CN113109325A - Pepsinogen I enzymatic chemiluminescence detection kit and preparation method and application thereof - Google Patents
Pepsinogen I enzymatic chemiluminescence detection kit and preparation method and application thereof Download PDFInfo
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- CN113109325A CN113109325A CN202110280853.5A CN202110280853A CN113109325A CN 113109325 A CN113109325 A CN 113109325A CN 202110280853 A CN202110280853 A CN 202110280853A CN 113109325 A CN113109325 A CN 113109325A
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- 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
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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
Abstract
The invention discloses a pepsinogen I enzymatic chemiluminescence detection kit and a preparation method and application thereof. The kit comprises a solution of a magnetic microsphere conjugate marked with a PGI monoclonal antibody, a solution of an enzyme-labeled conjugate marked with a PGI antibody and a substrate solution. The kit is characterized in that a pepsinogen antibody is connected to the surface of a magnetic bead microsphere as a solid phase reagent, and an enzyme-labeled antibody sandwich immune complex is formed after pepsinogen in a sample is captured and an enzyme-labeled anti-pepsinogen monoclonal antibody reagent is added. The invention has the advantages that the high-sensitivity chemiluminescence technology and the high-stability magnetic particle technology are combined together, and the prepared kit has high sensitivity and good stability and can meet the clinical requirements; meanwhile, the whole reaction detection process can be matched with a full-automatic detection analyzer, continuous sample adding is realized, the operation time is shortened, the method can be used for large-scale community health examination, and a certain application prospect is realized.
Description
Technical Field
The invention belongs to the technical field of diagnostic kits, and particularly relates to a pepsinogen I enzymatic chemiluminescence detection kit, and a preparation method and application thereof.
Background
In recent years, the incidence of gastric cancer has been on the rise, mainly due to changes in people's lifestyle and diet. 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. Meanwhile, gastric cancer cannot be diagnosed until it reaches an advanced stage in most cases.
Pepsinogen (PG), an inactive precursor of pepsin (a proteolytic enzyme) in gastric juice, is a protein polypeptide chain consisting of 375 amino acids with an average relative molecular mass of 42000, and has 7 groups of pepsin isozymes in human gastric mucosa. The PGI is mainly secreted by the mucous neck cells of the main cells of the gastric gland; PGII, in addition to being secreted by the major cells of the acid-secreting glands of the gastric body and the gastric fundus mucosa, the mucous neck cells of the acid-secreting glands, the mucous cells of the pyloric glands of the cardiac and gastric antrum, and the Brunner gland of the upper duodenum also produce PGII, which is synthesized by the gastric mucosa in an amount of about 25% of the total amount. Pepsinogen i (pgi), an inactive precursor of pepsin, is one of the important indicators of gastric mucositis. The level of PGI may reflect the morphological and functional state of the gastric mucosa. Studies have found that the PGI of gastric cancer patients will be significantly reduced. Measurement of PGI levels may provide a non-invasive and convenient method for large-scale screening for gastric cancer, as compared to conventional endoscopy.
To date, a number of methods for detecting PGI have been developed, including enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), time-resolved fluoroimmunoassay (TRFIA), electrochemiluminescence immunoassay (ECLIA) and direct chemiluminescence immunoassay (dCLIA). However, ELISA is time consuming and poorly reproducible and cannot be used for high throughput assays, RIA assays are radioactive, are prone to environmental contamination, and the cost of instruments and reagents, ECLIA and dCLIA, are high, making them impractical for large-scale health examinations in resource-limited areas.
The existing PGI detection kit needs longer test time for determination, mostly adopts semi-automatic test, is easy to cause sample pollution, cannot continuously sample adding test, has larger influence on storage time by environmental conditions, and has poor stability and sensitivity in long-term storage.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a pepsinogen I enzymatic chemiluminescence detection kit and a preparation method and application thereof. The pepsinogen I (PGI) detection kit is ready to use after being opened, does not need to be prepared, has good measurement repeatability and high sensitivity, can be stored for a long time, can realize full-automatic sample adding, has less sample consumption and saves samples.
