Method of detecting targeted immunosuppressant in whole blood without centrifugation by combination of reagents

Abstract

To provide a combination of reagents that is low in toxicity and safe, and a method of detecting targeted immunosuppressants in whole blood.SOLUTION: The present invention relates to the field of in vitro detection technology, specifically to a method of detecting targeted immunosuppressants in whole blood without centrifugation by a combination of reagents. The present invention is to provide a safe combination of reagents with low toxicity and a method of detecting targeted immunosuppressants in whole blood; the method according to the present invention can be used for performing immunodetection, and the detection results can be highly consistent with mass spectrometry analytical detection results, which shows that the whole blood treatment agent of the present invention can be used for treating a specimen, and bound medicines can be fully released, and the obtained medicines in free state can meet the requirements of immunodetection. The detection method provided by the present invention truly realizes detection without centrifugation and greatly improves detection efficiency.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the field of in vitro detection technology, and specifically, to a method for detecting a target immunosuppressant in whole blood without centrifugation by a combination of reagents.

Background Art

[0002] An immunosuppressant is a drug having an immunosuppressive effect, and is mainly used clinically for organ transplant rejection reactions and autoimmune diseases.

[0003] After a drug is absorbed into the blood by various administration routes, it reaches the action site or receptor site through blood circulation. The free drug in the blood enters the extracellular fluid by diffusion or further diffuses into the cells to bind to the receptor and produce a pharmacological effect. The distribution of the drug in tissues or the binding of the drug to the receptor at the action site is a reversible physiological and biochemical process, and a dynamic balance is taken. When the distribution of the drug in vivo is balanced, the drug concentrations in the blood and the target site are not always equal, but for most drugs, especially those transported in vivo by the passive transport method, the ratio of the drug concentration in the blood to the drug concentration in the target site is constant, and there is a correlation between the drug potency and the blood drug concentration. Therefore, the measurement of the blood drug concentration is generally accepted clinically and has become the main method for monitoring therapeutic drugs.

[0004] On the one hand, in order to avoid the occurrence of organ transplant rejection reactions, organ transplant recipients need to use immunosuppressants throughout their lives after transplantation. However, these drugs have a narrow therapeutic range and significant side effects. When the effect of immunosuppressants is enhanced, the immunodeficiency reaction is also enhanced, weakening the patient's overall immune function, reducing the patient's resistance to infection and tumors, and increasing the incidence of infection and tumors. When the immunosuppressive effect weakens, the rejection reaction cannot be effectively controlled and prevented, and the function of the graft will be lost. Therefore, in order to improve the medication compliance, safety, effectiveness, rationality, and economy of organ transplant recipients, doctors need to directly participate in the pharmaceutical supervision of patients after organ transplantation.

[0005] Generally, immunosuppressants required for monitoring therapeutic drugs in clinical practice mainly include cyclosporine (CsA), tacrolimus (FK506), sirolimus, etc. Most of these drugs exist in a bound form in the blood. For example, CsA has a very high binding rate to both blood cells and plasma proteins in the blood. 50% - 75% is taken up by red blood cells, and the rest is distributed in the plasma, with most binding to lipoproteins. After FK506 is absorbed, 75% - 80% of the FK506 in whole blood exists inside red blood cells, and the FK506 in plasma mainly binds to plasma proteins, with a binding rate of over 98%. In the human body, 95% of sirolimus binds to red blood cells.

[0006] Due to their hydrophobic structure, immunosuppressants form relatively stable complexes with various macromolecules in the blood and blood cells. Therefore, when performing whole blood detection, it is necessary to disrupt the blood cells to release these stable complexes and extract the free immunosuppressants by substitution.

[0007] Currently, it is common to detect immunosuppressants in blood or blood cells using chemiluminescence methods. Whole blood affects the signal measurement of the luminescent group in subsequent chemiluminescence methods. Therefore, the conventional method first lyses the blood, centrifuges it, removes impurities, and releases the immunosuppressant from the blood cells. However, the centrifugation process increases the workload of laboratory physicians. This step is time-consuming and requires controlling conditions to ensure the quality of the centrifuged samples. Generally, a toxic whole blood precipitant also needs to be added, which also poses a safety risk to the laboratory staff.

[0008] For example, the disclosed method for detecting immunosuppressants includes the following steps. Mix a whole blood specimen, a whole blood lysing agent, and a whole blood precipitant (a methanol and ethylene glycol solution containing zinc sulfate) in an XSYSTEMS centrifuge tube at a ratio of 2:1:4. Immediately vortex the sample for 5 - 10 s, then centrifuge for 4 min. Transfer the supernatant to a pretreatment tube and detect it with a device. Methanol and ethylene glycol in this solution are toxic. Moreover, this method still requires a centrifugation process, and the procedure is complicated.

[0009] Therefore, it is necessary to develop a combination of low-toxicity and safe reagents and a method for detecting immunosuppressants based on this reagent combination. When detecting immunosuppressants in whole blood using this reagent combination, it can meet the requirements of immunoassay without the need for a centrifugation process.

Summary of the Invention

Problems to be Solved by the Invention

[0010] The present invention aims to solve at least one of the technical problems in the related art to some extent. For this purpose, one object of the present invention is to provide a combination of reagents with low toxicity and safety, and a method for detecting a target immunosuppressant in whole blood. By using the method of the present invention for immunoassay, after centrifugation, it can maintain a high degree of consistency with the mass spectrometry detection results, indicating that the drug in the bound state in the sample is sufficiently released, and the obtained free drug can meet the requirements of immunoassay.

