A teicoplanin detection reagent combination and its preparation method and application

Abstract

The present invention provides a teicoplanin detection reagent combination and its preparation method and application. Specifically, the reagent combination includes: (i) a first test liquid, including a teicoplanin-carrier protein complex; and (ii) a second test liquid, including a teicoplanin polyclonal antibody. The reagent combination of the present invention can be used to detect the concentration of teicoplanin in an immunosuppressive turbidimetric method, with strong specificity, fast analysis speed, and high accuracy.

Description

Technical Field

[0001] The present invention relates to the field of medical testing, and in particular to a teicoplanin detection reagent combination, a preparation method and an application thereof. Background Art

[0002] Teicoplanin, also known as taicoplanin, was first discovered in 1975. It is a new type of bactericidal glycopeptide antibacterial drug produced by fermentation of Actinomycetes. It is a vancomycin family glycopeptide antibiotic obtained by fermentation and extraction of specific Actinomycetes. It is an antibiotic mixture composed of multiple compounds with very similar chemical structures. It is widely used in clinical practice to treat severe infections caused by Gram-positive bacteria such as Staphylococcus aureus and Streptococcus, such as endocarditis, osteomyelitis, sepsis, and infections of the respiratory tract, urinary tract, skin, and soft tissue. Teicoplanin has the following characteristics: 1. Side chain group protection, dense molecular spatial structure, binding site in the crack, not easy to be hydrolyzed and inactivated; 2. High protein binding rate is more conducive to stable blood drug concentration and improve bactericidal efficacy; 3. Good water solubility under physiological pH conditions; This makes teicoplanin have obvious clinical advantages compared with drugs of the same category.

[0003] The trough concentration of teicoplanin is greater than 10 mg / L. For different diseases, the trough concentration of teicoplanin varies greatly. When the trough concentration of teicoplanin is less than 10 mg / L, the effective therapeutic concentration is not reached; the trough concentration of 10-20 mg / L is for uncomplicated Gram-positive bacterial infections; the trough concentration of 20-60 mg / L is for complicated Gram-positive bacterial infections such as severe Staphylococcus aureus infections including endocarditis; when the trough concentration of teicoplanin is greater than 60 mg / L, the risk of poisoning is high. Moreover, the absorption and metabolism of teicoplanin in different patients often show great individual differences. Even if the same drug dose is used, the blood concentration of teicoplanin in different patients often varies significantly. Therefore, accurate detection of teicoplanin blood concentration has important reference value for improving individualized medication regimens, increasing drug efficacy, and controlling teicoplanin drug toxicity.

[0004] At present, the methods for detecting the blood concentration of teicoplanin mainly include high performance liquid chromatography (HPLC), fluorescence polarization immunoassay (FPIA), microbiological method and latex turbidimetry. HPLC can accurately determine the blood concentration of teicoplanin, but it is necessary to pre-treat the clinical samples and it usually takes nearly an hour for HPLC to determine a sample, which is inefficient and cumbersome to operate, and is not suitable for routine clinical testing. Fluorescence polarization immunoassay is difficult to promote clinically due to the high cost of reagents. The microbiological method requires bacterial culture for 14-15 hours, which is time-consuming and not accurate. The latex turbidimetry method requires the antibody to be coupled to the surface of latex microspheres, which is complicated to operate and time-consuming and laborious to develop reagents.

[0005] Therefore, there is an urgent need to provide a teicoplanin detection method and / or detection reagent with strong specificity, fast analysis speed and high accuracy. Summary of the invention

[0006] The purpose of the present invention is to provide a teicoplanin detection reagent combination with strong specificity, fast analysis speed and high accuracy.

[0007] In a first aspect, the present invention provides a teicoplanin detection reagent combination, the reagent combination comprising:

[0008] (i) a first test fluid comprising a teicoplanin-carrier protein complex; and

[0009] (ii) A second test solution comprising polyclonal antibody to teicoplanin.

[0010] In another preferred embodiment, in the teicoplanin-carrier protein complex, the carrier protein is selected from the following group: serum albumin, serum gamma globulin, thyroglobulin, ovalbumin or keyhole limpet hemocyanin.

[0011] In another preferred embodiment, the teicoplanin-carrier protein complex is soluble.

[0012] In another preferred embodiment, the teicoplanin-carrier protein complex and the teicoplanin polyclonal antibody can bind to each other and produce an insoluble complex.

[0013] In another preferred embodiment, the second test solution does not contain antibodies to the carrier protein.

[0014] In another preferred embodiment, the teicoplanin to be detected is selected from the following group: teicoplanin TA 2-1 , Teicoplanin TA 2-2 , Teicoplanin TA 2-3 , Teicoplanin TA 2-4 , Teicoplanin TA 2-5 , or a combination thereof.

[0015] In another preferred embodiment, the teicoplanin polyclonal antibody is selected from: teicoplanin TA 2-1 Polyclonal antibody, Teicoplanin TA 2-2 Polyclonal antibody, Teicoplanin TA 2-3 Polyclonal antibody, Teicoplanin TA 2-4 Polyclonal antibody, Teicoplanin TA 2-5 polyclonal antibodies, or combinations thereof.