The purpose of the invention is realized by at least one of the following technical solutions.
The pepsinogen I determination kit provided by the invention comprises:
the kit comprises a solution of a magnetic microsphere conjugate labeled with a PGI monoclonal antibody, a solution of an enzyme-labeled conjugate labeled with a PGI antibody and a substrate solution.
Further, the magnetic microsphere conjugate labeled with the PGI monoclonal antibody is a conjugate of a magnetic microparticle and a PGI antibody; the diameter of the magnetic particles is 1000-2000 nm; in the solution of the PGI monoclonal antibody-labeled magnetic microsphere conjugate, the concentration of the PGI monoclonal antibody-labeled magnetic microsphere conjugate is 3-5 mug/mL. The magnetic microsphere conjugate marked with the PGI monoclonal antibody is prepared by washing, connecting, sealing and diluting activated magnetic particles with the diameter of 1000-2000 nanometers and the PGI antibody.
Further, the enzyme-labeled conjugate labeled with the PGI antibody is a conjugate of alkaline phosphatase and the PGI antibody; in the solution of the enzyme-labeled conjugate labeled with the PGI antibody, the concentration of the enzyme-labeled conjugate labeled with the PGI antibody is 0.4 to 0.6 mu g/mL.
Further, the substrate solution is AMPDD substrate solution; the manufacturer of the substrate liquid is Wanfu biotechnology, Inc.
Further preferably, the pepsinogen I assay kit provided by the invention further comprises: PGI calibrators. The PGI calibrator is prepared by diluting PGI antigen. Preparing the PGI calibrator, comprising: PGI antigen was diluted with 0.05mol/L Tris-HCl calibrator diluent at pH 7.5 containing 0.15mol/L NaCl, 1% BSA, 0.08% Tween-20, 0.05% ProCline300 at concentrations of 15ng/mL and 80ng/mL and stored at 2-8 ℃.
The method for preparing the pepsinogen I determination kit provided by the invention comprises the following steps:
(1) preparation of magnetic microsphere conjugate labeled with PGI monoclonal antibody:
washing the magnetic particles with borate buffer solution (the washing times are more than 3 times for activation) to obtain activated magnetic particles; adding the activated magnetic particles into an ammonium sulfate buffer solution, uniformly dispersing to obtain a mixed solution 1, then adding a PGI monoclonal antibody to obtain a mixed solution 2, placing the mixed solution 2 in an incubator at 37 ℃ for primary incubation treatment, placing the incubator on a magnetic frame, separating supernatant and precipitate by using magnetic force, taking the supernatant, adding a BSA solution into the supernatant, and performing secondary incubation treatment (sealing the magnetic particles and the antibody) to obtain a solution of a magnetic microsphere conjugate marked with the PGI monoclonal antibody;
(2) preparation of enzyme-labeled conjugates labeled with PGI antibody:
adding alkaline phosphatase and PGI antibody into water, and uniformly mixing to obtain a mixed solution 3; adding a phosphate buffer solution containing glutaraldehyde into the mixed solution, performing first oscillation incubation, then adding a monoethanolamine solution (a termination solution of a PGI antibody and alkaline phosphatase), and performing second oscillation incubation to obtain a mixed solution 4; and (3) dialyzing the mixed solution to obtain an enzyme-labeled conjugate labeled with the PGI antibody.
Further, the magnetic particles in the step (1) are Tosyl magnetic particles; the pH value of the borate buffer solution is 9.0-10.0, and the concentration of the borate buffer solution is 0.8-1.2 mol/L; the pH value of the ammonium sulfate buffer solution is 9.0-10.0, and the concentration of the ammonium sulfate buffer solution is 2-4 mol/L; in the mixed liquid 1, the concentration of the activated magnetic particles is 9-11 mg/mL; in the mixed solution 2, the concentration of the PGI monoclonal antibody is 150-250. mu.g/mL.
