Kit for quantitative detection of IGF-1 (Insulin-like Growth Factor-1) and preparation and detection methods thereof

Abstract

The invention relates to a kit for quantitative detection of IGF-1 (Insulin-like Growth Factor-1) and a preparation and detection method thereof. The kit comprises a calibrator, a quality control product, an internal standard solution, an immune enrichment solution, a dissociation solution, a leacheate, an eluent, a mobile phase 1 and a mobile phase 2. The complete IGF-1 protein can be fully automatically detected from top to bottom, and the sample treatment efficiency is remarkably improved; wherein the matrix liquid used for preparing the kit adopts human serum without endogenous IGF-1, so that the in-vivo environment can be simulated to the greatest extent, and the detection deviation caused by different matrixes is reduced; an eluent of the kit adopts a formic acid-hydrochloric acid mixed acid-acetonitrile system, the system can provide a mild ionization environment through formic acid, and hydrochloric acid synergistically stabilizes the synergistic effect of the pH value of the solution, so that the ion inhibition effect is reduced, the response signal of IGF-1 is improved, and the signal-to-noise ratio of detection is further improved.

Description

Technical Field

[0001] This invention belongs to the field of IGF-1 detection technology, specifically, it relates to a kit for quantitative detection of IGF-1 and its preparation and detection method. Background Technology

[0002] Insulin-like growth factor-1 (IGF-1) is a key regulator of growth hormone (GH) activity, playing a crucial role in cell proliferation, differentiation, apoptosis, and various physiological and pathological processes. Due to its long half-life and high stability, IGF-1 detection has significant clinical value in diagnosing diseases such as growth hormone deficiency and acromegaly. Currently, the main methods for IGF-1 detection include immunoassays (such as chemiluminescence and electrochemiluminescence) and mass spectrometry (MS). While immunoassays are widely used, easily achieving high throughput, automation, and low cost, they rely on specific antibody binding and are susceptible to interference from IGF-binding proteins (IGFBPs), leading to inconsistent results and a risk of misdiagnosis.

[0003] Mass spectrometry, with its high specificity, accuracy, and traceability, is gradually becoming an important alternative method for IGF-1 detection. Currently, mass spectrometry strategies are mainly divided into two types: bottom-up and top-down. The former detects characteristic peptides after enzymatic digestion, while the latter directly analyzes the intact protein using mass spectrometry. Chinese patent CN117630385A discloses a MRM-targeted measurement method for insulin-like growth factor IGF-1 expression, which involves a bottom-up detection method. The sample undergoes protein extraction, reductive alkylation, and trypsin digestion to obtain the IGF-1 marker peptide APQTGIVDECCFR. The relative content of IGF-1 is calculated by detecting the MRM characteristic peak area of ​​this peptide. Although this method effectively avoids interference from IGFBPs and improves detection specificity, its core enzymatic digestion step is complex and prone to errors. The digestion process is time-consuming, requiring overnight incubation (8-16 hours), and the detection process lacks a stable isotope-labeled internal standard to correct for matrix effects, resulting in poor reproducibility.

[0004] Chinese patent CN114910549A discloses a quantitative detection kit and method for insulin-like growth factor I (IGF-1) based on MALDI-TOF MS. This method is a top-down quantitative detection method for IGF-1 based on MS. It utilizes compounds such as urea and sodium dodecyl sulfate (SDS) to lyse the IGF-1-IGFBP3-ALS ternary complex in serum / plasma, releasing IGF-1. IGF-1 is then captured using biotin-labeled IGF-1 antibody-streptavidin magnetic beads. After multiple elutions, the eluent is spotted onto a target plate and heated for crystallization, followed by MALDI-TOF analysis. However, the sample pretreatment step uses surfactants such as SDS, which can easily cause secondary contamination to the mass spectrometer. Furthermore, the pretreatment process requires two centrifugations, and after the second centrifugation, residual eluent needs to be carefully aspirated with a micropipette. This makes the accuracy of the experimental results highly dependent on the meticulous operation of skilled technicians, making it difficult to integrate the sample pretreatment step into an automated module and significantly reducing sample processing efficiency.

