CN111398591A - Pepsinogen I and pepsinogen II combined detection kit and application thereof - Google Patents
Pepsinogen I and pepsinogen II combined detection kit and application thereof Download PDFInfo
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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
- G01N33/577—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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
- G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
- G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
- G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
- G01N2333/96472—Aspartic endopeptidases (3.4.23)
- G01N2333/96475—Aspartic endopeptidases (3.4.23) with definite EC number
- G01N2333/96477—Pepsin (3.4.23.1; 3.4.23.2; 3.4.23.3)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/06—Gastro-intestinal diseases
Abstract
The invention discloses a combined detection kit for pepsinogen I and pepsinogen II and application thereof. The invention discloses a pepsinogen I and II combined detection kit for the first time, which comprises a coating antibody and a labeled antibody, wherein the coating antibody is an anti-PG I monoclonal antibody named monoclonal antibody IA and an anti-PG II monoclonal antibody named monoclonal antibody IIA; the labeled antibodies are an anti-PG I monoclonal antibody named monoclonal antibody IB and an anti-PG II monoclonal antibody named monoclonal antibody IIB. Further discloses application of the kit. The invention utilizes the pepsinogen I and II combined detection kit to combine the high-sensitivity inductively coupled plasma mass spectrometry detection technology on the basis of time-resolved fluorescence immunoassay, can simultaneously complete the detection of two indexes of PG I and PG II in one cycle, shortens the time for obtaining the results of PG I value and PG I/PG II ratio in clinic and greatly reduces the detection cost.
Description
Technical Field
The invention belongs to the technical field of in-vitro diagnostic reagents, and particularly relates to a combined detection kit for pepsinogen I and pepsinogen II and application thereof.
Background
Gastric cancer is one of the most common malignant tumors, and the mortality rate of gastric cancer is the second place of the mortality rate of common malignant tumors worldwide. Early discovery, early diagnosis and early treatment are one of the key measures for reducing the death rate of the gastric cancer. At present, the diagnosis of the gastric cancer and other gastric diseases is still determined by relying on gastroscopy and histopathology, but the gastroscopy has certain pain and is not accepted by patients, so that the diagnosis and treatment are easily delayed.
In recent years, the relationship between the content changes of serum pepsinogen I (PG II) and pepsinogen II (PG II) and the ratio thereof (PG I/PG II) and gastric diseases and the role thereof in the early diagnosis of gastric cancer have attracted more and more attention. Pepsinogen (PG), a digestive enzyme precursor secreted by the stomach, is an inactive precursor of pepsin in gastric juice and can be divided into two subtypes PG i and PG ii according to biochemical properties and immunogenicity. PG I and PG II are good diagnostic indexes of Helicobacter Pylori (HP) infection and atrophic gastritis, and the content of PG I and PG II can reflect the secretion function of gastric mucosa. The PG I mainly reacts on the mucous membrane states of the gastric fundus and the gastric corpus, the gastric acid secretion is increased, the PG I for gastritis and peptic ulcer is increased, the gastric acid secretion is reduced or the PG I for atrophy of gastric mucosal glands is reduced; when atrophic gastritis occurs, the gastric mucosa main cells are lost, the gastric mucosa secretory capacity is reduced, and the PG I level is obviously reduced; PG II has a large correlation with the pathological changes of the gastric fundus mucosa (relative to the gastric antral mucosa), and the increase of PG II is related to atrophy of the gastric fundus ducts, intestinal metaplasia or pseudopyloric metaplasia and dysplasia; moreover, PG II is the most sensitive index of Helicobacter Pylori (HP) infection, the raising amplitude of PG II is more obvious than that of PG I, and the ratio of PG I to PG II is reduced; therefore, the joint determination of the ratio of PG I to PG II plays a role of serological biopsy of gastric fundus mucosa, and the diseases in the stomach are changed to different degrees according to different serum measurement values, thereby providing reliable diagnostic value for clinic.
In the current immunoassay technology, no matter the earliest Radioimmunoassay (RIA) or the non-radioactive immunoassay methods developed gradually later, such as enzyme-linked immunoassay (E L ISA) and chemiluminescence immunoassay (C L IA), only one disease marker result can be obtained in each detection, if a plurality of disease markers need to be detected, serum samples are respectively added to a plurality of small holes for reaction and detection, the work is complex, time and labor are wasted, and the simultaneous detection and analysis of multiple indexes cannot be realized.
