CN112129933B - Reagent, kit and method for resisting biological interference in immunoassay system - Google Patents
Reagent, kit and method for resisting biological interference in immunoassay system Download PDFInfo
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- CN112129933B CN112129933B CN201910554114.3A CN201910554114A CN112129933B CN 112129933 B CN112129933 B CN 112129933B CN 201910554114 A CN201910554114 A CN 201910554114A CN 112129933 B CN112129933 B CN 112129933B
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- biotin
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Classifications
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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
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The invention discloses an anti-biotin interference reagent, a kit and a method in an immunoassay system. The anti-biotin interference reagent comprises biotin or biotin derivatives, and the kit comprises the anti-biotin interference reagent; and the method of anti-biotin interference is to use the anti-biotin interference agent. By using the reagent, the kit and the method for resisting the biotin interference, the detection sensitivity can be improved and the endogenous biotin interference can be avoided on the premise of not influencing the normal detection and measurement results; the reagent of the invention has low cost and is suitable for industrial production.
Description
Technical Field
The invention relates to the field of medicine inspection, in particular to an anti-biotin interference reagent in an immunoassay system, a kit and a method thereof.
Background
The immune analyzer is one of the most frequently used instruments in clinical application in hospitals, and clinicians often diagnose diseases of patients according to detection results of the immune analyzer on corresponding physiological indexes of body fluids of clinical patients.
The biotin-avidin system is a biological reaction amplification system widely used in immunoassay systems, which greatly improves the sensitivity of immunoassay by firmly binding to a tracer or biomarker and a multistage amplification effect. The biotin-avidin system is widely used in various immunoassay techniques such as enzyme immunoassay, fluorescence immunoassay, radioimmunoassay, colloidal gold and chemiluminescent immunoassay, and automated analysis. Based on this, manufacturers at home and abroad develop a series of immunoassay products, such as Cobas series of Roche company, architct series of Abbott company, TALENT series of Fenghua company, and IS 1200 of Maccura company.
Biotin (Biotin) is one of the B vitamins, also called vitamin H, vitamin B7, coenzyme R (Coenzyme R), a water-soluble vitamin widely distributed in every living cell of animals and plants, and is a necessary nutrient substance for maintaining natural growth, development and human skill health of the human body. Generally, the human body does not lack biotin, and the biotin content in normal blood of the human body is less than 0.1-0.8 ng/mL. (Paul Grimsey et al proposal pharmacokinetics OF exogenous biotin and the relationship between biotin serum levels and in vitroimmunoassay interface. INTERNATIONAL JOURNAL OF PHARMACOKINETICSVOL.2, NO. 4). However, biotin is widely used as a health product for maintaining hair and skin health, such as preventing hair loss and premature grey hair; in addition, biotin can be used for treating biotin deficiency, seborrheic dermatitis, infantile enuresis, etc. Biotin is thus now also used as an exogenous supplement, and about 30-60 μg of biotin can be ingested with a normal intake of the microbial preparation. The oral biotin can be rapidly absorbed from the stomach and intestinal tract, and 80% of biotin in blood exists in a free form, so that if free endogenous biotin exists in a sample to be tested, the capability of capturing a target of avidin in a biotin-avidin immunoassay system is interfered, thereby causing the false increase or the false decrease of a detection result and affecting clinical judgment. The research finds that: the sample to be tested is detected by a biotin-avidin immunoassay system, and endogenous biotin can interfere with the detection results of conventional detection items (hormone, myocardial markers, tumor markers), such as: free thyronine, thyroglobulin antibody, thyroid stimulating hormone, luteinizing hormone, prolactin, urinary human chorionic gonadotropin, troponin, carcinoembryonic antigen, and the like.
In view of the above problems, the existing strategies for eliminating the interference of endogenous biotin to the biotin-avidin immunoassay system are as follows: the patient pauses the administration of biotin, changes the immunoassay system to a non-biotin-avidin system, re-measurement after dilution of the sample, neutralization (removal of biotin from the sample using avidin), etc. The above method first requires identifying whether the sample is subject to biotin interference; secondly, the sample needs to be preprocessed, so that a lot of extra workload is added, time and effort are wasted-! And the problem of biotin interference in a sample cannot be fundamentally solved on the basis of the existing kit.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a reagent, a kit and a method thereof which can directly solve the problem of endogenous biotin interference in a sample so as to cause abnormal detection results on the premise of not influencing the normal detection and measurement results in a biotin-avidin immunoassay system.
