CN112129955B - Testosterone detection kit - Google Patents
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- CN112129955B CN112129955B CN201910554532.2A CN201910554532A CN112129955B CN 112129955 B CN112129955 B CN 112129955B CN 201910554532 A CN201910554532 A CN 201910554532A CN 112129955 B CN112129955 B CN 112129955B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
- G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
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Abstract
The application relates to a testosterone detection kit and a using method thereof, the kit comprises a reagent R1, a reagent R2 and a reagent R3, wherein the reagent R1 contains streptavidin magnetic particles, the reagent R2 contains an anti-testosterone antibody marked by a chemiluminescent substance, the reagent R3 contains biotinylated testosterone, and the reagent R2 and/or the reagent R3 also contain an anti-interference agent. The kit provided by the invention has the advantages of high stability, high sensitivity, high accuracy and strong anti-interference capability, and can meet the use requirements.
Description
Technical Field
The invention belongs to the technical field of testosterone detection, and particularly relates to a testosterone detection kit.
Background
Testosterone (Testosterone, T) is a steroid with a molecular weight of 288.4D, and is the most important androgen in the circulatory system. In males, testosterone is predominantly synthesized in leydig cells (also known as leydig cells), regulated by luteinizing hormone, and plays an important role in the development of secondary male sexual characteristics and the differential diagnosis of hypogonadism. Elevated testosterone levels in men are seen in complete sex hormone resistance, while the common causes of decreased testosterone levels are: hypogonadism, orchiectomy, estrogen therapy, klinefelter syndrome, hypopituitarism and cirrhosis, among others. In women, testosterone is produced and secreted by the ovaries and adrenal glands. Women normally have much lower testosterone levels than healthy men. Causes of elevated testosterone levels in women include: polycystic ovarian syndrome, adrenal hyperplasia and the like. Women with excessive testosterone levels are clinically manifested as infertility, hirsutism, amenorrhea, and the like.
At present, testosterone is mostly detected by a radioimmunoassay method and an enzyme-linked immunosorbent assay method. However, these methods all have some disadvantages: the enzyme-linked immunosorbent assay has low sensitivity and narrow detection range, is easy to interfere, and cannot meet the requirement of accurate detection of a large number of clinical samples; radioactive elements in the radioimmunoassay cause environmental pollution, and the short half-life of the radioactive elements causes inconvenience to operation and use and limits the application of the radioactive elements.
Chemiluminescence immunoassay is a non-radioactive immunoassay technique that has been developed very rapidly worldwide in recent years. The detection principle is that the luminescent substance is used as a signal amplification system and the immunological binding is directly measured by virtue of the luminous intensity. The method has high sensitivity and wide detection range, and is an important development direction of immunological detection.
But the chemiluminescence immunoassay kit which is suitable for testosterone determination and has the advantages of high stability, high sensitivity, high accuracy and strong anti-interference capability still has the problem to be solved at present.
Disclosure of Invention
In view of this, the invention provides a testosterone detection kit with high stability, high sensitivity, high accuracy and strong anti-interference capability, which comprises a reagent R1, a reagent R2 and a reagent R3. The reagent R1 comprises streptavidin magnetic particles and a basic buffer solution; the reagent R2 comprises an anti-testosterone antibody marked by a detectable marker and a basic buffer solution; the reagent R3 contains biotinylated testosterone and a basic buffer solution; at least one of the reagent R2 and the reagent R3 also comprises an anti-interference agent, and the anti-interference agent is selected from one or more of biotin and biotin derivatives.
In the present invention, the "streptavidin magnetic particle" refers to a streptavidin protein-coated magnetic particle. The amount of the "streptavidin magnetic particle" to be used may be selected by those skilled in the art, and may be, for example, 0.01 to 1.0mg/mL, or 0.01mg/mL, 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL, or 1.0mg/mL.
In the present invention, "detectable label" refers to a kind of substance detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means, such as enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β -galactosidase and other enzymes commonly used in ELISA), fluorescent dyes (e.g., fluorescein, texas red, rhodamine and green fluorescent protein, etc., see, for example, molecular Probes, eugene, oreg., USA), radioactive labels (e.g., 3H, 125I, 35S, 14C or 32P), luminescent substances (e.g., chemiluminescent substances, further, e.g., acridine (e.g., acridine-9-carboxamide), phenanthridine (phenanthridinium), dioxetane (dioxetanes) and luminol, etc.), colorimetric labels (e.g., colloidal gold, further, gold particles, e.g., having a particle size of 40nm to 80nm, efficiently scatter green light) or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
Preferably, the "detectable label" is a chemiluminescent substance. "chemiluminescent substance" refers to a substance that forms an excited intermediate by catalysis by a catalyst and/or oxidation by an oxidant, and that emits photons when the excited intermediate returns to a stable ground state.