[0005] Furthermore, the accurate quantification of IGF-1 is highly dependent on the accuracy of the calibrator calibration system, which in turn is closely related to the calibrator matrix. This is because IGF-1 (molecular weight approximately 7.6 kDa), as an endogenous protein, not only undergoes natural post-translational modifications but also forms complex interactions with other proteins and lipids in serum. These characteristics directly affect its detection "accessibility." If the calibrator matrix differs significantly from actual serum and cannot simulate the aforementioned natural state, the "concentration-response" relationship will mismatch with the actual sample, thus introducing quantitative bias. Therefore, the calibrator matrix should be as close as possible to actual human samples to ensure detection accuracy. Currently, most publicly disclosed IGF-1 related patents use animal substitute matrices, including widely used rat serum, 1% bovine serum albumin (patent CN114910549A), and rabbit serum (patent CN119827692A). However, existing studies have shown that when calibrating apolipoproteins, using natural serum as a matrix is ​​significantly more effective than using animal serum as a matrix with purified apolipoproteins. Therefore, real human serum matrix should be the preferred choice for preparing IGF-1 related calibrators. However, human serum has an inherent problem: it contains IGF-1 background, which needs to be removed through pretreatment. Currently, there is no publicly available technology that discloses a simple, rapid, and large-scale production method for removing IGF-1 background from human serum, which severely limits the practical application of human serum matrix. Summary of the Invention

[0006] To overcome the technical problems existing in the prior art, this invention provides a kit for the quantitative detection of IGF-1 and its preparation and detection method. It enables fully automated "top-down" detection of intact IGF-1 protein, significantly improving sample processing efficiency. The matrix solution used in the kit preparation employs human serum with endogenous IGF-1 removed, which can maximally simulate the in vivo internal environment and reduce detection bias caused by different matrix components. The elution buffer of the kit uses a formic acid-hydrochloric acid mixed acid-acetonitrile system. This system can reduce ion inhibition effects and improve the IGF-1 response signal by "formic acid providing a mild ionization environment and hydrochloric acid synergistically stabilizing the solution pH," thereby improving the signal-to-noise ratio of detection.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A kit for quantitative detection of IGF-1, the kit comprising calibrators, quality controls, internal standard, immunoenrichment solution, dissociation solution, eluent, elution solution, mobile phase 1 and mobile phase 2; The calibrators include multiple concentrations of IGF-1; The quality control material includes IGF-1 at one or more concentrations; The internal standard solution includes IGF-1-N 15 ; The immunoenrichment solution includes IGF-1 specific antibody conjugated with Tosyl magnetic beads, which are obtained by coupling IGF-1 specific antibody with Tosyl magnetic beads. The dissociation solution includes acetic acid; The rinsing solution includes PBS buffer; The eluent includes formic acid, hydrochloric acid, and acetonitrile; The mobile phase 1 comprises an aqueous formic acid solution; The mobile phase 2 comprises a formic acid acetonitrile solution.

[0008] Preferably, the concentration of the calibrator is 10~1000ng / mL, and the concentration of the quality control sample is 50~500ng / mL.

[0009] Preferably, the internal standard solution is IGF-1-N at a concentration of 80~300 ng / mL. 15 The immunoenrichment buffer was 0.2–0.5 mg / mL LIFG-1 specific antibody conjugated with Tosyl magnetic beads; the dissociation buffer was 0.8%–1.2% acetic acid; the eluent was PBS buffer with a pH of 6.9–7.5; and the elution buffer included 0.05%–0.15% formic acid, 0.005%–0.01% hydrochloric acid, and 25%–35% acetonitrile. Mobile phase 1 was a 5%–15% aqueous formic acid solution, and mobile phase 2 was a 5%–15% formic acid-acetonitrile solution.