Preferably, the concentration of the borate buffer in the step (1) is 0.1 mol/L.
Preferably, the concentration of the ammonium sulfate buffer solution in the step (1) is 3 mol/L.
Preferably, the pH of the borate buffer of step (1) is 9.5.
Preferably, the pH value of the ammonium sulfate buffer solution in the step (1) is 9.5.
Further, the temperature of the first incubation treatment in the step (1) is 37 ℃, and the time of the first incubation treatment is 16-20 hours; the concentration of the BSA solution is 1-1.5 wt%, and the volume ratio of the BSA solution to the supernatant is 1:1-1: 2; the temperature of the second incubation treatment is 37 ℃, and the time of the second incubation treatment is 5-7 hours.
Preferably, the concentration of the BSA solution in step (1) is 1 wt%.
Preferably, the time of the first incubation treatment in step (1) is 18 hours.
Preferably, the time of the second incubation treatment in step (1) is 6 hours.
Further, after the second incubation treatment in step (1), the magnetic microsphere conjugate labeled with the PGI monoclonal antibody may be washed with a Tris-HCl buffer. The Tris-HCl buffer solution is 0.025mol/L Tris-HCl buffer solution (pH 7.0-8.0) containing 0.15mol/L NaCl and 0.05% Tween 20.
Further, after the second incubation treatment in step (1), the magnetic microsphere conjugate labeled with the PGI monoclonal antibody may be diluted with a Tris-HCl buffer. The Tris-HCl buffer solution is 0.05mol/L Tris-HCl buffer solution (pH 7.0-8.0) containing 0.15mol/L NaCl, 0.5% BSA, 0.08% Tween-20, and 0.05% ProCline 300.
Further, in the mixed solution 3 in the step (2), the concentration of alkaline phosphatase is 1-3mg/mL, and the concentration of PGI antibody is 3-5 mg/mL; in the phosphate buffer solution containing glutaraldehyde, the concentration of glutaraldehyde is 1-2%; the volume ratio of the phosphate buffer solution containing glutaraldehyde to the mixed solution 3 is 1:1-1: 2; the temperature of the first shaking incubation is 37 ℃, and the time of the first shaking incubation is 3-5 h; the concentration of the monoethanolamine solution is 1-2 mol/L; the volume ratio of the monoethanolamine solution to the phosphate buffer solution containing glutaraldehyde is 1:1-1: 2; the time of the second shaking incubation is 1-3h, and the temperature of the second shaking incubation is room temperature; the cut-off molecular weight of a dialysis bag adopted in the dialysis treatment is 40kD, and the dialysis treatment time is 3-5 h.
Preferably, the concentration of glutaraldehyde in the glutaraldehyde-containing phosphate buffer is 1%, and the pH of the glutaraldehyde-containing phosphate buffer is 7.0 to 8.0.
Further, the concentration of the monoethanolamine solution in the step (2) is 1 mol/L.
Preferably, the protective agent of the PGI antibody-labeled enzyme-labeled conjugate in step (2) may be glycerol and 1% BSA in equal volumes.
Preferably, the enzyme-labeled conjugate labeled with the PGI antibody of step (2) may be diluted to a desired concentration using a buffer; the buffer solution formula contains 0.15mol/L NaCl, 1% BSA, 0.08% Tween-20 and 0.1mmol/LZnCl2，5mmol/LMgCl2·6H2O, 0.05% ProCline300 in 0.05mol/LMES buffer, pH 5-7.
Preferably, the time for the first shaking incubation in step (2) is 4 h.
Preferably, the time for the second shaking incubation in step (2) is 2 h.
