

[0149] Sample preparation: Mixed whole blood (sirolimus 10.92 ng / mL) assigned by mass spectrometry was used as the positive sample; mixed whole blood from drug-free subjects was used as the negative sample.

[0150] Indicator verification method: Whole blood samples were pretreated according to different examples / comparative examples, and the test was repeated multiple times on the instrument to obtain relative light intensity (RLU).

[0151] Indicator interpretation method: The RLU imprecision (CV) of positive samples reflects the detection repeatability of the pretreatment scheme. The smaller the CV, the better the homogeneity of the supernatant and the better the detection repeatability.

[0152] The evaluation results of sensitivity and repeatability are shown in Table 3: Testing process Negative titer average Average positive titer RLU ratio CV Example 2.1 Method 1 16356.6 223094.4 13.6 4.56% Example 2.1 Method 2 2609.4 47155.2 18.1 3.57% Example 2.2 Method 1 15387.4 308680.6 20.1 3.05% Example 2.2 Method 2 2357.8 64996.0 27.6 2.36% Example 2.3 Method 1 15580.8 232410.6 14.9 2.34% Example 2.3 Method 2 2621.2 46046.0 17.6 3.68% Example 2.4 Method 1 15574.0 243497.8 15.6 2.73% Example 2.4 Method 2 2263.0 44699.0 19.8 3.14% Example 2.5 Method 1 15435.2 298477.0 19.3 10.34% Example 2.5 Method 2 2423.4 76722.0 31.7 10.52% Example 2.6 Method 1 16043.0 256486.4 16.0 4.74% Example 2.6 Method 2 2111.0 41538.0 19.7 4.10% Example 2.7 Method 1 15218.4 254593.4 16.7 10.38% Example 2.7 Method 2 2509.0 46765.0 18.6 8.81% Example 2.8 Method 1 15366.2 267725.2 17.4 9.53% Example 2.8 Method 2 2264.8 40686.4 18.0 8.09% Example 2.9 Method 1 15151.4 240284.8 15.9 2.87% Example 2.9 Method 2 2462.6 49559.0 20.1 3.71% Example 2.10 Method 1 21363.8 216141.2 10.1 35.72% Example 2.10 Method 2 3685.8 39531.6 10.7 24.25% Example 2.11 Method 1 15615.4 303913.0 19.5 4.34% Example 2.11 Method 2 2376.6 67775.4 28.5 4.66%

[0153] Based on the experimental results in Table 3.

[0154] The RLU ratios for Examples 2.1-2.11 were all greater than 10, demonstrating the ability to distinguish between positive and negative samples and demonstrating reasonable sensitivity. Furthermore, the sandwich assay based on the quaternary immune complex performed better than the sandwich assay based on the ternary immune complex. This demonstrates that the dissociation agent treatment followed by dilution method of the present invention can separate sirolimus from samples and can be used in both immunoassays described above.

[0155] As can be seen from Example 2.2 and Example 2.11, when the centrifugation step is performed under the same dissociation agent and dilution conditions, the effect of taking the supernatant is consistent. Compared with the centrifugation method, no additional equipment is required, and instrument automation pretreatment can be achieved.

[0156] From Examples 2.1 and 2.2, it can be seen that as the working concentration of the dissociation agent increases, the RLU ratio increases and the CV value decreases, indicating that the dissociation effect is improved. Compared with Example 2.2 and Example 2.9, the RLU decreases slightly, indicating that too high a concentration of the dissociation agent will affect the titer of subsequent reactions and have a certain inhibitory effect on the immune response.

[0157] As can be seen from Examples 2.2, 2.7, and 2.8 and Example 2.10, the addition of a defoaming agent can improve sensitivity and repeatability. Common silicone substances including dimethylsiloxane, polyether-modified silicone, and fluorosilicone-modified polyether can all achieve similar effects. Among them, dimethylsiloxane has the best defoaming effect and has less impact on normal immunoassay reactions.

[0158] As can be seen from the results of Examples 2.2, 2.3, and 2.4 compared to Example 2.11 using the centrifugation method, the fully automatic implementation scheme of the present invention can meet or even exceed the results of centrifugation. The data fluctuations also indicate that the dissociation agent (guanidine thiocyanate) has an impact on the subsequent immunoassay reaction. If the dilution factor is too small, the titer will be reduced due to the inhibitory effect of the high concentration of the dissociation agent; if the dilution factor is too large, the titer will also be reduced due to the decrease in analyte concentration.

[0159] From Example 2.2, Example 2.5, and Example 2.6 and Example 2.10, it can be seen that as the concentration of the defoaming agent increases, the repeatability becomes better, indicating that adding a defoaming agent helps to improve the defoaming effect. Figure 4 It seems that the difference in repeatability may be due to the fact that the precipitated particles adhered to the bubbles on the liquid surface affect the sample aspiration; by comparing Example 2.2 and Example 2.6, it can be seen that further increasing the defoaming agent concentration will affect the RLU ratio, which may be that the defoaming agent itself affects the immunoassay reaction, resulting in a decrease in the detection titer.