[0016] In another preferred embodiment, the teicoplanin polyclonal antibody is of animal origin, preferably mammals, more preferably rabbits, sheep, horses, dogs or mice.

[0017] In another preferred embodiment, in the first test solution, the concentration of the teicoplanin-carrier protein complex is 10 μg / L-10 mg / L, preferably, 50 μg / L-5 mg / L, more preferably, 100 μg / L-1 mg / L, and most preferably, 200 μg / L-1 mg / L.

[0018] In another preferred embodiment, the teicoplanin polyclonal antibody is added to the second test solution in the form of antiserum, and the concentration of the antiserum in the second test solution is 0.01-10 g / L, preferably 0.05-5 g / L, more preferably 0.1-1 g / L, and most preferably 0.2-0.5 g / L.

[0019] In another preferred embodiment, the ratio of the molar concentration of the teicoplanin-carrier protein complex in the first test solution to the molar concentration of the teicoplanin polyclonal antibody in the second test solution is 1:5-20, preferably, 1:8-15, more preferably, 1:9-12.

[0020] In another preferred embodiment, the solvent of the first test liquid is water or substantially water. Preferably, the weight content of water in the solvent is ≥80%, more preferably, ≥90% or ≥95%.

[0021] In another preferred embodiment, the solvent of the second test liquid is water or substantially water. Preferably, the weight content of water in the solvent is ≥80%, more preferably, ≥90% or ≥95%.

[0022] In another preferred embodiment, the first test liquid and / or the second test liquid further comprises an aggregation promoter.

[0023] In another preferred embodiment, the polymerization promoter is selected from: polyethylene glycol, preferably, polyethylene glycol 35000, 20000, 12000, 10000, 8000, 6000, 4000, 2000, or a combination thereof, more preferably, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, or a combination thereof.

[0024] In another preferred embodiment, in the first test liquid and / or the second test liquid, the content of the polymerization promoter is independently 1-200 g / L, preferably 10-100 g / L, and more preferably 20-80 g / L.

[0025] In another preferred embodiment, the first test solution and the second test solution each independently further include: an osmotic pressure regulator, a solubilizing agent, a pH regulator, a preservative, or a combination thereof.

[0026] In another preferred embodiment, the osmotic pressure regulator is selected from: sodium chloride, potassium chloride, or a combination thereof.

[0027] In another preferred embodiment, in the first test liquid and / or the second test liquid, the content of the osmotic pressure regulator is independently 50-300 mmol / L, preferably, 100-200 mM.

[0028] In another preferred embodiment, the preservative is selected from: sodium azide, Proclin 300, thimerosal, gentamicin sulfate, or a combination thereof.

[0029] In another preferred embodiment, in the first test liquid and / or the second test liquid, the content of the preservative is independently 0.01-0.2wt%, preferably 0.02-0.1wt%, and more preferably 0.04-0.08wt%, based on the total weight of the test liquid.

[0030] In another preferred embodiment, the cosolvent is selected from: Tween 20, Tween 40, Tween 60, Tween 80, or a combination thereof

[0031] In another preferred embodiment, in the first test liquid and / or the second test liquid, the content of the co-solvent is independently 0.01-0.2 v / v%, preferably 0.02-0.1 v / v%, preferably 0.04-0.08 v / v%, based on the total weight of the test liquid.

[0032] In another preferred embodiment, the pH adjuster is tris(hydroxymethylaminomethane) buffer, phosphate buffer, citric acid buffer solution, or a combination thereof.

[0033] In another preferred embodiment, the pH adjuster in the first test solution is tris(hydroxymethyl)aminomethane buffer.

[0034] In another preferred embodiment, the concentration of the Tris buffer is 10-50 mmol / L, preferably 20-40 mmol / L, and more preferably 25-30 mmol / L.

[0035] In another preferred embodiment, the final pH value of the first test solution is 7-9, preferably 7-8.5, and more preferably 7.5-8.5.

[0036] In another preferred embodiment, the pH adjuster in the second test solution is a phosphate buffer.

[0037] In another preferred embodiment, the concentration of the phosphate buffer is 10-80 mmol / L, preferably 30-70 mmol / L, and more preferably 40-60 mmol / L.

[0038] In another preferred embodiment, the final pH value of the second test solution is 6.5-9, preferably 7-8.5, and more preferably 7-8.

[0039] In another preferred embodiment, the first test liquid and the second test liquid are placed separately.

[0040] In a second aspect of the present invention, a teicoplanin detection kit is provided, the kit comprising:

[0041] The reagent combination as described in the first aspect of the present invention, and at least two containers containing the first test liquid and the second test liquid respectively.

[0042] In another preferred embodiment, the kit further comprises: a teicoplanin calibrator.

[0043] In another preferred embodiment, the teicoplanin calibrator is a teicoplanin standard or a teicoplanin standard solution.