The detection method of the pepsinogen I (PGI) determination kit provided by the invention comprises the following steps:
adding a solution of a magnetic microsphere conjugate labeled with a PGI monoclonal antibody and a solution of an enzyme-labeled conjugate labeled with a PGI antibody into a reaction container, adding a sample to be tested, performing primary incubation treatment, cleaning, adding a substrate solution, performing secondary incubation treatment, cleaning, measuring a luminescence value, calculating a formula according to a master batch (see fig. 2), and calculating the concentration.
Further, the volume ratio of the solution of the magnetic microsphere conjugate labeled with the PGI monoclonal antibody to the solution of the enzyme-labeled conjugate labeled with the PGI antibody is 1:0.8-1-: 1.2; the volume ratio of the solution of the enzyme-labeled conjugate labeled with the PGI antibody to the sample to be detected is 4.8:1-5.2: 1; the time of the first incubation treatment is 5-10 min; the volume ratio of the substrate liquid to the sample to be detected is 18: 1-20: 1; the time of the second incubation treatment is 5-10 min.
The kit provided by the invention is characterized in that a pepsinogen antibody is connected to the surface of a magnetic bead microsphere as a solid phase reagent, and an enzyme-labeled antibody sandwich immune complex is formed after pepsinogen in a sample is captured and an enzyme-labeled anti-pepsinogen monoclonal antibody reagent is added.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the pepsinogen I determination kit provided by the invention is ready to use after being opened, does not need to be prepared, has good measurement repeatability and high sensitivity, can be stored for a long time, can realize full-automatic sample adding, has small sample consumption and saves samples.
(2) The pepsinogen I determination kit provided by the invention combines a high-sensitivity chemiluminescence technology with a strong-stability magnetic particle technology, and the prepared kit has high sensitivity and good stability and can meet clinical requirements; meanwhile, the whole reaction detection process can use a full-automatic detection analyzer in a matching way, continuous sample adding is realized, the operation time is shortened, and the method can be used for large-scale community health examination and has a certain application prospect.
Drawings
FIG. 1 is a schematic diagram illustrating the detection principle of the immunoassay kit according to the embodiment of the present invention;
FIG. 2 is a standard curve of the pepsinogen I (PGI) assay kit in an embodiment of the present invention;
FIG. 3 is a diagram illustrating the results of a PGI comparison test according to an embodiment of the present invention.
Detailed Description
The following examples are presented to further illustrate the practice of the invention, but the practice and protection of the invention is not limited thereto. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.
Example 1
A pepsinogen I assay kit comprising (1) a solution of magnetic microsphere conjugates labeled with PGI monoclonal antibodies; (2) a solution of an enzyme-labeled conjugate labeled with a PGI antibody; (3) substrate solution; (4) PGI calibrators.
The magnetic microsphere conjugate marked with the PGI monoclonal antibody is a conjugate of a magnetic particle and a PGI antibody; the diameter of the magnetic particles is 1000-2000 nm; in the solution of the magnetic microsphere conjugate labeled with the PGI monoclonal antibody, the concentration of the magnetic microsphere conjugate labeled with the PGI monoclonal antibody is 4 mug/mL.
In the solution of the enzyme-labeled conjugate labeled with the PGI antibody, the concentration of the enzyme-labeled conjugate labeled with the PGI antibody was 0.5. mu.g/mL.
The substrate solution is AMPDD substrate solution; the manufacturer of the substrate liquid is Wanfu biotechnology, Inc.
The solution of the magnetic microsphere conjugate marked with the PGI monoclonal antibody is prepared by washing, connecting, sealing and diluting activated magnetic particles with the diameter of 1000-2000 nanometers and the PGI antibody; the enzyme-labeled conjugate labeled with the PGI antibody is prepared by labeling the PGI antibody with alkaline phosphatase; the PGI calibrator is prepared by diluting a PGI antigen;
the formula of the buffer solution for magnetic particle activation is 0.1mol/L borate buffer solution, and the pH value is 9.5.
The formula of the buffer solution for combining the magnetic particles and the PGI monoclonal antibody is 3mol/L ammonium sulfate buffer solution, and the pH value is 9.5.