In particular, in a first aspect, the invention provides an anti-biotin interference reagent in an immunoassay system, the reagent comprising biotin or a derivative thereof.
Biotin is understood as being a naturally occurring purified compound (CAS: 58-85-5) or coincidental to biotin. Biotin derivatives are understood as modified biotin molecules; more specifically, a biotin derivative refers to a class of compounds in which a hydrogen atom or radical of biotin is replaced with another atom or radical, for example, biotin esters.
In some embodiments, the concentration of biotin or biotin derivative in the reagent is 50 to 100ug/mL.
In other embodiments, the reagent further comprises a solvent that can solubilize biotin or a biotin derivative; preferably, the solvent is a solvent which does not affect the detection result of the immunoassay; more preferably, the solvent is dimethyl maple.
Because biotin is slightly soluble in water and ethanol and insoluble in other common organic solvents, the use of dimethyl maple can dissolve biotin or biotin derivatives without affecting the immunoassay detection results, and the solvent is less toxic.
In other embodiments, the immunoassay system is an immunoassay system that uses a biotin-avidin system.
In a second aspect, the invention also provides a kit for immunoassay comprising the above-described anti-biotin-interference reagent.
In some embodiments, the kit is used to detect large and small molecular species in a sample, the small molecular species being natural compounds having a molecular weight of less than 1000 Da.
In some preferred embodiments, the kit is preferably used for detecting small molecule compounds having a molecular weight of less than 1000 Da.
In some preferred embodiments, the small molecule compounds include estradiol, testosterone, 25 hydroxy vitamin D, progesterone, cortisol, and the like. Immunoassays for small molecule substances are typically competitive immunoassays.
In some embodiments, the kit is an enzyme immunoassay kit, a fluorescent immunoassay kit, a radioimmunoassay kit, a colloidal gold immunoassay kit, a chemiluminescent immunoassay kit, or an automated immunoassay kit. Preferably, the kit is an enzyme immunoassay kit or a chemiluminescent immunoassay kit. More preferably, the kit is a chemiluminescent immunoassay kit.
In a third aspect, the use of biotin or a biotin derivative in an immunoassay system for anti-biotin interference.
In some embodiments, the biotin or biotin derivative is preferably used in immunoassays using a biotin-avidin system.
Preferably, the immunoassay is an enzyme-linked immunoassay, a fluorescent immunoassay, a radioimmunoassay, a colloidal gold immunoassay, or a chemiluminescent immunoassay; more preferably, the immunoassay is a chemiluminescent immunoassay.
In a fourth aspect, a method of anti-biotin interference in an immunoassay system, the method comprising:
when the test substance within the sample is contacted simultaneously with (i) the specific binding substance for capture and (ii) the specific binding substance labeled with the detectable label; pre-adding biotin or a biotin derivative to a solution containing (i) a specific binding substance for capture, and/or (ii) a specific binding substance labeled with a detectable label; or,
when the test substance within the sample is contacted simultaneously with (i) the specific binding substance for capture and (ii) the specific binding substance labeled with the detectable label; pre-adding biotin or a biotin derivative to one or more of (i) a solution containing a specific binding substance for capture, (ii) a solution containing a specific binding substance labeled with a detectable label, and a third solution;
the third solution is a solution to be added together when the analyte in the sample is simultaneously contacted with (i) the specific binding substance for capture and (ii) the specific binding substance labeled with the detectable label.
Specific implementation manners of the method can be exemplified: a kit comprising a solution R1 containing (i) a specific binding substance for capture, a solution R2 containing (ii) a specific binding substance labeled with a detectable label, and a solution R3;
when the sample and the solution R1, the solution R2 and the solution R3 are added into the reaction container simultaneously or in a divided manner, the sample is not washed in the process; that is, biotin or a biotin derivative may be added to one or more of the solutions R1, R2, R3 in advance when the substance to be detected in the sample is simultaneously contacted with (i) the specific binding substance for capturing and (ii) the specific binding substance labeled with the detectable label;
when the sample is mixed with the solution R1 for a certain period of time, washed, and then added with R2 and R3 for the mixing reaction, biotin or a biotin derivative may be added to the solution R2 and/or the solution R3 in advance.