[0044] In another preferred embodiment, the teicoplanin content in the teicoplanin standard is > 90% by weight, preferably 95% by weight, more preferably 98% by weight, and most preferably 99% by weight.

[0045] In another preferred embodiment, the kit further comprises instructions.

[0046] The third aspect of the present invention provides a use of the reagent combination as described in the first aspect of the present invention and the kit as described in the second aspect of the present invention, for preparing a kit for detecting teicoplanin by immunoturbidimetry.

[0047] In a fourth aspect, the present invention provides a method for preparing the reagent combination according to the first aspect of the present invention, comprising the steps of:

[0048] A) preparing a first teicoplanin-carrier protein complex;

[0049] B) preparing a first test solution using the first teicoplanin-carrier protein complex;

[0050] C) preparing a second teicoplanin-carrier protein complex; and

[0051] D) using the second teicoplanin-carrier protein complex as an immunogen to immunize an animal to obtain teicoplanin polyclonal antibodies, and using the teicoplanin polyclonal antibodies to prepare a second test solution;

[0052] The carrier protein in the first teicoplanin-carrier protein complex is different from that in the second teicoplanin-carrier protein complex.

[0053] In another preferred embodiment, in step A) or step C), the method for preparing the first or second teicoplanin-carrier protein complex comprises the steps of:

[0054] i) activating teicoplanin with EDCI or EDCI-NHS; and

[0055] ii) coupling the activated teicoplanin with a carrier protein to obtain a teicoplanin-carrier protein complex.

[0056] In another preferred embodiment, the activation is to activate the carboxyl group on the teicoplanin molecule.

[0057] In another preferred embodiment, in step D), the second teicoplanin-carrier protein complex is used as an immunogen to immunize an animal to obtain teicoplanin polyclonal antibodies, comprising the steps of:

[0058] i) immunizing the animal for the first time using a mixed solution of teicoplanin-carrier protein complex and Freund's complete adjuvant;

[0059] ii) then immunizing the same animal once or more times with a mixed solution of teicoplanin-carrier protein complex and Freund's incomplete adjuvant; and

[0060] iii) Collecting antiserum.

[0061] In another preferred embodiment, the antiserum is used directly or after the teicoplanin polyclonal antibody is separated and used.

[0062] In another preferred embodiment, each immunization is separated by 5-15 days, preferably 7-14 days.

[0063] In another preferred embodiment, the animal is a mammal, preferably a rabbit, sheep, horse, dog or mouse.

[0064] In a fifth aspect, the present invention provides an immunosuppressive turbidimetric method for detecting teicoplanin, comprising the steps of:

[0065] 1) Providing the reagent combination as described in the first aspect of the present invention or the kit as described in the second aspect of the present invention;

[0066] 2) mixing the teicoplanin calibrator or sample with the first test solution and incubating the mixture; and

[0067] 3) The mixed solution is mixed with the second test solution respectively, and the initial absorbance A1 after mixing is tested. After incubation for a certain period of time, the absorbance A2 is tested, and ΔA=A2-A1 is calculated.

[0068] In another preferred embodiment, the detection is qualitative detection or quantitative detection.

[0069] In another preferred embodiment, the method further comprises the steps of:

[0070] Perform steps (1) to (3) on calibrators of different concentrations to prepare concentration-ΔA 标准 Standard curve, obtain the standard curve equation; and

[0071] The sample is subjected to steps (1)-(3) to obtain ΔA 样本 , substituted into the standard curve equation to calculate the concentration of teicoplanin in the sample.

[0072] In another preferred embodiment, the absorbance is the absorbance at 330-350 nm, preferably 335-345 nm, more preferably 340 nm.

[0073] In another preferred embodiment, in step 2), the incubation time is 1-10 min, preferably 2-8 min, and more preferably 3-5 min.

[0074] In another preferred embodiment, in step 3), the incubation time is 1-10 min, preferably 2-8 min, and more preferably 4-6 min.

[0075] In another preferred embodiment, in step 3), the absorbance A1 is the absorbance value at 1-10 s after mixing, preferably 3-5 s.

[0076] In another preferred embodiment, the incubation temperature is room temperature, preferably, 10-40°C, more preferably, 25-40°C, and most preferably, 35-38°C.

[0077] In another preferred embodiment, the concentration range of the standard curve is 0-500 mg / L, preferably, 0-200 mg / L, more preferably, 0-150 mg / L, and most preferably, 0-100 mg / L.

[0078] In another preferred embodiment, the sample is selected from: whole blood, plasma, and serum.

[0079] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0080] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 is the structural formula of teicoplanin;

[0082] Figure 2 is a calibration curve based on teicoplanin-BSA;

[0083] Figure 3 This is a correlation diagram between the teicoplanin kit and the HPLC method for measuring 50 clinical samples. DETAILED DESCRIPTION

[0084] The inventors have conducted extensive and in-depth research, and through a large number of screenings and tests, have provided a teicoplanin detection reagent combination. The reagent combination of the present invention can be used to detect the concentration of teicoplanin by immunosuppression turbidimetry, and has strong specificity, fast analysis speed, high accuracy, safety and environmental protection. The present invention was completed on this basis.