The formula of the buffer solution for blocking the magnetic particles and the PGI monoclonal antibody is 1 wt% BSA solution.
The formulation of the washing buffer solution after the magnetic particles are combined with the PGI monoclonal antibody is 0.15mol/L NaCl, 0.025mol/L Tris-HCl buffer solution of 0.05% Tween20, and the pH value is 7.5.
The magnetic particle buffer solution formula is 0.05mol/L Tris-HCl buffer solution containing 0.15mol/L NaCl, 0.5% BSA, 0.08% Tween-20 and 0.05% ProCline300, and the pH value is 7.5.
The buffer formula for combining the PGI antibody and the alkaline phosphatase is 0.1mol/L phosphate buffer containing 1 wt% of glutaraldehyde and the pH value is 7.5.
The formulation of the PGI antibody and alkaline phosphatase stop solution is 1mol/L monoethanolamine solution.
The protective agent of the enzyme-labeled conjugate labeled with the PGI antibody is glycerol and 1 wt% BSA with equal volume.
The buffer solution formula of the enzyme-labeled conjugate labeled with the PGI antibody during dilution comprises 0.15mol/L NaCl, 1% BSA, 0.08% Tween-20 and 0.1mmol/LZnCl2，5mmol/LMgCl2·6H2O, 0.05% ProCline300 in 0.05mol/LMES buffer, pH 6.0.
The PGI calibrator diluent buffer solution formula is 0.05mol/L Tris-HCl buffer solution containing 0.15mol/L NaCl, 1% BSA, 0.08% Tween-20 and 0.05% ProCline300, and the pH value is 7.5.
Example 2
A preparation method of a PGI-based kit comprises the following steps:
preparation of a solution of magnetic microsphere conjugates labeled with PGI monoclonal antibodies: 1mL of Tosyl magnetic microparticles were washed 3 times with 0.1mol/L of a borate buffer pH 9.5 for activation; suspending the activated magnetic particles in 550 mu L of 3mol/L ammonium sulfate buffer solution with the pH value of 9.5; then 200. mu.L of 1mg/mLPGI monoclonal antibody solution is added into the ammonium sulfate binding buffer solution; placing the test tube in an incubator at 37 ℃ for 18 hours of rotary incubation, after reaction, placing the test tube on a magnetic frame to separate supernatant, then adding 100 mu L of 1 wt% BSA solution to the supernatant, and continuing to perform rotary incubation for 6 hours at 37 ℃; after the reaction, washing 5 times with 0.025mol/L Tris-HCl buffer solution with pH 7.2 containing 0.15mol/L NaCl and 0.05% Tween 20; finally, the magnetic particles labeled with PGI monoclonal antibody were diluted with 0.05mol/L Tris-HCl buffer solution containing 0.15mol/L NaCl, 0.5% BSA, 0.08% Tween-20, 0.05% ProCline300 at pH 7.5 to a bead concentration of 4. mu.g/mL and stored at 2-8 ℃ for later use.
Preparation of a solution of an enzyme-labeled conjugate labeled with PGI antibody: adding alkaline phosphorusThe enzyme and PGI antibody were dissolved in ultrapure water and diluted to 4mg/mL and 8mg/mL, respectively. mu.L of 4mg/mL alkaline phosphatase solution was transferred to a 1.5mL test tube and mixed with 250. mu.L of 8mg/mL PGI antibody solution, and secondly, 0.5mL of 0.1mol/L phosphate buffer solution of pH 7.4 containing 1 wt% glutaraldehyde was added to the solution. The resulting mixture was incubated at 37 ℃ for 4h with gentle shaking. Then 0.1mL of a 1mol/L monoethanolamine solution was added to the mixture followed by incubation at room temperature for 2h with shaking; dialyzing the mixture at 4 ℃ with a PBS solution, wherein the dialysis treatment adopts a dialysis bag with the molecular weight cutoff of 40kD, and the dialysis treatment time is 4 h; after dialysis, the enzyme-labeled PHI antibody was transferred to a test tube and mixed with an equal volume of glycerol and 1 wt% BSA. Finally, the mixture was washed with a pH of 6.0 solution containing 0.15mol/L NaCl, 1 wt% BSA, 0.08% Tween-20, 0.1mmol/LZnCl2，5mmol/LMgCl2·6H2O, 0.05 wt% ProCline300 in 0.05mol/LMES buffer solution the enzyme-labeled complex was diluted 2000-fold to obtain a solution of 0.5. mu.g/mL of enzyme-labeled conjugate labeled with PGI antibody, which was placed at 2-8 ℃ for later use.