[0010] The present invention also provides a method for preparing the above-mentioned kit for quantitative detection of IGF-1, comprising the following steps: (1) Pretreatment of matrix solution: Human serum was acidified with acetic acid, then adsorbed by macroporous resin and neutralized with ammonia water to obtain blank matrix solution; (2) Preparation of calibrators and quality control products: The standard is dissolved and diluted with methionine-acetic acid to prepare intermediate solutions for calibrators and quality control products. The intermediate solutions are then diluted with blank matrix solutions to obtain calibrator and quality control product solutions. (3) Preparation of internal standard solution: The internal standard is dissolved and diluted with methionine-acetic acid to prepare an internal standard intermediate solution. The intermediate solution is then diluted with blank matrix solution to obtain the internal standard solution. (4) Preparation of protein enrichment solution: IGF-1 specific antibody was coupled with Tosyl magnetic beads to obtain IGF-1 specific antibody coupled with Tosyl magnetic beads; (5) Preparation of dissociation solution: Take acetic acid, add water, mix well to obtain acetic acid solution; (6) Preparation of rinsing solution: Take PBS buffer powder and put it into a clean beaker, add purified water and dissolve it completely to obtain the solution; (7) Preparation of eluent: Take formic acid, hydrochloric acid, acetonitrile and purified water into a beaker, mix thoroughly to obtain the eluent; (8) Preparation of mobile phase 1: Take purified water and formic acid into a centrifuge tube and mix thoroughly to obtain the mobile phase 1. (9) Preparation of mobile phase 2: Take acetonitrile and formic acid into a centrifuge tube and mix thoroughly to obtain the mobile phase 2.

[0011] The elution buffer of the kit of this invention uses a formic acid-hydrochloric acid-acetonitrile system, which has the following advantages compared with the published trifluoroacetic acid (TFA) system: (1) It has better ionization efficiency; formic acid provides milder ionization conditions, which improves the ionization efficiency of target proteins in mass spectrometry detection. (2) It has stronger elution ability; the synergistic effect of trace amounts of hydrochloric acid and formic acid provides a low pH environment, which can more effectively dissociate antigen-antibody complexes and ensure the complete elution of IGF-1. (3) It has a higher detection response; the system effectively avoids the ion suppression of TFA and improves the response value and signal-to-noise ratio of IGF-1 in mass spectrometry detection.

[0012] As a preferred embodiment, step (1) is as follows: take human serum, use a magnetic stirrer, set the temperature to 32~40℃ and the speed to 600~1000 rpm, add acetic acid during the liquid mixing process, stir for 3~8 min, then add activated macroporous resin, use an electric stirrer, set the speed to 60~90 rpm, stir at room temperature for 0.5~2 h, and filter; transfer the filtrate to a beaker, use a magnetic stirrer, set the speed to 600~1000 rpm, add ammonia water during the liquid mixing process, stir for 3~8 min, and centrifuge to obtain a blank matrix solution.

[0013] Preferably, the activated macroporous resin is prepared by the following steps: place the macroporous resin in a beaker, add ethanol to soak it, and then filter off the solvent; then wash it with ethanol and purified water respectively.

[0014] Ethanol soaking: Deeply swells the resin, opens the network channels inside the resin polymer, and dissolves organic impurities such as pore-forming agents and pigments; Ethanol washing: removes pore-forming agents, pigments and other organic impurities; Washing with purified water: Removes ethanol and prepares for subsequent serum loading.

[0015] This invention employs a three-step pretreatment method—acetic acidification, macroporous resin adsorption, and ammonia neutralization—to achieve a 98% IGF-1 removal rate, meeting the production requirements for matrix quality. It is an effective, simple, and large-scale method for removing IGF-1 from human serum, and can well meet the requirements for preparing debased human serum.