The foregoing examples are for illustrative purposes and are not intended to limit the scope of the invention.
In some embodiments, the immunoassay is an immunoassay using a biotin-avidin system.
In other embodiments, the biotin or biotin derivative is added in an amount such that the final concentration in each solution is 0.3ng/mL to 5 mg/mL.
In the present invention, the effect of the anti-biotin interference can be obtained only when the added amount of biotin or a biotin derivative is 0.3ng/mL to 5mg/mL of the final concentration in each solution.
In other preferred embodiments, the biotin or biotin derivative is added in an amount of 00001% to 1% (v/v) of the volume of each solution.
In another embodiment, the specific binding substance for capture is an avidin or streptavidin coated solid support; the detectable label is an enzyme, a fluorescent dye, a radiolabel, an isotopic label, or a luminescent substance.
The present invention will be explained in detail below.
When an immunoassay reagent analyzes a substance to be detected in a sample, endogenous biotin in the sample tends to cause inaccurate detection results. The present invention surprisingly found that by using biotin or a biotin derivative in an immunoassay reagent, the biotin or biotin derivative can avoid the interference of endogenous biotin during the immunoassay, and particularly when using a biotin-avidin system, the interference of endogenous biotin can be remarkably avoided. The invention therefore also relates to the use of agents that avoid interference with endogenous biotin in assays using the biotin-avidin system. According to the invention, biotin or a biotin derivative is used in soluble form.
The reagent, the kit and the method thereof can resist endogenous biotin interference in a sample and improve the sensitivity of a detection result on the premise of not affecting the accuracy of the detection result, and the capability of resisting the biotin interference of the reagent can be regulated by adding the reagent; the reagent has the advantages of easily available raw materials and low cost; the kit is suitable for industrial production, has good anti-interference effect and high detection sensitivity; the anti-interference method is simple and easy to operate.
Detailed Description
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, 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. In case of conflict, the present specification will control.
Definition of the definition
For the description of numerical ranges herein, each intermediate value with the same degree of precision in the range is expressly contemplated. For example, for ranges 6 to 9, the numbers 7 and 8 are covered in addition to 6 and 9, and for ranges 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly covered.
The term "immunoassay" as used herein is an analysis of a substance to be tested using the principle of specific binding of an antigen and an antibody. Specifically, the immunoassay includes an enzyme-linked immunoassay, a fluorescent immunoassay, and a chemiluminescent immunoassay.
The detection method of the test substance referred to herein includes a double antibody sandwich method, a double antigen sandwich method, and an indirect method.
The term "test substance" used herein includes, for example, antibodies, antigens, nucleic acids, biologically active substances, bacteria, viruses, peptides, therapeutic agents, and the like, but is not particularly limited thereto. Furthermore, antibodies can also become antigens. Examples of the antibody include an antibody against an antigen, and the like, but are not particularly limited. Examples of the antigen include nucleic acids, physiologically active substances, bacteria, viruses, peptides, and the like, but are not particularly limited. Examples of the nucleic acid include, but are not particularly limited to, a nucleic acid encoding a disease-causing gene or the like, a nucleic acid encoding a gene of a bacterium or a virus, and the like. Examples of the physiologically active substance include, but are not particularly limited to, a cell growth factor, a differentiation induction factor, a cell adhesion factor, an enzyme, a cytokine, a hormone, and a sugar chain.
The term "sample" as used herein refers to any composition containing a substance to be detected, the sample being derived from a biological source ("biological sample"), such as tissue (e.g., biopsy sample), extract or culture, biological or physiological fluids, and the like. For example, the sample typically comprises a bodily fluid, such as blood, serum, or plasma, saliva, urine, or other bodily fluid.
