Detection method of gastrin 17

Abstract

The invention relates to the technical field of biology, in particular to a detection method of gastrin 17. According to the invention, the gastrin 17 in serum / plasma is quantified by an immune enrichment method. According to the pretreatment method, the gastrin 17 in the serum is enriched by adopting the magnetic beads coated with the gastrin 17 specific antibody, the enrichment rate close to 100% can be obtained by uniformly mixing and incubating for 20 minutes at a low speed, the magnetic bead immune enrichment process is optimized in detail, the pretreatment operation is simple and convenient, the enrichment effect is good, and the detection sensitivity of the method is high (2pg / ml). Liquid phase conditions are optimized and determined, and a detection method with excellent performance is developed; according to the method, the magnetic bead enrichment efficiency and the absolute recovery rate are carried out, linearity, sensitivity and standard recovery performance index confirmation is carried out, and the performance is excellent.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for detecting gastrin 17. Background Art

[0002] Gastrin is a mixture of peptides and amino acid derivatives of different lengths, such as gastrin 14, gastrin 17, gastrin 34, gastrin 52, and gastrin 71. The total amount of gastrin 17 and gastrin 34 in peripheral blood circulation accounts for more than 90% of the total gastrin. The above-mentioned gastrin subtypes usually exist in sulfate and non-sulfate forms. Gastrin 17 is one of the sensitive indicators reflecting the endocrine function of the gastric antrum, which can indicate the atrophy of the gastric antrum mucosa or the presence of abnormal proliferation. Gastrin 17 itself also promotes the occurrence and development of gastric cancer.

[0003] For the clinical detection of gastrin 17, the common methods currently include radioimmunoassay, enzyme immunoassay, and magnetic particle chemiluminescence. Radioimmunoassay has been gradually eliminated due to shortcomings such as long reporting time, unstable results, and isotope contamination; the latter three detection methods are fast and have high throughput, and they are dominant. However, because the active sites (the four terminal amino acids) of various gastrin subtypes and their sulfate structures are the same, they are subject to cross-interference during the detection process. Liquid chromatography-tandem mass spectrometry has the characteristics of high sensitivity and high specificity, which can overcome problems such as cross-reaction of analogs in immunological methods. Among them, isotope dilution liquid chromatography-tandem mass spectrometry has the highest traceability level, and the measured values ​​can be directly traced back to SI units. At present, this technology is considered to be the first reference measurement principle for the establishment of reference measurement procedures in laboratory medicine standardization.

[0004] At present, the liquid chromatography-mass spectrometry detection of gastrin 17 in biological samples has a method published by Peking Union Medical College Hospital: the joint detection of gastrin 17 and gastrin 34 in human serum using solid phase extraction MAX and WAX pre-treatment; the recovery of gastrin spiked with 85% to 115%, and the precision of 4% to 15%, meet the requirements of rapid and joint detection of clinical mass spectrometry methods, but do not meet the standards of reference methods.

[0005] From the current research literature, it can be found that the current clinical mass spectrometry detection of gastrin mostly adopts the standard curve method; the absolute recovery rate of solid phase extraction is as low as 15% to 30% due to polypeptide adsorption, and the sensitivity is insufficient.

[0006] Therefore, it is necessary to provide a detection method for accurately determining the content of gastrin 17 in human serum. Summary of the invention

[0007] In view of this, the present invention provides a method for detecting gastrin 17.

[0008] The present invention provides a method for detecting gastrin 17. When establishing a gastrin 17 liquid-mass spectrometry method, the present invention adopts affinity chromatography pretreatment technology (magnetic microspheres coated with gastrin 17-specific antibodies) to enrich gastrin 17 in human blood, with little pretreatment loss (absolute recovery rate of about 80%). Performance confirmation shows that the gastrin 17 spiked recovery rate of the patented technology is 98.85% to 99.77%, the precision is 1.56% to 2.86%, and the quantitative limit is 5.14 pg / ml on a Waters TQ-S instrument, and it is expected to become a good candidate reference measurement procedure.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a method for detecting gastrin 17, comprising the following steps:

[0011] Step 1: Weigh the sample / standard and internal standard on a balance, and gently shake to mix to obtain the processed sample / standard;

[0012] Step 2: taking the treated sample / standard to be tested and incubating it with the magnetic bead suspension coated with gastrin 17-specific antibody after the first washing, and then washing it for the second time, eluting it, re-dissolving it, and detecting it;

[0013] The target substances include gastrin-17.