Preferably, the "detectable label" is an "acridinium chemiluminescent substance". The "acridine chemiluminescent substance" refers to acridine ester and its saltDerivatives, acridine sulfonamide chemiluminescent substances and lucigenins, which are suitable for use in chemiluminescent immunoassay systems. In the alkaline state H 2 O 2 In solution, when molecules are attacked by hydrogen peroxide ions, unstable ethylene dioxide is formed, which is decomposed into CO 2 And an electronically excited state of N-methylacridone which emits a photon when it returns to the ground state.
Preferably, the "detectable label" is an "acridinium ester derivative". Acridinium ester derivatives "are well known in the field of chemiluminescence immunoassay, and for example, acridinium ester derivatives for chemiluminescence immunoassay and a preparation method thereof are described in chinese patent 201510090045.7; law et al (Novel poly-mutated aryl acid esters and the use in immunoassays, J Biolumin Chemicalin 1989Jul;4 (1): 88-98) synthesized a series of acridinium ester analogs that could be used in immunoassays.
In the present invention, "anti-testosterone antibodies" refer to immunoglobulin molecules and fragments thereof that specifically bind testosterone, including but not limited to chimeric antibodies, humanized antibodies, fully human antibodies, CDR-grafted antibodies, and antibody constructs, such as single chain Fv (scFv) or antibody fusion proteins; recombinant or synthetic antibodies are also contemplated. And, also, to "fragments of an antibody," i.e., generally comprising the antigen binding region, light and/or heavy chain variable regions, at least a portion of one or more (e.g., six) CDRs of an antibody, which retain at least some of the binding specificity of the parent antibody. Examples of antibody fragments include, but are not limited to, fab ', F (ab') 2, and Fv fragments; a dimeric molecule; a linear antibody; single chain antibody molecules, e.g., sc-Fv; and multispecific antibodies formed from antibody fragments. Typically, a fragment retains at least 50% of the binding activity to a small molecule compound when the activity is expressed on a molar basis. Preferably, the fragment retains at least 60%, 70%, 80%, 90%, 95%, or 100% of the binding activity to the small molecule compound as compared to the parent antibody.
In the present invention, the "anti-testosterone antibody" contained in the reagent R2 refers to an "anti-testosterone antibody" labeled with a "detectable marker". Labeling antibodies with detectable labels is well known in the art. The amount of the anti-testosterone antibody labeled with a detectable label and the amount thereof can be selected by those skilled in the art according to the actual circumstances, and for example, a murine anti-testosterone monoclonal antibody labeled with acridinium ester can be used at a concentration of, for example, 0.01 to 1.0. Mu.g/mL, or at a concentration of 0.01. Mu.g/mL, 0.02. Mu.g/mL, 0.03. Mu.g/mL, 0.04. Mu.g/mL, 0.05. Mu.g/mL, 0.08. Mu.g/mL, 0.1. Mu.g/mL, 0.2. Mu.g/mL, 0.3. Mu.g/mL, 0.4. Mu.g/mL, 0.5. Mu.g/mL, 0.6. Mu.g/mL, 0.7. Mu.g/mL, 0.8. Mu.g/mL, 0.9. Mu.g/mL, or 1.0. Mu.g/mL.
In the present invention, "biotinylated testosterone" refers to biotin-modified testosterone or a derivative thereof. "Testosterone derivative" refers to testosterone that contains an active group that can be used for conjugation, and whose molecular modification does not affect its immunoreactivity. The amount of biotinylated testosterone used may be selected, for example, from 0.2 to 200ng/mL, or from 0.2ng/mL, 2ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 80ng/mL, 100ng/mL, 120ng/mL, 140ng/mL, 160ng/mL, 180ng/mL, or 200ng/mL.
In the present invention, "basic buffer" refers to a type of solution having salt balance, pH buffering, protection of the structure and biological properties of the active ingredient. The basic buffer solution comprises a pH value regulator, inorganic salt ions, a surfactant and a stabilizer.