The terms "specific binding", "specific binding" herein characterize the interaction between two molecules (e.g. antigen and antibody) that have the ability to react selectively with each other as a pair. The term "specifically binds to … …" refers, for example, to the ability of an antibody to specifically bind to its target antigen, but not to other entities. The term "specific binding" refers to a binding preference (e.g., affinity) for a target molecule/sequence that is at least 2-fold, more preferably at least 5-fold, and most preferably at least 10-fold or 20-fold that of a non-specific target molecule (a randomly generated molecule lacking a specific recognition site). Specific binding herein includes, but is not limited to, antigen-antibody binding, avidin binding.
Solid phase as used herein refers to any material that is insoluble or capable of becoming insoluble by subsequent reactions. The solid phase may be selected for its inherent ability to attract and immobilize the capture agent. Alternatively, the solid phase may be immobilized with a cross-linking agent that has the ability to attract and immobilize the capture agent, which enables indirect collection of the capture agent to the solid phase material. The solid phase may be, for example, plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon, including, but not limited to, for example, test tubes, microtiter wells, plates, beads, microparticles, chips, and other configurations known to those of ordinary skill in the art.
As used herein, "detectable label" includes any composition that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Markers useful in the present invention include magnetic beads (e.g., dynabeads (TM), fluorescent dyes (e.g., fluorescein, texas Red, rhodamine, and Green fluorescent proteins, etc.), see, e.g., molecular Probes, eugene, oreg., USA); chemiluminescent compounds such as acridine (e.g., acridine-9-carboxamide), phenanthridine (phenanthridine), dioxetane (dioxetanes), luminol, and the like; a radiolabel (e.g., 3H, 125I, 35S, 14C, or 32P); catalysts, such as enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and other enzymes commonly used in ELISA); and colorimetric labels such as colloidal gold (e.g., gold particles having a particle size of 40nm to 80nm efficiently scatter green light) or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
In the present specification, avidin includes streptavidin (streptavidin), avidin (avidin) and NeutrAvidin proteins, each of which is capable of binding four biotin molecules with high affinity and specificity. Among them, streptavidin is most commonly used, which is not glycosylated and has a very low level of non-specific binding. Preferably, the biotin-avidin system comprises biotin-avidin system (BAS), biotin-streptavidin system. More preferably, the biotin-avidin system is a biotin-streptavidin system.
Avidin (avidin), an alkaline glycoprotein consisting of 4 identical subunits, having a molecular weight of 68kDa and an isoelectric point pI of 10.5; heat-resistant and resistant to the action of various proteolytic enzymes. While Streptavidin (SA) is a protein with similar biological characteristics to Avidin (AV), is a secretion of streptomyces avidinii bacteria, has molecular weight and biotin binding capacity similar to those of avidin in egg white, has isoelectric point of 6.0, and has non-specific binding far lower than that of avidin.
The present disclosure also provides a test kit for testing a test sample. The kit includes one or more reagents for performing one or more immunoassays according to the present disclosure. Kits typically include a package having one or more containers holding the reagents as one or more separate components, or alternatively, as a mixture in which the reagents are compatible. The kit also includes other materials that may be desirable from a user standpoint, such as buffers, diluents, standards, and/or any other material used in sample processing, washing, or performing any other assay step.
In order that those skilled in the art will better understand the technical solutions of the present application, the present invention will be further described with reference to examples, and it is apparent that the described examples are only some of the examples of the present application, not all the examples. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments herein without making any inventive effort, shall fall within the scope of the present application.
The immunoassay kit herein is preferably exemplified by a small molecule assay kit having a detection molecular weight of less than 1000 Da.
Taking a chemiluminescent detection kit of estradiol as an example, the kit comprises a reagent R1, a reagent R2 and a reagent R3; the reagent R1 comprises a solid phase carrier coated by avidin or streptavidin; reagent R2 comprises a specific binding partner labeled with a detectable label; reagent R3 comprises biotin-labeled estradiol.