[0014] In some specific embodiments of the present invention, the target-free serum comprises target-free human serum.

[0015] In some specific embodiments of the present invention, the volume ratio of the processed sample to be tested in step 2 to the magnetic bead suspension coated with gastrin 17-specific antibody is (20 μl-1 ml): 150 μl.

[0016] The magnetic bead suspension coated with gastrin 17-specific antibody is 100-300 μl;

[0017] Preferably, the magnetic bead suspension coated with gastrin 17-specific antibody is fixed at 100-200 μl;

[0018] In some specific embodiments of the present invention, the volume of the magnetic bead suspension coated with gastrin 17-specific antibody is 100-200 μl, preferably 150 μl.

[0019] In some specific embodiments of the present invention, step 2 is specifically as follows: blow the magnetic microparticle suspension evenly, take 150 μl and let it stand for 1 min, discard the supernatant, add 500 μl PBS solution to wash the magnetic beads and residual sample, let it stand for 1 min, discard the supernatant, and use 100 μl blank serum to transfer the washed magnetic beads to the treated sample / standard to be tested.

[0020] In some specific embodiments of the present invention, the incubation in step 2 includes shaking incubation at 18°C ​​to 26°C and 500 rpm for 20 min.

[0021] In some specific embodiments of the present invention, the detection in step 2 comprises mass spectrometry detection and / or liquid phase detection;

[0022] The mobile phase A of the liquid phase detection is acetonitrile solution, the mobile phase B is 0.2% ammonia aqueous solution, and the time is 11 minutes.

[0023] In some specific embodiments of the present invention, the elution solution used in step 2 is an aqueous solution containing 0.5% formic acid and 30% acetonitrile.

[0024] In some specific embodiments of the present invention, the volume of the elution solution used for elution is 100 μl.

[0025] In some specific embodiments of the present invention, the re-dissolving solution used in step 2 comprises an aqueous solution containing 0.5% ammonia water and 10% acetonitrile.

[0026] In some specific embodiments of the present invention, the volume of the reconstitution solution used for the reconstitution is 75 μl to 90 μl.

[0027] In some specific embodiments of the present invention, the first cleaning uses a 10 mM PBS solution; the second cleaning uses a 10 mM PBS solution and pure water, respectively.

[0028] In some specific embodiments of the present invention, when the sample to be tested in step 1 is less than 500 μl, blank serum is added to 500 μl. The purpose is to reduce adsorption and weaken the matrix effect.

[0029] In some specific embodiments of the present invention, the mass spectrometry detection: capillary voltage 2.8 kV, ion source temperature 150° C., desolvation gas temperature 550° C., desolvation gas flow 1000 L / Hr;

[0030] The injection volume of the liquid phase detection was 20 μl, the flow rate was 0.2 ml / min, and the column temperature was 45° C.

[0031] In some specific embodiments of the present invention, the method for detecting gastrin 17 specifically comprises the following steps:

[0032] Step 1): After weighing the sample / standard and the internal standard on a balance, gently shake and mix to obtain the processed sample / standard. When the sample / standard is less than 500 μl, fill it with blank serum to 500 μl;

[0033] Step 2): Place 150 μl of magnetic bead suspension (coated with gastrin 17-specific antibody) on a magnetic rack and discard the supernatant after 1 min. Then wash and mix with 0.5 ml PBS solution (10 mM), place on the magnetic rack again for 1 min and discard the supernatant;

[0034] Step 3): Use 100 μl of blank serum to transfer the washed magnetic beads to the processed sample / standard to be tested; obtain the incubated sample to be tested;

[0035] The incubation includes: incubating at 18°C ​​to 26°C and 500 rpm for 20 minutes;

[0036] Step 4): After incubation, the sample to be tested is placed on a magnetic plate. After 1 minute, the sample is taken out and discarded. 0.5 ml (10 mM) PBS solution is added to wash the residual serum. After discarding the PBS solution in the tube, 0.5 ml of pure water is added to wash the residual PBS. The sample is placed on a magnetic plate for 1 minute, and the pure water in the tube is discarded.