[0085] the term

[0086] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0087] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0088] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0089] Teicoplanin, protein complex, polyclonal antibody

[0090] Teicoplanin is usually a mixture of antibiotics consisting of multiple compounds with very similar chemical structures (such as Figure 1 The teicoplanin of the present invention refers to teicoplanin TA 2-1 , Teicoplanin TA 2-2 , Teicoplanin TA 2-3 , Teicoplanin TA 2-4 , Teicoplanin TA 2-5 A combination of one or more of .

[0091] Accordingly, the concentration of teicoplanin detected in the present invention refers to the total concentration (sum of concentrations) of one or more teicoplanin species present in the sample.

[0092] The term "teicoplanin-carrier protein complex" refers to the product of one or more teicoplanins complexed with a carrier protein. When it is the product of multiple teicoplanins complexed with a carrier protein, the teicoplanin-carrier protein complex is a mixture. Preferably, the teicoplanin-carrier protein complex is soluble.

[0093] Typically, in the teicoplanin-carrier protein complex, the carrier protein is selected from the group consisting of serum albumin, serum gamma globulin, thyroglobulin, ovalbumin or keyhole limpet hemocyanin.

[0094] The term "teicoplanin multi-antibody" refers to a drug that is resistant to teicoplanin. 2-1 , Teicoplanin TA 2-2 , Teicoplanin TA 2-3 , Teicoplanin TA 2-4 , Teicoplanin TA 2-5 There are combinations of one or more antibodies that have resistance. The polyclonal antibody to teicoplanin of the present invention can also form an immune complex with the teicoplanin-carrier protein complex.

[0095] Teicoplanin and the teicoplanin-carrier protein complex competitively bind to the teicoplanin polyclonal antibody. Preferably, teicoplanin and the teicoplanin polyclonal antibody form a soluble complex after binding, while the teicoplanin-carrier protein complex and the teicoplanin polyclonal antibody produce an insoluble complex after binding. Preferably, the teicoplanin polyclonal antibody does not complex with a single carrier protein.

[0096] Preferably, the teicoplanin polyclonal antibody is selected from: teicoplanin TA 2-1 Polyclonal antibody, Teicoplanin TA 2-2 Polyclonal antibody, Teicoplanin TA 2-3 Polyclonal antibody, Teicoplanin TA 2-4 Polyclonal antibody, Teicoplanin TA 2-5 polyclonal antibodies, or combinations thereof.

[0097] In another preferred embodiment, the teicoplanin polyclonal antibody is of animal origin, preferably mammals, more preferably rabbits, sheep, horses, dogs or mice.

[0098] Teicoplanin detection reagent combination, kit

[0099] A teicoplanin detection reagent combination, comprising: (i) a first test solution comprising a teicoplanin-carrier protein complex; and (ii) a second test solution comprising a teicoplanin polyclonal antibody.

[0100] In another preferred embodiment, in the first test solution, the concentration of the teicoplanin-carrier protein complex is 10 μg / L-10 mg / L, preferably, 50 μg / L-5 mg / L, more preferably, 100 μg / L-1 mg / L, and most preferably, 200 μg / L-1 mg / L.

[0101] In another preferred embodiment, the teicoplanin polyclonal antibody is added to the second test solution in the form of antiserum, and the concentration of the antiserum in the second test solution is 0.01-10 g / L, preferably 0.05-5 g / L, more preferably 0.1-1 g / L, and most preferably 0.2-0.5 g / L.

[0102] In another preferred embodiment, the ratio of the molar concentration of the teicoplanin-carrier protein complex in the first test solution to the molar concentration of the teicoplanin polyclonal antibody in the second test solution is 1:5-20, preferably, 1:8-15, more preferably, 1:9-12.

[0103] In another preferred embodiment, the solvent of the first test liquid is water or substantially water.

[0104] In another preferred embodiment, the first test liquid and / or the second test liquid further comprises an aggregation promoter.

[0105] Typically, the polymerization promoter is selected from: polyethylene glycol, preferably, polyethylene glycol 35000, 20000, 12000, 10000, 8000, 6000, 4000, 2000, or a combination thereof, more preferably, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, or a combination thereof.

[0106] In another preferred embodiment, in the first test liquid and / or the second test liquid, the content of the polymerization promoter is independently 1-200 g / L, preferably 10-100 g / L, and more preferably 20-80 g / L.

[0107] In another preferred embodiment, the first test solution and the second test solution each independently further include: an osmotic pressure regulator, a solubilizing agent, a pH regulator, a preservative, or a combination thereof.

[0108] The osmotic pressure regulator is used to adjust the osmotic pressure of the test solution. There is no special requirement for the selection of the osmotic pressure regulator, such as various inorganic salts. Preferably, the osmotic pressure regulator is selected from: sodium chloride, potassium chloride, or a combination thereof. The concentration of the osmotic pressure regulator preferably adjusts the diluent to a concentration close to the osmotic pressure of human plasma, such as a concentration close to that of physiological saline.