[0016] As a preferred option, step (4) specifically involves: (4-1) Prepare ultrafiltration centrifuge tubes: Take ultrafiltration centrifuge tubes, add water, and centrifuge; add magnetic bead buffer solution to replace the buffer solution; the magnetic bead buffer solution is sodium bicarbonate-sodium carbonate buffer salt, pH 9.0~9.5; (4-2) Antibody preparation: Take IGF-1 specific antibody, add the antibody in batches to the prepared ultrafiltration centrifuge tube at room temperature, centrifuge, add magnetic bead buffer, and centrifuge again; transfer the antibody in the ultrafiltration centrifuge tube to a 1.5 mL centrifuge tube, wash the ultrafiltration centrifuge tube with magnetic bead buffer, transfer the washing solution to a 1.5 mL centrifuge tube, and detect the antibody concentration. (4-3) Preparation of magnetic bead solution: After thoroughly mixing the Tosyl magnetic bead solution, add a volume to a 5 mL centrifuge tube, separate the magnetic beads using a magnetic rack, retain the magnetic beads, discard the supernatant, and then wash with magnetic bead washing solution; separate the magnetic beads using magnetic rack, retain the magnetic beads, discard the supernatant, and then wash with magnetic bead buffer solution at least twice, and finally resuspend with magnetic bead buffer solution; wherein, the components of the magnetic bead washing solution include Tween-20 and sodium bicarbonate-sodium carbonate buffer, pH 9.0~9.5; (4-4) Antibody-magnetic bead conjugation: Mix the antibody and magnetic bead solution, add conjugation buffer, and incubate at 35-40℃ for 6-16 hours by rotating. The conjugation buffer is ammonium sulfate. (4-5) Blocking reaction: Add blocking buffer and mix by rotating at room temperature for 2-6 hours; (4-6) Washing: Magnetic separation retains magnetic beads, discards supernatant, washes several times with washing buffer, and resuspends in washing buffer to make the antibody concentration 0.2~0.5 mg / mL; the washing buffer components include Tween-20, bovine serum albumin, proclin300 and PBS buffer, pH 7.0~7.4.

[0017] In addition, the present invention also provides a non-diagnostic method for quantitative detection of IGF-1, using the above-mentioned kit, comprising the following steps: (I) Reagent preparation; (II) Automated pretreatment using a fully automated magnetic solid phase extraction instrument: Place several 96-well deep well plates into the designated positions in the magnetic solid phase extraction instrument, add different components to different columns automatically according to the preset pretreatment program, extract samples according to the set time sequence, and after the operation is completed, automatically transfer the eluent to the 96-well plate for mass spectrometry analysis.

[0018] In this invention, the serum sample is simply transferred directly into a fully automated magnetic solid-phase extraction instrument, and the instrument is run according to the set time sequence. During operation, the IGF protein in the serum is first dissociated into a free state by acetic acid. Then, tosyl-modified magnetic beads coupled with the IGF-1 specific antibody bind specifically to the antigen, accurately capturing the free IGF-1 protein. At the same time, other impurities and interfering substances in the serum are removed, achieving efficient enrichment and purification of the target protein. Subsequently, the IGF-1 bound to the magnetic beads is eluted with a mixed acid elution buffer to obtain a high-purity target protein solution. The eluted target protein solution is then directly introduced into a mass spectrometer to complete the qualitative and quantitative analysis of IGF-1.

[0019] Preferably, step (I) specifically includes: (I-1) Remove all components of the kit and let stand at room temperature; (I-2) Reconstitute the calibrators, quality control samples, and internal standard to obtain calibrators J1~J6, quality control samples Z7~Z8, and internal standard solution; (I-3) Dilute mobile phase 1 with purified water, sonicate, and mix thoroughly to obtain an aqueous phase; dilute mobile phase 2 with acetonitrile, sonicate, and mix thoroughly to obtain an organic phase.