Means for Solving the Problems

[0011] For this purpose, a first aspect of the present invention provides a whole blood treatment agent. According to the embodiments of the present invention, the whole blood treatment agent contains butanol and optionally saponin and / or ascorbic acid.

[0012] According to the embodiments of the present invention, when treating whole blood with the whole blood treatment agent, the working concentration of the butanol is 0.1-1.0% v / v.

[0013] According to the embodiments of the present invention, when treating whole blood with the whole blood treatment agent, the working concentration of the saponin is 0.3-3% w / v, Optionally, the working concentration of the ascorbic acid is 0.5-1.0% w / v.

[0014] A second aspect of the present invention provides a combination of reagents. According to the embodiments of the present invention, the combination of reagents includes the whole blood treatment agent described in the first aspect and a buffer solution, The buffer solution includes at least one selected from PBS buffer solution, Tris buffer solution, and MES buffer solution, and further includes at least one of Tween-20, Triton, and hydrophilic amino acids.

[0015] According to the embodiments of the present invention, the hydrophilic amino acid contains at least one selected from arginine, asparagine, aspartic acid, glutamine, serine, threonine, cysteine, glutamic acid, lysine, and histidine.

[0016] According to the embodiments of the present invention, when the buffer contains Triton, the concentration of Triton in the buffer is 0.065 - 0.3% v / v. The third aspect of the present invention provides the use of the whole blood treatment agent described in the first aspect and the combination of reagents described in the second aspect in the detection of target molecules in whole blood.

[0017] According to the embodiments of the present invention, the target molecule contains an immunosuppressant.

[0018] According to the embodiments of the present invention, the immunosuppressant contains at least one selected from cyclosporine, sirolimus, and FK506.

[0019] The fourth aspect of the present invention provides a method for detecting target molecules in whole blood. According to the embodiments of the present invention, the detection method includes: Step (1) of treating the whole blood to be detected with the whole blood treatment agent described in the first aspect and obtaining a sample of the measurement target after treatment; Step (2) of detecting the target molecule for the sample of the measurement target after treatment based on an immunoassay method.

[0020] According to the embodiments of the present invention, the target molecule contains an immunosuppressant.

[0021] According to the embodiments of the present invention, the immunosuppressant contains at least one selected from cyclosporine, sirolimus, and FK506.

[0022] According to the embodiments of the present invention, the volume ratio of the whole blood to the whole blood treatment agent in step (1) is (1 - 50):(1 - 3).

[0023] The fifth aspect of the present invention provides a method for detecting a target immunosuppressant in whole blood. According to the implementation means of the present invention, providing a signal detection reagent and a magnetic carrier coated with a first antibody, provided that the signal detection reagent contains a signal product bound to a second antibody, and the first antibody can specifically recognize a target molecule in a sample to be measured and bind to form a target molecule-first antibody complex, and the target molecule in the complex induces the first antibody to form at least one new conformational epitope, and the second antibody can specifically bind to the at least one new conformational epitope (step 1); treating the whole blood to be detected with the whole blood treatment agent according to the first aspect and obtaining a sample to be measured after treatment (step 2); contacting the sample to be measured after treatment with the magnetic carrier coated with the first antibody to obtain a complex of target immunosuppressant - first antibody - magnetic carrier (step 3); contacting the complex of target immunosuppressant - first antibody - magnetic carrier with the signal detection reagent to obtain a complex of target immunosuppressant - first antibody - magnetic carrier - second antibody - signal product (step 4); detecting the signal of the complex of target immunosuppressant - first antibody - magnetic carrier - second antibody - signal product obtained in 4) by immunoassay and detecting the target immunosuppressant contained in the whole blood (step 5).

[0024] According to the implementation means of the present invention, the affinity constant KD value between the second antibody and the target immunosuppressant - first antibody is 1×10 -10 ~9×10 -9 .

[0025] According to the implementation means of the present invention, the magnetic carrier includes any one of electromagnetic beads and microspheres.

[0026] According to the implementation means of the present invention, the magnetic carrier is a microsphere having a particle size of 0.3 to 9.0 μm.

[0027] According to the embodiments of the present invention, the input mass ratio of the first molecule to the microsphere coated with the first molecule is 1000:(8 - 20).

[0028] According to the embodiments of the present invention, the surface of the magnetic carrier is coated with a hydrophilic group.

[0029] According to the embodiments of the present invention, the hydrophilic group includes those selected from a hydroxyl group, a carboxyl group, and an amino group.

[0030] According to the embodiments of the present invention, the concentration of the hydrophilic group coating the surface of the magnetic carrier is 0.5 - 2.0 mmol / g.

[0031] According to the embodiments of the present invention, the target immunosuppressant includes at least one selected from cyclosporine, sirolimus, and FK506.

[0032] According to the embodiments of the present invention, the detection method further includes providing a buffer solution, and contacting the processed sample to be measured and the magnetic carrier coated with the first antibody in the buffer solution.

[0033] According to the embodiments of the present invention, the buffer solution includes at least one selected from PBS buffer solution, Tris buffer solution, and MES buffer solution.

[0034] According to the embodiments of the present invention, the buffer solution further includes at least one of Tween - 20, Triton, and hydrophilic amino acids.