[0037] Step 5): Add 100 μl of 0.5% formic acid and 30% acetonitrile aqueous solution and mix well, let stand for 10 min to elute the target, place on a magnetic rack and let stand for 1 min, remove the supernatant and transfer to a new 1.5 ml low adsorption protein centrifuge tube;

[0038] Step 6): blow dry with nitrogen for 20 min, add 90 μl of 0.5% ammonia and 5% acetonitrile solution to re-dissolve;

[0039] Step 7): After centrifugation at 13000 r / min for 10 min, liquid chromatography-mass spectrometry analysis was performed.

[0040] In some specific embodiments of the present invention, the magnetic bead suspension is a carboxyl magnetic bead stock solution.

[0041] In some specific embodiments of the present invention, liquid-mass analysis includes mass spectrometry detection and / or liquid phase detection.

[0042] In some specific embodiments of the present invention, the mobile phase A of the liquid phase detection is acetonitrile solution, the mobile phase B is 0.2% ammonia aqueous solution, and the time is 11 minutes.

[0043] In some specific embodiments of the present invention, the mass spectrometry detection: capillary voltage 2.8 kV, ion source temperature 150° C., desolvation gas temperature 550° C., desolvation gas flow 1000 L / Hr;

[0044] The injection volume of the liquid phase detection was 20 μl, the flow rate was 0.2 ml / min, and the column temperature was 45° C.

[0045] The present invention provides a method for detecting gastrin 17. The present invention uses an immunoenrichment method to quantify gastrin 17 in serum / plasma. In terms of the pretreatment method, the present invention uses magnetic beads coated with gastrin 17-specific antibodies to enrich gastrin 17 in serum. 20 minutes of low-speed mixing can obtain an enrichment rate close to 100%. The present invention optimizes the magnetic bead immunoenrichment process in detail, and the pretreatment operation is simple, the enrichment effect is good, and the method has high detection sensitivity (2pg / ml). The liquid phase conditions are optimized and determined, and a detection method with excellent performance is developed; the method is confirmed by the magnetic bead enrichment efficiency, absolute recovery rate, linearity, sensitivity, and spike recovery performance indicators, and the performance is excellent. In terms of result processing and calculation, the gastrin 17 liquid mass spectrometry method uses the three-isotope dilution method, i.e., the bracket method, which is unique to the reference method, to process and calculate the results in a quantitative manner, thereby ensuring the accuracy and reliability of the calculation results of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0047] Figure 1 14 shows the linear graph of the standard curve in Example 7. DETAILED DESCRIPTION

[0048] The present invention discloses a method for detecting gastrin 17. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0049] The present invention uses a pretreatment method of enriching gastrin 17 in human blood by magnetic microspheres coated with gastrin 17-specific antibodies, combines the quantitative technology of an isotope dilution liquid chromatography tandem mass spectrometry platform, and adopts a bracket method measurement model to accurately determine the gastrin 17 content in human blood.

[0050] Explanation of terms: 1. Reference measurement procedure: A measurement procedure that has been thoroughly analyzed and studied and produces values ​​with measurement uncertainties commensurate with its intended use, in particular when evaluating the correctness of other measurement procedures for measuring the same quantity and when characterizing reference materials.

[0051] 2. Affinity chromatography: refers to a chromatography technique that uses a chromatographic medium covalently linked to a specific ligand to separate the target protein or other molecules that can specifically bind to the ligand in a protein mixture.

[0052] When establishing the gastrin 17 liquid-mass spectrometry method, the present invention adopts affinity chromatography pretreatment technology (magnetic microspheres coated with gastrin 17-specific antibodies) to enrich gastrin 17 in human blood, with small pretreatment loss (absolute recovery rate of about 80%). Performance confirmation shows that the gastrin 17 spike recovery rate of the present invention technology is 98.85% to 99.77%, the precision is 1.56% to 2.86%, and the quantitative limit is 5.14 pg / ml on the Waters TQ-S instrument. It is expected to become a good candidate reference measurement procedure.

