CN112684166A - Magnetic particle chemiluminescence detection kit for determining content of human gastrin G17 - Google Patents
Magnetic particle chemiluminescence detection kit for determining content of human gastrin G17 Download PDFInfo
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Abstract
The invention relates to a magnetic particle chemiluminescence detection kit for determining the content of human gastrin G17, which comprises: magnetic separation reagent, R1 reagent, R2 reagent, calibrator liquid series and chemiluminescence substrate liquid; the R2 reagent is alkaline phosphatase-labeled gastrin G17 monoclonal antibody diluent, the chemiluminescence substrate solution is alkaline phosphatase-catalyzed luminescence substrate solution, the R1 reagent is sample diluent, and the calibrator liquid is diluent containing gastrin G17 antigens with different concentrations; the method is characterized in that the magnetic separation reagent is immunomagnetic beads coated with a gastrin G17 monoclonal antibody. The invention greatly improves the signal intensity and sensitivity of immunoreaction, enables low-content substances to generate strong chemiluminescent signals when carrying out immune combination, and provides a more accurate, precise, convenient, rapid and simple method for detecting the gastrin G17.
Description
Technical Field
The invention relates to the field of medical diagnostic reagents, in particular to a magnetic particle chemiluminescence detection kit for detecting the content of human gastrin G17 by adopting a magnetic particle chemiluminescence technology and an antigen-antibody combination technology.
Background
China is a countless "stomach illness kingdom" worldwide, peptic ulcer, chronic gastritis, gastric cancer are common stomach diseases, gastric cancer is the most common digestive tract tumor, and is the main cause of cancer-related death. China is a high-incidence country of stomach cancer and accounts for 1/2 of the number of stomach cancers in the world. Related documents report that the five-year survival rate of gastric cancer at different periods greatly differs after endoscopic minimally invasive therapy or surgical therapy. Therefore, early detection and early treatment are important channels for improving the survival quality of gastric cancer patients.
Gastrin is a gastrointestinal hormone secreted mainly by G cells of the antrum and duodenum and plays an important role in regulating the function of the digestive tract and maintaining its structural integrity. Gastrin molecules currently found include various types, including progastrin, glycine-extended gastrin, and amidated gastrin. Wherein, more than 95 percent of the bioactive gastrin of human bodies is alpha-amidated gastrin which mainly comprises two isomers G-17 and G-34, wherein 80 to 90 percent of the isomers are 6 to 17.
Gastrin 17, a brand new index of the stomach applied to clinical medicine, has received attention from many experts both at home and abroad. Not only does the assistant diagnosis of gastric mucosa diseases by combining gastrin 17 and pepsinogen write the consensus opinion of Chinese early gastric cancer screening and endoscopic diagnosis and treatment in China, but also the project is regarded as a scientific research subject by the national ministry of science and technology and the Chinese health promotion foundation. It has been listed in the stomach cancer prevention and treatment program in japan, korea, finland.
Gastrin 17 is a gastrointestinal hormone secreted by G cells of the mucosa near the antrum and duodenum. The physiological action of G-17 in human body is mainly to stimulate the secretion of gastric acid and pepsin, promote the growth of mucous membrane and regulate the function of gastrointestinal tract, thus having very important function for maintaining the normal function of gastrointestinal tract. G-17 levels vary differently under different pathological conditions. When atrophy of gastric mucosa occurs, the number of G cells in the gastric antrum is reduced due to the loss of the gland of the gastric antrum, and the level of G-17 entering the blood circulation is reduced; gastric atrophy results in decreased gastric acid secretion, decreased inhibition of gastric antral G cells, and increased serum G-17 secretion. Serum G-17 was therefore considered to be a serological marker of antral atrophy.
The magnetic particle chemiluminescence method is a new development of immunoassay technology, and the technology applies the magnetic microspheres to a solid phase of immunoassay, thereby greatly improving the surface area of reaction, increasing the adsorption quantity of antigen or antibody, accelerating the reaction speed and improving the sensitivity of detection. Compared with time-based immune-resolved fluorescence, immunochromatography and ELI SA methods, the method has the characteristics of high accuracy, good specificity, high precision, wide linear range, good reagent stability and the like; the magnetic particle chemiluminescence immunoassay technology is the most widely used analysis method in the immunodiagnosis field all over the world at present and is one of the important development directions of immunological reagents. The prior art detection methods of gastrin 17 mainly comprise an enzyme-linked immunosorbent assay, a time-resolved fluorescence immunoassay, a chemiluminescence method and the like, but the methods have the defects of narrow linear range, long detection time and the like, and cannot meet the clinical requirements.
Therefore, a gastrin G17 detection kit and a use method thereof, which have the advantages of small pollution, high sensitivity, simple operation, high automation degree, good specificity and low cost, are needed at present.
Disclosure of Invention
The invention aims to develop a magnetic particle chemiluminescence detection kit for determining the content of human gastrin G17, which has the advantages of high sensitivity, no pollution, simple operation, good specificity and low cost.