[0109] In another preferred embodiment, in the first test liquid and / or the second test liquid, the content of the osmotic pressure regulator is independently 50-300 mmol / L, preferably, 100-200 mM.

[0110] The preservative is used for antibacterial and anti-mold, preventing microorganisms (such as bacteria or mold) from decomposing drug molecules and / or contaminating the test liquid, and can extend the storage time of the test liquid.

[0111] The test solution of the present invention has no special requirements for preservatives, and preservatives commonly used in the art can be used. Preferred preservatives are selected from: sodium azide, Proclin 300, thimerosal, gentamicin sulfate, or a combination thereof.

[0112] In another preferred embodiment, the cosolvent is selected from: Tween 20, Tween 40, Tween 60, Tween 80, or a combination thereof

[0113] In another preferred embodiment, in the first test liquid and / or the second test liquid, the content of the co-solvent is independently 0.01-0.2 v / v%, preferably 0.02-0.1 v / v%, preferably 0.04-0.08 v / v%, based on the total weight of the test liquid.

[0114] The present invention has no special requirements for the pH regulator, and a commonly used pH regulator in the art can be used to maintain the pH of the test solution within a certain range. Typically, the pH regulator is tris buffer, phosphate buffer, citric acid buffer solution, or a combination thereof.

[0115] In another preferred embodiment, the final pH value of the first test solution is 7-9, preferably 7-8.5, and more preferably 7.5-8.5.

[0116] In another preferred embodiment, the final pH value of the second test solution is 6.5-9, preferably 7-8.5, and more preferably 7-8.

[0117] According to the method for using the reagent combination of the present invention, the first test liquid and the second test liquid are placed separately before use and are mixed during use.

[0118] The reagent combination of the present invention can also be made into a teicoplanin detection kit, which includes the reagent combination.

[0119] In another preferred embodiment, the kit further comprises: a teicoplanin calibrator.

[0120] Preparation method

[0121] The present invention also provides a method for preparing the reagent combination, comprising the steps of:

[0122] A) preparing a first teicoplanin-carrier protein complex;

[0123] B) preparing a first test solution using the first teicoplanin-carrier protein complex;

[0124] C) preparing a second teicoplanin-carrier protein complex; and

[0125] D) using the second teicoplanin-carrier protein complex as an immunogen to immunize an animal to obtain teicoplanin polyclonal antibodies, and using the teicoplanin polyclonal antibodies to prepare a second test solution;

[0126] The carrier protein in the first teicoplanin-carrier protein complex is different from that in the second teicoplanin-carrier protein complex.

[0127] In another preferred embodiment, in step A) or step C), the method for preparing the first or second teicoplanin-carrier protein complex comprises the steps of:

[0128] i) activating teicoplanin with EDCI or EDCI-NHS; and

[0129] ii) coupling the activated teicoplanin with a carrier protein to obtain a teicoplanin-carrier protein complex.

[0130] In another preferred embodiment, the activation is to activate the carboxyl group on the teicoplanin molecule.

[0131] In another preferred embodiment, in step D), the second teicoplanin-carrier protein complex is used as an immunogen to immunize an animal to obtain teicoplanin polyclonal antibodies, comprising the steps of:

[0132] i) immunizing the animal for the first time using a mixed solution of teicoplanin-carrier protein complex and Freund's complete adjuvant;

[0133] ii) then immunizing the same animal once or more times with a mixed solution of teicoplanin-carrier protein complex and Freund's incomplete adjuvant; and

[0134] iii) Collecting antiserum.

[0135] In another preferred embodiment, the antiserum is used directly or after the teicoplanin polyclonal antibody is separated and used.

[0136] In another preferred embodiment, each immunization is separated by 5-15 days, preferably 7-14 days.

[0137] In another preferred embodiment, the animal is a mammal, preferably a rabbit, sheep, horse, dog or mouse.

[0138] Immunosuppression turbidimetry for the detection of teicoplanin

[0139] During the detection process, the teicoplanin small molecules in the sample compete with the teicoplanin-carrier protein complex in the reagent for binding to the teicoplanin polyantibody. The higher the teicoplanin concentration in the sample, the greater the degree of binding of the teicoplanin polyantibody to it, and the lower the turbidity of the immune complex formed by the binding of the teicoplanin-carrier protein complex to the teicoplanin polyantibody. The turbidity of the reaction system is negatively correlated with the teicoplanin concentration in the sample. By measuring the absorbance at a certain wavelength (such as 340nm), a calibration curve is established, and the concentration of teicoplanin in the sample is calculated based on the sample absorbance measurement value.

