CN111505273A - Non-competitive chemiluminescence immunoassay kit for digoxin and application thereof - Google Patents
Non-competitive chemiluminescence immunoassay kit for digoxin and application thereof Download PDFInfo
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- CN111505273A CN111505273A CN202010315792.7A CN202010315792A CN111505273A CN 111505273 A CN111505273 A CN 111505273A CN 202010315792 A CN202010315792 A CN 202010315792A CN 111505273 A CN111505273 A CN 111505273A
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- 229960005156 digoxin Drugs 0.000 title claims abstract description 85
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- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 title claims abstract description 56
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- XYIPYISRNJUPBA-UHFFFAOYSA-N [3-(3'-methoxyspiro[adamantane-2,4'-dioxetane]-3'-yl)phenyl] dihydrogen phosphate Chemical compound O1OC2(C3CC4CC(C3)CC2C4)C1(OC)C1=CC=CC(OP(O)(O)=O)=C1 XYIPYISRNJUPBA-UHFFFAOYSA-N 0.000 description 1
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- Urology & Nephrology (AREA)
- Analytical Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention provides a digoxin non-competitive chemiluminescence immunoassay kit and application thereof.A constructed detection system comprises a coating antibody and a detection antibody; the coating antibody is a monoclonal antibody of an anti-small molecule compound; the detection antibody is an anti-immune complex antibody; the immune complex is formed from a small molecule compound and a monoclonal antibody to the small molecule compound. The invention takes the digoxin monoclonal antibody as a coating antibody, combines with digoxin in a sample to form a digoxin-anti-digoxin monoclonal antibody immune complex, and then takes the anti-immune complex antibody as a detection antibody to construct a chemiluminescence detection system, thereby realizing non-competitive detection of small molecular compounds and remarkably improving the detection sensitivity, accuracy and linear range.
Description
Technical Field
The invention belongs to the technical field of immunodetection, relates to a non-competitive detection kit for a small molecular compound and application thereof, and particularly relates to a non-competitive chemiluminescent immunoassay kit for digoxin and application thereof.
Background
Digoxin (Digoxin) is a bioactive substance extracted from Digitalis purpurea (Digitalis), and is a steroid glycoside with cardiotonic effect. The origin of digitalis preparation as a cardiotonic goes back to the 18 th century, and until now, the position of digitalis preparation as a heart failure (HFrEF) treatment accompanied by a decline in left ventricular ejection fraction stands out and maintains a high recommended level. Digitalis is one of the most effective therapeutic drugs, particularly in cases with tachycardia-associated upper ventricular heart rate disorders. But digoxin is easy to cause digitalis poisoning (vomiting, dizziness, visual disturbance, and atrioventricular block and ventricular arrhythmia in severe cases) due to the narrow therapeutic concentration range of digoxin; in addition, the individual difference is large, the incidence of poisoning is high, digitalis has become one of the most difficult drugs to be mastered in clinic, and is listed as a TDM (therapeutic Drug monitoring) Drug.
The mechanism of action of digoxin is to inhibit Na+、K+ATPase, and therefore in the case of a concomitant hypokalemia, is liable to cause intoxication; in the course of treatment, in order to improve the conditions of fluid retention, edema and the like caused by severe cardiac insufficiency, diuretics are often used in combination, and the side effect is that hypokalemia is easy to occur. Therefore, it is very important to detect the in vivo concentration of the homoxins. Even when the amount of digoxin to be administered is determined, it is recommended that the blood concentration of digoxin be measured in real time as the drug combination varies.
Until 12 months 2019, kits for measuring blood levels of digoxin based on chemiluminescence enzyme immunoassay (chemiluminescence enzyme immunoassay, C L EIA) all employ a competition method because digoxin is a small molecular compound, is small in size and contains only one epitope, and is physically limited so that it does not support the conventional sandwich method in principle, in the competition method, the amounts of labeled hapten and antibody are limited, an analyte competes with the labeled hapten for binding a small amount of solid-phase antibody, after the reaction is balanced, bound and free hapten is separated, and finally the signal intensity is read in inverse proportion to the content of the analyte in a sample, for example, CN110618261A discloses a chemiluminescence immunoassay kit for quantitatively measuring digoxin, which includes reagents R1, R2 and R3, wherein reagent R1 is a suspension containing streptavidin magnetic particles, reagent R5 is a monoclonal antibody containing a chemiluminescent labeled digoxin label, and reagent R64 is a monoclonal antibody containing a monoclonal antibody, and the principle of detecting digoxin is a competitive solution using a competitive method.