In view of the above, the present invention provides a magnetic particle chemiluminescence assay kit for determining the content of human gastrin G17, comprising: magnetic separation reagent, R1 reagent, R2 reagent, calibrator liquid series and chemiluminescence substrate liquid; the magnetic separation reagent comprises immunomagnetic beads coated with a gastrin G17 monoclonal antibody, the R1 reagent is a sample diluent, the R2 reagent is an alkaline phosphatase-labeled gastrin G17 monoclonal antibody diluent, the calibrator liquid is a diluent containing gastrin G17 antigens with different concentrations, and the chemiluminescent substrate liquid is a substrate liquid for catalyzing and emitting light by alkaline phosphatase.
The immunomagnetic bead coating method for the coated gastrin G17 monoclonal antibody comprises the following steps:
(1) washing the magnetic beads: taking 100mg/mL magnetic beads 100uL, adding 1mL of 0.1M MES buffer solution, mixing uniformly for 5 minutes, carrying out magnetic separation, removing supernatant, and repeatedly washing for 3 times;
(2) and (3) activation: taking the washed magnetic beads, adding 50ul of EDC solution (20mg/mL, used within 5 minutes after being prepared by using refrigerated MES buffer solution) and 50ul of NHS solution (20mg/mL, used within 5 minutes after being prepared by using refrigerated MES buffer solution), fully mixing, and carrying out oscillation reaction at room temperature for 2 hours;
(3) and (3) washing after activation: after the reaction is finished, carrying out magnetic separation, removing supernatant, and carrying out heavy suspension washing for 3 times by using MES solution;
(4) coating: adding the gastrin G17 monoclonal antibody diluted by 0.05M boric acid buffer solution into the washed magnetic beads, fully and uniformly mixing, and performing shaking table incubation at 37 ℃ for 18 h;
(5) and (3) sealing: magnetically separating the incubated magnetic beads, removing supernatant, adding 1mL of confining liquid, uniformly mixing for 5 minutes, magnetically separating, removing supernatant, and repeatedly washing for 3 times to obtain immunomagnetic beads coated with the gastrin G17 monoclonal antibody, wherein the concentration is 10 mg/mL;
(6) and (3) volume fixing: finally, the confining liquid is used for fixing the volume to 10mL, the concentration is 1mg/mL, and the solution is placed at the temperature of 2-8 ℃ for balancing for 24 hours to obtain the magnetic separation reagent coated by the gastrin G17 monoclonal antibody;
the pH value of the confining liquid is 7.0-8.0, and the confining liquid comprises: PBS, concentration 0.02M; magnesium chloride solution with molar concentration of 1M; zinc chloride at a molar concentration of 0.1M; bovine serum albumin with the concentration of 10.0-50.0 g/L; sucrose: the concentration is 20.0-100.0 g/L; proclin300 with the concentration of 1.05-4.20 g/L; BRO (Bronidox) with the concentration of 1.05-4.20 g/L; the rest components are deionized water.
The sealing liquid also comprises glycerol with the concentration of 10.0-20.0 g/L.
Preferably, the blocking solution has a pH of 7.4 and comprises: PBS, concentration 0.02M; magnesium chloride solution with molar concentration of 1M; zinc chloride at a molar concentration of 0.1M; bovine serum albumin with a concentration of 10.0 g/L; sucrose: the concentration is 100.0 g/L; glycerol with the concentration of 20.0 g/L; proclin300 with the concentration of 2.1 g/L; bronidox with concentration of 2.1 g/L; the rest components are deionized water.
In another scheme, the R1 reagent is a sample diluent and comprises a buffer solution and a blocking agent, wherein the pH value of the buffer solution is 5.8-8.5, and the concentration of the blocking agent is 50-200 ug/mL; the buffer solution comprises: tris with the concentration of 5.98-6.52 g/L; proclin-300 with the concentration of 0.98-1.99 g/L; sodium chloride with the concentration of 8.5-9.5 g/L; bovine serum albumin with the concentration of 10-30 g/L; EDTA-2Na with the concentration of 1-10 g/L; the balance of deionized water.
In another embodiment, the R1 reagent is a sample diluent, the pH value of the buffer solution is 7.4, and the buffer solution comprises Tris with the concentration of 6.05 g/L; proclin-300 with the concentration of 1.99 g/L; sodium chloride with the concentration of 9 g/L; bovine serum albumin with a concentration of 10 g/L; EDTA-2Na with the concentration of 2 g/L; the concentration of the blocking agent is 100 ug/mL; the balance of deionized water.
In another embodiment, the R2 reagent, namely the alkaline phosphatase-labeled gastrin G17 monoclonal antibody diluent is prepared by uniformly mixing the SMCC-activated alkaline phosphatase and the 2-iminosulfane hydrochloride-activated gastrin G17 monoclonal antibody in a molar ratio to perform a coupling reaction, then balancing with a Tris buffer solution, and performing separation and purification of fragments with different molecular sizes by using a gel column to obtain the alkaline phosphatase-labeled gastrin G17 labeled antibody.