Disclosure of Invention
The invention aims to solve the technical problem of how to efficiently and low-cost finish the detection of two indexes of pepsinogen I (PG II) and pepsinogen II (PG II) in serum.
In order to solve the technical problems, the invention firstly provides a combined detection kit for pepsinogen I and pepsinogen II.
The combined detection kit for the pepsinogen I and the pepsinogen II comprises a coating antibody and a labeled antibody, wherein the coating antibody is an anti-PG I monoclonal antibody named monoclonal antibody IA and an anti-PG II monoclonal antibody named monoclonal antibody IIA;
the labeled antibodies are an anti-PG I monoclonal antibody named monoclonal antibody IB and an anti-PG II monoclonal antibody named monoclonal antibody IIB;
the monoclonal antibody IA and the monoclonal antibody IB are two different monoclonal antibodies, and the monoclonal antibody IIA and the monoclonal antibody IIB are two different monoclonal antibodies.
Specifically, the kit comprises the magnetic beads coated by the monoclonal antibody IA, the magnetic beads coated by the monoclonal antibody IB, the monoclonal antibody IB marked by rare earth element C1 and the monoclonal antibody IIB marked by rare earth element C2, wherein the rare earth element C1 and the rare earth element C2 are two different rare earth elements.
In the kit, the magnetic beads coated with the monoclonal antibody IA and the magnetic beads coated with the monoclonal antibody IB are obtained by respectively performing coupling reaction on the biotin-modified monoclonal antibody IA and the monoclonal antibody IB and streptavidin-modified magnetic beads through biotin-streptavidin. Specifically, the reaction solution used in the biotin-streptavidin coupling reaction was 0.01M PBS buffer at pH 7.4.
In the kit, the biotin-modified monoclonal antibody IA and monoclonal antibodyThe monoclonal antibody IB is biotin activated by an NHS active group and NH on the monoclonal antibody IA and the monoclonal antibody IB respectively2Obtained by reaction. In particular, the NHS active group activated biotin and NH on the antibody2The reaction solution used for the reaction was 0.01MPBS buffer solution at pH 7.4, and the molar ratio of the antibody to biotin was 1: 20.
In the kit, the monoclonal antibody IB marked by the rare earth element C1 and the monoclonal antibody IIB marked by the rare earth element C2 are obtained by respectively reacting a chloride hydrate of the rare earth element C1 and a chloride hydrate of the rare earth element C2 with a metal chelating agent to obtain a rare earth element C1-metal chelate probe and a rare earth element C2-metal chelate probe; the rare earth element C1-metal chelate probe reacts with the monoclonal antibody IB to obtain the monoclonal antibody IB marked by the rare earth element C1, and the rare earth element C2-metal chelate probe reacts with the monoclonal antibody IIB to obtain the monoclonal antibody IIB marked by the rare earth element C2. Specifically, the reaction liquid of the chloride hydrate of the rare earth element C1 and the reaction liquid of the chloride hydrate of the rare earth element C2 and the metal chelating agent are both 0.5M acetic acid-sodium acetate buffer solution with the pH of 5.8, and the molar ratio of the metal chelating agent to the chloride hydrate of the rare earth element C1 and the chloride hydrate of the rare earth element C2 is 1: 1.2.
In the kit, the reaction solution of the reaction between the rare earth element C1-metal chelate probe and the monoclonal antibody ib, the reaction between the rare earth element C2-metal chelate probe and the monoclonal antibody iib is 0.1M carbonate buffer solution with pH of 9.6, the reaction conditions are that the reaction is carried out at 25 ℃ for 20 hours, and the molar ratio of the rare earth element C1-metal chelate probe to the monoclonal antibody ib, the molar ratio of the rare earth element C2-metal chelate probe to the monoclonal antibody iib are all 30: 1.
in the kit, the rare earth element C1 and the rare earth element C2 are thulium and lutetium respectively, or the metal chelating agent is DTPA. The chloride hydrate of the rare earth element thulium is thulium chloride hexahydrate, and the chloride hydrate of the rare earth element lutetium is lutetium chloride hexahydrate.