PGI calibrator preparation: PGI antigen was diluted with 0.05mol/L Tris-HCl calibrator diluent at pH 7.5 containing 0.15mol/L NaCl, 1 wt% BSA, 0.08% Tween-20, 0.05 wt% ProCline300 at concentrations of 15ng/mL and 80ng/mL and stored at 2-8 ℃.
The substrate solution is AMPDD substrate solution; the manufacturer of the substrate liquid is Wanfu biotechnology, Inc.
Example 3 evaluation of the Performance of the kit of the present invention
1. Standard curve
Diluting PGI antigen with standard substance diluent to obtain calibrator solutions S0-S6 with different concentrations of 0, 3, 6, 12, 25, 50, and 100ng/mL, and storing at-20 deg.C. Then, the kit provided by the embodiment of the invention is used for detecting the calibration products, and the corresponding luminous intensity values of the calibration products are respectively read. The concentration is used as the abscissa and the luminous intensity is used as the ordinate to perform fitting to obtain a standard curve, and the result is shown in fig. 2.
2. Minimum limit of detection
The method comprises the steps of detecting by using a zero-concentration calibrator diluent as a sample, repeatedly measuring for 20 times to obtain an optical signal value of 20 measurement results, calculating an average value (M) and a Standard Deviation (SD) by using a calculator to obtain M +2SD, carrying out two-point regression fitting according to the concentration between a zero-concentration enterprise linear reference product and an adjacent concentration calibrator and the optical signal value result to obtain a linear equation (y is 7882x +554.2), substituting the optical signal value of the M +2SD into an equation y is 7882x +554.2, and obtaining a corresponding concentration value which is a minimum detection limit, wherein the result is shown in Table 1.
2. Accuracy of
A low-value sample of PGI antigen (namely, serum containing PGI antigen at a concentration of 80ng/mL, hereinafter collectively referred to as low-value serum) was added to high-value serum (serum containing PGI antigen at a concentration of 150 ng/mL), the volume ratio between the high-value serum and the low-value serum was 1:9, the detection with the kit of the present embodiment was repeated twice, the average value was taken, and the results were calculated according to the formula (1), and the results are shown in Table 1.
In the formula: r-recovery rate; v0-volume of low value serum; v-volume of high value serum; c, adding the high-value serum into the low-value serum and then detecting the concentration; c 0-concentration of low value serum; cs-concentration of high value serum.
3. Precision degree
The kits provided in the examples of the invention were subjected to an intra-and inter-batch precision assay using two different concentrations of PGI calibrators (15ng/mL, 80ng/mL) over a period of 3 days, with 6 replicate assays for intra-batch precision and 6 different day assays for inter-batch precision, with the results given in table 1.
4. Interfering substances
4 serum samples containing PGI antigen with the concentration of 16ng/mL are selected, the following interference substances bilirubin (0.22mg/mL), triglyceride (33mg/mL), hemoglobin (5mg/mL) and total protein (13.2mg/mL) are respectively added to the detection samples, and the kit provided by the embodiment of the invention is used for detecting each sample and reading the luminous intensity. Whether the deviation of the detection result from the control sample (the control sample is a serum sample containing PGI antigen at a concentration of 16ng/ml without adding interfering substances) is observed to be less than or equal to 10% is shown in Table 1.