In another scheme, the R2 reagent, namely the alkaline phosphatase-labeled gastrin G17 monoclonal antibody diluent is prepared by uniformly mixing the SMCC-activated alkaline phosphatase and the 2-iminosulfane hydrochloride-activated gastrin G17 monoclonal antibody according to a molar ratio, carrying out coupling reaction, balancing by using a Tris buffer solution, carrying out separation and purification on fragments with different molecular sizes by using a gel column, and preparing the alkaline phosphatase-labeled gastrin G17 labeled antibody, wherein the alkaline phosphatase-labeled gastrin G17 monoclonal antibody with the concentration of 0.3-0.6 mug/mL is diluted by the buffer solution.
In another scheme, the pH value of the R2 reagent buffer solution is 5.8-8.5, the buffer solution comprises Tris, and the concentration is 5.98-6.52 g/L; proclin-300 with the concentration of 0.98-1.99 g/L; thimerosal with the concentration of 0.99-2.01 g/L; sodium chloride with the concentration of 8.5-9.5 g/L; bovine serum albumin with the concentration of 10-30 g/L; gelatin with the concentration of 5-10 g/L; newborn bovine serum with the concentration of 30-50 g/L; n-ethyl maleimide with the concentration of 0.85-2.05 g/L; TritonX-100 with the concentration of 0.98-1.99g/L and Tween-20 with the concentration of 0.98-1.99 g/L; the balance of deionized water.
In another scheme, the R2 reagent, namely the alkaline phosphatase-labeled gastrin G17 monoclonal antibody diluent is prepared by diluting an alkaline phosphatase-labeled gastrin G17 monoclonal antibody with the concentration of 0.5 mu G/mL by using a buffer solution.
In another embodiment, the R2 reagent buffer has a pH of 7.4 and comprises Tris at a concentration of 6.05 g/L; proclin-300 with the concentration of 1.99 g/L; thimerosal, concentration 0.99 g/L; sodium chloride with concentration of 8.98 g/L; bovine serum albumin with a concentration of 29.88 g/L; gelatin with concentration of 9.86 g/L; newborn bovine serum with the concentration of 50 g/L; n-ethylmaleimide with a concentration of 1.05 g/L; TritonX-100 with the concentration of 0.98 g/L; tween-20 with concentration of 1.99 g/L; the balance of deionized water.
In another embodiment, the calibrator liquid is a diluent containing gastrin G17 antigen with different concentrations.
In another scheme, the pH value of the buffer solution of the calibrator liquid series is 7.0-8.0, and the buffer solution comprises PBS with the concentration of 0.02M; newborn bovine serum with the concentration of 250-500 g/L; casein (Casein), the concentration is 10-30 g/L; n-ethyl maleimide with the concentration of 0.85-2.05 g/L; proclin-300 with the concentration of 1.98-1.99 g/L; tetracycline, the concentration is 0.98-0.99 g/L; the rest components are deionized water.
In another scheme, the concentration range of the calibrator liquid is 0-100 pmol/L, the pH value of the buffer solution is 7.4, and the buffer solution comprises PBS with the concentration of 0.02M; newborn bovine serum with the concentration of 500 g/L; casein, the concentration is 20 g/L; n-ethylmaleimide with a concentration of 1.05 g/L; proclin-300 with the concentration of 1.98 g/L; tetracycline, with a concentration of 0.99 g/L; the rest components are deionized water.
Alternatively, the calibrator liquid may be used at a concentration of 0pmol/L, 1pmol/L, 5pmol/L, 10pmol/L, 50pmol/L, or 100 pmol/L.
In another embodiment, the chemiluminescent substrate solution is a chemiluminescent substrate solution catalyzed and luminous by alkaline phosphatase with a molar concentration of 0.1-0.3M, a Tris-HCl buffer solution with a molar concentration of 0.2M and a pH value of 8-10, and contains dioxane compound (APCL) with a concentration of 0.2-0.4 mg/mL.
In another embodiment, the chemiluminescent substrate solution is Tris-HCl buffer 0.2M at 0.2M molar concentration, pH 9.3 molar concentration, and contains 0.3mg/mL dioxane compound (APCL).
Compared with the prior art, the invention has the beneficial effects that:
the kit is designed aiming at the detection of human gastrin G17, is a magnetic particle chemiluminescence detection kit, the R1 reagent does not need to be marked by fluorescein, the magnetic separation reagent is coated at the constant temperature of 37 ℃, polyhydroxy substances are added into a sealing liquid, sucrose can generate a glass state effect in the solution, and the stability of magnetic beads containing G17 is improved. Meanwhile, the blocking agent is added into the reagent R1, so that the influence of complement, heterophilic antibodies and the like is effectively eliminated, and the sample detection accuracy is improved; n-ethyl maleimide is added into the reagent R2, so that the stability of the kit is effectively improved.