The kit also comprises a PG compound calibrator solution, wherein the PG compound calibrator solution consists of PG I, PG II and a standard diluent, namely PG I and PG II are diluted by the standard diluent to obtain PG compound calibrator solutions of PG I and PG II with different concentrations, so that the concentration range of PG I is 1.0-200ng/m L, and the concentration range of PG II is 0.5-100ng/m L. in the specific embodiment of the invention, the PG compound calibrator solution comprises PG compound calibrator solution A, B, C, D, E, F, and the concentrations of PG I and PG II in the PG compound calibrator solution A, B, C, D, E, F are 0ng/m L and 0ng/m L, 3.1ng/m L and 1.7ng/m L, 12.5ng/m L and 6.3ng/m L, 25ng/m L and 12.5ng/m L, 100ng/m L and 50ng/m 6345, 50ng/m 828925 and 8536 respectively.
The kit also comprises a PG composite quality control product, wherein the PG composite quality control product is composed of PG I, PG II and a standard substance diluent, namely PG I and PG II with different concentrations are obtained by diluting PG I and PG II with the standard substance diluent, the concentrations of PG I and PG II in the PG composite quality control product are fixed so as to verify the detection accuracy of the kit.
In the kit, the standard substance diluent comprises protective protein, the concentration of the protective protein is 2 g/L, the protective protein is one or the mixture of any two of bovine serum albumin, casein and animal serum, and the protective protein can provide a good stable reaction environment and keep the antigen/antibody in a natural conformation.
In a specific embodiment of the invention, the standard dilution is prepared by the following preparation method:
1) weighing 6.05g of tris (hydroxymethyl) aminomethane (tris) and 9g of sodium chloride, adding into 800ml of deionized water, and fully stirring to completely dissolve the tris (hydroxymethyl) aminomethane (tris) and the sodium chloride to obtain a solution;
2) adding hydrochloric acid solution to adjust the pH value of the solution to 7.8;
3) weighing 2g of bovine serum albumin, adding the bovine serum albumin into the solution, and uniformly stirring to fully dissolve the bovine serum albumin;
4) deionized water is added to a constant volume of 1L, filtered by a 0.2 μm filter, and stored in an aseptic environment at 2-8 ℃.
The kit also comprises a dissociation solution, wherein the dissociation solution consists of a solute and a solvent, the solvent is a dilute nitric acid solution with the volume percentage content of 1%, the solute is rhenium (Re) element, the content of the rhenium (Re) element in the dissociation solution is 1ng/m L, and the rhenium (Re) is an internal standard element of inductively coupled plasma mass spectrometry (ICP-MS).
The kit also comprises an immune cleaning solution,
the kit also comprises an immune cleaning solution, wherein the immune cleaning solution is 50mM Tris-HCl buffer solution with the pH value of 7.8, and contains 0.05% of Tween 20 by volume percentage.
In a specific embodiment of the invention, the immune cleaning solution is prepared by the following preparation method:
1) weighing 6.05g of tris (hydroxymethyl) aminomethane (tris) and 9g of sodium chloride, adding into 800ml of deionized water, and fully stirring to completely dissolve the tris (hydroxymethyl) aminomethane (tris) and the sodium chloride to obtain a solution;
2) adding hydrochloric acid solution to adjust the pH value of the solution to 7.8;
3) adding Tween 20 liquid 500 mu L into the solution, and stirring uniformly to dissolve completely;
4) deionized water is added to a constant volume of 1L, filtered by a 0.2 μm filter, and stored in an aseptic environment at 2-8 ℃.
In the pepsinogen I and II combined detection kit, the monoclonal antibody IA is a PG I antibody with the serial number of 8015 of the medix Biochemica company, the monoclonal antibody IIA is an antibody with the serial number of 8101 of the medix Biochemica company, the monoclonal antibody IB is a PG I antibody with the serial number of 8003 of the medix Biochemica company, and the monoclonal antibody IIB is a PG II antibody with the serial number of 8103 of the medix Biochemica company.
In order to solve the technical problems, the invention further provides the application of the pepsinogen I and II combined detection kit in combined detection of pepsinogen I and pepsinogen II in a sample to be detected.
In the above application, the sample to be tested is isolated serum.
The application of the combined detection kit for pepsinogen I and pepsinogen II in preparing products for detecting stomach diseases is also within the protection scope of the invention.
In the present invention, the gastric disease may be a gastric disease infected with helicobacter pylori and/or atrophic gastritis and/or a tumor.
The invention utilizes the pepsinogen I and II combined detection kit to combine the high-sensitivity inductively coupled plasma mass spectrometry (ICP-MS) detection technology on the basis of time-resolved fluorescence immunoassay, can simultaneously complete the detection of two indexes of PG I and PG II in one cycle, shortens the time for obtaining the results of PG I value and PG I/PG II ratio in clinic, and greatly reduces the detection cost. In addition, the rare earth elements are adopted for marking, so that the ICP-MS detection is not influenced by optical, electrochemical and other characteristics of a marker, and the detection result is more stable and reliable.