The term "pH regulator" refers to an agent capable of offsetting and reducing the influence of an external strong acid or strong base on the pH value of a solution to a certain extent, thereby keeping the pH value of the solution relatively stable. For the type of the pH value regulator, the invention is selected from one or more of phosphate buffer, citrate buffer, acetate buffer, tris buffer and HEPES buffer; the amount of the pH adjustor to be used may be selected by those skilled in the art according to the actual conditions, and for example, a concentration of 0.5 to 20g/L may be used when a phosphate buffer is used.
"inorganic salt ions" refers to a class of agents that maintain the ionic concentration of a solution in a relatively stable equilibrium. For the types of inorganic salt ions, the invention is selected from one or more of sodium chloride and potassium chloride; the amount of the inorganic salt ion to be used may be selected by those skilled in the art according to the actual circumstances, and for example, a concentration of 5 to 20g/L may be used when sodium chloride is used.
The "surfactant" refers to a substance which is added in a small amount and can make the interface state of a solution system change obviously, has fixed hydrophilic and lipophilic groups and can be arranged on the surface of the solution in an oriented way. For the types of surfactants, the invention can be selected from Tween, SPAN, triton, EMULGEN series surfactants (such as Tween 20, tween 40 and Triton 100), and one or more of laurylglycerol, anionic alkylpolyglycoside, dodecyl dimethyl betaine, 3ARAMT1, SHO62C and AMPHITOL; the amount of the surfactant to be used can be selected by those skilled in the art according to the actual situation, and for example, when tween 20 or triton 100 is used, a concentration of 0.01 to 5.0mL/L, preferably 0.1 to 0.5mL/L, can be used.
"stabilizer" refers to a class of agents for their stabilizing action, preferably selected from one or more of mannitol, bovine serum albumin, glycerol, trehalose, casein, polyvinylpyrrolidone and hydroxyethyl starch; the amount of stabilizer used can be selected by the person skilled in the art as a function of the circumstances, for example a concentration of 5 to 40g/L, for example a concentration of 5g/L, 10g/L, 20g/L, 30g/L or 40 g/L.
In the present invention, the "biotin derivative" refers to a compound in which a hydrogen atom or atomic group of biotin is substituted with another atom or atomic group, for example, biotin ester.
Preferably, the anti-interference agent is added to the testosterone detection kit provided by the invention at a concentration of 5-100 ng/mL, i.e., the concentration of the anti-interference agent in reagent R2 or reagent R3 is 5-100 ng/mL, such as 5ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL or 100ng/mL.
Preferably, in the testosterone detection kit provided by the invention, the ratio of the concentration of the anti-interference agent to the concentration of biotinylated testosterone is 0.5. In the test process, the applicant finds that the addition of anti-interference agents such as biotin and the like can obviously resist the interference of biotin in a sample and improve the accuracy of a detection result.
Preferably, in the testosterone detecting kit provided by the invention, the reagent R1 further comprises an antifoaming agent. The type of the defoaming agent can be selected from one or more of polyether defoaming agent and silicon defoaming agent; the amount of the defoaming agent can be selected from 0.01-1 mu L/L.
Preferably, in the testosterone detection kit provided by the invention, the reagent R2 further comprises a testosterone releasing agent, preferably estradiol and derivatives thereof, and the dosage is 0.1-10 μ g/mL, for example, 0.1 μ g/mL, 0.5 μ g/mL, 1 μ g/mL, 1.5 μ g/mL, 2 μ g/mL, 3 μ g/mL, 4 μ g/mL, 5 μ g/mL, 6 μ g/mL, 7 μ g/mL, 8 μ g/mL, 9 μ g/mL, 10 μ g/mL.
Preferably, the testosterone detection kit provided by the invention further comprises a calibrator, and the calibrator comprises a testosterone antigen and a calibrator diluent.
In the invention, the 'calibrator diluent' is a solution for diluting the testosterone antigen and protecting the structure and function of the testosterone antigen. The calibrator diluent comprises a pH value regulator, inorganic salt ions, a surfactant and a stabilizer.
The selection of the types of the pH value regulator, the inorganic salt ions, the surfactant and the stabilizer is similar to the selection of corresponding components in the basic buffer solution, wherein the dosage range of the pH value regulator is 2-20 g/L; the dosage range of the inorganic salt ions is 1.0-10 g/L; the dosage range of the surfactant is 0.1-10 mL/L; the dosage range of the stabilizer is 3-300 g/L.