The magnetic particle chemiluminescence detection kit containing human gastrin G17 provided by the invention can be used together with a full-automatic chemiluminescence analyzer, the operation steps are greatly simplified, the detection speed and the detection flux are increased, the detection efficiency is improved, and errors caused by manual operation are avoided. The kit has the advantages of good specificity, good repeatability, low cost, high detection precision and good stability, can be well used for detecting the human gastrin G17, and can meet the comprehensive performance requirements of market use.
Drawings
Fig. 1 is a graph of concentration-luminescence of gastrin G17 in the kit of example 2 of the present invention;
FIG. 2 is a graph showing the results of correlation between the detection of samples in the Biohit kit in the kit according to example 2 of the present invention.
Detailed Description
The present invention is further explained with reference to the following examples and drawings, but the scope of the present invention is not limited thereto.
The invention relates to a magnetic particle chemiluminescence detection kit for determining the content of human gastrin G17, which comprises a magnetic separation reagent, an R1 reagent, an R2 reagent, a calibrator liquid series and a chemiluminescence substrate liquid.
The magnetic separation reagent comprises: 1) magnetic particles: immunomagnetic beads coated with the gastrin G17 monoclonal antibody, wherein the concentration is 10 mg/mL; 2) sealing liquid: PBS, concentration 0.02M; magnesium chloride solution with molar concentration of 1M; zinc chloride at a molar concentration of 0.1M; bovine serum albumin with the concentration of 10.0-50.0 g/L; sucrose: the concentration is 20.0-100.0 g/L; proclin300 with the concentration of 1.05-4.20 g/L; bronidox with the concentration of 1.05-4.20 g/L; the rest components are deionized water. The pH value of the confining liquid of the magnetic separation reagent is 7.0-8.0.
The R1 reagent includes: tris with the concentration of 5.98-6.52 g/L; proclin-300 with the concentration of 0.98-1.99 g/L; sodium chloride with the concentration of 8.5-9.5 g/L; bovine serum albumin with the concentration of 10-30 g/L; sodium dodecyl sulfate with the concentration of 1-5 g/L; EDTA-2Na with the concentration of 1-10 g/L; the concentration of the blocking agent is 50-200 ug/mL; the balance of deionized water. The buffer of the R1 reagent has a pH of 7.4, and the buffer of the R1 reagent refers to the remaining components that do not contain a blocking agent.
The R2 reagent includes: 1) r2 antibody: the concentration of the basic phosphatase-labeled gastrin G17 monoclonal antibody is 0.3-0.6 mu G/ml; 2) buffer solution: comprises Tris with the concentration of 5.98-6.52 g/L; proclin-300 with the concentration of 0.98-1.99 g/L; thimerosal with the concentration of 0.99-2.01 g/L; sodium chloride with the concentration of 8.5-9.5 g/L; bovine serum albumin with the concentration of 10-30 g/L; gelatin with the concentration of 5-10 g/L; newborn bovine serum with the concentration of 30-50 g/L; n-ethyl maleimide with the concentration of 0.85-2.05 g/L; TritonX-100 with the concentration of 0.98-1.99g/L and Tween-20 with the concentration of 0.98-1.99 g/L; the balance of deionized water. The pH value of the buffer solution of the R2 reagent is 5.8-8.5.
The calibrator liquid comprises: 1) a dilution of gastrin G17 antigen; 2) buffer solution: the buffer solution component comprises PBS with the concentration of 0.02M; newborn bovine serum with the concentration of 250-500 g/L; casein with the concentration of 10-30 g/L; n-ethyl maleimide with the concentration of 0.85-2.05 g/L; proclin-300 with the concentration of 1.98-1.99 g/L; tetracycline, the concentration is 0.98-0.99 g/L; the rest components are deionized water. The pH of the buffer of the calibrator liquid series was 7.4. The calibrator liquid series was a series of reagents containing different concentrations (0, 1, 5, 10, 50, 100 pmol/L).
The substrate solution is chemiluminescence substrate solution with the molar concentration of 0.1-0.3M and catalyzed luminescence by alkaline phosphatase, wherein the concentration of a dioxane compound (APCL) in the chemiluminescence substrate solution is 0.2-0.4 mg/mL, the buffer solution is Tris-HCl with the molar concentration of 0.2M, and the pH value is 8-10.
A preparation method of a magnetic particle chemiluminescence detection kit for determining the content of human gastrin G17 comprises the following steps:
preparing a magnetic separation reagent: 1) preparing a sealing liquid according to the component content of the magnetic particle sealing liquid, washing and activating magnetic beads, and then washing, coupling and sealing the magnetically separated supernatant-removed substances to obtain immunomagnetic beads coated with a gastrin G17 monoclonal antibody; 2) and (3) carrying out constant volume on the immunomagnetic beads coated with the gastrin G17 monoclonal antibody by using a confining liquid of the magnetic separation reagent to prepare the magnetic separation reagent coated with the gastrin G17 monoclonal antibody.