Drawings
FIG. 1 is a graph of the PG I standard provided in example 1 of the present invention.
FIG. 2 is a graph of the PG II standard provided in example 1 of the present invention.
FIG. 3 is a graph showing the results of the serum alignment test using PG I in example 2 of the present invention.
FIG. 4 is a graph showing the results of the serum alignment test using PG II in example 2 of the present invention.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The selection criteria and purchase sources of the raw materials in the following examples of the invention are as follows:
the invention discloses an anti-PG I monoclonal antibody and an anti-PG II monoclonal antibody, which are characterized in that antibodies with colorless transparent clear liquid, no visible foreign matters and no shaking-proof precipitates are selected by verifying the appearance, concentration, purity and titer of the antibodies, the protein content of the antibodies is detected by an ultraviolet absorption method, and the antibodies with the protein content of not less than 4mg/m L are selected.
The pure pepsinogen I (PG I) and pepsinogen II (PG II) antigens are products of Medix Biochemica, and are purchased from Shanghai Youning vitamin science and technology Co.
Magnetic beads: through analyzing the aspects of appearance, particle size uniformity, adsorption consistency and the like of the magnetic beads, and through multiple times of analytical research, the magnetic beads with regular appearance, uniform particle size and adsorption consistency CV less than or equal to 8 percent are selected. The magnetic bead of the invention is a magnetic bead modified by streptavidin, which is a product of Suzhou beaver biomedical engineering limited company.
The thulium chloride hexahydrate, lutetium chloride hexahydrate and DTPA are redissolved and uniformly mixed by analyzing the aspects of appearance, purity and the like of thulium chloride (Tm) hexahydrate, lutetium chloride (L u) hexahydrate and S-2- (4-isothiocyanato-zyl) -diethylenetriamine pentaacetic acid (DTPA), and thulium chloride hexahydrate, lutetium chloride hexahydrate and DTPA which are clear and transparent liquid, free of impurities and particles are selected.
Internal standard elements for inductively coupled plasma mass spectrometry (ICP-MS): by verifying the appearance, concentration and purity of an internal standard element rhenium (Re) standard, the Re standard which is colorless transparent clear liquid, has no visible foreign matters and has no shaking and non-scattering precipitate is selected. The Re standard substance selected by the invention is the Re standard substance of the national standard substance center.
EXAMPLE 1 Combined detection kit for pepsinogen I (PG I) and pepsinogen II (PG II)
The combined detection kit for pepsinogen I (PG I) and pepsinogen II (PG II) comprises the following components:
magnetic beads coated with an anti-PG I monoclonal antibody (PG I8015 antibody) and magnetic beads coated with an anti-PG II monoclonal antibody (PG II 8101 antibody);
an anti-PG I monoclonal antibody (PG I8003 antibody) marked by rare earth element thulium (Tm) and an anti-PG II monoclonal antibody (PG II 8103 antibody) marked by rare earth element lutetium (L u);
a PG composite calibrator;
PG composite quality control products;
a dissociation liquid;
and (4) immune cleaning fluid.
The preparation method of the components comprises the following steps:
1. the preparation method of the magnetic beads coated with the anti-PG I monoclonal antibody (PG I8015 antibody) and the anti-PG II monoclonal antibody (PG II 8101 antibody) comprises the following steps:
1) the biotin modified antibody is prepared by weighing a certain amount of biotin activated by NHS active groups, dissolving the biotin in a DMSO organic solvent to prepare a biotin mother solution with the concentration of 10mM for standby, diluting 1mg of antibody with a carbonate solution to obtain an antibody solution with the concentration of 2mg/M L, adding the antibody solution and the biotin mother solution into a 0.01M PBS solution (pH 7.4) to ensure that the molar ratio of the antibody to the biotin is 1:20, and reacting at normal temperature for 30min to ensure that the biotin activated by the NHS active groups and NH on the antibody2Reacting to obtain biotin modificationThe antibody of (1). And classifying and purifying the biotin-modified antibody by using a chromatographic purification system, and monitoring the peak of the protein by using an ultraviolet detector to obtain the purified biotin-modified antibody.