[0160] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the concept of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims, and the description and drawings may be used to interpret the content of the claims.

Claims

1. A whole blood sample analysis method applied to a sample analyzer, the method comprising: Step 1: Controlling a pipette to draw a whole blood sample containing small molecules from a sample storage mechanism and a dissociation agent from a reagent storage mechanism, respectively, and adding them to a first reaction cup to form a first mixture, wherein the working concentration of the dissociation agent in the mixed first mixture is 2-4 mol / L, and the dissociation agent is guanidine thiocyanate; Step 2: After the first mixture in step 1 is allowed to react for 1-15 minutes, the pipette is controlled to draw a diluent from the reagent storage mechanism and add it to the first reaction cup to form a second mixture, wherein the diluent is selected from PBS, Tris, MES, HEPES, and Citrate buffer, and the amount of the diluent in the second mixture is more than twice the amount of the first mixture; Step 3: After the second mixture is precipitated for 5-20 minutes in step 2 to obtain a supernatant, the pipette is controlled to draw the supernatant from the first reaction cup to the second reaction cup; Step 4: controlling the detection of the concentration of the small molecule in the second reaction cup, wherein the concentration of the small molecule in the second reaction cup reflects the concentration of the small molecule in the whole blood sample; Wherein, the small molecule is an organic compound with a molecular weight of less than 5000 Daltons; Optionally, in step 1, the ratio of the volume of the whole blood sample to the volume of the dissociation agent in the first mixture in the first reaction cup is 1:0.5-2; Optionally, in step 2, the volume of the diluent in the second mixture is 2-4 times the volume of the first mixture; Optionally, the small molecule is selected from any one of drugs, biotoxins, environmental pollutants, hormones, and metabolites; Optionally, the small molecule is an immunosuppressant selected from any one of sirolimus, everolimus, tacrolimus, and cyclosporine; Optionally, the diluent further comprises a defoaming agent, and the concentration of the defoaming agent in the diluent is in the range of 0.005%-0.02%; Further optionally, the main component of the defoaming agent is an organosilicon substance selected from any one of polydimethylsiloxane, polyether-modified organosilicon, and fluorosilicone-modified polyether.

2. According to the method according to claim 1, step 3 also includes controlling the pipette to draw one or more detection reagents from the reagent storage mechanism into the second reaction cup to form a multi-component immune complex, and the controlling the detection of the concentration of the small molecule in the second reaction cup in step 4 includes controlling the detection of the multi-component immune complex to obtain the concentration of the small molecule in the second reaction cup.

3. The method according to claim 2, wherein the detection reagent comprises a small molecule binding partner reagent and a complex antibody reagent, the multi-component immune complex is a ternary immune complex with a structure of small molecule / small molecule binding partner complex-complex antibody, the small molecule binding partner can specifically bind to the small molecule to obtain the small molecule / small molecule binding partner complex, and form a new conformational epitope that is different from the free small molecule and the free small molecule binding partner, and the complex antibody specifically binds to at least one of the new conformational epitopes.

4. The method according to claim 2, wherein the detection reagent comprises a small molecule binding partner reagent and a complex antibody reagent, the complex antibody reagent comprising a first complex antibody reagent and a second complex antibody reagent; the multi-component immune complex is a quaternary immune complex having a structure of "first complex antibody-small molecule / small molecule binding partner complex-second complex antibody"; wherein the small molecule binding partner has at least two antigen binding sites, which can bind to at least two of the small molecules to obtain the small molecule / small molecule binding partner complex, and form at least two new conformational epitopes that are different from the free small molecule and the free small molecule binding partner; the first complex antibody and the second complex antibody each specifically bind to at least one new conformational epitope.

5. The method according to claim 3 or 4, wherein the affinity constant between the complex antibody and the new conformational epitope is K D1 , the affinity constant between the small molecule and the binding partner is K D2 , then K D2 / K D1 ≥10 2 :1, or ≥10 3 :1, or ≥10 4 :1; Optionally, the affinity constant K between the small molecule binding partner and the small molecule D3 ≤9×10 -9 ; Optionally, K D1 ≤9×10 -9 ; Optionally, K D2 ≥1×10 -6 .

6. The method according to claim 4, wherein the complex antibody is a complete immunoglobulin or an antibody fragment; Optionally, the first complex antibody and the second complex antibody have the same or different CDR sequences; Optionally, the first complex antibody or the second complex antibody comprises one or more antibody variable regions, which may further be Fv, Fab, Fab' or F(ab')2 fragments.