However, the sensitivity of competitive immunoassays is largely limited by the affinity constant of antibodies, which is typically no more than 10 for monoclonal antibodies to small molecule compounds12In addition, low-concentration reaction signals are difficult to distinguish from zero, so that the relative error of a zero dose point is large, the analysis process needs repeated detection, and the reaction incubation time is too long, so that the competitive immunoassay method is inferior to non-competitive immunoassay in the aspects of sensitivity, precision, dynamics, detection linear range and the like.
Disclosure of Invention
Aiming at the defects and practical requirements of the prior art, the invention provides a digoxin non-competitive chemiluminescence immunoassay kit and application thereof, wherein the kit takes an immune complex of digoxin and an anti-digoxin monoclonal antibody as a detection object, and detects the content of the immune complex by using a sandwich method, thereby indirectly realizing non-competitive sensitive and specific detection of digoxin.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a non-competitive assay system for a small molecule compound, the system comprising a coating antibody and a detection antibody;
the coating antibody is a monoclonal antibody of an anti-small molecule compound;
the detection antibody is an anti-immune complex antibody;
the immune complex is formed from a small molecule compound and a monoclonal antibody to the small molecule compound.
In the invention, the monoclonal antibody of the anti-small molecular compound is used as a coating antibody and is combined with the small molecular compound to form a small molecular compound-anti-small molecular compound monoclonal antibody immune complex, and then the anti-immune complex antibody is used as a detection antibody to construct a detection system, so that the non-competitive detection of the small molecular compound is realized.
Preferably, the coated antibody is coated directly and/or indirectly on a solid support.
Preferably, the small molecule compound comprises any one of digoxin, thyroxine, triiodothyronine, estradiol, progesterone or cortisol.
In a second aspect, the present invention provides a non-competitive chemiluminescent immunoassay kit for digoxin, the kit comprising a detection antibody;
the detection antibody is an anti-immune complex antibody;
the immune complex is formed from digoxin and an anti-digoxin monoclonal antibody.
Preferably, the anti-immune complex antibody is modified with a labeling group.
In the invention, common labeling groups such as chemiluminescence labeling molecules, fluorescence labeling molecules, biotin, streptavidin and the like are modified on the anti-immune complex antibody, so that the application range of the antibody can be expanded, and the antibody can be applied to the field of digoxin detection.
Preferably, the anti-immune complex antibody is modified with alkaline phosphatase.
In the invention, the alkaline phosphatase modified antibody can be used as a detection antibody in the chemiluminescence-based digoxin detection.
Preferably, the kit further comprises a coating antibody.
Preferably, the coating antibody comprises an anti-digoxin monoclonal antibody.
In the invention, the digoxin monoclonal antibody is used as a coating antibody and combined with digoxin in a sample to form a digoxin-anti-digoxin monoclonal antibody immune complex, and then the anti-immune complex antibody is used as a detection antibody to construct a chemiluminescence detection system, so that the non-competitive detection of small molecular compounds is realized, and the detection sensitivity, specificity and accuracy are obviously improved.
Preferably, the coated antibody is coated directly and/or indirectly on a solid support.
Preferably, the solid phase carrier comprises any one of a cell plate, a magnetic particle, an affinity membrane or a liquid phase chip or a combination of at least two of them, preferably a magnetic particle.
Preferably, the magnetic particles are streptavidin magnetic particles.
Preferably, the anti-digoxin monoclonal antibody is modified with biotin.
In a third aspect, the present invention provides a non-competitive chemiluminescent immunoassay system for digoxin, said system comprising an anti-immune complex antibody;
the immune complex is formed by digoxin and an anti-digoxin monoclonal antibody;
the anti-immune complex antibody is modified with alkaline phosphatase.