[0020] Preferably, in step (II), the chromatographic parameters are controlled as follows: Chromatographic column: Sepax Bio C4 reversed-phase liquid chromatography column with online filter; Flow rate: 0.4 mL / min; Column temperature: 55℃; Injection volume: 20 μL; HPLC elution gradient: From 0 to 2.0 min, the proportion of mobile phase A is 95% and the proportion of mobile phase B is 5%. 2.0–3.1 min, mobile phase A ratio 40–95%, mobile phase B ratio 5–60%; 3.1–3.2 min, mobile phase A proportion 40–95%, mobile phase B proportion 5–60%; 3.2–4.3 min, with mobile phase A comprising 40–95% and mobile phase B comprising 5–60%; 4.3–4.4 min, mobile phase A accounts for 40–95%, and mobile phase B accounts for 5–60%; 4.4–5.0 min, mobile phase A accounts for 95% and mobile phase B accounts for 5%; Mass spectrometry parameters are controlled as follows: The ion source is an electrospray ion source in positive ion mode; The capillary voltage is 2.0 kV, the desolventizing temperature is 600℃, the desolventizing gas flow rate is 1000 L / hr, and the conical backflush gas flow rate is 150 L / hr. The scanning mode is multi-response monitoring mode; the cone voltage is 20-50V, and the collision voltage is 20-50V.

[0021] This invention, in its selection of chromatographic columns, does not employ the conventional combination of a BEH C18 column (2.1mm × 150mm, C18 1.7μm) + online filter. Instead, it chooses a Sepax Bio C4 reversed-phase liquid chromatography column (2.1*50mm, 3μm) + online filter. The C4 column has the lowest IGF-1 column residue, and this residue level has been assessed to not interfere with the accuracy of subsequent low-concentration sample detection. Furthermore, in practical application, this column maintained excellent performance after 6000 consecutive injections in the target elution system. The chromatographic system pressure remained within the normal range without abnormal fluctuations; the chromatographic peaks were regular, with stable symmetry and resolution, without peak broadening or tailing; and the linearity fully met the experimental method requirements, satisfying the standard usage for column life in routine analysis. In addition, this column is a domestically produced product, with a single column costing only 1500-2000 RMB. While ensuring performance and lifespan, the cost of experimental analysis is also relatively low, demonstrating a significant cost-performance advantage.

[0022] The beneficial effects of this invention are as follows: 1. This invention provides a method for immunoenrichment and automated detection of IGF-1 and its matching kit, which can realize fully automated "top-down" detection of complete IGF-1 protein. Compared with conventional detection methods, it significantly improves its sample processing efficiency, with a detection throughput of only 1 hour / 96 samples. 2. The matrix solution used in the preparation of the kit of the present invention is debaseted human serum (i.e., human serum from which endogenous IGF-1 and related interfering components have been removed). Its composition is highly consistent with real serum, which can simulate the natural environment of IGF-1 in the body to the greatest extent, effectively avoid the deviation of detection results caused by differences in matrix composition, and improve detection accuracy. 3. The eluent of the kit of the present invention adopts a formic acid-hydrochloric acid-acetonitrile system, which has better ionization efficiency, stronger elution ability, and higher detection response; this system effectively avoids the ion suppression of TFA and improves the response value and signal-to-noise ratio of IGF-1 in mass spectrometry detection. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the automated pre-processing flow of the present invention; Figure 2 This is a graph of the linear verification regression equation for this invention; Figure 3 This is a comparison diagram between the automated preprocessing of this invention and conventional methods. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and the technical solutions of the present invention will be further described in detail. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any modifications and / or alterations made to the present invention will fall within the protection scope of the present invention.

[0025] In this invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used are commercially available or commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art. Unless otherwise specified, the components or equipment in the following embodiments are general standard parts or components known to those skilled in the art, and their structures and principles can be learned by those skilled in the art through technical manuals or conventional experimental methods.