Preferably, the testosterone detecting kit provided by the invention comprises bovine serum albumin in a calibrator diluent; the bovine serum albumin content is preferably 20 to 90g/L, more preferably 20 to 80g/L, 30 to 70g/L, 30 to 60g/L, or 30 to 50g/L. Through experiments, the applicant finds that the bovine serum albumin with the concentration range is added into the diluent of the calibrator, so that the stability of the calibrator can be improved, the specificity of a calibration curve can be improved, and the accuracy of a detection result can be improved.
Another object of the invention is to provide a method of using a testosterone detection kit, comprising the following: and mixing the sample to be tested with the reagent R2, and after the incubation is finished, adding the reagent R1 and the reagent R3 for incubation.
Compared with the prior art, the invention has the following beneficial effects: according to the testosterone detection kit provided by the invention, the reagent R2 and/or the reagent R3 contain an anti-interference agent, so that the interference of biotin in a sample can be resisted, and the accuracy of a detection result is improved; the reagent R1 contains the defoaming agent, so that the influence of bubbles on sample addition when the reagent is uniformly mixed with magnetic particles is prevented, and the stability of a detection system is improved; the calibration product contains bovine serum albumin with specific concentration, has good stability, is convenient to use and store, and can improve the accuracy of detection results. In addition, the sensitivity and the accuracy of testosterone detection can be further improved by adopting the testosterone detection kit provided by the invention and combining the use method provided by the invention.
Detailed Description
In order to make the technical solutions in the present application better understood, the present invention is further described below with reference to examples, and it is obvious that the described examples are only a part of the examples of the present application, but not all examples. All other embodiments, such as component selections and dosage range selections of conventional reagents (such as pH regulators, inorganic salt ions, surfactants, and stabilizers) in the kit, which can be obtained by the platform skilled in the art without creative efforts based on the embodiments in the present application, shall fall within the protection scope of the present application.
The reagent components referred to in the following examples are commercially available, for example, streptavidin magnetic particles are available from Sichuan Michael BioNew Material technology, inc. (Cat: XCL 1026), biotinylated testosterone is available from Sichuan Michael BioNew Material technology, inc. (Cat: XCL 1495), biotin (also known as vitamin H) is available from Kyoto Koron Chemicals, inc., antifoaming agent is available from Sichuan Michael BioNew Material technology, inc. (Cat: XCL 1602), estradiol is available from Toronto Research Chemicals, and bovine serum albumin is available from Sichuan Michael BioNew Material technology, inc. (Cat: XCL 1353). The acridinium ester-labeled anti-testosterone antibody used in the following examples is a reagent component obtained by labeling a commercially available acridinium ester-labeled mouse anti-testosterone monoclonal antibody, wherein the mouse anti-testosterone monoclonal antibody is purchased from Sichuan Michael Biotech materials Co., ltd. (product number: XCL 1343).
Example 1
1.1 preparation of Testosterone detection kit 1
Reagent R1, reagent R2 and reagent R3 were prepared according to the following tables, respectively:
1.2 preparation of Testosterone detection kit 2
Reagent R1, reagent R2 and reagent R3 were prepared according to the following tables, respectively:
1.3 investigation of anti-interference Effect
Taking 5 samples to be detected, and respectively detecting by using the testosterone detection kit 1 and the testosterone detection kit 2; then, respectively adding 10ng/mL biotin into the detection samples, and respectively detecting by adopting the testosterone detection kit 1 and the testosterone detection kit 2; the results of the measurements are shown in the following table:
in the above table, "control measurements" indicate the concentration of testosterone measured in the sample without added biotin interference; "Interferometric value" means the concentration of testosterone determined when 10ng/mL biotin was added to the corresponding sample to produce biotin interference. It can be seen from the above table that, the difference between the values measured by adding and not adding biotin in the same sample by the testosterone detection kit 1 is small (the deviation is less than 10%), while the difference between the results obtained by detecting by the testosterone detection kit 2 is large (the deviation is more than 10%), which indicates that the antibiotic interference effect of the testosterone detection kit 1 is good, and the antibiotic interference effect of the testosterone detection kit 2 is poor.
1.4 stability Studies
Preparing main calibrators, wherein the concentrations of testosterone antigens added into each calibrator are as follows: calA:0-0.1ng/mL; and (4) CalB:0.2-0.6ng/mL; and (3) CalC:0.5-1.5ng/mL; and (4) CalD:3.0-7.0ng/mL; and (3) CalE:20.0-30.0ng/mL.