TABLE 1 results of performance tests of the kit of the invention
Example 4 clinical Performance of the kits of the invention
Serum samples are collected from blood donors subjected to normal physical examination in a physical examination center, and 95 physical examination samples are screened and detected by using the kit provided by the embodiment of the invention. The detection method (shown in fig. 1) includes:
adding a solution of a magnetic microsphere conjugate marked with a PGI monoclonal antibody and a solution of an enzyme-labeled conjugate marked with a PGI antibody into a reaction container, wherein the volume ratio of the solution of the magnetic microsphere conjugate marked with the PGI monoclonal antibody to the solution of the enzyme-labeled conjugate marked with the PGI antibody is 1:1, adding a sample to be tested, carrying out primary incubation treatment, wherein the time of the primary incubation treatment is 5min, cleaning, adding a substrate solution, wherein the volume ratio of the substrate solution to the sample to be tested is 20:1, carrying out secondary incubation treatment, wherein the time of the secondary incubation treatment is 5min, cleaning, measuring a luminous value, and calculating the concentration according to a master curve calculation formula (the formula is shown in figure 2).
The concentration of the sample was measured using the Yapek kit (measurement was carried out according to the instructions of the kit).
The detection concentration obtained by the kit prepared in the embodiment of the invention is analyzed and compared with the detection result concentration of an Yapei company kit (model ARCHITECT Pepsinogen I), wherein the PGI clinical relevance is R20.977, indicating that the kit of the present invention has a good correlation with the international well-known brand kit (yapei corporation kit). The clinical relevance results are shown in figure 3.
The PGI quantitative detection kit provided by the invention has the advantages of rapidness, high flux, high sensitivity, high precision, high specificity and the like, can meet the requirement of clinical PGI quantitative detection, provides a tool for large-scale screening of stomach function health, has relatively low price and high cost performance compared with imported reagents, can relieve the economic burden of patients, and has certain application value.
The above examples are only preferred embodiments of the present invention, which are intended to be illustrative and not limiting, and those skilled in the art should understand that they can make various changes, substitutions and alterations without departing from the spirit and scope of the invention.
Claims (10)
1. A pepsinogen I determination kit, which is characterized by comprising:
the kit comprises a solution of a magnetic microsphere conjugate labeled with a PGI monoclonal antibody, a solution of an enzyme-labeled conjugate labeled with a PGI antibody and a substrate solution.
2. The pepsinogen I assay kit as claimed in claim 1, wherein said magnetic microsphere conjugate labeled with a PGI monoclonal antibody is a conjugate of a magnetic microparticle and a PGI antibody; the diameter of the magnetic particles is 1000-2000 nm; in the solution of the PGI monoclonal antibody-labeled magnetic microsphere conjugate, the concentration of the PGI monoclonal antibody-labeled magnetic microsphere conjugate is 3-5 mug/mL.
3. The pepsinogen I assay kit as claimed in claim 1, wherein said enzyme-labeled conjugate labeled with PGI antibody is a conjugate of alkaline phosphatase and PGI antibody; in the solution of the enzyme-labeled conjugate labeled with the PGI antibody, the concentration of the enzyme-labeled conjugate labeled with the PGI antibody is 0.4 to 0.6 mu g/mL.
4. The pepsinogen I assay kit as claimed in claim 1, wherein said substrate solution is AMPDD substrate solution; the manufacturer of the substrate liquid is Wanfu biotechnology, Inc.