[0026] The Tosyl magnetic beads used in this invention were purchased from Suzhou Nanomicro Technology Co., Ltd., the human serum was obtained from Baichen Medical Laboratory, and the IGF-1 specific antibody was purchased from Blue Torch Biotechnology (Hangzhou) Co., Ltd.

[0027] Example 1 A quantitative detection kit for insulin-like growth factor 1 and its preparation The kit includes calibrators, quality controls, internal standard, dissociation buffer, immunoenrichment buffer, rinsing buffer, elution buffer, mobile phase 1, and mobile phase 2. Specific components are shown in Table 1. Table 1 ; (1) Pretreatment of matrix solution: Human serum was pretreated by acidification with acetic acid, adsorption with macroporous resin, and neutralization with ammonia. Take 500 g of macroporous resin and place it in a beaker. Add ethanol to soak it and then filter out the solvent using a Buchner funnel and medical degreased cotton gauze. Wash it twice with ethanol and purified water to obtain the activated macroporous adsorption resin. The macroporous resin is a non-polar hydrophobic interaction type resin, preferably a resin with a polystyrene skeleton structure. In this example, HPD-100 is used. Take 1 L of human serum. Using a magnetic stirrer, set the temperature to 37℃ and the speed to 800 rpm. While mixing the liquid, slowly add 6.5 mL of acetic acid using a power pipette, controlling the pipette speed setting to 1. Add the acetic acid to the serum in a "speckled" manner, not a continuous stream. Stir for 5 min. Then, add 500 g of activated macroporous resin at a ratio of 0.5 g / mL. Use a power stirrer, setting the speed to 75 rpm, and stir at room temperature for 1 h. Filter once using a vacuum filtration device. Transfer the filtrate to a beaker. Using a magnetic stirrer, set the speed to 800 rpm (ensuring all liquid is agitated), slowly add 8 mL of ammonia water (concentration 25%~28%) using a power pipette, controlling the pipette speed setting to 1. Add the liquid to the filtrate in a "speckled" manner, not a continuous stream. Stir for 5 min. Centrifuge to obtain a blank matrix solution.

[0028] (2) Preparation of calibrators and quality control products: IGF-1 standard was dissolved and diluted with 2mM methionine-2% acetic acid to prepare intermediate solution for calibrators and quality control products. The intermediate solution was diluted 10 times with blank matrix solution to obtain calibrator and quality control product solutions. The solutions were dispensed into low adsorption vials, lyophilized, and stored at 2~8℃. The concentrations of calibrators and quality control products (unit: ng / mL) are shown in Table 2 below.

[0029] Table 2 ; (3) Preparation of internal standard solution: The internal standard is dissolved and diluted with 2mM methionine-2% acetic acid to prepare the internal standard intermediate solution. The intermediate solution is diluted 10 times with blank matrix solution to obtain the internal standard solution. It is dispensed into low adsorption vials, freeze-dried, and stored at 2~8℃.