The testosterone detection kit 1 and the testosterone detection kit 2 which are placed at 4 ℃ are used for detecting the main calibrator, then the two kits are placed in a 37 ℃ environment and placed for 7 days, the main calibrator is detected, and the obtained calibration curve signal value and the calibration curve signal retention rate are shown in the following table:
in the above table, "signal value at 4 ℃" refers to a calibration curve signal value measured after placing the testosterone detection kit 1 or the testosterone detection kit 2 in an environment at 4 ℃; the "signal value at 37 ℃" refers to a signal value of a calibration curve measured after placing the testosterone detection kit 1 or the testosterone detection kit 2 in an environment at 37 ℃ for 7 days; "Signal retention" refers to the percentage of "signal value at 37 ℃ to" signal value at 4 ℃ in the same calibrator, and a higher signal retention indicates better stability. As can be seen from the above table, the stability of testosterone assay kit 1 is better than testosterone assay kit 2.
1.5 immunoassay examination
20 random samples are respectively detected by using a testosterone detection kit 1 and a testosterone detection kit 2, and the detection results are as follows:
as can be seen from the above table, compared with the testosterone detection kit 2, the testosterone detection kit 1 has the advantages that biotin is added into the reagent R3, but the difference of the detection results of the two kits is small, which indicates that the immunoreactivity of the reagent R3 is not influenced after the biotin is added into the reagent R3.
Example 2
2.1 preparation of Testosterone detection kit 3
Reagent R1, reagent R2 and reagent R3 were prepared according to the following tables, respectively:
2.2 investigation of anti-interference Effect
The same method as that of the 1.3 anti-interference effect investigation in the embodiment 1 is adopted, but the testosterone detection kit 1 is replaced by the testosterone detection kit 3, and the result shows that the anti-interference effect of the testosterone detection kit 3 with the biotin added in the reagent R2 is obviously better than that of the testosterone detection kit 2 without the biotin.
2.3 stability Studies
The same method as in "1.4 stability test" in example 1 was adopted except that the testosterone test kit 1 was replaced with the testosterone test kit 3, and as a result, it was found that the stability of the testosterone test kit 3 in which biotin was added to the reagent R2 was better than that of the testosterone test kit 2 in which no biotin was added.
2.4 Immunoassay examination
The same method as that of the "1.5 immunoassay investigation" in example 1 was used, except that the testosterone detection kit 1 was replaced with the testosterone detection kit 3, and as a result, it was found that the immunogenicity of the testosterone detection kit 3 was equivalent to that of the testosterone detection kit 2, indicating that the immunogenicity of the kit was not affected by the addition of biotin to the reagent R2.
2.5 examination of biotin addition amount
On the basis of a testosterone detection kit 3, the addition amounts of biotin in a reagent R2 of the testosterone detection kit are considered, the addition amounts of the biotin are respectively 5ng/mL, 30ng/mL and 50ng/mL, so that the testosterone detection kit with 3 biotin addition amounts is obtained, 3 samples are detected by the 3 kits by adopting the same method of '1.3 anti-interference effect determination' in example 1, and the results are shown in the following table:
similar to the "1.3 assay for anti-interference effects", the "control assay" indicates the concentration of testosterone measured in the sample without biotin interference; "Interferometric value" means the testosterone concentration determined for the same sample to which 10ng/mL biotin was added to produce biotin interference. As can be seen from the above table, under the condition that the biotinylated testosterone in the reagent R3 is the same, the addition amount of biotin in the reagent R2 has different effects on the anti-interference effect, and based on the disclosure of the present application, a person skilled in the art can reasonably adjust the content of biotin in the reagent R2 according to specific conditions (for example, the content of biotinylated testosterone) to achieve a better anti-interference effect. The Applicant has also found that the addition of biotin in reagent R3 has a similar effect to that of reagent R2.
Example 3
3.1 preparation of dilution of calibrator
3 different dilutions of the calibrator were prepared according to the following table:
3.2 stability Studies
Preparing a main calibrator by using the calibrator diluent 1, the calibrator diluent 2 and the calibrator diluent 3 respectively, wherein the concentrations of testosterone antigens added into each calibrator are respectively as follows: and (4) CalA:0-0.1ng/mL; and (3) CalB:0.2-0.6ng/mL; and (3) CalC:0.5-1.5ng/mL; calD:3.0-7.0ng/mL; and (3) CalE:20.0-30.0ng/mL.