[0053] The present invention is based on magnetic bead immunoenrichment pretreatment combined with isotope dilution liquid chromatography tandem mass spectrometry platform, and the dilution liquid chromatography tandem mass spectrometry platform is used to accurately determine the content of gastrin 17 in human serum.

[0054] 1. After comparison, the extended elution gradient liquid phase condition was adopted, which has the advantage of a carryover contamination rate of <0.1%.

[0055] 2. The entire pre-treatment process of using a magnetic particle suspension coated with anti-gastrin 17 monoclonal antibody to enrich G17 in human serum.

[0056] 3. In the sample pretreatment method, the amount of magnetic microparticle suspension (150 μl), the concentration and amount of PBS solution, the amount of water, the room temperature incubation time (20 min), and the elution of the target analyte on the magnetic beads are achieved. During the pretreatment process, 100 μl of the above elution solution is added, and after nitrogen drying, 60 μl to 200 μl of the reconstitution solution is added for reconstitution.

[0057] 4. The triple isotope dilution method, i.e., the bracket method, which is unique to the reference method, was used for G17 liquid quality testing.

[0058] 1) A liquid quality detection method for G17, comprising a. weighing samples: according to the initial concentration of the sample to be tested, weighing appropriate amounts of calibration solution and the sample to be tested, adding a fixed amount (e.g., 60 μl) of internal standard solution to each. Adding human serum without analyte to the calibration solution and the sample to be tested to make up to 500 μl, mixing and waiting for processing.

[0059] b. Cleaning before enrichment: Mix the magnetic bead suspension, take 150 μl into a low-adsorption centrifuge tube, place it on a magnetic plate, remove the supernatant, add 0.5 ml PBS (10 mM) solution, mix for 1 min, place it on a magnetic plate, remove the supernatant, add 0.5 ml water, mix for 1 min, place it on a magnetic plate, and remove the supernatant.

[0060] c. Enrichment reaction: Add the pre-prepared standard solution to be treated and the sample solution to be tested; incubate at room temperature (18°C to 26°C) and 500 rpm for 20 minutes.

[0061] d. Post-enrichment washing: Place on a magnetic plate and remove the supernatant after 5 minutes; add 0.5 ml PBS (10 mM) solution to the centrifuge tube to wash the magnetic beads for 1 minute, and then aspirate the supernatant again; add 0.5 ml water, mix for 2 minutes, place on a magnetic plate, and aspirate the supernatant.

[0062] e. Elution of target substance: Add 100 μl of 0.5% formic acid and 30% acetonitrile aqueous solution, mix well and let stand for 10 minutes, then place on a magnetic rack for 1 minute, remove the eluate and add it to a new low-adsorption centrifuge tube.

[0063] f. Solution replacement: blow dry with nitrogen for 20 min, add 90 μl of 0.5% ammonia 10% acetonitrile solution to re-dissolve.

[0064] g. Centrifuge at 13000r / min for 10min, transfer the supernatant to a 96-well sample plate for liquid-mass analysis.

[0065] 2) The magnetic beads are carboxyl magnetic beads stock solution.

[0066] 3) Liquid phase detection: 10% acetonitrile and 0.5% ammonia in the G17 complex solution.

[0067] 4) The volume of magnetic microparticle suspension is 100μl-200μl.

[0068] The sample reconstitution solution is 0.1%-2% ammonia and 0%-10% acetonitrile aqueous solution.

[0069] In some schemes, the eluent can adjust the acid concentration and acid type (0.5% to 1% formic acid, 0.5% to 2% acetic acid, 0.1% trifluoroacetic acid), and achieve elution with different proportions of organic phase (10% to 60% methanol, 20% to 60% acetonitrile).

[0070] In some embodiments, gastrin 17-specific antibodies or non-specific antibodies or solid phase extraction materials are linked to the surface of magnetic particles to achieve this.

[0071] In some approaches, gastrin 17-specific or non-specific antibodies can be combined with different carriers to achieve immunoaffinity targets.

[0072] In some protocols, the one or more ion pairs measured include different ion pairs of gastrin 17;

[0073] In some schemes, mass spectrometers such as high-resolution mass spectrometers may be combined with the ion pairs of the present invention to achieve the same quantitative performance;

[0074] In some protocols, magnetic bead enrichment can also be achieved using fully automated equipment.