[0035] According to the embodiments of the present invention, when the buffer solution contains Triton, in the buffer solution, the concentration of the Triton is 0.065 - 0.3% v / v. According to the embodiments of the present invention, the hydrophilic amino acid includes at least one selected from arginine, asparagine, aspartic acid, glutamine, serine, threonine, cysteine, glutamic acid, lysine, and histidine.

[0036] According to the implementation means of the present invention, the signal product includes at least one selected from ABEI, acridinium ester, luminol, isoluminol, AHEI, ITCI, and lucigenin.

Advantages of the Invention

[0037] The drug obtained by the specimen processing means provided by the present invention can perform immunoassay. The drug can not only react with the first antibody, but also form an accurate antigen recognition epitope after the reaction with the first antibody and can be recognized by the second antibody. By detecting the formed sandwich complex, a linear detection result can be obtained. The detection means further provided by the present invention can reduce the difference in detection results before and after whole blood detection centrifugation, is advantageous for realizing detection without centrifugation, and extremely improves the detection efficiency.

[0038] Additional aspects and advantages of the present invention are partially shown in the following description, partially will become apparent from the following description, or can be understood by the practice of the present invention.

Modes for Carrying Out the Invention

[0039] Hereinafter, embodiments of the present invention will be described in detail. The embodiments described below are exemplary and are only used for interpreting the present invention and cannot be understood as limiting the present invention.

[0040] It should be noted that the terms "first" and "second" are only for the purpose of explanation and should not be understood as indicating relative importance or implying or implicitly indicating the number of technical features shown. Therefore, the features limited by "first" and "second" may explicitly or implicitly include one or more of those features. Furthermore, in the description of the present invention, "a plurality" means two or more unless otherwise specified.

[0041] The endpoints of the ranges and any values disclosed in this specification are not limited to the exact ranges or values themselves, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each individual range, between the endpoint values of each individual range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this specification.

[0042] In the present invention, unless otherwise explained, the scientific terms and technical terms used in this specification have meanings generally understood by those skilled in the art. Also, the protein and nucleic acid chemistry, molecular biology, related terms and laboratory operation steps used in this specification are all terms widely used in the corresponding fields and normal steps. For example, the standard recombinant DNA and molecular cloning techniques used in the present invention are well-known to those skilled in the art and are described in more detail in the following literature. Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter referred to as "Sambrook").

[0043] In this specification, the term "comprising" or "including" is a non-limiting expression, that is, it includes the content specified in the present invention but does not exclude the content of other aspects.

[0044] In this specification, the terms "optionally", "optional" or "option" usually mean that the event or situation described thereafter may occur but not necessarily, and the description includes the case where the event or situation occurs and the case where the event or situation does not occur.

[0045] As used herein, the term "and / or" encompasses all combinations of the items joined by that term, and each individual combination should be considered as being separately described herein. For example, "A and / or B" encompasses "A", "A and B", and "B". For example, "A, B and / or C" encompasses "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0046] According to one specific embodiment of the present invention, the present invention provides a whole blood treatment agent containing butanol and optionally saponin and / or ascorbic acid.

[0047] According to one specific embodiment of the present invention, the whole blood treatment agent provided by the present invention can contain, for example, butanol individually, or butanol and saponin, or butanol and ascorbic acid. According to a preferred embodiment of the present invention, the whole blood treatment agent provided by the present invention contains butanol, saponin and ascorbic acid. As long as the main substances in the whole blood that solubilize cell fragments of blood cells in the whole blood treatment agent are butanol and optionally saponin and / or ascorbic acid, other components may be included, and any other auxiliary components are also included within the protection scope of the whole blood treatment agent of the present invention.

[0048] The inventors have found that by adding the organic solvent butanol to whole blood using the principle of "like dissolves like", large cell fragments can be further lysed. For example, the addition amount is whole blood:butanol = 5:1 to 50:1. Although phenol can also achieve a similar technical effect, since the solubility of phenol is small and its preparation is difficult, butanol is preferred. A free immunosuppressant can be obtained by that treatment method and can be used in immunological tests.

[0049] According to one specific embodiment of the present invention, when treating whole blood with the whole blood treatment agent, the working concentration of the butanol is 0.1 to 1.0% v / v, The working concentration of the saponin is 0.3 to 3.0% w / v, and the working concentration of the ascorbic acid is 0.5 to 1% w / v.

[0050] In addition, saponin or ascorbic acid may be directly mixed with butanol in solid form to form a whole blood treatment agent, or butanol, saponin or ascorbic acid, which is a whole blood treatment agent, may be stored in solution form. When it is necessary to use it for detecting target molecules in whole blood, butanol, saponin or ascorbic acid may be added to the whole blood. Regarding the addition amount of butanol, saponin or ascorbic acid, it may be adjusted according to the concentration of the butanol, saponin or ascorbic acid solution and the volume of the whole blood, as long as the final working concentration is within the above range. For example, when the storage concentration of the butanol solution is high, when adding butanol to the whole blood accordingly, the added volume may be reduced.

[0051] According to one specific embodiment of the present invention, the present invention includes the aforementioned whole blood treatment agent and a buffer solution, and the buffer solution includes at least one selected from PBS buffer solution, Tris buffer solution, and MES buffer solution, and further provides a combination of reagents including at least one of Tween-20, Triton, and hydrophilic amino acids.