Preferably, the concentration of the anti-immune complex antibody is 1-10 ng/m L, for example, 1ng/m L, 2ng/m L0, 3ng/m L1, 4ng/m L, 5ng/m L, 6ng/m L, 7ng/m L, 8ng/m L, 9ng/m L or 10ng/m L, preferably 10ng/m L.
Preferably, the system further comprises a biotinylated anti-digoxin monoclonal antibody.
Preferably, the concentration of the biotinylated anti-digoxin monoclonal antibody is 1-10 ng/m L, and can be, for example, 1ng/m L, 2ng/m L0, 3ng/m L1, 4ng/m L, 5ng/m L, 6ng/m L, 7ng/m L, 8ng/m L, 9ng/m L or 10ng/m L, preferably 10ng/m L.
Preferably, the system further comprises streptavidin magnetic particles.
Preferably, the concentration of the streptavidin magnetic particles is 0.01% to 0.1%, and may be, for example, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%.
In a fourth aspect, the present invention provides a noncompetitive chemiluminescent immunoassay method for digoxin, comprising the steps of:
(1) mixing a sample to be detected with streptavidin magnetic particles and a biotinylated anti-digoxin monoclonal antibody for incubation;
(2) adding alkaline phosphatase modified anti-immune complex antibody, mixing and incubating, and magnetically separating to remove supernatant;
(3) the substrate solution was added and the chemiluminescence intensity was measured.
Preferably, the incubation temperature in step (1) and step (2) is 20-40 ℃, for example, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃ or 40 ℃, preferably 20-25 ℃.
Preferably, the incubation time in step (1) and step (2) is 1-10 min, for example, 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min or 10min, preferably 3-5 min.
In a fifth aspect, the invention provides a kit of the second aspect or a system of the third aspect for use in preparing a digoxin detection reagent and/or a drug for detection.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention aims to solve the problems of low sensitivity and poor accuracy of the traditional competitive detection method of the small molecular compound, and constructs the detection method of the sandwich method, wherein the method takes an immune compound formed by digoxin and an anti-digoxin monoclonal antibody as a detection object, and takes an anti-immune compound antibody as a detection antibody, thereby indirectly realizing the sensitive and specific detection of the small molecular compound;
(2) the invention takes the digoxin monoclonal antibody as a coating antibody, combines with digoxin in a sample to form a digoxin-anti-digoxin monoclonal antibody immune complex, and then takes the anti-immune complex antibody as a detection antibody to construct a chemiluminescence detection system, thereby realizing non-competitive detection of small molecular compounds and remarkably improving the detection sensitivity, specificity and accuracy;
(3) the digoxin non-competitive chemiluminescence immunoassay system and the detection kit constructed by the invention have high sensitivity, the lowest detection limit reaches 0.07ng/m L, the accuracy is high, the linear range reaches 0.1-5.0 ng/m L, the repeatability is good, and the digoxin non-competitive chemiluminescence immunoassay system and the detection kit have wide application prospects and great market values in the field of detection of small molecular compounds.
Detailed Description
To further illustrate the technical means and effects of the present invention, the present invention is further described with reference to the following examples. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Example 1 preparation of digoxin anti-immune Complex antibody
This example refers to the method of patent JP2793587, which takes immune complex formed by digoxin and anti-digoxin monoclonal antibody as immunogen to prepare anti-immune complex antibody, and the steps are as follows:
carrying out in vitro immune reaction on digoxin and a mouse anti-digoxin monoclonal antibody in advance, and taking an immune complex formed by combining the digoxin and the mouse anti-digoxin monoclonal antibody as an antigen to immunize the mouse; after 15 days, 35 days and 97 days of the initial immunization, additional immunization was carried out, and 3 days after the final immunization, hybridomas having anti-monoclonal antibody production ability were established according to a conventional method;
performing binding force screening on the monoclonal antibody produced by the hybridoma, and selecting an antibody which is reactive to the immune complex and non-reactive to digoxin;
the screened anti-immune complex antibody was purified by protein A column, Fab' treated, labeled with SMCC-conjugated alkaline phosphatase, and then prepared to a concentration of 10ng/m L containing 25mM PBS, 0.1% Proclin300, and 0.1% ZnCl2、0.01％MgCl2And an alkaline phosphatase-labeled anti-immune complex antibody working solution with a pH of 6.8.