[0075] Materials and Methods

[0076] Reagents and consumables

[0077] Pure gastrin 17 was purchased from Bachem, Switzerland, and the internal standard of gastrin 17 G17-13C915N was synthesized by Shanghai Ketide.

[0078] Methanol and acetonitrile were purchased from Merck, formic acid was purchased from Fisher, ammonia water was purchased from Aladdin, and PBS buffer solution was purchased from Sigma.

[0079] Bovine serum albumin (BSA) was purchased from Sigma; Trina commercial human serum, batch number 20230825 / 23ESP290685 (3000 ml / bottle).

[0080] The experimental water was prepared by the Millipore pure water system in our laboratory. Low adsorption protein tube; Waters QuanRecovery with MaxPeak low adsorption receiving plate, gastrin 17 detection kit (Zhengzhou Antu Bioengineering Co., Ltd., magnetic particle chemiluminescence method, reagent batch number 20240126) and the magnetic particle suspension component therein, the coating antibody of the magnetic particle suspension is a gastrin 17-specific antibody, the coating titer is 25ug / ml, produced by Zhengzhou Imino Biotechnology Co., Ltd., batch number 20230310.

[0081] Main instruments

[0082] The instrument used in this study was an ACQUITY UPlC I-Class chromatograph tandem with a TQ-S mass spectrometer, equipped with an XBridge Premier BEH C18 Column ( 2.5μm, 2.1*100mm). Mettler XPE-205 electronic balance was used for the preparation of standard solutions and the weighing of samples.

[0083] Mass spectrometry methods

[0084] Electrospray ionization (ESI) negative ion mode and multiple reaction monitoring (MRM) mode were used for analysis, with a capillary voltage of 2.8 kV, an ion source temperature of 150°C, a desolvation gas temperature of 550°C, and a desolvation gas flow of 1000 l / Hr.

[0085] Table 1 Mass spectrometry conditions for gastrin 17

[0086]

[0087] Result calibration and calculation method

[0088] Before the formal analysis, add a pre-experimental test sample to obtain the approximate concentration of gastrin 17 in the sample. According to the initial concentration, select the concentration of the calibrator / internal standard. According to the amount of internal standard and the initial concentration of the sample, weigh and record the mass of the appropriate sample so that the ratio with the internal standard is approximately 1:1.

[0089] The prepared standard solution and internal standard working solution were sampled at a ratio of 0.8 to 1.2, and their accurate masses were weighed on a balance. The concentration of gastrin 17 in the sample was calculated based on the sample peak area ratio, the mass of the sample and the internal standard.

[0090] The raw materials and reagents used in the gastrin 17 detection method provided by the present invention can all be purchased from the market.

[0091] The present invention will be further described below in conjunction with embodiments:

[0092] Example 1

[0093] The liquid phase square gradient of the present invention: mobile phase A is acetonitrile solution, mobile phase B is 0.2% ammonia aqueous solution; the gradient elution conditions are shown in Table 2, the entire analysis time is 11min, the injection volume is 20μl, the flow rate is 0.2ml / min, the column temperature is 45°C, and the automatic sampler temperature is 10°C.

[0094] During the development of the G17 candidate reference method, the liquid phase gradient 2 with extended elution had a lower contamination rate than the mobile phase gradient in Comparative Example 1. Therefore, the present invention adopts the extended elution gradient in Table 2.

[0095] Table 2 Mobile phase gradient of the present invention

[0096]

[0097] Table 3 Gradient carryover contamination rate of extended elution of the present invention

[0098]

[0099] Note: Carryover rate = (L3-L2) / (H-L2)*100%; carryover rate is required to be less than 0.1%.

[0100] Comparative Example 1

[0101] The liquid phase gradient of the control group: mobile phase A was acetonitrile solution, and mobile phase B was 0.2% ammonia aqueous solution; the gradient elution conditions were shown in Table 4, the entire analysis time was 8 min, the injection volume was 20 μl, the flow rate was 0.2 ml / min, the column temperature was 45°C, and the automatic sampler temperature was 8°C.