Preparing a reagent R1: 1) preparing a buffer solution according to the component content of the buffer solution of the reagent R1, and adjusting the pH value; 2) a blocking agent (blocking agent not a component of the buffer) is added.
Preparing a reagent R2: 1) preparing a buffer solution according to the component content of the buffer solution of the reagent R2, and adjusting the pH value; 2) preparing an alkaline phosphatase-labeled gastrin G17 monoclonal antibody; 3) the alkaline phosphatase-labeled gastrin G17 monoclonal antibody was diluted with a reagent R2 buffer.
Preparing a calibrator: 1) preparing a phosphate buffer solution according to the component content of the calibrator buffer solution; 2) the gastrin G17 antigen was dissolved in the calibrator buffer to be configured at various concentrations.
Preparing a chemiluminescent substrate solution: 1) preparing 0.2M Tris-HCl buffer solution with the pH value of 9.3, and adding 0.3mg/mL dioxane; 2) a chemiluminescent substrate that catalyzes luminescence by alkaline phosphatase is dissolved in a buffer.
Method for measuring magnetic particle chemiluminescence detection kit for human gastrin G17 content and drawing standard curve
(1) Firstly, 30 μ L of calibrator series (with the concentration of 0, 1, 5, 10, 50, 100pmol/L respectively) and 50 μ L of reagent R1 and 25 μ L of magnetic separation reagent are added into a reaction tube in sequence, and mixed and incubated for 15min at 37 ℃;
(2) washing with a washing solution 3 times to remove unbound antibodies and impurities;
(3) then 100 mul of reagent R2 is added, mixed and incubated for 15min at 37 ℃;
(4) washing with a washing solution 3 times to remove unbound antibodies and impurities;
(5) adding 150 μ L of luminous substrate solution, performing ALP catalytic substrate luminescence, and measuring relative luminescence intensity (RLU) with chemiluminescence detector of Lidmann to obtain gastrin G17 concentration-luminescence value series;
(6) and fitting according to the series of values of the concentration-luminescence value of the gastrin G17 to obtain a standard curve of the concentration-luminescence value of the gastrin G17.
(7) And calculating the concentration value of the sample to be detected according to the fitted gastrin G17 concentration-luminous value standard curve.
Within a certain range, the RLU is in direct proportion to the concentration of the gastrin G17 antigen, and the content of the gastrin G17 of the sample to be detected can be read from the standard curve by an interpolation method.
Example 1 a magnetic particle chemiluminescence assay kit for determining the amount of human gastrin G17 comprises a magnetic separation reagent, an R2 reagent, a calibrator liquid set, and a chemiluminescence substrate liquid.
In this embodiment, the magnetic separation reagent includes: 1) magnetic particles: immunomagnetic beads coated with the gastrin G17 monoclonal antibody, wherein the concentration is 10 mg/mL; 2) sealing liquid: PBS, concentration 0.02M; magnesium chloride solution with molar concentration of 1M; zinc chloride at a molar concentration of 0.1M; bovine serum albumin with a concentration of 10.0 g/L; sucrose: the concentration is 100.0 g/L; proclin300 with the concentration of 2.1 g/L; bronidox with concentration of 2.1 g/L; the rest components are deionized water. The pH value of the confining liquid of the magnetic separation reagent is 7.4.
In this example, the R1 reagent includes: 1) buffer Tris, the concentration is 6.05 g/L; proclin-300 with the concentration of 1.99 g/L; sodium chloride with the concentration of 9 g/L; bovine serum albumin with a concentration of 10 g/L; sodium dodecyl sulfate, the concentration is 1 g/L; EDTA-2Na with the concentration of 2 g/L; the concentration of the blocking agent is 100 ug/mL; the balance of deionized water.
In this example, the R2 reagent includes: 1) r2 antibody: alkaline phosphatase-labeled gastrin G17 monoclonal antibody, the concentration is 0.5 mug/ml; 2) buffer solution: tris, the concentration is 6.05 g/L; proclin-300 with the concentration of 1.99 g/L; thimerosal, concentration 0.99 g/L; sodium chloride with concentration of 8.98 g/L; bovine serum albumin with a concentration of 29.88 g/L; gelatin with concentration of 9.86 g/L; newborn bovine serum with the concentration of 50 g/L; n-ethylmaleimide with a concentration of 1.05 g/L; TritonX-100 with the concentration of 0.98 g/L; tween-20 with concentration of 1.99 g/L; the balance of deionized water. The buffer pH of the R2 reagent is preferably 7.4.
In this embodiment, the calibration liquid includes: 1) dilutions containing different concentrations of gastrin G17 antigen; 2) buffer solution: PBS, concentration 0.02M; newborn bovine serum with the concentration of 500 g/L; casein, the concentration is 20 g/L; n-ethylmaleimide with a concentration of 1.05 g/L; proclin-300 with the concentration of 1.98 g/L; tetracycline, with a concentration of 0.99 g/L; the rest components are deionized water. The pH of the buffer of the calibrator liquid series was 7.4. The calibration lines were listed as containing different concentrations (0, 1, 5, 10, 50, 100pmol/L) of the actual series.