5. A method for preparing the pepsinogen I assay kit as defined in any one of claims 1 to 4, comprising the steps of:
(1) preparation of magnetic microsphere conjugate labeled with PGI monoclonal antibody:
washing the magnetic particles with borate buffer solution to obtain activated magnetic particles; adding the activated magnetic particles into an ammonium sulfate buffer solution, uniformly dispersing to obtain a mixed solution 1, then adding a PGI monoclonal antibody to obtain a mixed solution 2, carrying out primary incubation treatment, magnetically separating a supernatant and a precipitate, taking the supernatant, adding a BSA solution into the supernatant, and carrying out secondary incubation treatment to obtain a solution of a magnetic microsphere conjugate labeled with the PGI monoclonal antibody;
(2) preparation of enzyme-labeled conjugates labeled with PGI antibody:
adding alkaline phosphatase and PGI antibody into water, and uniformly mixing to obtain a mixed solution 3; adding a phosphate buffer solution containing glutaraldehyde into the mixed solution, performing first oscillation incubation, then adding a monoethanolamine solution, and performing second oscillation incubation to obtain a mixed solution 4; and (3) dialyzing the mixed solution to obtain a solution of the enzyme-labeled conjugate labeled with the PGI antibody.
6. The method for preparing a pepsinogen I assay kit according to claim 5, wherein the magnetic particles in step (1) are Tosyl magnetic particles; the pH value of the borate buffer solution is 9.0-10.0, and the concentration of the borate buffer solution is 0.8-1.2 mol/L; the pH value of the ammonium sulfate buffer solution is 9.0-10.0, and the concentration of the ammonium sulfate buffer solution is 2-4 mol/L; in the mixed liquid 1, the concentration of the activated magnetic particles is 9-11 mg/mL; in the mixed solution 2, the concentration of the PGI monoclonal antibody is 150-250. mu.g/mL.
7. The method for preparing a pepsinogen I assay kit according to claim 5, wherein the temperature of the first incubation treatment in the step (1) is 37 ℃, and the time of the first incubation treatment is 16-20 hours; the concentration of the BSA solution is 1-1.5 wt%, and the volume ratio of the BSA solution to the supernatant is 1:1-1: 2; the temperature of the second incubation treatment is 37 ℃, and the time of the second incubation treatment is 5-7 hours.
8. The method for preparing a pepsinogen I assay kit according to claim 5, wherein in the mixed solution 3 of step (2), the concentration of alkaline phosphatase is 1-3mg/mL, and the concentration of PGI antibody is 3-5 mg/mL; in the phosphate buffer solution containing glutaraldehyde, the concentration of glutaraldehyde is 1-2%; the volume ratio of the phosphate buffer solution containing glutaraldehyde to the mixed solution 3 is 1:1-1: 2; the temperature of the first shaking incubation is 37 ℃, and the time of the first shaking incubation is 3-5 h; the concentration of the monoethanolamine solution is 1-2 mol/L; the volume ratio of the monoethanolamine solution to the phosphate buffer solution containing glutaraldehyde is 1:1-1: 2; the time of the second shaking incubation is 1-3h, and the temperature of the second shaking incubation is room temperature; the cut-off molecular weight of a dialysis bag adopted in the dialysis treatment is 40kD, and the dialysis treatment time is 3-5 h.
9. The method for detecting a pepsinogen I assay kit as claimed in any one of claims 1 to 4, characterized in that it comprises the following steps:
adding a solution of a magnetic microsphere conjugate marked with a PGI monoclonal antibody and a solution of an enzyme-labeled conjugate marked with a PGI antibody into a reaction container, adding a sample to be tested, performing primary incubation treatment, cleaning, adding a substrate solution, performing secondary incubation treatment, cleaning, measuring a luminous value, and calculating the concentration according to a master batch calculation formula.
10. The detection method of the pepsinogen I detection kit according to the claim 9, characterized in that the volume ratio of the solution of the magnetic microsphere conjugate labeled with the PGI monoclonal antibody to the solution of the enzyme-labeled conjugate labeled with the PGI antibody is 1:0.8-1: 1.2; the volume ratio of the solution of the enzyme-labeled conjugate labeled with the PGI antibody to the sample to be detected is 4.8:1-5.2: 1; the time of the first incubation treatment is 5-10 min; the volume ratio of the substrate liquid to the sample to be detected is 18: 1-20: 1; the time of the second incubation treatment is 5-10 min.
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