The PG I8015 antibody and the PG II 8101 antibody are labeled with biotin respectively according to the steps to obtain the purified PG I8015 antibody modified by the biotin and the PG II 8101 antibody modified by the biotin.
2) The biotin-streptavidin coupling reaction comprises the steps of transferring 100u L streptavidin modified magnetic beads into a centrifuge tube by a liquid transfer machine, separating the magnetic beads by a magnetic separator, sucking supernatant by the liquid transfer machine, removing the centrifuge tube from the magnetic separator, adding 1m L0.01.01 MPBS buffer solution (pH 7.4) into the centrifuge tube, adding 20 mu L biotin modified antibody, fully shaking the resuspended magnetic beads, placing the centrifuge tube on a rotary mixer, rotating and mixing for 60min at room temperature, magnetically separating, sucking the supernatant, washing the magnetic beads for 3 times by washing buffer solution (PBS containing 0.05% Tween-20) to obtain antibody coated Tris magnetic beads, suspending the antibody coated magnetic beads in-HCl buffer solution, and storing at 4 ℃.
The biotin-modified PG I8015 antibody and the biotin-modified PG II 8101 antibody respectively carry out biotin-streptavidin coupling reaction with streptavidin-modified magnetic beads according to the steps to respectively obtain PG I8015 antibody-coated magnetic beads and PG II 8101 antibody-coated magnetic beads.
3) Determining the optimal working concentration of the PG I8015 antibody coated magnetic bead and the PG II 8101 antibody coated magnetic bead to be 0.5mg/m L by adopting a chessboard method, simultaneously diluting with an antibody diluent, uniformly mixing to obtain the PG I8015 antibody coated magnetic bead and the PG II 8101 antibody coated magnetic bead, and storing at the temperature of 2-8 ℃.
2. The preparation method of the rare earth element thulium (Tm) labeled anti-PG I monoclonal antibody (PG I8003 antibody) and the rare earth element lutetium (L u) labeled anti-PG II monoclonal antibody (PG II 8103 antibody) comprises the following steps:
1) respectively dissolving DTPA and thulium chloride (Tm) hexahydrate or lutetium chloride (L u) hexahydrate in 0.5M acetic acid-sodium acetate buffer solution (pH is 5.8), wherein the molar ratio of DTPA to the rare earth element Tm or L u is 1:1.2, and the reaction condition is that the reaction is carried out for 30 minutes at 37 ℃ to obtain a Tm-DTPA chelate probe or a L u-DTPA chelate probe;
2) respectively reacting the Tm-DTPA chelate probe with a PG I8003 antibody, L u-DTPA chelate probe and a PG II 8103 antibody in 0.1M carbonate buffer solution (pH is 9.6), wherein the molar ratio of the Tm-DTPA chelate probe to the PG I8003 antibody, the molar ratio of the L u-DTPA chelate probe to the PG II 8103 antibody are both 30: 1, the reaction condition is that the reaction is carried out for 20 hours at 25 ℃, obtaining a PG I8003 antibody marked by rare earth element Tm and a PG II 8103 antibody marked by rare earth element L u, purifying the PG I8003 antibody marked by rare earth element Tm and the PG II 8103 antibody marked by rare earth element L u by chromatography, monitoring the peak of the protein by an ultraviolet detector, obtaining the purified PG I8003 antibody marked by rare earth element and PG II 8103 antibody marked by rare earth element L u, freezing and drying, and storing at-20 DEG C
3) The optimal working concentration of the rare earth element Tm-labeled PG I8003 antibody and the rare earth element L u-labeled PG II 8103 antibody is determined to be 1 mu g/m L by adopting a chessboard method, the antibodies are simultaneously diluted by an antibody diluent and uniformly mixed to obtain the rare earth element Tm-labeled PG I8003 antibody and the rare earth element L u-labeled PG II 8103 antibody, and the antibodies are stored at the temperature of 2-8 ℃.
3. The preparation method of the standard substance diluent comprises the following specific steps:
1) weighing 6.05g of tris (hydroxymethyl) aminomethane (tris) and 9g of sodium chloride in a container of 1L, and adding 800ml of deionized water to fully stir to completely dissolve the tris (hydroxymethyl) aminomethane (tris) and the sodium chloride;
2) adding hydrochloric acid solution into the container 1L with a plastic dropper, and adjusting pH to 7.8;
3) weighing 2g of bovine serum albumin, adding the bovine serum albumin into the 1L container, and stirring uniformly to fully dissolve the bovine serum albumin;
4) diluting to volume of 1L, filtering with 0.2 μm filter, labeling, and storing at 2-8 deg.C under sterile condition.