[0052] According to one specific embodiment of the present invention, the hydrophilic amino acid includes at least one selected from arginine, asparagine, aspartic acid, glutamine, serine, threonine, cysteine, glutamic acid, lysine, and histidine.

[0053] According to one specific embodiment of the present invention, when the buffer solution contains Triton, the concentration of Triton in the buffer solution is 0.065 to 0.3% v / v.

[0054] The whole blood treatment agent or reagent combination provided by the present invention can be used to detect target molecules in whole blood. The target molecules include immunosuppressants, and may be, for example, cyclosporine, sirolimus, FK506, etc. Naturally, it can further include other types of immunosuppressants that need to be clinically detected, and all can be detected using the whole blood treatment agent or reagent combination of the present invention.

[0055] According to one specific embodiment of the present invention, the present invention Step (1) of treating the whole blood to be detected with the above-mentioned whole blood treatment agent and obtaining a sample of the measurement target after treatment; Based on an immunoassay method, step (2) of detecting target molecules for the sample of the measurement target after the treatment, and a method for detecting target molecules in whole blood is provided.

[0056] According to one specific embodiment of the present invention, the volume ratio of the whole blood to the whole blood treatment agent in step (1) is (1 to 50):(1 to 3). For example, when the whole blood treatment agent is 5% butanol or contains 5% butanol, the volume ratio of the whole blood to 5% butanol may be 5:1 to 50:1. When the whole blood treatment agent contains 30% saponin, the volume ratio of the whole blood to 30% saponin may be 10:1 to 100:1. When the whole blood treatment agent contains 2% ascorbic acid, the volume ratio of the whole blood to 2% ascorbic acid may be 1:1 to 3:1.

[0057] Immunoassay is a biochemical analysis method that utilizes an immune reaction to detect and measure specific substances. By detecting a substance using an antibody / antigen, such a method can rapidly and accurately measure the content of the substance. These substances may be proteins, nucleic acids, hormones, antigens, etc. In the present invention, the target molecule detected by the immunoassay is an immunosuppressant, and the immunosuppressant may be, for example, cyclosporine, sirolimus, FK506, etc. Any type of immunosuppressant that needs to be detected in whole blood clinically known in this field can be detected by the above method for detecting the target molecule in whole blood. Immunoassay is performed by the detection method of the present invention, and the detection result can maintain a high degree of consistency with the mass spectrometry detection result, indicating that the drug in the bound state in the sample is sufficiently released, and the obtained free drug can meet the requirements of immunoassay.

[0058] According to one specific embodiment of the present invention, the present invention provides a signal detection reagent and a magnetic carrier coated with a first antibody, wherein the signal detection reagent contains a signal product bound to a second antibody, the first antibody can specifically recognize a target molecule in a sample to be measured and bind to form a target molecule - first antibody complex, and the target molecule in the complex induces the first antibody to form at least one new conformational epitope, and the second antibody can specifically bind to the at least one new conformational epitope (step 1); processing the whole blood to be detected with the aforementioned whole blood treatment agent to obtain a sample to be measured after processing (step 2); contacting the sample to be measured after processing with the magnetic carrier coated with the first antibody to obtain a complex of the target immunosuppressant - first antibody - magnetic carrier (step 3); contacting the complex of the target immunosuppressant - first antibody - magnetic carrier with the signal detection reagent to obtain a complex of the target immunosuppressant - first antibody - magnetic carrier - second antibody - signal product (step 4); By immunoassay, step 5) of performing optical signal detection on the complex of the target immunosuppressant - first antibody - magnetic carrier - second antibody - signal product obtained in 4) to detect the target immunosuppressant contained in whole blood, and a method for detecting a target immunosuppressant in whole blood is provided.

[0059] According to one specific embodiment of the present invention, the reagents required for the immunological test include an electromagnetic bead reagent, an ABEI reagent, and a buffer. Among them, the electromagnetic bead is coated with a CsA antibody, and the ABEI reagent contains another antibody that specifically recognizes the CsA - CsA antibody complex. The buffer contains Triton.

[0060] During detection, the sample is mixed with a whole blood treatment agent (containing saponin / ascorbic acid / butanol), the mixed sample is mixed with the electromagnetic bead reagent and the buffer, washed after sufficient reaction, then the ABEI reagent is added and reacted sufficiently, and then washed again. By detecting the ternary complex, the amount of CsA in the sample is reflected.

[0061] It is experimentally verified that the CsA obtained by the above sample treatment not only reacts with the first antibody, but also can form an accurate antigen recognition epitope recognized by the complex antibody (second antibody) after the first antibody reacts. By detecting the formed sandwich complex, a linear detection result can be obtained.

[0062] By processing and detecting the sample by the above method, the problem that there is a small difference in the detection results before and after centrifugation still exists, which is for further simplifying the detection step of the immunosuppressant.

[0063] Therefore, another aspect of the present invention provides an immunosuppressant detection reagent system method that can reduce the difference in detection results before and after centrifugation, which is advantageous for realizing the detection of immunosuppressants without centrifugation.

[0064] Regarding the reagent components, the inventors further performed the following specific optimizations. (I) Regarding the electromagnetic beads, the inventors have discovered that the performance of the electromagnetic beads can be further optimized, for example, by increasing the hydrophilic groups (500 - 2000 mmol / g) such as carboxyl groups, hydroxyl groups, and amino groups on the surface of the electromagnetic beads. (II) Regarding the buffer, add T - 20 to increase the viscosity of the reaction system, reduce the sedimentation rate of cell debris, and accelerate the further separation of the electromagnetic beads from the cell debris. (III) Regarding the buffer, add hydrophilic substances such as amino acids (arginine, lysine, asparagine, histidine), surfactants (Tween, Brij - 35), etc., to reduce the binding between the cell debris and the electromagnetic beads.