Placing main calibrators with different testosterone antigen concentrations, which are prepared by different calibrator diluents, in an environment at 4 ℃, and then detecting the diluted main calibrators by using the testosterone detection kit 1 in the embodiment 1; then, the main calibrator with different concentrations of testosterone antigens prepared by the different calibrator dilutions is placed in an environment at 37 ℃ for 7 days, and then the testosterone detection kit 1 in example 1 is used for detection, and the obtained calibration curve signal value and the retention rate of the calibration curve signal are shown in the following table:
in the above table, "signal value at 4 ℃" refers to a calibration curve signal value measured after different calibrator dilutions are prepared into main calibrators with different testosterone antigen concentrations and placed in an environment at 4 ℃; the '37 ℃ signal value' refers to a calibration curve signal value measured after a main calibrator with different testosterone antigen concentrations prepared from different calibrator diluents is placed in a 37 ℃ environment for 7 days; "Signal retention" refers to the percentage of "signal value at 37 ℃ to" signal value at 4 ℃ in the same calibrator, and a higher signal retention indicates better stability. As can be seen from the above table, the stability of the calibrator prepared from calibrator diluent 2 and calibrator diluent 3 is comparable, the stability is significantly better than that of the calibrator prepared from calibrator diluent 1, and the specificity of the calibration curve obtained from calibrator diluent 2 and calibrator diluent 3 is higher.
3.3 investigation of accuracy
Preparing a main calibrator from the 3.1 calibrator dilutions, respectively, determining 8 random samples by using the testosterone detection kit 1 in example 1, wherein the calibrator is determined before sample determination, and the result of the determined sample is based on the calibration result of the calibrator according to the calibration rule.
In order to compare the accuracy of the detection results, 8 samples were tested using a testosterone determination kit (chemiluminescence microparticle immunoassay) from yapei corporation (kit cat # 2p13-28; calibrator cat # 2p 13-01), and the components of each reagent in the kit from yapei corporation were as follows:
the testosterone detection kit 1 in example 1 is matched with calibrators prepared by 3 calibrators diluents in 3.1 respectively, and the test results are shown in the following table by taking the kit of Yapei company as comparison:
in the above table, "measured value of the comparison manufacturer" means the content of testosterone in the sample obtained by the comparison manufacturer (yapei corporation), and "measured value" means the content of testosterone measured by using the calibrator prepared by using the calibrator diluent 1/2/3 respectively matched with the calibrator diluent in the testosterone detection kit 1 of example 1. It can be seen from the above table that the bovine serum albumin content in the calibrator diluent affects the detection accuracy of the kit, and the result of the sample measured after the calibration by using the main calibrator prepared from calibrator diluent 2 is more accurate.
Example 4
In this embodiment, the testosterone detection kit 3 in embodiment 2 is used, and the main calibrator prepared from the calibrator diluent 2 in embodiment 3 is used in a matching manner, wherein the concentrations of testosterone antigens added to the calibrator are as follows: and (4) CalA:0-0.1ng/mL; and (4) CalB:0.2-0.6ng/mL; and (4) CalC:0.5-1.5ng/mL; calD:3.0-7.0ng/mL; and (3) CalE:20.0-30.0ng/mL.
4.1 methods of use of Testosterone detection kits 1
Adding the sample, the reagent R2 and the reagent R3 for co-incubation, then adding the reagent R1 for incubation, and after incubation is finished, washing to remove unbound sample reagent. A luminescent substrate is added and the relative luminescence value (RLU) is measured.
4.2 methods of Using Testosterone detection kits 2
Adding a sample and the reagent R2 for incubation, then adding the reagent R3 and the reagent R1 for incubation, and after incubation is finished, washing to remove unbound sample reagent. A luminescent substrate is added and the relative luminescence value (RLU) is measured.
4.3 sensitivity investigation
The main calibrator was tested by methods of "method of use 1 of the 4.1 testosterone assay kit" and "method of use 2 of the 4.2 testosterone assay kit", respectively, and the calibration curves obtained are shown in the following table:
as can be seen from the above table, the change degree of the signal value measured by using the method 2 of the testosterone detection kit is larger with the change of the concentration of the testosterone antigen in the calibrator, that is, the detection sensitivity of the method 2 of the testosterone detection kit is higher.
4.4 accuracy survey
The method of using the 4.1 testosterone detection kit 1 and the method of using the 4.2 testosterone detection kit 2 are respectively adopted to detect 20 random samples, the calibrator is determined before the sample is determined, the result is calibrated according to the calibration rule, and the determined sample result is based on the calibration result of the calibrator.