[0030] (4) Preparation of protein enrichment solution: (4-1) Prepare ultrafiltration centrifuge tubes: Take an ultrafiltration centrifuge tube (Amicon Ultra-0.5 centrifuge filter), add 400 μL of water, centrifuge at 12000g for 10 min; add 400 µL of magnetic bead buffer (0.1 M sodium bicarbonate-sodium carbonate buffer, pH 9.0~9.5) and replace twice; (4-2) Antibody preparation: Take 1 mg of IGF-1 specific antibody, bring it to room temperature, add the antibody in batches to the prepared ultrafiltration centrifuge tube, centrifuge at 12000g for 10 min, then add 350µL of magnetic bead buffer, centrifuge at 12000g for 10 min, repeat twice. Transfer the antibody from the ultrafiltration centrifuge tube to a 1.5 mL centrifuge tube, wash the ultrafiltration centrifuge tube once with 200 μL of magnetic bead buffer, transfer the washing buffer to a 1.5 mL centrifuge tube, and detect the antibody concentration; (4-3) Preparation of magnetic beads: After thoroughly mixing Tosyl magnetic beads (10 mg / mL), take 2.5 mL and add it to a 5 mL centrifuge tube. Separate the beads using a magnetic rack, retain the magnetic beads, discard the supernatant, and then add 1 mL of magnetic bead washing buffer (0.05% Tween-20, 0.1 M sodium bicarbonate-sodium carbonate buffer, pH 9.0~9.5) to wash once; separate the beads using a magnetic rack, retain the magnetic beads, discard the supernatant, and then add 1 mL of magnetic bead buffer to wash twice. Finally, resuspend the beads in 500 µL of magnetic bead buffer. (4-4) Antibody-magnetic bead conjugation: Mix 200 μL of antibody and 500 μL of magnetic bead solution, add 350 μL of conjugation buffer (3M ammonium sulfate), and incubate overnight at 37°C by rotating the mixture. (4-5) Blocking reaction: Add 1.25 mL of blocking buffer (Blockmaster™ (JSR LifeScience, CE510)) and mix by rotation at room temperature for 4 h; (4-6) Washing: Magnetic beads are retained after magnetic separation. The supernatant is discarded. 1 mL of washing buffer (containing 0.05% Tween-20, 0.5% bovine serum albumin (BSA), 0.05% proclin 300, PBS buffer, pH 7.0~7.4) is added and the mixture is washed 3 times. The suspension is then placed in 5 mL of washing buffer to achieve an antibody concentration of 0.2 mg / mL. The suspension is stored at 2~8℃. (5) Preparation of dissociation solution: Take 10 mL of acetic acid, add 990 mL of water, mix well to obtain a 1% acetic acid solution.

[0031] (6) Preparation of rinsing solution: Take one bag of PBS buffer powder (Solarbie, P1010) and put it into a 2L clean beaker. Add 2L of purified water and dissolve it completely. (7) Preparation of eluent: Accurately measure 1 mL formic acid, 0.22 mL hydrochloric acid (concentration 37%), 300 mL acetonitrile and 700 mL purified water into a beaker, mix thoroughly, dispense and store at 2~8℃; (8) Preparation of mobile phase 1: Accurately measure 90 mL of purified water and 10 mL of formic acid into a 50 mL centrifuge tube using a pipette, mix thoroughly, dispense, and store at 2~8℃; (9) Preparation of mobile phase 2: Accurately measure 90 mL of acetonitrile and 10 mL of formic acid into a 50 mL centrifuge tube using a pipette, mix thoroughly, dispense into portions, and store at 2~8℃.

[0032] 2. Automated extraction and detection using reagent kits: (1) Reagent preparation: 1. Remove each component of the kit from the 2-8℃ refrigerator and allow it to stand at room temperature for 10 min; 2. Reconstitution of calibrators, quality control products and internal standard: At room temperature, reconstitute the calibrators, quality control products and internal standard with purified water, tighten the rubber stopper, invert 10 times, and vortex mix for 5 minutes until the lyophilized powder is completely dissolved to obtain calibrators J1~J6, quality control products Z7~Z8 and internal standard solution; 3. Mobile phase dilution: Dilute mobile phase 1 100 times with purified water, sonicate for 30 min, and mix thoroughly to obtain the aqueous phase. Dilute mobile phase 2 100 times with acetonitrile, sonicate for 30 min, and mix thoroughly to obtain the organic phase. (2) Automated pretreatment using a fully automated magnetic solid-phase extraction (MSPE) system: Place six 96-well deep-well plates into the designated positions in the MSPE system. Follow the preset pretreatment program. The sample loading process for the 96-well deep-well plates is shown in Table 3, and the preset automated pretreatment sequence is shown in Table 4. Automatically add 30 μL of immunoenrichment solution and 70 μL of 10% methanol-water to columns 1 and 7. Add 100 μL of calibrator, quality control solution, or serum, 45 μL of internal standard solution, and 100 μL of dissociation agent to columns 2 and 8. Add 300 μL of eluent to columns 3-4 and 9-10. Add 300 μL of purified water to columns 5 and 11. Add 150 μL of elution solution to columns 6 and 12. Extract samples according to the set sequence. After the operation is complete, the elution solution from columns 6 and 12 can be automatically transferred to the 96-well plate for mass spectrometry analysis. The instrument can process 96 samples at a time, taking 1 hour.