[0065] Experimental verification proves that by optimizing the above - mentioned reagent components, the problem that the detection results before and after centrifugation by the sandwich method do not remain consistent can be solved. From this, since the blood cell - disrupted membrane fragments are hydrophobic substances, they may non - specifically adsorb onto the electromagnetic beads, preventing the FK506 / CsA small molecules from binding to the first antibody, or after CsA binds to the first antibody, the hydrophobic substances may shield the recognition epitope of the complex antibody. The inventors have speculated.

[0066] According to one specific embodiment of the present invention, the affinity constant KD value of the second antibody is 1×10 -10 ~9×10 -9 .

[0067] According to one specific embodiment of the present invention, the magnetic carrier includes any one of electromagnetic beads and microspheres. When the magnetic carrier is a microsphere, the particle size of the microsphere is preferably 0.3 - 9.0 μm.

[0068] According to one specific embodiment of the present invention, the input mass ratio of the first molecule to the microsphere in the microsphere coated with the first molecule is 1000:(8-20). For example, the administration mass ratio of the first molecule to the microsphere in the microsphere coated with the first molecule is 1 mg:8 μg to 1 mg:20 μg.

[0069] According to one preferred embodiment of the present invention, the surface of the magnetic carrier is coated with a hydrophilic group. According to one specific embodiment of the present invention, the hydrophilic group includes those selected from a hydroxyl group, a carboxyl group, and an amino group. Of course, in addition to these hydrophilic groups, other types of hydrophilic groups may be coated on the surface of the magnetic carrier of the present invention. Preferably, the hydrophilic group is a hydroxyl group.

[0070] According to one preferred embodiment of the present invention, the concentration of the hydrophilic group coating the surface of the magnetic carrier is 0.5-2 mmol / g. The concentration here means that an average of 0.5-2 mmol of hydrophilic groups are coated per 1 g of the surface of the magnetic carrier.

[0071] According to one specific embodiment of the present invention, the target immunosuppressant includes at least one selected from cyclosporine, sirolimus, and FK506.

[0072] According to one specific embodiment of the present invention, step 3) further includes contacting the processed sample to be measured with the magnetic carrier coated with the first molecule in a buffer solution.

[0073] According to one specific embodiment of the present invention, the buffer solution includes at least one selected from PBS buffer solution, Tris buffer solution, and MES buffer solution.

[0074] According to one specific embodiment of the present invention, the buffer solution further includes at least one of T-20, Triton, and hydrophilic amino acids.

[0075] According to one specific embodiment of the present invention, when Triton is contained in the buffer solution, the concentration of Triton in the buffer solution is 0.065 to 0.30% v / v.

[0076] According to one specific embodiment of the present invention, the hydrophilic amino acid contains at least one selected from arginine, asparagine, aspartic acid, glutamine, serine, threonine, cysteine, glutamic acid, lysine, and histidine.

[0077] According to one specific embodiment of the present invention, the signal product contains at least one selected from ABEI, acridinium ester, luminol, isoluminol, AHEI, ITCI, and lucigenin. Preferably, the signal product is ABEI.

Example

[0078] Hereinafter, the means of the present disclosure will be interpreted with reference to examples. As will be understood by those skilled in the art, the following examples are merely for explaining the present disclosure and should not be construed as limiting the scope of the present disclosure. When specific techniques and conditions in the examples are not specified, they are carried out according to the techniques and conditions described in the literature in this field or according to the product specifications. The reagents and instruments used are all ordinary products that are commercially available when the manufacturer is not specified.

[0079] Verification of antibody binding affinity The binding affinity between the CsA antibody and the CsA-CsA antibody complex and the CsA complex antibody was detected using the BIAcore technique (shown in Table 1). The experimental process was carried out according to the Biacore R3000 and Biacore AB specifications. The equilibrium dissociation constant Kd (M) represents the degree of dissociation of the binding affinity at equilibrium. The smaller the Kd (M), the stronger the affinity.

Table 1

[0080] Table 2 shows the detection of the binding affinity between the FK506 antibody and the FK506-FK506 antibody complex and the FK506 complex antibody using the same technique as described above.

Table 2

[0081] (Example 1) Detection of immunosuppressive agents 1. Reagents for CsA detection (1) For the electromagnetic bead reagent, it is an electromagnetic bead coated with a first antibody that specifically recognizes CsA. The input ratio of the electromagnetic bead to the antibody is 1 mg: 12 μg, and the suspension concentration of the electromagnetic bead is 20 mg / mL. (2) For the labeling reagent, a second antibody that specifically recognizes CsA and the CsA antibody complex binds to ABEI. The input ratio of ABEI to the second antibody is 0.5 mg: 12.5 μg. For the whole blood treatment agent, it is shown in Table 1. For the Buffer, the MES buffer system contains 0.5% Triton.

[0082] Regarding the sample injection process Mix whole blood and the whole blood treatment agent at the addition ratio shown in Table 3, and perform detection using the chemiluminescent immunoassay analyzer MAGLUMIX8 of Xinchanye Biotechnology Co., Ltd. in Shenzhen. The sample injection process parameters of the instrument are 10 μL of the sample mixed with the whole blood treatment agent + 20 μL of the electromagnetic bead + 200 μL of the Buffer, incubate for 4 min, wash 3 times + 150 μL of ABEI, incubate for 6 min, wash 3 times, and detect.