The formulations of the resulting standard dilutions are shown in table 1.
TABLE 1 formulation of standard dilutions
4. The preparation method of the PG composite calibration product and the PG composite quality control product comprises the following specific steps:
1) diluting PG I and PG II antigen pure products with standard product diluent to obtain PG compound calibrator solution with a working concentration range of 1.0-200ng/m L II and a working concentration range of 0.5-100ng/m L, and specifically preparing PG compound calibrator solution A, B, C, D, E, F, wherein the concentration of each PG compound calibrator solution is set as shown in Table 2.
TABLE 2 concentration of PG I and PG II in the PG complex calibrator solutions
| PG composite calibration article | PGⅠ(ng/mL) | PGⅡ(ng/mL) |
| A | 0 | 0 |
| B | 3.1 | 1.7 |
| C | 12.5 | 6.3 |
| D | 25 | 12.5 |
| |
100 | 50 |
| |
200 | 100 |
2) Diluting pure PG I and PG II antigens by standard product diluent to obtain PG composite quality control products, and specifically preparing PG composite quality control products 1 and PG composite quality control products 2, wherein the concentration of PG I in the PG composite quality control products 1 is 20ng/m L II and is 5ng/m L, and the concentration of PG I in the PG composite quality control products 2 is 80ng/m L II and is 30ng/m L.
5. The preparation method of the dissociation liquid comprises the following specific steps:
1) diluting high-purity concentrated nitric acid into 1% (volume percentage content) dilute nitric acid solution by using ultrapure water;
2) taking a certain amount of rhenium (Re) standard substance, and preparing a Re solution with the concentration of 1ng/m L by using the 1% dilute nitric acid solution to obtain the dissociation solution.
6. The preparation method of the immune cleaning solution comprises the following specific steps:
1) weighing 6.05g of tris (hydroxymethyl) aminomethane (tris) and 9g of sodium chloride in a container of 1L, and adding 800ml of deionized water to fully stir to completely dissolve the tris (hydroxymethyl) aminomethane (tris) and the sodium chloride;
2) adding hydrochloric acid solution into the container 1L with a plastic dropper, and adjusting pH to 7.8;
3) transferring Tween 20 liquid 500 mu L, adding into the container 1L, and stirring to dissolve completely;
4) diluting to volume of 1L, filtering with 0.2 μm filter, labeling, and storing at 2-8 deg.C under sterile condition.
The formulation of the resulting immune-cleansing solution is shown in Table 3.
TABLE 3 formulation of the immune cleaning solution
Secondly, the method for jointly detecting the pepsinogen I and the pepsinogen II by using the pepsinogen I and pepsinogen II joint detection kit comprises the following steps:
A) making a standard curve:
a1) 50u L of magnetic beads coated by PG I8015 antibody and 50u L of magnetic beads coated by PG II 8101 antibody, PG I8003 antibody marked by rare earth element Tm and PG II 8103 antibody marked by rare earth element L u are mixed with PG composite calibrator solution A, B, C, D, E, F50 & mu & L respectively, then added into a plate hole of a 96-well plate, and incubated for 20 minutes at 37 ℃;
a2) adding immune cleaning solution according to 200 mu L/hole, washing the plate by adopting an automatic plate washing machine, and repeatedly washing for 3 times;
a3) adding dissociation liquid according to 100 mu L/hole, introducing the supernatant of the dissociation liquid into inductively coupled plasma mass spectrometry (ICP-MS) for detection after 1 minute, respectively collecting response values of a rare earth element Tm and a rare earth element L u and an internal standard element rhenium element Re, namely corresponding ion count values (cps) per second, and respectively drawing a standard curve of PG I (shown in figure 1) and a standard curve of PG II (shown in figure 2) by taking the concentration of PG I or PG II in the PG composite calibrator solution as an abscissa and the response value ratio of the rare earth element Tm or the rare earth element L u to the internal standard element Re as an ordinate.