The chemiluminescent detection step of the estradiol comprises the following steps: firstly, adding a sample, namely adding biotinylated estradiol (reagent R3) and acridinium ester marked anti-estradiol monoclonal antibody (reagent R2) in a first step, incubating the sample with two components, and releasing the estradiol in the sample by using an estradiol releasing agent so that the estradiol in the sample is competitively combined with the biotinylated estradiol and the acridinium ester marked antibody; in the second step, streptavidin magnetic particles (reagent R1) are added, incubated, and then immune complexes are formed with the streptavidin magnetic particles, and the unbound sample reagent is removed by washing. The luminescent substrate is added and the relative luminescence value (RLU) is detected, wherein the detection device is a michigan i3000 full-automatic chemiluminescence apparatus.
The volume ratio of the sample to the reagent R2 to the reagent R3 is 1:1:1, specifically, after the sample volume is 50 mu L and incubation is carried out for 10min, adding the reagent R1 with the same volume as the sample and further incubating for 10 min; after washing, a luminescent substrate was added and the relative luminescence value (RLU) was detected. More specifically, the process is carried out,
the specific components of the chemiluminescent detection kit for estradiol are shown in the following table:
in the invention, the buffer salt is a phosphate buffer system, a Tris buffer system, a Mes buffer system or a Hepes buffer system; the inorganic salt is sodium chloride; the stabilizer is preferably protein substances and/or saccharide substances, wherein the protein substances are selected from one or more of BSA, casein, bovine serum and sheep serum; the saccharide is selected from mannitol, trehalose, glucose, fructose, lactose, galactose or sucrose; the surfactant is a nonionic surfactant; the preservative is ProClin series preservative, kroen series preservative, kathon or sodium azide.
Experimental materials:
the methods and kits described above were carried out using each of examples 1 to 4 and comparative examples 1 to 2.
The specific formulation of the estradiol assay kit used herein is as follows:
first, a solution A having a concentration of 50ug/mL was prepared using biotin and dimethyl sulfoxide.
1. By addition of biotin
Example 1:
an estradiol assay kit comprising 3 separate but non-intersecting three reagents (R1, R2 and R3). Solution A was added to reagent R1 to give a final biotin concentration of 50ng/mL in the reagent.
Example 2:
an estradiol assay kit comprising 3 separate but non-intersecting three reagents (R1, R2 and R3). Solution A was added to reagent R2 to give a final biotin concentration of 50ng/mL in the reagent.
Example 3:
an estradiol assay kit comprising 3 separate but non-intersecting three reagents (R1, R2 and R3). Solution A was added to reagent R3 to give a final biotin concentration of 50ng/mL in the reagent.
Example 4:
an estradiol assay kit comprising 3 separate but non-intersecting three reagents (R1, R2 and R3). Adding the solution A into the reagent R2 to make the final concentration of biotin in the reagent be 30ng/mL; solution A was added to reagent R3 to give a final biotin concentration of 30ng/mL in the reagent.
Comparative example 1:
an estradiol assay kit comprising 3 separate but non-intersecting three reagents (R1, R2 and R3). Solution A was added to reagent R2 to give a final biotin concentration of 25ng/mL in the reagent.
Comparative example 2:
an estradiol assay kit comprising 3 separate but non-intersecting three reagents (R1, R2 and R3). Solution A was added to reagent R2 to give a final biotin concentration of 100ng/mL in the reagent.
The following experiments were carried out with the kits of examples 1 to 4 and comparative examples 1 to 2, using as a control a kit for measuring estradiol without any addition, the experimental data being the average of the values after three measurements:
1. main calibrator signal value determination experiment
Performing calibration tests of different kits by using the calibrator, and detecting a calibration curve obtained by each kit, wherein the calibration curve is shown in table 1; and calculating the slope of the curve of each calibration point, wherein the method for calculating the slope of the curve is to use the absorbance values of two similar calibration points to carry out division operation, and the method is specifically shown in the table 2:
table 1 shows the signal values of the primary calibrator for the different estradiol measurement kits
Table 2 shows the slope of the main calibrator curve for the different estradiol assay kits
As can be seen from tables 1 and 2, the addition of biotin to any one or more of the reagents in the kit can increase the measured signal value of the main calibrator of the whole kit without changing the curvature of the curve of the main calibrator of the kit, thereby increasing the sensitivity.