Table 3

[0083] Furthermore, the electromagnetic bead reagent and the labeling reagent of the above detection reagents were optimized as shown in Table 4.

Table 4

[0084] 2. Reagents for FK506 detection (1) Regarding the electromagnetic bead reagent, it is an electromagnetic bead coated with a first antibody that specifically recognizes FK506. The input ratio of the electromagnetic bead to the antibody is 1 mg: 10 μg, and the suspension concentration (Tris buffer) of the electromagnetic bead is 20 mg / mL. (2) Regarding the labeling reagent, a second antibody that specifically recognizes the FK506 and FK506 antibody complex is bound to ABEI, and the input ratio of ABEI to the complex antibody is 0.5 mg: 5 μg. Regarding the buffer, the MES buffer system contained 0.5% Triton.

[0085] Regarding the sample injection process, Whole blood and the whole blood treatment agent were mixed at the addition ratios shown in Table 5, and it was 10 μL of the sample mixed with the whole blood treatment agent + 100 μL of buffer + 20 μL of electromagnetic beads. Incubation was carried out for 6 min, + 150 μL of ABEI, incubation was carried out for 9 min, washed 3 times, and detected.

Table 5

[0086] Comparison of titer deviation before and after centrifugation of CsA samples in Verification Test 1 Regarding the CsA samples, CsA standards of 1500, 1352, 1204, 1056, 908, 760, 612, 464, 316, 168, 20 ng / mL were respectively added to whole blood samples (normal human blood without taking drugs), and 10 samples were obtained.

[0087] Regarding the calculation method of the deviation, Deviation % = titer after centrifugation / titer before centrifugation - 1 Average deviation % = average value of deviation % of all samples.

[0088] Table 6 shows the average deviation of the detection results of CsA samples before and after centrifugation in Examples 2 to 22.

Table 6

[0089] Comparison of the titer deviation before and after centrifugation of the verification test 2 FK506 specimens For the FK506 specimens, FK506 reference standards were added to whole blood specimens (normal human blood without taking the drug) at 50, 45.05, 40.1, 35.15, 30.2, 25.25, 20.3, 15.35, 10.4, 5.45, and 0.5 ng / mL respectively, and 10 specimens were obtained.

[0090] The average deviation of the detection results of the FK506 specimens before and after centrifugation in Example 23 and Example 24 was calculated by the same deviation calculation method as in the verification test 1 and shown in Table 7.

Table 7

[0091] Regarding the description of the verification test 1 and 2 data The detection results of Examples 2 to 4 were that 30% saponin was added in the specimen pretreatment process. When the addition amount of whole blood: 30% saponin was 10:1 to 100:1, the effect was the best. At this time, the average deviation of the titer before and after centrifugation was 70.81%, 62.65%, and 79.54% respectively, but this deviation was large before and after centrifugation, indicating that the consistency between the detection results with and without centrifugation could not be guaranteed.

[0092] The detection results of Examples 5 to 7 were that 2% ascorbic acid was added in the specimen pretreatment process. When the addition amount of whole blood: 2% ascorbic acid was 1:1 to 3:1, the effect was the best. At this time, the average deviation of the titer before and after centrifugation was 65.82%, 60.54%, and 79.23% respectively. Compared with Examples 2 to 4, the average deviation before and after centrifugation exceeded 60% in both cases, still indicating the problem of large deviation and that the consistency between the detection results with and without centrifugation could not be guaranteed.

[0093] The detection results of Examples 8 to 11 show that when 5% butanol was added during the specimen pretreatment process, the best effect was achieved when the addition ratio of whole blood to butanol was 5:1 to 50:1. At this time, the average deviations of the titers before and after centrifugation were 53.13%, 38.20%, 40.68%, and 58.20% respectively, which were significantly smaller than those before and after centrifugation in the example with only saponin added, indicating that the relative consistency between the detection results with and without centrifugation can be guaranteed.

[0094] The detection results of Examples 12 to 13 show that when evaluating by combining the excellent results in the above examples, the average deviation of the titer before and after centrifugation was 20.35% when the ratio of whole blood: 30% saponin: 5% butanol was 50:1:2, and the average deviation of the titer before and after centrifugation was 35.64% when the ratio of whole blood: 2% ascorbic acid: 5% butanol was 5:5:1. Compared with the example with only saponin added and Examples 5 to 7, the deviation before and after centrifugation was even smaller, indicating that the relative consistency between the detection results with and without centrifugation can be guaranteed.

[0095] The detection results of Examples 14 to 16 show that the hydrophilic groups cross-linked to the electromagnetic ball were improved, and the average deviations of the titers before and after centrifugation were 18.19%, 15.64%, and 20.98% respectively when the number of groups was 500, 1000, and 2000 μmol / g. Therefore, compared with Example 12, it shows that the consistency between the detection results with and without centrifugation can be further improved.

[0096] The detection results of Examples 17 and 18 show that when 0.05% and 0.02% T-20 were added to the Buffer, the average deviations of the titers before and after centrifugation were 15.02% and 19.53% respectively. Compared with Example 12, it shows that adding T-20 can further improve the consistency between the detection results with and without centrifugation.