In this example, the substrate solution was a chemiluminescent substrate solution in which luminescence was catalyzed by alkaline phosphatase, wherein the concentration of the dioxane compound (APCL) in the luminescent substrate solution was 0.3mg/mL, the buffer solution was Tris-HCl with a molar concentration of 0.2M, and the pH was 9.3.
EXAMPLE 2 preparation and measurement of magnetic particle chemiluminescence detection kit for measuring human gastrin G17 content
(1) First, 30. mu.l of the calibrator series of example 1 (concentrations of 0, 1, 5, 10, 50, 100pmol/L, respectively) and 50. mu.l of the reagent R1 of example 1 and 25. mu.l of the magnetic separation reagent were added to a reaction tube in this order, and mixed and incubated at 37 ℃ for 30 min;
(2) washing with a washing solution 3 times to remove unbound antibodies and impurities;
(3) then 100 mul of reagent R2 is added, mixed and incubated for 15min at 37 ℃;
(4) washing with a washing solution 3 times to remove unbound antibodies and impurities;
(5) after adding 150. mu.l of the luminescent substrate solution of example 1 and allowing the substrate to emit light by ALP catalysis, the relative luminescence intensity (RLU) was measured using a chemiluminescence detector from Ledmann, and the results are shown in the following table:
TABLE 1
| Calibrator (pmol/L) | 0 | 1 | 5 | 10 | 50 | 100 |
| RLU(100000) | 0.08 | 1.23 | 4.58 | 8.59 | 38.47 | 71.27 |
(6) Fitting according to the values in table 1 to obtain a gastrin G17 concentration-luminescence value standard curve (fig. 1), wherein the ordinate is the luminescence value and the abscissa is the gastrin G17 concentration;
(7) adding 30 μ l of sample to be tested, 50 μ l of reagent R1 in example 1 and 25 μ l of magnetic separation reagent in turn into a reaction tube, and mixing and incubating for 15min at 37 ℃;
(8) washing with a washing solution 3 times to remove unbound antibodies and impurities;
(9) then 100 mul of reagent R2 is added, mixed and incubated for 15min at 37 ℃;
(10) washing with a washing solution 3 times to remove unbound antibodies and impurities;
(11) adding 150 μ l of luminescent substrate solution of example 1, and measuring relative luminescence intensity RLU of 1566471 with a Lindman self-developed chemiluminescence detector after ALP catalysis substrate luminescence;
(12) and (5) calculating the concentration value of the gastrin G17 corresponding to the sample 1566471 to be detected to be 18.26pmol/L according to the standard curve of the concentration of the gastrin G17 and the luminescence value.
The data of the methodology identification when the detection kit is used for determining the content of the human gastrin G17 can reach the following indexes:
the blank limit is less than or equal to 0.1 pmol/L;
the linear detection range of the gastrin G17 is 1.0-100 pmol/L;
the precision-the intra-analysis precision is 2.37% on average (n is 10), the inter-analysis precision is 4.02% on average (n is 10), and the precision is far lower than the national requirement, which shows that the kit has good repeatability in the experimental process;
accuracy-the recovery rate is 90-100% after high-value serum with known gastrin G17 concentration is diluted by low-value serum with known gastrin G17 concentration according to the proportion of 1: 9;
endogenous interference-hemoglobin of 800mg/dl, fat emulsion of 2000mg/dl, bilirubin of 20mg/dl and rheumatoid factor of 1500IU/ml have deviation of less than 10 percent;
the crossing rate of specificity-with TG and Gastrin-34 is less than 10%;
the HOOK-Gastrin-17 concentration is as high as 100000pmol/L, and no high-dose HOOK effect exists;
the kit of the embodiment has better consistency with the Biohit detection result, R20.9929 (fig. 2).
The steps show that the reaction mode of the double-antibody sandwich method adopted by the invention utilizes the principle of combining the chemiluminescence detection technology with the magnetic particle immune separation technology to quantitatively detect the content of the gastrin G17 in the human serum or plasma sample, ensures the detection sensitivity, has no pollution, strong specificity, simple operation, low requirement on the pretreatment of the sample, can quickly detect a large number of samples with high flux, and is convenient for the application of clinical reagents. The invention provides a more accurate, precise, convenient, rapid and simple method for clinically detecting the gastrin G17 in human serum.
Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to those examples; within the idea of the invention, also technical features in the above embodiments or in different embodiments may be combined and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity. Therefore, any omissions, modifications, substitutions, improvements and the like that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.
Nothing in this specification is said to apply to the prior art.