Therefore, it is hypothesized that in the detection of small molecule substances, the signal value may be increased due to endogenous biotin in the sample, so that the obtained result is low; after biotin is added to the detection reagent, the signal value of each calibration point is increased by about 1.6 times and then reaches a peak value to a certain height without increasing; the addition of an amount of biotin to the reagent masks the interference of the biotin in the sample with the test result by increasing the signal value of the test result.
2. Quality control product concentration determination experiment
The estradiol measurement values of the composite quality control products 1, 2 and 3 were measured, and then the relative deviation between the detection results of the biotin-added and non-biotin-added estradiol kits was calculated. Specifically, the results are shown in Table 3.
The composite quality control product is a quality control product for various detection items. The composite quality control product is a protein-containing buffer produced by Michael organism, and different quality control products comprise detection substances such as estradiol and cortisol with different concentrations.
Table 3 shows the results of quality control of various estradiol measurement kits
From the detection results of the quality control product, biotin is added into one or more detection reagents in the kit, and the deviation of the detection results is less than 4%, so that the detection results of normal samples are not influenced.
3. Test of anti-interference Capacity of avidin
One serum sample without obvious hemolysis, lipidemia was prepared and divided equally into 4 parts, each 1mL. Biotin was added to the four samples to simulate a clinical sample (blood sample) containing endogenous biotin, so that the concentrations of endogenous biotin in each simulated sample were 0ng/mL, 10ng/mL, 20ng/mL and 30ng/mL, respectively. The four simulated blood samples were then tested for estradiol concentration using different estradiol assay kits, the specific test results being shown in table 4.
Table 4 shows the results of estradiol measurements from different estradiol assay kits on clinical mock samples
When the endogenous biotin content in the clinical simulation sample is 0ng/mL, biotin is added into one or more detection reagents in the kit, and the concentration of detected substances obtained by detection is slightly increased, but the overall data deviation is within 10%, so that the detection result of the clinical sample is not considered to be influenced.
When the endogenous biotin content in the simulated sample is increased, the biotin is added into one or more detection reagents in the kit, so that the interference of the endogenous biotin on the detection result can be effectively eliminated. And as can be seen from comparative example 1 and comparative example 2, in the test kit of estradiol, when the added solution a is too small or too large, the effect is not optimal although the effect of eliminating interference may be exhibited. Therefore, the addition amount of biotin or biotin derivatives needs to be adjusted according to the biological characteristics, reaction principles, etc. of the items to obtain the best anti-interference effect on the specific detection items.
The following uses a cortisol determination kit as an example, and specific components of the cortisol determination kit are as follows:
the material of the cortisol is as follows:
the detection principle of the cortisol kit is a magnetic particle chemiluminescence detection method, and the detection method specifically comprises the following steps: firstly, adding a sample, firstly adding biotinylated cortisol (reagent R3) and acridinium ester marked anti-cortisol monoclonal antibody (reagent R2), incubating the sample with two components, and releasing cortisol in the sample by a cortisol releasing agent so that the cortisol in the sample is competitively combined with the biotinylated cortisol and the acridinium ester marked antibody; in the second step, streptavidin magnetic particles (reagent 1) are added, incubated, and then immune complexes are formed with the streptavidin magnetic particles, and the unbound sample reagent is removed by washing. The luminescent substrate is added and the relative luminescence value (RLU) is detected, wherein the detection device is a michigan i3000 full-automatic chemiluminescence apparatus.
The volume ratio of the sample to the reagent R2 to the reagent R3 is 1:1:1, specifically, after the sample volume is 50 mu L and incubation is carried out for 10min, adding the reagent R1 with the same volume as the sample and further incubating for 10 min; after washing, a luminescent substrate was added and the relative luminescence value (RLU) was detected.
The above method and kit used in each of example 5 and comparative examples 3 to 4 were carried out.
Cortisol as used herein is specifically formulated as follows:
first, a solution B having a concentration of 100. Mu.g/mL was prepared using biotin and dimethyl sulfoxide.
Example 5
A cortisol immunoassay kit for a biotin-streptavidin system comprising three reagents (R1, R2 and R3) of 3 that are independent and do not cross. Solution B was added to reagent R2 to give a final biotin concentration of 100ng/mL in reagent R2.