Meanwhile, in order to compare the accuracy of the detection result, 20 random samples were detected by using a testosterone determination kit (chemiluminescence microparticle immunoassay) from yapei corporation (kit cat # 2p13-28; calibrator cat # 2p 13-01). The results of the measurements are shown in the following table:
as can be seen from the above table: by adopting the method of using the method 2 of the 4.2 testosterone detection kit, the consistency of the detected result and a comparison manufacturer is higher, namely, the sample result measured by using the method 2 of the testosterone detection kit is more accurate.
Example 5
5.1 basic buffer for reagent R1
The base buffer was prepared according to the following table:
5.2 magnetic particle buffer for reagent R1
Magnetic particle buffer was prepared according to the following table:
| starting materials | Amount of the composition |
| Disodium hydrogen phosphate | 2.56g/L |
| Sodium dihydrogen phosphate (dihydrogen phosphate) | 0.436g/L |
| Sodium chloride | 9.00g/L |
| Mannitol | 20.00g/L |
| Bovine serum albumin (B) | 10.00g/L |
| 4-aminoantipyrine | 0.02g/L |
| TX-100 | 0.10mL/L |
| Biological preservative PC-300 | 3.00mL/L |
| Glycerol | 10.00mL/L |
| Defoaming agent | 0.05μL/L |
| Purified water | Constant volume to 1L |
5.3 investigation of sedimentation under natural gravity:
taking 7mL of magnetic particles, respectively using two buffers of 5.1 and 5.2 to wash the magnetic particles for 3 times, then respectively using the two buffers to carry out resuspension on the magnetic particles, uniformly mixing, and evenly dividing into 7 parts with each part being 1mL. At intervals 100. Mu.L of supernatant was taken and its absorbance at 550nm was measured using a multifunctional reader (Thermo) and the results were as follows:
as seen from the above table: the natural sedimentation rate of the magnetic particles in the basic buffer solution is faster than that of the magnetic particles in the magnetic particle buffer solution, and at 30min, the supernatant close to the liquid level is clear and transparent when the magnetic particles naturally settle in the basic buffer solution for 30min, and the supernatant in the magnetic particle buffer solution is still a suspension without obvious layering boundary.
5.4 examination of magnetic separation Effect under the action of magnetic field
The procedure was essentially similar to the sedimentation under natural gravity of 5.3, except that 100. Mu.L of magnetically separated supernatant was taken at the time of magnetic separation for 0s, 10s, 20s, 30s and 45s under a magnetic field of 2800 Gauss, and the absorbance value at 550nm was determined as follows:
as can be seen from the above table: under the magnetic field environment, the magnetic separation rate difference of the magnetic particles in the basic buffer solution and the magnetic particle buffer solution is not obvious, and the magnetic separation rate at 30s is more than 92%.
5.5 defoaming Effect investigation
For a large-package chemiluminescence magnetic particle reagent, the components of the magnetic particles need to be uniformly mixed before each measurement, and when the residual volume is small, bubbles are easily generated after the magnetic particles are uniformly mixed, so that the phenomenon of probe empty detection of the reagent occurs in the subsequent measurement process. Magnetic particles were prepared using two buffers of 5.1 and 5.2, respectively, and a magnetic particle mixing procedure was performed on a full-automatic chemiluminescence apparatus of michael bio-inc, and the foaming ratio was observed with naked eyes within 5 seconds, with the following results:
| number of measurements | 5.1 basic buffer | 5.2 magnetic particle buffer |
| 1 | 50％ | Is free of |
| 2 | 90％ | Is free of |
| 3 | 100％ | Is composed of |
| 4 | 100％ | Is free of |
| 5 | 100％ | Is free of |
| 6 | 100％ | Is free of |
| 7 | 100％ | Is free of |
| 8 | 100％ | Is free of |
| 9 | 100％ | Is free of |
| 10 | 100％ | Is free of |
| 11 | 100％ | Is free of |
| 12 | 100％ | Is free of |
| 13 | 100％ | Is free of |
| 14 | 100％ | Is free of |
| 15 | 100％ | Is free of |
From the above table can be seen: the proportion of bubbles generated by the magnetic particle buffer solution in the process of uniformly mixing the magnetic beads is obviously lower than that of the basic buffer solution.