Claims (10)
1. A magnetic particle chemiluminescence detection kit for determining the content of human gastrin G17 comprises: magnetic separation reagent, R1 reagent, R2 reagent, calibrator liquid series and chemiluminescence substrate liquid; the R2 reagent is alkaline phosphatase-labeled gastrin G17 monoclonal antibody diluent, the chemiluminescence substrate solution is alkaline phosphatase-catalyzed luminescence substrate solution, the R1 reagent is sample diluent, and the calibrator liquid is diluent containing gastrin G17 antigens with different concentrations; it is characterized in that the magnetic separation reagent is immunomagnetic beads coated with a gastrin G17 monoclonal antibody,
the immunomagnetic bead coating method for the coated gastrin G17 monoclonal antibody comprises the following steps:
(1) washing the magnetic beads: taking 100mg/mL magnetic beads 100uL, adding 1mL of 0.1M MES buffer solution, mixing uniformly for 5 minutes, carrying out magnetic separation, removing supernatant, and repeatedly washing for 3 times;
(2) and (3) activation: adding 20mg/mL and 50uL EDC solution and 20mg/mL and 50uL NHS solution into the washed magnetic beads, fully and uniformly mixing, and carrying out oscillation reaction at room temperature for 2 h;
(3) and (3) washing after activation: after the reaction is finished, carrying out magnetic separation, removing supernatant, and carrying out heavy suspension washing for 3 times by using MES solution;
(4) coating: adding the gastrin G17 monoclonal antibody diluted by 0.05M boric acid buffer solution into the washed magnetic beads, fully and uniformly mixing, and performing shaking table incubation at 37 ℃ for 18 h;
(5) and (3) sealing: magnetically separating the incubated magnetic beads, removing supernatant, adding 1mL of confining liquid, uniformly mixing for 5 minutes, magnetically separating, removing supernatant, and repeatedly washing for 3 times to obtain immunomagnetic beads coated with the gastrin G17 monoclonal antibody, wherein the concentration is 10 mg/mL;
(6) and (3) volume fixing: finally, the confining liquid is used for fixing the volume to 10mL, the concentration is 1mg/mL, and the solution is placed at the temperature of 2-8 ℃ for balancing for 24 hours to obtain the magnetic separation reagent coated by the gastrin G17 monoclonal antibody;
the pH value of the confining liquid is 7.0-8.0, and the confining liquid comprises: PBS, concentration 0.02M; magnesium chloride solution with molar concentration of 1M; zinc chloride at a molar concentration of 0.1M; bovine serum albumin with the concentration of 10.0-50.0 g/L; sucrose with the concentration of 20.0-100.0 g/L; proclin300 with the concentration of 1.05-4.20 g/L; bronidox with the concentration of 1.05-4.20 g/L; the rest components are deionized water.
2. The kit according to claim 1, wherein the confining liquid further comprises glycerol at a concentration of 10.0-20.0 g/L.
3. The kit of claim 2, wherein the blocking solution has a pH of 7.4 and comprises: PBS, concentration 0.02M; magnesium chloride solution with molar concentration of 1M; zinc chloride at a molar concentration of 0.1M; bovine serum albumin with a concentration of 10.0 g/L; sucrose with a concentration of 100.0 g/L; glycerol with the concentration of 20.0 g/L; proclin300 with the concentration of 2.1 g/L; bronidox with concentration of 2.1 g/L; the rest components are deionized water.
4. The kit according to claim 5, wherein the R1 reagent is a sample diluent containing a blocking agent, has a pH value of 5.8-8.5, and comprises: tris with the concentration of 5.98-6.52 g/L; proclin-300 with the concentration of 0.98-1.99 g/L; sodium chloride with the concentration of 8.5-9.5 g/L; bovine serum albumin with the concentration of 10-30 g/L; EDTA-2Na with the concentration of 1-10 g/L; the concentration of the blocking agent is 50-200 ug/mL; the balance of deionized water;
preferably, the R1 reagent is a sample diluent containing a blocking agent, having a pH of 7.4, comprising: tris, the concentration is 6.05 g/L; proclin-300 with the concentration of 1.99 g/L; sodium chloride with the concentration of 9 g/L; bovine serum albumin with a concentration of 10 g/L; EDTA-2Na with the concentration of 2 g/L; the concentration of the blocking agent is 100 ug/mL; the balance of deionized water.
5. The kit according to claim 1, wherein the alkaline phosphatase-labeled gastrin G17 monoclonal antibody diluent as the reagent R2 is an alkaline phosphatase-labeled gastrin G17 labeled antibody prepared by uniformly mixing an alkaline phosphatase activated by SMCC and a gastrin G17 monoclonal antibody activated by 2-iminosulfane hydrochloride in a molar ratio, performing a coupling reaction, equilibrating the reaction with a Tris buffer solution, and separating and purifying different molecular size fragments with a gel column.