Comparative example 3
A cortisol assay kit for a biotin-streptavidin system comprising 3 separate but non-intersecting three reagents (R1, R2 and R3). Solution B was added to reagent R1 to give a final biotin concentration of 70ng/mL in reagent R1.
Comparative example 4
A cortisol assay kit for a biotin-streptavidin system comprising 3 separate but non-intersecting three reagents (R1, R2 and R3). Solution B was added to reagent R1 to give a final biotin concentration of 150ng/mL in reagent R1.
The following experiments were performed with example 5 and comparative examples 3 and 4, using the cortisol assay kit without any addition as a control.
1. Main calibrator signal value determination influence experiment
Performing calibration tests of different kits by using the calibrator, and detecting a calibration curve obtained by each kit, wherein the calibration curve is shown in Table 5; and calculating the slope of the curve of each calibration point, wherein the method for calculating the slope of the curve is to use the absorbance values of two similar calibration points to carry out division operation, and the method is specifically shown in table 6:
table 5 shows the signal values of the primary calibrator for the different cortisol determination kits
Table 6 shows the slope of the main calibrator curve for the different cortisol determination kits
As can be seen from tables 5 and 6, the addition of biotin to the various reagents in the cortisol assay kit can increase the assay signal value of the main calibrator of the kit, thereby increasing the sensitivity.
2. Quality control product concentration determination experiment
The cortisol measurement values for composite quality control 1, 2, 3 (which are the same as described above) were measured separately, and then the relative deviation of the measurement results for the biotin-added versus biotin-free cortisol kit was calculated. Specifically, the results are shown in Table 7.
Table 7 shows the results of quality control product measurements for different cortisol measurement kits
As can be seen from Table 7, the quality control test results of biotin added to different reagents in the cortisol test kit were not more than.+ -. 5% compared with those of the control kit not added, and thus it was considered that the measurement results of normal samples were not affected.
3. Test of anti-interference Capacity of avidin
One serum sample without obvious hemolysis, lipidemia was prepared and divided equally into 4 parts, each 1mL. Biotin was added to the four samples to simulate a clinical sample (blood sample) containing endogenous biotin, so that the concentrations of endogenous biotin in each simulated sample were 0ng/mL, 10ng/mL, 20ng/mL and 30ng/mL, respectively. The four simulated blood samples were then tested for cortisol concentration using different cortisol assay kits, the specific test results being shown in table 8.
Table 8 shows the results of cortisol detection on clinical simulation samples using different cortisol determination kits
As can be seen from Table 8, the addition of biotin to different reagents in the cortisol assay kit can effectively eliminate the interference of biotin in the sample to the detection result, and the addition of too much or too little biotin can not achieve the optimal interference elimination effect. The corresponding detection results of the conjugated estradiol kit can be further confirmed. The concentration of biotin to be added is appropriately adjusted according to the biological diagnosis and reaction principle of each item.
It is to be understood that this invention is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also encompassed by the appended claims.
Claims (3)
1. A method of anti-biotin interference in an immunoassay system, comprising:
contacting the test substance in the sample with a ligand labeled with a detectable label that specifically binds to the test substance prior to contacting the test substance with the avidin or streptavidin coated solid support; wherein biotin or a biotin derivative is added in advance to one or more of a solution containing an avidin-or streptavidin-coated solid carrier, a solution containing a ligand labeled with a detectable label that specifically binds to a substance to be detected, and a third solution;
the third solution is a solution which is added together when the detected substance in the sample is contacted with the solid phase carrier coated by avidin or streptavidin and the ligand which is marked by the detectable marker and specifically binds to the detected substance;
the biotin or the biotin derivative is added according to the final concentration of the biotin or the biotin derivative in each solution of 0.3 ng/mL-5 mg/mL.
2. The method of anti-biotin interference in an immunoassay system according to claim 1, wherein: the immunoassay is an immunoassay using a biotin-avidin system.
3. The method of anti-biotin interference in an immunoassay system according to claim 1, wherein: the detectable label is an enzyme, a radioactive label, an isotopic label, or a luminescent substance.
Priority Applications (1)
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