[0097] The detection results of Examples 19 to 21 showed that arginine, a hydrophilic substance with different contents, was added to the Buffer. When the addition amounts were 3%, 5%, and 7%, the average deviation of the titer before and after centrifugation at this time was 13.43%, 10.69%, and 17.35%, respectively. Compared with Example 12, it is shown that adding arginine, a hydrophilic substance, to the Buffer can further improve the consistency between the detection results with and without centrifugation.

[0098] The detection result of Example 22 was obtained by mixing and evaluating the optimal effects of the above examples. At this time, the average deviation of the titer before and after centrifugation was 5.61%, indicating that the problem of the difference in titer before and after centrifugation of the whole blood treatment specimen by sandwich method reagent detection was further solved.

[0099] The detection result of Example 23 showed that the titer deviation of FK506 before and after centrifugation in Example 24 was 68.51%. By applying the optimization means of CsA to FK506, the average deviation of the titer before and after centrifugation at this time was 1.96%, indicating that the problem of the difference before and after centrifugation of the whole blood treatment specimen by sandwich method reagent detection was further solved.

[0100] Verification Experiment 3 Comparison of Consistency between Before and After Centrifugation and Mass Spectrometry Detection Results For CsA and FK506, by mass spectrometry detection method, after adding an internal standard solution, a red blood cell lysate, a protein precipitant, and an electromagnetic bead solution to the whole blood specimen to be measured respectively and shaking, magnetic separation was performed. After magnetic separation, the supernatant was obtained to complete the specimen treatment process.

[0101] Regarding specific experimental parameters, (1) The addition ratio of whole blood sample: internal standard solution: red blood cell lysate: protein precipitant: electromagnetic bead solution = 50:1:1:30:10. After magnetic separation, the supernatant was taken, (2) Regarding the chromatography conditions of liquid chromatography tandem mass spectrometry, there are mobile phase A and mobile phase B. In the elution process, from the 0th to 0.5th minute, the volume fraction of mobile phase B is 100%; from the 0.5th to 1.5th minute, the volume fraction of mobile phase B is 80% - 100%; from the 1.5th to 2.0th minute, the volume fraction of mobile phase B is 100%; from the 2.0th to 3.0th minute, the volume fraction of mobile phase B is 50%. (3) Regarding the chromatography conditions, the analytical column is a C18 column, the flow rate is 0.5 mL / min, the column temperature is 45 °C, the sample injection chamber temperature is 2 - 8 °C, and the sample injection volume is 10 μL. (4) Regarding the mass spectrometry conditions, it is an electrospray ionization source (ESI) and positive ion MRM scanning. The specific parameters of the positive ion MTM scanning are ionization voltage 5500 (V), temperature 480 °C, curtain gas 30 psi, collision gas 6 psi, spray gas 45 psi, and auxiliary heating gas 45 psi. For the validation specimens, 100 whole blood specimens using CsA drug were used.

[0102] The validation detection results are shown in Tables 8, 9, and 10.

Table 8

[0103]

Table 9

[0104]

Table 10

[0105] The results in Table 8 show that, in any case where the whole blood treatment agent was optimized using the means of the present invention, or in addition to the optimization of the whole blood treatment agent, reagent components such as electromagnetic bead reagents and buffers were further optimized, the correlation with the detection results of mass spectrometry after centrifugation reached 0.9 or more. Therefore, it is shown that in the above means, the drug in the bound state in the specimen is sufficiently released, and the released free drug can meet the requirements of immunoassay. This solution provides a more environmentally friendly and safe alternative means compared to the toxic whole blood treatment means of the prior art.

[0106] In the results of Table 9 and Table 10, for Example 24 of the FK506 item, the mass spectrometry correlation was only 0.269 before centrifugation without performing centrifugation for specimen pretreatment, and the correlation after centrifugation improved to 0.985, indicating a large titer deviation before and after centrifugation and obvious improvement after centrifugation.

[0107] The detection results of Example 3 show that when a certain amount of saponin was added during the specimen pretreatment process and the addition amount of whole blood:saponin was 50:1, the correlations with mass spectrometry before and after centrifugation were 0.693 and 0.936 respectively, and the detection results after centrifugation show that the correlation with mass spectrometry reaches 90% or more. Therefore, it is explained that the specimen treatment method of Example 3 (the addition amount of whole blood:saponin is within the range of 10:1 to 100:1) can meet the requirements of immunoassay after centrifugation. However, the detection results of Example 22 evaluated by mixing the optimal effects of the above examples show that at this time, the correlations with mass spectrometry before and after centrifugation are almost equal, 0.986 and 0.984 respectively, indicating that the problem of the difference in the detection of whole blood treatment specimens by sandwich method reagents before and after centrifugation is completely solved.

[0108] The detection results of Example 23 show that the optimization means of CsA was applied to FK506, and at this time, the correlations with mass spectrometry before and after centrifugation are almost equal, 0.991 and 0.991 respectively, indicating that the problem of the difference in the detection of whole blood treatment specimens by sandwich method reagents before and after centrifugation is completely solved.

[0109] In the description of this specification, the description of terms such as "one embodiment", "some embodiments", "example", "specific example", "some implementation means", or "some examples" means that the specific features, structures, materials, or characteristics described in relation to the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example. Further, the described specific features, structures, materials, or characteristics can be combined in any one or more embodiments or examples in a suitable manner. Furthermore, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples without contradiction with each other.