7. The method according to claim 3 or 4, wherein the small molecule binding partner is an anti-small molecule antibody; Optionally, the anti-small molecule antibody is selected from any one of IgG, IgA, IgM, IgD, and IgE; Optionally, the anti-small molecule antibody is a complete immunoglobulin or an antibody fragment; Optionally, the anti-small molecule antibody comprises two or more antibody variable regions, which may further be F(ab')2; Optionally, the anti-small molecule antibody comprises two or more antibody variable regions, and the sequences of the two or more antibody variable regions are identical or inconsistent.

8. The method according to claim 3, wherein in the detection reagent, one of the small molecule binding partner and the complex antibody exists in a form conjugated to a solid phase, and the other of the complex antibody and the small molecule binding partner exists in a form coupled to a signal generator.

9. The method according to claim 4, wherein the small molecule binding partner is present in the small molecule binding partner reagent in a free state, the first complex antibody is present in the first complex antibody reagent in the form of being conjugated to a solid phase, and the second complex antibody is present in the second complex antibody reagent in the form of being coupled to a signal generator.

10. The method according to claim 9, wherein controlling the pipette to draw one or more detection reagents from the reagent storage mechanism into the second reaction cup to form a multiplexed immune complex comprises: controlling the pipette to draw the small molecule binding partner reagent from the reagent storage mechanism into the second reaction cup to form a small molecule / small molecule binding partner complex, Controlling the pipette to draw the first complex antibody reagent and the second complex antibody reagent from the reagent storage mechanism into the second reaction cup to obtain the multi-component immune complex containing the structure of "first complex antibody-small molecule / small molecule binding partner complex-second complex antibody"; Optionally, controlling the pipette to draw the first complex antibody reagent and the second complex antibody reagent from the reagent storage mechanism into the second reaction cup to obtain the multi-component immune complex containing the "first complex antibody-small molecule / small molecule binding partner complex-second complex antibody" structure also includes at least one washing step to separate unbound substances from the solid phase.

11. A sample analyzer for detecting a whole blood sample containing small molecules, comprising: A sample storage mechanism, used for storing sample tubes carrying whole blood samples; A reagent storage mechanism for storing reagents, wherein the reagents include a dissociation agent, a diluent, and one or more detection reagents; The reaction device has at least one placement position, wherein the placement position is used to place a reaction cup and incubate the reaction liquid in the reaction cup; A measuring device, used to perform optical measurement on the reaction solution after incubation to obtain the test result of the sample to be tested; a pipette, used to transfer the whole blood sample or the reagent into the reaction cup; The control device is electrically connected to the sample storage mechanism, the reagent storage mechanism, the reaction device, the measuring device and the pipette, and is configured to: receiving a test instruction, wherein the test instruction includes the type of small molecules to be tested in the whole blood sample; In response to the test instruction: Step 1: Controlling a pipette to draw a whole blood sample containing small molecules from a sample storage mechanism and a dissociation agent from a reagent storage mechanism, respectively, and adding them to a first reaction cup to form a first mixture, wherein the working concentration of the dissociation agent in the mixed first mixture is 2-4 mol / L, and the dissociation agent is guanidine thiocyanate; Step 2: After the first mixture in step 1 is allowed to react for 1-15 minutes, the pipette is controlled to draw a diluent from the reagent storage mechanism and add it to the first reaction cup to form a second mixture, wherein the diluent is selected from PBS, Tris, MES, HEPES, and Citrate buffer, and the amount of the diluent in the second mixture is more than twice the amount of the first mixture; Step 3: After the second mixture is precipitated for 5-20 minutes in step 2 to obtain a supernatant, the pipette is controlled to draw the supernatant from the first reaction cup to the second reaction cup; Step 4: controlling the calculation of the concentration of the small molecule in the second reaction cup according to the signal value in the second reaction cup measured by the measuring device, so as to reflect the concentration of the small molecule in the whole blood sample; Wherein, the small molecule is an organic compound with a molecular weight of less than 5000 Daltons; Optionally, in step 1, the ratio of the volume of the whole blood sample to the volume of the dissociation agent in the first mixture in the first reaction cup is 1:0.5-2; Optionally, in step 2, the volume of the diluent in the second mixture is 2-4 times the volume of the first mixture; Optionally, the small molecule is selected from any one of drugs, biotoxins, environmental pollutants, hormones, and metabolites; Optionally, the small molecule is an immunosuppressant selected from any one of sirolimus, everolimus, tacrolimus, and cyclosporine; Optionally, the diluent further comprises a defoaming agent, and the concentration of the defoaming agent in the diluent is in the range of 0.005%-0.02%; Further optionally, the main component of the defoaming agent is an organosilicon substance selected from any one of polydimethylsiloxane, polyether-modified organosilicon, and fluorosilicone-modified polyether.

Images