5.6 immunoassay examination
The reagent R1 is prepared by respectively adopting 5.1 basic buffer solution and 5.2 magnetic particle buffer solution, the reagent R2 and the reagent R3 in the testosterone detection kit have the same components as those in the example 1, 25 clinical samples are determined by adopting a full-automatic immunoassay chemiluminescence apparatus, whether the two buffer solutions influence the application of an immunoassay is observed, and the detection results are as follows:
as seen from the above table: compared with the 5.1 basic buffer solution, the 5.2 magnetic particle buffer solution has the advantages that the defoaming agent component is added, but the difference of the results measured by the detection kit prepared from the two buffer solutions is small, namely, the immunoreactivity of the reagent R1 is not influenced after the defoaming agent is added.
Claims (16)
1. The use method of the testosterone detection kit is characterized in that the use method comprises the following steps
Adding a sample, a reagent R2 and a reagent R3 for co-incubation, then adding a reagent R1 for incubation, washing after incubation is finished, and removing unbound sample reagents; or
Adding a sample and a reagent R2 for incubation, then adding a reagent R3 and a reagent R1 for incubation, washing after incubation is finished, and removing unbound sample reagent;
wherein the kit comprises a reagent R1, a reagent R2 and a reagent R3; the reagent R1 comprises streptavidin magnetic particles and a basic buffer solution;
the reagent R2 comprises an anti-testosterone antibody labeled with a detectable label and a basal buffer;
the reagent R3 comprises biotinylated testosterone and a basal buffer;
at least one of the reagent R2 and the reagent R3 further comprises an anti-interference agent, and the anti-interference agent is selected from one or more of biotin and biotin derivatives.
2. The method of using a testosterone detection kit according to claim 1, wherein the concentration of said anti-interference agent in reagent R2 or reagent R3 is 5-100 ng/mL.
3. The use method of the testosterone detection kit according to claim 1, wherein the concentration ratio of the anti-interference agent to biotinylated testosterone is 0.5.
4. The method of using a testosterone detection kit according to claim 1, wherein said detectable label is an enzyme, a fluorescent dye, a radioactive label, an isotopic label, or a luminescent substance.
5. The method of using a testosterone detection kit according to claim 4, wherein said detectable label is a chemiluminescent substance.
6. The method of using the testosterone detection kit according to claim 4, wherein said detectable label is an acridinium chemiluminescent substance.
7. The method of using the testosterone detection kit according to claim 4, wherein said detectable label is an acridinium ester derivative.
8. The method of using a testosterone detection kit according to claim 1, wherein said base buffer comprises a pH adjusting agent, inorganic salt ions, a surfactant, and a stabilizer.
9. The method of using the testosterone detection kit according to claim 1, wherein said reagent R1 further comprises an antifoaming agent.
10. The use method of the testosterone detection kit according to claim 9, wherein said antifoaming agent is one or more selected from polyether antifoaming agents and silicon antifoaming agents.
11. The method of using the testosterone detection kit according to claim 1, wherein said reagent R2 further comprises a testosterone releasing agent.
12. The method of using the testosterone detection kit according to claim 11, wherein said testosterone releasing agent is selected from the group consisting of estradiol, estradiol derivatives, and combinations thereof.
13. The method of using a testosterone detection kit according to claim 1, further comprising a calibrator comprising testosterone antigens and a calibrator diluent comprising a pH adjuster, inorganic salt ions, a surfactant, and a stabilizer.
14. The method of using a testosterone detection kit according to claim 13, wherein said calibrator diluent comprises bovine serum albumin.
15. The use method of the testosterone detection kit according to claim 14, wherein the content of bovine serum albumin is 20-90 g/L.
16. The method of using a testosterone detection kit according to any one of claims 2-15, comprising the following: and mixing the sample to be tested with the reagent R2, and after the incubation is finished, adding the reagent R1 and the reagent R3 for incubation.
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| DE4136010A1 (en) * | 1991-10-31 | 1993-08-12 | Boehringer Mannheim Gmbh | METHOD FOR DETERMINING AN IMMUNOLOGICALLY BINARY SUBSTANCE |
| JP3027770B2 (en) * | 1994-03-05 | 2000-04-04 | ロシュ ダイアグノスティックス ゲーエムベーハー | Interference removers for use in immunoassays |
| US5705353A (en) * | 1995-06-07 | 1998-01-06 | Beckman Instruments, Inc. | Method of reducing interferences in assays |
| US6096508A (en) * | 1995-08-16 | 2000-08-01 | Kirkegaard & Perry Laboratoies, Inc. | Method of reducing background in biotin-based assays |
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