[0110] As described above, the embodiments of the present invention have been illustrated and described. However, the above embodiments are illustrative and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions, and variations of the above embodiments within the scope of the present invention.

Claims

1. Use in the detection of a target molecule in whole blood by a whole blood treatment agent, wherein the whole blood treatment agent contains butanol and optionally saponin and / or ascorbic acid, and the target molecule contains an immunosuppressive agent.

2. When treating whole blood with the whole blood treatment agent, the working concentration of the butanol is 0.1% v / v to 1% v / v, optionally, the working concentration of the saponin is 0.3% w / v to 3% w / v, optionally, the working concentration of the ascorbic acid is 0.5% w / v to 1% w / v. The use according to claim 1, characterized in that.

3. Use in the detection of a target molecule in whole blood by a combination of reagents, comprising the whole blood treatment agent according to claim 1 or 2 and a buffer solution, wherein the buffer solution contains at least one selected from PBS buffer solution, Tris buffer solution, MES buffer solution, and further contains at least one of Tween-20, Triton, and hydrophilic amino acids, and the target molecule contains an immunosuppressive agent.

4. The use according to claim 3, characterized in that the hydrophilic amino acid contains at least one selected from arginine, asparagine, aspartic acid, glutamine, serine, threonine, cysteine, glutamic acid, lysine, and histidine.

5. The use according to claim 1 or 2, characterized in that the immunosuppressive agent contains at least one selected from cyclosporine, sirolimus, and FK506.

6. Step (1) of treating the whole blood to be detected with the whole blood treatment agent according to claim 1 or 2 and obtaining a sample of the measurement target after treatment; Step (2) of detecting the target molecule for the sample of the measurement target after treatment based on an immunoassay method. A method for detecting a target molecule in whole blood, characterized in that it comprises.

7. The immunoassay method includes using a solid-phase carrier coated with an antigen or an antibody, and the solid-phase carrier is a magnetic carrier, optionally, the magnetic carrier contains any one of electromagnetic beads and microspheres. The detection method according to claim 6, characterized in that.

8. The target molecule contains an immunosuppressive agent, optionally, the immunosuppressive agent contains at least one selected from cyclosporine, sirolimus, and FK506. Optionally, the volume ratio of the whole blood to the whole blood treatment agent in step (1) is (1 to 50):(1 to 3), and the detection method according to claim 6 is characterized in that.

9. Step 1) providing a signal detection reagent and a magnetic carrier coated with a first antibody, wherein the signal detection reagent comprises a signal product bound to a second antibody, and the first antibody can specifically recognize a target molecule in a sample to be measured and bind to form a target molecule - first antibody complex, and the target molecule in the complex induces the first antibody to form at least one new conformational epitope, and the second antibody can specifically bind to the at least one new conformational epitope; Step 2) treating the whole blood to be detected with the whole blood treatment agent according to claim 1 or 2, and obtaining a sample of the measurement target after treatment; Step 3) contacting the sample of the measurement target after treatment with the magnetic carrier coated with the first antibody to obtain a complex of the target immunosuppressant - first antibody - magnetic carrier; Step 4) contacting the complex of the target immunosuppressant - first antibody - magnetic carrier with the signal detection reagent to obtain a complex of the target immunosuppressant - first antibody - magnetic carrier - second antibody - signal product; Step 5) detecting the signal of the complex of the target immunosuppressant - first antibody - magnetic carrier - second antibody - signal product obtained in 4) by immunoassay to detect the target immunosuppressant contained in the whole blood, and a method for detecting a target immunosuppressant in whole blood, characterized in that it comprises.

10. The affinity constant KD value of the second antibody is 1×10 －１０ to 9×10 －９ and is Optionally, the magnetic carrier comprises any one of electromagnetic beads and microspheres; Optionally, the magnetic carrier is a microsphere having a particle size of 0.3 μm to 9.0 μm; Optionally, the input mass ratio of the first molecule to the microsphere in the microsphere coated with the first molecule is 1000:(8 to 20); Optionally, the surface of the magnetic carrier is coated with a hydrophilic group; Optionally, the hydrophilic group includes those selected from a hydroxyl group, a carboxyl group, and an amino group; Optionally, the concentration of the hydrophilic group coating the surface of the magnetic carrier is 0.5 mmol / g to 2 mmol / g, and the detection method according to claim 9 is characterized in that.

11. The detection method according to claim 9, wherein the target immunosuppressant comprises at least one selected from cyclosporine, sirolimus, and FK506.

12. The detection method further comprises providing a buffer solution, contacting the processed sample to be measured and the magnetic carrier coated with the first antibody in the buffer solution, and optionally, the buffer solution comprises at least one selected from PBS buffer solution, Tris buffer solution, and MES buffer solution. Optionally, the buffer solution further comprises at least one of T-20, Triton, and hydrophilic amino acids. Optionally, when Triton is contained in the buffer solution, in the buffer solution, the concentration of Triton is 0.065% v / v to 0.3% v / v. Optionally, the hydrophilic amino acid comprises at least one selected from arginine, asparagine, aspartic acid, glutamine, serine, threonine, cysteine, glutamic acid, lysine, and histidine. The detection method according to claim 9 is characterized by this.

13. The detection method according to claim 9, wherein the signal product comprises at least one selected from ABEI, acridinium ester, luminol, isoluminol, AHEI, ITCI, and lucigenin.

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