[0140] Typically, the immunosuppression turbidimetric assay for the detection of teicoplanin comprises the steps of:

[0141] 1) Providing the reagent combination as described in the first aspect of the present invention or the kit as described in the second aspect of the present invention;

[0142] 2) mixing the teicoplanin calibrator or sample with the first test solution and incubating the mixture; and

[0143] 3) The mixed solution is mixed with the second test solution respectively, and the initial absorbance A1 after mixing is tested. After incubation for a certain period of time, the absorbance A2 is tested, and ΔA=A2-A1 is calculated.

[0144] In another preferred embodiment, the method further comprises the steps of:

[0145] Perform steps (1) to (3) on calibrators of different concentrations to prepare concentration-ΔA 标准 Standard curve, obtain the standard curve equation; and

[0146] The sample is subjected to steps (1)-(3) to obtain ΔA 样本 , substituted into the standard curve equation to calculate the concentration of teicoplanin in the sample.

[0147] In another preferred embodiment, the absorbance is the absorbance at 330-350 nm, preferably 335-345 nm, more preferably 340 nm.

[0148] In another preferred embodiment, in step 2), the incubation time is 1-10 min, preferably 2-8 min, and more preferably 3-5 min.

[0149] In another preferred embodiment, in step 3), the incubation time is 1-10 min, preferably 2-8 min, and more preferably 4-6 min.

[0150] In another preferred embodiment, in step 3), the absorbance A1 is the absorbance value at 1-10 s after mixing, preferably 3-5 s.

[0151] In another preferred embodiment, the incubation temperature is room temperature, preferably, 10-40°C, more preferably, 25-40°C, and most preferably, 35-38°C.

[0152] In another preferred embodiment, the concentration range of the standard curve is 0-500 mg / L, preferably, 0-200 mg / L, more preferably, 0-150 mg / L, and most preferably, 0-100 mg / L.

[0153] In another preferred embodiment, the sample is selected from: whole blood, plasma, and serum.

[0154] The main advantages of the present invention include:

[0155] 1. The raw materials of the teicoplanin detection reagent of the present invention are simple and easy to obtain, low in cost, good in stability and good in homogeneity.

[0156] 2. The teicoplanin detection reagent combination of the present invention has strong specificity, high detection result accuracy, simple and rapid operation, can improve detection efficiency, and generates less waste liquid during detection, which is more environmentally friendly and safe.

[0157] 3. The teicoplanin reagent combination of the present invention can be used in conjunction with a fully automatic biochemical analyzer to achieve automated analysis, and is suitable for timely and accurate detection of large-scale samples.

[0158] The present invention will be further described below in conjunction with specific implementation. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0159] Reagents

[0160]

[0161] Abbreviations:

[0162] BSA: bovine serum albumin;

[0163] KLH: keyhole limpet hemocyanin;

[0164] EDCI: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0165] NHS: N-hydroxysuccinimide 1-hydroxypyrrolidine-2,5-dione;

[0166] PBS: phosphate buffered saline.

[0167] Example 1

[0168] Synthesis of Teicoplanin-Carrier Protein Complex

[0169] Preparation of Teicoplanin-BSA Complex

[0170] Weigh 20 mg of teicoplanin and dissolve it in 10 mL of 0.1 M PBS (pH 8.0), add 10 mg of EDCI and 50 mg of NHS, and stir the reaction at room temperature for 1 hour to completely activate the carboxyl group of teicoplanin. Then slowly add 100 mg of BSA in batches and stir the reaction at room temperature for 16 hours. Filter with a 0.45 μm filter membrane, and purify the cross-linked product by ultrafiltration or dialysis to obtain a teicoplanin-BSA complex.

[0171] Example 2

[0172] Preparation of Teicoplanin-KLH Complex

[0173] The method is substantially the same as Example 1, except that KLH is used instead of BSA to prepare the teicoplanin-KLH complex.

[0174] Example 3

[0175] Preparation of polyclonal antibody against teicoplanin

[0176] Take the teicoplanin-BSA complex synthesized in Example 1 and use it to immunize rabbits. New Zealand white rabbits were selected as rabbits. 0.5 mg of teicoplanin-BSA complex was mixed with an equal volume of Freund's complete adjuvant per rabbit and injected subcutaneously at multiple points. The next immunization was performed every two weeks. After the first immunization, the adjuvant was Freund's incomplete adjuvant. One week after the fourth immunization, the rabbit's carotid artery was bled to collect whole blood. The blood was placed at room temperature to coagulate for about 1 hour, and then placed at 4°C overnight to shrink the blood clot. Centrifuge at 2500g for 10 minutes, discard the precipitate and collect the serum to obtain teicoplanin rabbit antiserum (rabbit polyclonal antibody).

[0177] According to the present invention, if the test solution R1 is added with a teicoplanin-BSA complex, the teicoplanin polyclonal antibody in the test solution R2 cannot be immunized with the teicoplanin-BSA complex, but should be a polyclonal antibody obtained by immunization with another complex coupled with a carrier protein. This is because the polyclonal antibody obtained by immunization with the teicoplanin-BSA complex may also contain anti-BSA antibodies. If the test solution R2 is a polyclonal antibody obtained by immunization with the teicoplanin-BSA complex, part of the signal value is the result caused by the anti-BSA antibodies and the teicoplanin-BSA complex, and not entirely the result caused by the anti-teicoplanin polyclonal antibody and the teicoplanin-BSA complex.