B) Detecting a sample to be tested
a1) 50u L of each magnetic bead coated by the PG I8015 antibody and 50u L of each magnetic bead coated by the PG II 8101 antibody, 20 mu L of a sample to be detected and 50u 8003 antibody labeled by rare earth element Tm and PG II 8103 antibody labeled by rare earth element L u are mixed and then added into a plate hole of a 96-well plate, and the incubation is carried out for 20 minutes at 37 ℃;
a2) adding immune cleaning solution according to 200 mu L/hole, washing the plate by adopting an automatic plate washing machine, and repeatedly washing for 3 times;
a3) adding dissociation liquid according to a hole of 100 mu L/hole, introducing the supernatant of the dissociation liquid into inductively coupled plasma mass spectrometry (ICP-MS) for detection after 1 minute, respectively collecting response values of a rare earth element Tm and a rare earth element L u as well as an internal standard element rhenium element Re, namely a corresponding ion count value per second (cps), calculating the concentrations of PG I and PG II in a sample to be detected according to a standard curve, and simultaneously obtaining the ratio of PG I/PG II.
Example 2 Performance testing and applications of the Propepsinogen I (PG I) and pepsinogen II (PG II) Joint test kit
First, performance index
1. Minimum detection limit
According to the method for detecting the sample to be detected in the step B) in the step two in the embodiment 1, the PG complex calibrator solution with zero concentration (namely, the PG complex calibrator solution A in the embodiment) is used as a sample to be detected for detection, the detection is repeated for 20 times, the response value of the detection result of 20 times, namely the counting value (cps) of corresponding ions per second, is obtained, and the average value (M) and the Standard Deviation (SD) are calculated, so that M +2SD is obtained. And (3) detecting by taking the adjacent concentration PG composite calibrator solution (namely PG composite calibrator solution B) as a sample to be detected, repeatedly detecting for 3 times, and averaging to obtain a response value-corresponding ion count value (cps) per second.
And performing two-point regression fitting according to the concentration-cps result between the zero-concentration PG compound calibrator solution and the adjacent concentration PG compound calibrator solution to obtain a linear equation, substituting the cps of M +2SD into the equation, and calculating the corresponding PG concentration value, namely the lowest detection limit, wherein the detection result is shown in Table 4.
2. Accuracy of
A serum sample (solution B) was added to a sample (solution A) containing PG I at 150ng/m L and PG II at 80ng/m L in the form of a dilution of a standard sample (solution A), and the volume of the solution A added was preferably not more than 10% of the total volume (solution A + solution B), and the measurement was repeated 3 times for each sample in accordance with the "method of step two in example 1", and the results were calculated in accordance with the formula (1), and the results are shown in Table 4.
In the formula: r-recovery rate; v-volume of addition of liquid A; v0-volume of liquid B; c-detecting PG I of liquid B after adding liquid AOr the concentration of PG II; c0Detecting the concentration of PG I or PG II in solution B; cs-detecting the concentration of PG I or PG II in solution A.
3. Linearity
Diluting the high value sample close to the upper limit of the linear range to at least 5 concentrations according to a certain proportion, wherein the lowest concentration sample is close to the lower limit of the linear interval. The detection of each concentration gradient sample was repeated 3 times in accordance with "method II in example 1", and the average value was taken to obtain the detected concentrations of PG I and PG II in each concentration gradient sample. The average values of the measured concentrations and the theoretical concentrations were fitted to each other by a straight line using the least square method, and the linear correlation coefficients r corresponding to PG I and PG ii were calculated, respectively, and the results are shown in table 4.
4. Precision in batch
The concentrations of PG i and PG ii in the PG complex quality control 1 and the PG complex quality control 2 of example 1 were measured with the same kit according to the "method of step two in example 1", and the measurement was repeated 10 times, and the average value M and the standard deviation SD of the 10 measurements were calculated, and the coefficient of variation CV was calculated according to the formula (2), and the results are shown in table 4.
CV＝SD/M×100％………………………(2)
In the formula: CV — coefficient of variation; SD-standard deviation of detection results; m-mean of the results
5. Inter-batch precision
The concentrations of PG i and PG ii in the PG complex quality control 1 and the PG complex quality control 2 of example 1 were measured with three different batches of kits according to the "method of step two in example 1", respectively, and the measurement was repeated 10 times, respectively, and the average value (M) and the Standard Deviation (SD) of the measurement results were calculated 30 times for the same concentration. The Coefficient of Variation (CV) was calculated according to equation (2), and the results are shown in table 4.
The results of the performance index measurements obtained by the above method are shown in Table 4.