Example 2 preparation of biotinylated anti-digoxin monoclonal antibodies
Dissolving biotin-NHS in DMSO, adding into the anti-digoxin monoclonal antibody according to the molar ratio of 1:20, fully mixing uniformly, reacting at room temperature for 1.8h, and dialyzing to obtain a biological anti-digoxin monoclonal antibody;
the working solution of the biochemical anti-digoxin monoclonal antibody was diluted to 10ng/m L with a buffer containing 25mM PBS, 0.1% BSA, 0.1% Proclin300 at pH 6.8.
Example 3 kit Assembly
Taking 1m L streptavidin magnetic particles (the particle size is 3 μm) with the concentration of 100mg/m L, adding into 30m L buffer solution containing 25mMPBS and 0.1% Proclin300 and having the pH value of 6.8, fully mixing uniformly, placing on a magnetic separator for magnetic separation, repeating for 3 times until the supernatant is not turbid, removing the supernatant, and keeping the magnetic particles;
adding the magnetic particles into 25mM PBS, 0.1% BSA, and 0.1% Proclin300 pH 6.8 magnetic particle buffer solution to prepare 0.1% magnetic particle working solution, which is marked as reagent R1;
the working solution of the biotinylated anti-digoxin monoclonal antibody prepared in example 2 was recorded as reagent R2;
the alkaline phosphatase anti-immune complex antibody working solution prepared in example 1 was designated as reagent R3;
the reagents R1, R2 and R3 are combined to form a digoxin non-competitive chemiluminescence immunoassay kit which is used for digoxin chemiluminescence immunoassay.
EXAMPLE 4 evaluation of the Performance of the kit
In this embodiment, an AI-3000 full-automatic chemiluminescence immunoassay analyzer manufactured by blue-yi science and technology group ltd is used as a detection instrument, and digoxin in a sample is detected according to a delayed one-step sandwich method, which comprises the following steps:
incubating a 25 mu L sample with 50 mu L reagent R1 and 50 mu L reagent R2 in the instrument for 4min to form magnetic particle-anti-digoxin monoclonal antibody-digoxin complexes, wherein the molar quantity of the complexes depends on the concentration of digoxin in the sample, continuously adding 50 mu L reagent R3, performing magnetic separation after incubating for 4min, adding alkaline phosphatase substrate AMPPD, and measuring chemiluminescence value to indicate the concentration of digoxin in the sample.
(1) Minimum detection limit
The assay was repeated 20 times using the calibrator (level 1) and the mean calculated
And standard deviation SD according to
Obtaining R L U value, and according to calibration curve equation of calibrator
The corresponding R L U value was substituted into the above equation to find the corresponding concentration value as the lowest detection limit of the kit.
The results are shown in Table 1, with the lowest detection limit of the kit being 0.07ng/m L.
TABLE 1
(2) Accuracy of
Adding high-value sample A with known concentration into serum matrix B (concentration is close to detection limit), repeatedly detecting for 3 times at volume ratio of 1:9, and calculating recovery rate according to formula (1), wherein R is recovery rate, and C is liquid BAverage value of the detected concentration after adding the solution A, V0Volume of liquid B, volume of liquid A and volume of liquid C0Is the average value of the concentration of the solution B, CsThe concentration of the solution A is shown.
As shown in Table 2, the recovery rate was 92%, and the recovery rate was 85% to 115%.
TABLE 2
(3) Linear range
Mixing a high-value sample close to the upper limit of a linear interval and a low-value sample close to the lower limit of the linear interval into 5 gradients according to a certain proportion, repeatedly testing the sample of each concentration gradient for 2 times, calculating the average value of the samples, performing straight line fitting on the average value of the measured concentration and the theoretical concentration by using a least square method to obtain a linear regression equation, and calculating a linear correlation coefficient r.
As shown in Table 3, the linear correlation coefficient r was not less than 0.990 and was 1.000 in the linear range of 0.1 to 5.0ng/m L.