[0178] Example 4

[0179] Use the conventional solution preparation method to prepare the test solution R1 according to Table 1. The percentages are based on the total weight of the test solution R1:

[0180] Table 1

[0181]

[0182] Example 5

[0183] The test solution R2 was prepared according to Table 2 using a conventional solution preparation method. The percentages are based on the total weight of the test solution R2. The amount of teicoplanin polyclonal antibody used refers to the amount of the antiserum obtained in Example 3 used directly.

[0184] Table 2

[0185] Disodium hydrogen phosphate 50mM Sodium dihydrogen phosphate 50mM Sodium chloride 150mM Teicoplanin 0.3g / L Twain 20 0.05% (v / v%) Sodium Azide 0.05% (wt%) pH 7.4

[0186] Example 6

[0187] Calibrators:

[0188] The teicoplanin small molecule was dissolved and diluted to concentrations of 0, 6.25, 12.5, 25, 50, and 100 mg / L using a calibration test solution (phosphate buffer 30 mM, sodium chloride 150 mM, Tween 20 0.05%, sodium azide 0.05%), and then sterilized by filtration using a 0.22 μm filter membrane and stored at 2 to 8°C.

[0189] Example 7

[0190] Methodological validation

[0191] During the detection process, the teicoplanin small molecules in the sample compete with the teicoplanin-carrier protein complex in the reagent for binding to teicoplanin polyantibody. The higher the teicoplanin concentration in the sample, the greater the degree of binding of teicoplanin polyantibody to it, and the lower the turbidity of the immune complex formed by the binding of teicoplanin-carrier protein complex to teicoplanin polyantibody. The turbidity of the reaction system is negatively correlated with the teicoplanin concentration in the sample (i.e., the smaller the turbidity, the higher the teicoplanin concentration). By measuring the absorbance at 340 nm, a calibration curve is established to calculate the concentration of teicoplanin in the plasma sample.

[0192] Table 3 Experimental parameters of Hitachi 7080 fully automatic biochemical analyzer

[0193] wavelength 340nm / None (Main / Secondary) specimen 6μL Reaction direction Two-point endpoint method Colorimeter light path 1cm R1 180μL Calibration Type Spline Reaction temperature 37℃ R2 60μL Reaction direction down

[0194] The dosage of each reagent during the detection process is shown in Table 4:

[0195] Table 4 Reagent dosage

[0196] Calibrators Determination Calibrators 6μL - sample - 6μL R1 180μL 180μL R2 60μL 60μL

[0197] Add the above test solution R1 to the calibration solution, incubate at 37°C for 5 minutes, then add the test solution R2, incubate at 37°C for 5 seconds, and measure the sample absorbance A1 at a certain wavelength; continue to incubate at 37°C for 5 minutes, then measure the absorbance A2 at the above wavelength, and calculate ΔA=A2-A1; a total of six groups of calibration solution concentration values ​​(0, 6.25, 12.5, 25, 50, 100 mg / L) were measured and nonlinearly fitted with the corresponding absorbance values ​​ΔA of the calibration solution, and the fitting equation was obtained as the calibration curve. The absorbance value ΔA of the plasma sample was determined in the same way. Substitute the absorbance value ΔA obtained from the test plasma sample into the above fitting equation, and the concentration value calculated is the content of teicoplanin in the sample.

[0198] Example 8

[0199] Correlation experiment:

[0200] According to the reagents obtained in Examples 1-6 and the method shown in Example 7, 50 plasma samples with teicoplanin measured values ​​from Tianjin First People's Hospital (HPLC method) were subjected to comparative determination of teicoplanin content by the method of the present invention. The results are as follows: Figure 3 The results show that the present invention has high accuracy and high consistency with the HPLC method, with a correlation coefficient of r=0.982 (y=0.9001x+2.1788), indicating that the teicoplanin reagent combination of the present invention can meet clinical performance requirements (generally, a correlation r>0.975 is considered to meet clinical requirements).

[0201] In summary, the reagent combination of the present invention can specifically measure teicoplanin without being affected by other substances on the basis of meeting the accuracy requirements of teicoplanin detection, and has strong specificity. It also has the advantages of low detection cost, fast detection speed, and simple operation. It can meet the requirements of automated analysis (such as being able to be used in conjunction with instruments such as fully automatic biochemical analyzers to achieve automated analysis), and is suitable for timely and accurate detection of large-scale samples. Compared with the HPLC method, the reagent combination of the present invention generates very little waste liquid during the detection process, which is more environmentally friendly and safe.