6. The kit according to claim 5, wherein the R2 reagent, namely the alkaline phosphatase-labeled gastrin G17 monoclonal antibody diluent is prepared by diluting an alkaline phosphatase-labeled gastrin G17 monoclonal antibody with a concentration of 0.3-0.6 μ G/mL with a buffer solution;
the pH value of the R2 reagent buffer solution is 5.8-8.5, the buffer solution comprises Tris, and the concentration is 5.98-6.52 g/L; proclin-300 with the concentration of 0.98-1.99 g/L; thimerosal with the concentration of 0.99-2.01 g/L; sodium chloride with the concentration of 8.5-9.5 g/L; bovine serum albumin with the concentration of 10-30 g/L; gelatin with the concentration of 5-10 g/L; newborn bovine serum with the concentration of 30-50 g/L; n-ethyl maleimide with the concentration of 0.85-2.05 g/L; TritonX-100 with the concentration of 0.98-1.99g/L and Tween-20 with the concentration of 0.98-1.99 g/L; the balance of deionized water.
7. The magnetic particle chemiluminescence kit for detecting the content of human gastrin G17, according to claim 10, wherein the R2 reagent, namely alkaline phosphatase-labeled gastrin G17 monoclonal antibody diluent is prepared by diluting alkaline phosphatase-labeled gastrin G17 monoclonal antibody with a concentration of 0.5 μ G/mL with a buffer solution;
the pH value of the R2 reagent buffer solution is 7.4, the buffer solution comprises Tris, and the concentration is 6.05 g/L; proclin-300 with the concentration of 1.99 g/L; thimerosal, concentration 0.99 g/L; sodium chloride with concentration of 8.98 g/L; bovine serum albumin with a concentration of 29.88 g/L; gelatin with concentration of 9.86 g/L; newborn bovine serum with the concentration of 50 g/L; n-ethylmaleimide with a concentration of 1.05 g/L; TritonX-100 with the concentration of 0.98 g/L; tween-20 with concentration of 1.99 g/L; the balance of deionized water.
8. The kit according to claim 1, wherein the buffer solution of the calibrator liquid series has a pH value of 7.0-8.0, and the buffer solution comprises: PBS, concentration 0.02M; newborn bovine serum with the concentration of 250-500 g/L; casein with the concentration of 10-30 g/L; n-ethyl maleimide with the concentration of 0.85-2.05 g/L; proclin-300 with the concentration of 1.98-1.99 g/L; tetracycline, the concentration is 0.98-0.99 g/L; the rest components are deionized water.
9. The kit of claim 8, wherein the calibrator solution is in a concentration range of 0 to 100pmol/L, the pH of the buffer is 7.4, the buffer component comprises PBS in a concentration of 0.02M; newborn bovine serum with the concentration of 500 g/L; casein, the concentration is 20 g/L; n-ethylmaleimide with a concentration of 1.05 g/L; proclin-300 with the concentration of 1.98 g/L; tetracycline, with a concentration of 0.99 g/L; the rest components are deionized water;
the different concentrations of the calibrator liquid series were 0pmol/L, 1pmol/L, 5pmol/L, 10pmol/L, 50pmol/L, and 100 pmol/L.
10. The kit of claim 8, wherein the blank limit of the kit is 0.1pmol/L or less;
the linear detection range of the gastrin G17 is 1.0-100 pmol/L;
the intra-assay precision was 2.37% (n ═ 10) on average, and the inter-assay precision was 4.02% (n ═ 10) on average;
diluting high-value serum with known gastrin G17 concentration by low-value serum with known gastrin G17 concentration according to a ratio of 1:9, and then obtaining a recovery rate of 90-100%;
the detection result deviations of 800mg/dl hemoglobin, 2000mg/dl fat emulsion, 20mg/dl bilirubin and 1500IU/ml rheumatoid factor are less than 10%;
the crossing rate with TG and Gastrin-34 is less than 10 percent;
the HOOK-Gastrin-17 concentration was 100000pmol/L with no high dose HOOK effect.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN113391073A (en) * | 2021-06-10 | 2021-09-14 | 中秀科技股份有限公司 | Magnetic particle chemiluminescence immunoassay kit for NPTX2 |
| CN113391068A (en) * | 2021-06-10 | 2021-09-14 | 中秀科技股份有限公司 | Magnetic particle chemiluminescence immunoassay kit for BACE1 |
| CN113804896A (en) * | 2021-09-14 | 2021-12-17 | 北京利德曼生化股份有限公司 | Magnetic particle chemiluminescence kit for measuring interleukin-8 content in human serum |
| CN113933521A (en) * | 2021-09-26 | 2022-01-14 | 北京贝尔医疗设备有限公司 | Magnetic particle chemiluminescence detection kit and preparation method and application thereof |
| CN114184603A (en) * | 2021-11-11 | 2022-03-15 | 宁波海壹生物科技有限公司 | Kit for determining copeptin by magnetic particle chemiluminescence method |
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| CN114184603A (en) * | 2021-11-11 | 2022-03-15 | 宁波海壹生物科技有限公司 | Kit for determining copeptin by magnetic particle chemiluminescence method |
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