TABLE 3
(4) Repeatability of
Repeating the detection 10 times by using precision reference substances P1 and P2, calculating the average value (M) and the Standard Deviation (SD), and obtaining the Coefficient of Variation (CV) according to the formula (2);
CV＝SD/M×100％ (2)
as shown in Table 4, the coefficient of variation was 3.04%, which meets the requirement that the coefficient of variation is not more than 4.00%.
TABLE 4
(5) Difference between batches
The precision reference products P1 and P2 were measured 10 times with 3 different lot number kits, respectively, and the mean value (M) and Standard Deviation (SD) of the 30 measurement results were calculated to obtain the Coefficient of Variation (CV) according to the formula (2).
The results are shown in Table 5, wherein the coefficient of variation of P1 is 2.11%, and the coefficient of variation of P2 is 4.06%, which meets the requirement that the Coefficient of Variation (CV) is less than or equal to 5.00%.
TABLE 5
In conclusion, the digoxin monoclonal antibody is used as a coating antibody, combined with digoxin in a sample to form a digoxin-anti-digoxin monoclonal antibody immune complex, and then the anti-immune complex antibody is used as a detection antibody to construct a chemiluminescence detection system, so that the non-competitive detection of small molecular compounds is realized, the detection sensitivity, specificity and accuracy are remarkably improved, and the method has a wide application prospect and a huge market value.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (10)
1. A non-competitive assay system for a small molecule compound, said system comprising a coating antibody and a detection antibody;
the coating antibody is a monoclonal antibody of an anti-small molecule compound;
the detection antibody is an anti-immune complex antibody;
the immune complex is formed from a small molecule compound and a monoclonal antibody to the small molecule compound.
2. The system of claim 1, wherein the coated antibody is coated directly and/or indirectly on a solid support;
preferably, the small molecule compound comprises any one of digoxin, thyroxine, triiodothyronine, estradiol, progesterone or cortisol.
3. A non-competitive chemiluminescent immunoassay kit for digoxin, wherein the kit comprises a detection antibody;
the detection antibody is an anti-immune complex antibody;
the immune complex is formed by digoxin and an anti-digoxin monoclonal antibody;
preferably, the anti-immune complex antibody is modified with a labeling group;
preferably, the anti-immune complex antibody is modified with alkaline phosphatase.
4. The kit of claim 3, further comprising a coating antibody;
preferably, the coating antibody comprises an anti-digoxin monoclonal antibody;
preferably, the coated antibody is coated directly and/or indirectly on a solid support;
preferably, the solid phase carrier comprises any one of a cell plate, a magnetic particle, an affinity membrane or a liquid phase chip or a combination of at least two of the above, preferably a magnetic particle;
preferably, the magnetic particles are streptavidin magnetic particles.
5. The kit according to claim 3 or 4, wherein the anti-digoxin monoclonal antibody is modified with biotin.
6. A non-competitive chemiluminescent immunoassay system for digoxin, wherein the system comprises an anti-immune complex antibody;
the immune complex is formed by digoxin and an anti-digoxin monoclonal antibody;
the anti-immune complex antibody is modified with alkaline phosphatase;
preferably, the concentration of the anti-immune complex antibody is 1-10 ng/m L.
7. The system of claim 6, further comprising a biotinylated anti-digoxin monoclonal antibody;
preferably, the concentration of the biotinylated anti-digoxin monoclonal antibody is 1-10 ng/m L.
8. The system of claim 6 or 7, further comprising streptavidin magnetic particles;
preferably, the concentration of the streptavidin magnetic particles is 0.01% to 0.1%.
9. A noncompetitive chemiluminescent immunoassay for digoxin, comprising the steps of:
(1) mixing a sample to be detected with streptavidin magnetic particles and a biotinylated anti-digoxin monoclonal antibody for incubation;
(2) adding alkaline phosphatase modified anti-immune complex antibody, mixing and incubating, and magnetically separating to remove supernatant;
(3) adding a substrate solution, and measuring the chemiluminescence intensity;
preferably, the incubation temperature of the step (1) and the step (2) is 20-40 ℃;
preferably, the incubation time of the step (1) and the step (2) is 1-10 min.
10. Use of a kit according to any one of claims 3 to 5 or a system according to any one of claims 6 to 8 for the preparation of a digoxin detection reagent and/or a drug for detection.
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