[0202] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A teicoplanin detection reagent combination, characterized in that: The reagent combination comprises: (i) a first test solution comprising 200 μg / L to 1 mg / L of a teicoplanin-carrier protein complex; The final concentration of Tris buffer is 20-40mmol / L; Sodium chloride 100-200 mM; Polyethylene glycol 6000 20-80 g / L; Tween 20 0.04-0.08v / v%; pH 7.5-8.5; preservatives; and The solvent is water; and (ii) a second test solution comprising polyclonal antibody to teicoplanin, The final concentration of phosphate buffer is 40-60mmol / L; Sodium chloride 100-200 mM; Tween 20 0.04-0.08v / v%; pH 7-8; preservatives; and The solvent is water; and the second test solution does not contain antibodies to the carrier protein; Wherein, the teicoplanin to be detected is a combination of teicoplanin TA2-1, teicoplanin TA2-2, teicoplanin TA2-3, teicoplanin TA2-4 and teicoplanin TA2-5; In the teicoplanin-carrier protein complex, the carrier protein is selected from the group consisting of serum albumin or keyhole limpet hemocyanin; and The ratio of the molar concentration of the teicoplanin-carrier protein complex in the first test solution to the molar concentration of the teicoplanin polyclonal antibody in the second test solution is 1:5-20.

2. The detection reagent combination according to claim 1, characterized in that In the teicoplanin-carrier protein complex, the carrier protein is bovine serum albumin.

3. The detection reagent combination according to claim 1, characterized in that The first test fluid is: Tris 30 mM; Sodium chloride 150 mM; Polyethylene glycol 6000 40 g / L; Teicoplanin-carrier protein complex 500 μg / L; Tween 20 0.05 v / v %; Sodium azide 0.05 wt %; and pH 8.0; The second test fluid is: Sodium hydrogen phosphate 50 mM; Sodium dihydrogen phosphate 50 mM; Sodium chloride 150 mM; Teicoplanin multi-antibody 0.3 g / L; Tween 20 0.05 v / v %; Sodium azide 0.05 wt %; and pH 7.

4.

4. The detection reagent combination according to claim 1, characterized in that In the first test solution, the concentration of the teicoplanin-carrier protein complex was 500 μg / L.

5. The detection reagent combination according to claim 1, characterized in that The ratio of the molar concentration of the teicoplanin-carrier protein complex in the first test solution to the molar concentration of the teicoplanin polyclonal antibody in the second test solution is 1:8-15.

6. The detection reagent combination according to claim 1, characterized in that The ratio of the molar concentration of the teicoplanin-carrier protein complex in the first test solution to the molar concentration of the teicoplanin polyclonal antibody in the second test solution is 1:9-12.

7. The detection reagent combination according to claim 1, characterized in that The final pH value of the first test solution is 8.0; and / or The final pH value of the second test solution was 7.

4.

8. A teicoplanin detection kit, characterized in that: The kit comprises: the detection reagent combination according to any one of claims 1 to 6, and at least two containers for respectively containing the first test liquid and the second test liquid.

9. The teicoplanin detection kit according to claim 8, characterized in that: The kit also includes: a teicoplanin calibrator.

10. A method for preparing the reagent combination according to claim 1, characterized in that: Includes steps: A) preparing a first teicoplanin-carrier protein complex; B) preparing a first test solution using the first teicoplanin-carrier protein complex; C) preparing a second teicoplanin-carrier protein complex; and D) using the second teicoplanin-carrier protein complex as an immunogen to immunize an animal to obtain teicoplanin polyclonal antibodies, and using the teicoplanin polyclonal antibodies to prepare a second test solution; wherein the carrier protein in the first teicoplanin-carrier protein complex is different from the carrier protein in the second teicoplanin-carrier protein complex; In step A) or step C), the method for preparing the first or second teicoplanin-carrier protein complex comprises the steps of: i) activating teicoplanin with EDCI or EDCI-NHS; and ii) coupling the activated teicoplanin with a carrier protein to obtain a teicoplanin-carrier protein complex; The carrier protein is selected from the group consisting of serum albumin and keyhole limpet hemocyanin.

11. An in vitro non-diagnostic turbidimetric method for detecting immunosuppression of teicoplanin, characterized in that: Includes steps: (1) Providing a detection reagent combination as described in any one of claims 1 to 6 or a kit as described in claim 8 or 9; (2) mixing the teicoplanin calibrator or sample with the first test solution and incubating the mixture; and (3) The mixed solution is mixed with the second test solution respectively, and the initial absorbance A1 after mixing is tested. After incubation for a certain period of time, the absorbance A2 is tested, and ΔA=A2-A1 is calculated.

12. The immunosuppression turbidimetric method for detecting teicoplanin according to claim 11, characterized in that: Also includes the steps: Perform steps (1) to (3) on calibrants of different concentrations to prepare concentration - △A 标准 Standard curve, get the standard curve equation; and The sample is subjected to steps (1)-(3) to obtain △A 样本 , substituted into the standard curve equation to calculate the concentration of teicoplanin in the sample.

13. The immunosuppression turbidimetric method for detecting teicoplanin according to claim 11, characterized in that: The sample is selected from: whole blood, plasma, and serum.

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