[0102] During the development of the G17 candidate reference method, the use of a control group mobile phase gradient would cause problems such as increased column pressure, carryover contamination, and peak tailing due to adsorption of analytes and sample impurities.

[0103] Table 4

[0104]

[0105] Table 5 Mobile phase gradient carryover contamination rate in the control group

[0106]

[0107] Note: Carryover rate = (L3-L2) / (H-L2)*100%; carryover rate is required to be less than 0.1%.

[0108] Example 2 Sample pretreatment method

[0109] Calibration solution: Prepare 200 ng / ml of pure gastrin 17 diluted with 0.3% ammonia and 5% acetonitrile water solution, dispense into low adsorption tubes and freeze at -80°C. Take one tube for each experiment and dilute it to the commonly used working solution concentration, such as 2 ng / ml, with blank human serum by weighing on a balance.

[0110] Internal standard solution: prepare gastrin 17 internal standard (2 ng / ml) with 0.3% ammonia water and 5% acetonitrile water, dispense into low adsorption tubes and freeze at -80°C for use.

[0111] a. Weigh the sample: According to the initial concentration of the sample to be tested, weigh the appropriate amount of calibration solution and the sample to be tested, and add a fixed amount (such as 60μl) of internal standard solution to each. If the sample is less than 500ul, add human serum without the target analyte to the sample to be tested / calibration solution to make up to 500μl, and mix at a low speed for processing.

[0112] b. Cleaning before enrichment: Mix the magnetic bead suspension, take 150μl into a low-adsorption centrifuge tube, place it on a magnetic plate, remove the supernatant, add 0.5ml of 10mM PBS buffer solution, mix well, place it on the magnetic plate for 1min, remove the supernatant, add 0.5ml of pure water, mix well, place it on the magnetic plate for 1min, and remove the supernatant.

[0113] c. Enrichment reaction: Use 100ul blank serum to transfer the washed magnetic beads to the pre-prepared calibration solution and the sample to be tested; mix and enrich at a low speed of 500rpm for 20 minutes at room temperature (18℃~26℃).

[0114] d. Post-enrichment washing: After standing on the magnetic plate for 1 minute, remove the supernatant; add 0.5 ml of PBS buffer solution to the centrifuge tube to wash the magnetic beads, and aspirate the supernatant again; add 0.5 ml of pure water to mix, stand on the magnetic plate for 1 minute, and aspirate the supernatant.

[0115] e. Elution target: Add 100 μl of eluent (0.5% formic acid 30% acetonitrile aqueous solution), mix well and let stand for 10 minutes, then place on a magnetic rack and let stand for 1 minute, aspirate the eluent and add it to a new low-adsorption centrifuge tube.

[0116] f. Solution replacement: blow dry with nitrogen for 20 min, add 90 μl of 0.5% ammonia and 10% acetonitrile aqueous solution to re-dissolve.

[0117] g. Centrifuge at 13000r / min for 10min, place the sample on the magnetic rack for 1min, transfer the supernatant to a low-adsorption 96-well plate for sample analysis.

[0118] Example 3 Optimization of the amount of magnetic beads

[0119] Two medium and high value samples (commercial human serum without target analytes, Trina) were selected, with concentrations of 0.2 ng / ml and 2 ng / ml, respectively. The amounts of magnetic beads were 100 μl, 200 μl, and 300 μl, respectively. 400 μl of sample was added to the centrifuge tubes with different amounts of magnetic beads, and each condition was tested in parallel. 60 μl of internal standard solution (internal standard concentration 5 ng / ml) was added to each tube of sample. The samples were pretreated according to the pretreatment method of Example 2, and the samples were re-dissolved and tested on the machine (for specific mass spectrometry conditions, refer to Table 1, and for liquid phase conditions, refer to Table 2 liquid phase gradient of Example 1). The results are shown in Table 6.

[0120] Table 6 Optimization of magnetic bead dosage

[0121]

[0122] From the data analysis in Table 4, there is little difference in sample response and response after enrichment with 100μl, 200μl, and 300μl of magnetic beads, so 150μl of magnetic beads is sufficient.