TABLE 4 Performance index test results
Second, clinical Properties
120 parts of human serum samples are collected from a 301 hospital clinical laboratory, 120 parts of human serum samples are detected by adopting a kit prepared in the first step of example 1 and a method in the second step of example 1, the concentrations of PG I and PG II detected by the kit disclosed by the invention and the concentrations of PG I and PG II detected by the kit disclosed by the invention are subjected to fitting analysis and comparison by adopting a least square method, wherein the serum comparison test result of PG I is shown in figure 3, the fitting correlation r is 0.9803, the serum comparison test result of PG II is shown in figure 4, and the fitting correlation r is 0.9907, so that the kit disclosed by the invention has good correlation with the kit disclosed by the invention, namely the kit disclosed by the invention can accurately detect the concentrations of PG I and PG II.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (10)
1. Pepsinogen I and pepsinogen II joint detection kit, its characterized in that: the kit comprises: the antibody comprises a coating antibody and a labeled antibody, wherein the coating antibody is an anti-PG I monoclonal antibody named monoclonal antibody IA and an anti-PG II monoclonal antibody named monoclonal antibody IIA;
the labeled antibodies are an anti-PG I monoclonal antibody named monoclonal antibody IB and an anti-PG II monoclonal antibody named monoclonal antibody IIB;
the monoclonal antibody IA and the monoclonal antibody IB are two different monoclonal antibodies, and the monoclonal antibody IIA and the monoclonal antibody IIB are two different monoclonal antibodies.
2. The combined pepsinogen I and pepsinogen II detection kit as claimed in claim 1, wherein: the kit comprises the magnetic beads coated by the monoclonal antibody IA, the magnetic beads coated by the monoclonal antibody IB, the monoclonal antibody IB marked by rare earth element C1 and the monoclonal antibody IIB marked by rare earth element C2, wherein the rare earth element C1 and the rare earth element C2 are two different rare earth elements.
3. The combined pepsinogen i and pepsinogen ii detection kit as claimed in claim 2, wherein: the magnetic beads coated with the monoclonal antibody IA and the magnetic beads coated with the monoclonal antibody IB are obtained by performing coupling reaction on the biotin-modified monoclonal antibody IA and the monoclonal antibody IB and streptavidin-modified magnetic beads respectively.
4. The combined pepsinogen I and pepsinogen II detection kit as claimed in claim 3, wherein: the biotin-modified monoclonal antibody IA and the monoclonal antibody IB are obtained by reacting biotin activated by an NHS active group with NH2 on the monoclonal antibody IA and the monoclonal antibody IB respectively.
5. The combined pepsinogen I and pepsinogen II detection kit as claimed in any one of claims 2 to 4, characterized in that: the monoclonal antibody IB marked by the rare earth element C1 and the monoclonal antibody IIB marked by the rare earth element C2 are respectively obtained by reacting rare earth element C1 and rare earth element C2 with a metal chelating agent to obtain a rare earth element C1-metal chelate and a rare earth element C2-metal chelate; the rare earth element C1-metal chelate is obtained by reacting with the monoclonal antibody IB, the rare earth element C2-metal chelate and the monoclonal antibody IIB.
6. The combined pepsinogen I and pepsinogen II detection kit as claimed in any one of claims 2 to 5, characterized in that: the rare earth element C1 and the rare earth element C2 are thulium and lutetium respectively, or the metal chelating agent is DTPA.
7. The combined pepsinogen I and pepsinogen II detection kit as claimed in any one of claims 1 to 6, characterized in that: the kit also comprises a PG composite calibrator solution, wherein the PG composite calibrator solution consists of PG I, PG II and a standard dilution.
8. The combined pepsinogen i and pepsinogen ii detection kit as claimed in any one of claims 1 to 7, characterized in that: the kit also comprises dissociation liquid, a PG composite quality control product and/or immune cleaning liquid.
9. The combined pepsinogen i and ii detection kit as claimed in any one of claims 1 to 8, wherein: the monoclonal antibody IA is a PG I antibody with the number of 8015 of the company media Biochemica, the monoclonal antibody IIA is a PG II antibody with the number of 8101 of the company media Biochemica, the monoclonal antibody IB is a PG I antibody with the number of 8003 of the company media Biochemica, and the monoclonal antibody IIB is a PG II antibody with the number of 8103 of the company media Biochemica.
10. The use of a combined pepsinogen i and pepsinogen ii detection kit as claimed in any one of claims 1 to 9 in any one of the following applications:
1) the application in combined detection of pepsinogen I and pepsinogen II in a sample to be detected;
2) application in preparing products for detecting stomach diseases.
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