[0123] Example 4 Study on the enrichment time of gastrin 17 specific antibody magnetic beads

[0124] Magnetic beads are used for enrichment in order to increase the sample signal value and obtain a higher detection sensitivity. In order to study the optimal enrichment time of magnetic beads, medium and low concentration serum samples were used, with a sample volume of 400μl, and enriched with 100μl and 200μl magnetic beads, respectively. The enrichment effect of gastrin 17-specific antibody-coated magnetic microparticle suspension at 0min, 10min, 20min, 30min, and 60min was studied. The concentration of the original sample before enrichment and the concentration of the supernatant sample at each subsequent enrichment time period were detected on the A2000 instrument using the magnetic microparticle luminescent gastrin 17 detection kit. The measurement results are shown in Table 7:

[0125] Table 7 Supernatant sample concentration after magnetic bead enrichment

[0126]

[0127]

[0128] According to the measured concentrations of the samples and supernatants in Table 7, the magnetic bead enrichment efficiency was calculated by the formula: magnetic bead enrichment rate = (1-supernatant concentration / stock solution concentration)*100%.

[0129] Table 8 Magnetic beads enrichment sample efficiency

[0130]

[0131] Comparing the data in Table 7 and Table 8, the enrichment effect of more than 98% was achieved by shaking the reaction at a low speed of 500 rpm for 20 minutes at room temperature (18°C to 26°C), and the target was nearly completely enriched. Because of the isotope internal standard correction, the enrichment of less than 100% does not affect the detection value, but the enrichment rate will affect the sensitivity and quantitative limit, so this patent selects a shorter reaction time of 20 minutes to obtain a higher enrichment rate.

[0132] Example 5 Pretreatment Absolute Recovery Comparison

[0133] Commercial human serum (Trina, batch number 2023ESP290685) without the target analyte was selected as the blank matrix, and 20 μl of 5 ng / ml G17 standard solution (standard solvent: 0.5% ammonia water 5% acetonitrile aqueous solution) was added. After the pretreatment method of Example 2, 0.5% ammonia water 10% acetonitrile aqueous solution was added for reconstitution to obtain 100 μl of sample. Among them, in the pretreatment method of Example 2, e. the eluent for eluting the target substance is to add 20 μl of 5 ng / g G17 standard solution (standard solvent: 0.5% ammonia 5% acetonitrile aqueous solution) to 80 μl of blank eluent (0.5% formic acid 30% acetonitrile aqueous solution), blow dry with nitrogen, add 100 μl of 0.5% ammonia 10% acetonitrile aqueous solution for re-dissolution, mix well and inject for analysis, the response ratio of the two results is the absolute recovery rate of pretreatment, and the absolute recovery rate of gastrin 17 in the magnetic particle enriched sample is 72.27% (Table 9), which is 2 to 4 times the absolute recovery rate of solid phase extraction MAX in Example 2.

[0134] Table 9

[0135]

[0136] Comparative Example 2

[0137] The absolute recovery rate of gastrin 17 was detected according to the solid phase extraction MAX method described in the Peking Union Medical College article Analytical and Clinical Performance of a liquid Chromatography--Tandem Mass Spectrometry Method for Measuring Gastrin Subtypes G34 and G17 in Serum. The results are shown in Table 10. The absolute recovery rate using solid phase extraction MAX was 26.64%.

[0138] Table 10

[0139]

[0140] Example 6 Limit of Detection and Limit of Quantification (LOD & LOQ)

[0141] Serum samples without gastrin 17 were selected as the matrix, and a series of low-value samples were prepared to assess the analytical sensitivity of the method. Three samples were processed in parallel for each level test sample, and two batches were tested, with a total of 6 data, and the deviation and variation from the theoretical value were calculated. The concentration when the average signal-to-noise ratio (S / N) was ≥3 was used as the detection limit of the method; the lowest concentration sample that met the conditions of S / N ≥ 10, total sample variation CV ≤ 20% (imprecision), and the deviation between the mean concentration determination and the theoretical concentration < 15% (accuracy) was used as the quantitative limit of the method. The concentration of 2.59 pg / ml is now used as the detection limit of the method, and 5.14 pg / ml is used as the quantitative limit of the method (Table 11).

[0142] Table 11 LOD & LOQ

[0143]

[0144] Taking into account the concentration range of gastrin 17 in human serum, mixed samples at three concentration levels, low, medium and high, were selected. The precision (pg / ml) was examined by conducting three batches of experiments on the samples at the three concentration levels, with five parallels in each batch, and the data were analyzed using one-way ANOVA.

[0145] Table 12 Precision investigation results

[0146]

[0147] Example 7 Linear

[0148] Commercial human serum without gastrin 17 was selected as blank matrix, and pure gastrin 17 was added to prepare samples with concentrations of 0.010, 0.050, 0.198, 0.502, 0.987, 2.007, 4.861, and 10.016 ng / ml, respectively. The same amount of internal standard solution was added to each sample for pretreatment. Each sample was injected three times continuously, and the mean response ratio (response) and mass ratio were plotted to obtain the linear equation ( Figure 1 ).

[0149] The linear equation is: y = 1.50814x + 0.0065073, R 2 =0.999096, with good linearity in the range of 0.010ng / g~10.016ng / ml, meeting the linearity requirements.

[0150] Example 8 Spiked Recovery

[0151] The spike recovery rate is calculated by adding standard substances to commercial human serum (Trina, batch number 2023ESP290685) without the target analyte to prepare gradient serum samples, and the recovery rate is calculated by instrument detection. It can verify the accuracy of the established method, and to a certain extent, it can indicate whether there is interference from other substances in the detection. It can also be used as a simple and direct method to reflect the size of the matrix effect.

[0152] The theoretical concentrations of standard additions were approximately 50 pg / ml, 200 pg / ml, and 800 pg / ml, respectively. The spike recovery experiment was tested for 2 days, with 3 parallel runs per day. The corresponding spike recoveries were 98.85%, 99.55%, and 99.77% (Table 13), which met the requirements for spike recovery in method validation (95% to 105%).

[0153] Table 13 Spike recovery test results

[0154]

[0155] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting gastrin 17, characterized in that: The steps include: Step 1: Weigh the sample / standard and internal standard on a balance, and gently shake to mix to obtain the processed sample / standard; Step 2: taking the treated sample / standard to be tested and incubating it with the magnetic bead suspension coated with gastrin 17-specific antibody after the first washing, and then washing it for the second time, eluting it, re-dissolving it, and detecting it; The target substances include gastrin-17.

2. The detection method according to claim 1, characterized in that The volume ratio of the processed sample to be tested in step 2 to the magnetic bead suspension coated with gastrin 17-specific antibody is (20 μl-1 ml): 150 μl.

3. The detection method according to claim 1 or 2, characterized in that: Step 2 is as follows: blow the magnetic microparticle suspension evenly, take 150 μl and let it stand for 1 min, discard the supernatant, add 500 μl PBS solution to wash the magnetic beads and residual sample, let it stand for 1 min, discard the supernatant, and use 100 μl blank serum to transfer the washed magnetic beads to the treated sample / standard to be tested.

4. The detection method according to any one of claims 1 to 3, characterized in that: The incubation conditions in step 2 are 18°C ​​to 26°C, shaking at 500 rpm for 20 min.

5. The detection method according to any one of claims 1 to 4, characterized in that: The detection in step 2 includes mass spectrometry detection and / or liquid phase detection; The mobile phase A of the liquid phase detection is acetonitrile solution, the mobile phase B is 0.2% ammonia aqueous solution, and the time is 11 minutes.

6. The detection method according to claim 5, characterized in that The elution solution used in step 2 is an aqueous solution containing 0.5% formic acid and 30% acetonitrile.

7. The detection method according to any one of claims 1 to 6, characterized in that: The reconstitution solution in step 2 is an aqueous solution containing 0.5% ammonia water and 10% acetonitrile.

8. The detection method according to any one of claims 1 to 7, characterized in that: The first cleaning uses a 10 mM PBS buffer solution; The second cleaning uses 10 mM PBS buffer solution and pure water respectively.

9. The detection method according to any one of claims 1 to 8, characterized in that: The volume of the elution solution used in step 2 is 100 μl; The volume of the reconstitution solution used in step 2 is 75ul to 90μl.

10. The detection method according to any one of claims 1 to 9, characterized in that: When the sample / standard substance to be tested in step 1 is less than 500 μl, fill it up with blank serum to 500 μl.

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