[0033] Table 3 ; Table 4 ; Reference Figure 1In this invention, the IGF-1 protein complex (serum sample) in the serum is directly transferred into a fully automated magnetic solid phase extraction instrument, and the instrument is run according to the set time sequence. During operation, the IGF protein in the serum is first dissociated into a free state by acetic acid, and then the tosyl-modified magnetic beads coupled with the IGF-1 specific antibody bind to the antigen through antigen-antibody specific binding, accurately capturing the free IGF-1 protein, while removing other impurities and interfering substances in the serum, achieving efficient immunoenrichment and purification of the target protein. Subsequently, the IGF-1 bound to the magnetic beads is eluted with a mixed acid elution buffer to obtain a high-purity target protein solution. The eluted target protein solution is directly introduced into a mass spectrometer (for mass spectrometry detection) to complete the qualitative and quantitative analysis of IGF-1.

[0034] (3) Chromatographic conditions: The chromatographic column was Sepax Bio C4 (2.1*50mm, 3μm) reversed-phase liquid chromatography column with an online filter; the mobile phase composition was 0.1% formic acid water (phase A) and 0.1% formic acid acetonitrile (phase B); the flow rate was 0.4 mL / min; the column temperature was 55℃; the injection volume was 20 μL; the needle washing solvent was 80% acetonitrile water; the gradient elution program was shown in Table 5, and the retention time of IGF-1 was 3.0~3.2 min.

[0035] Table 5 ; (4) Mass spectrometry settings: The ion source was an electrospray ionization (ESI) source in positive ion mode; the capillary voltage of the ion source was 2.0 kV, the desolventizing temperature was 600℃, the desolventizing gas flow rate was 1000 L / hr, and the cone backflush gas flow rate was 150 L / hr; the scanning mode was multiple reaction monitoring (MRM) mode, and the specific MRM parameters for IGF-1 and IGF-1 internal standard are shown in Table 6. Table 6 ; 3. Method validation of the reagent kit (1) Precision verification: The detection method of this invention was used to test samples at two levels of concentration, low and high. Three parallel samples were tested every day for five consecutive days. The measured values ​​were recorded, and the intra-batch and inter-batch coefficients of variation (CV) were calculated. The results are shown in Table 7.

[0036] Precision validation results: As shown in Table 7, the kit has good precision in detecting both low and high concentrations, meeting the clinically permissible imprecision requirements.

[0037] Table 7 ; (2) Linearity Validation: Using the detection method of this invention, high-concentration sample H (close to the upper limit of the linear interval) and low-concentration sample L (close to the lower limit of the linear interval) were diluted into five series of gradient concentration samples at L:H volume ratios of 4:0, 3:1, 2:2, 1:3, and 0:4. Three replicates were performed for each concentration, and the mean (yi) of the detection results was calculated. Using the dilution concentration (xi) as the independent variable and the mean (yi) of the detection results as the dependent variable, a linear regression equation was derived, and the linear regression correlation coefficient was calculated. The linear validation results are shown in Table 8, and the linear validation regression equation graph is shown below. Figure 2 As shown.

[0038] Linearity validation results: The regression equation for the linear validation of the kit is y=1.0221x-11.426, the regression coefficient is 1.0221, and the R2 is 0.9904, which meets the requirements, indicating that the linearity validation of the kit is successful.

[0039] Table 8 ; (3) Accuracy verification: The standard was dissolved and diluted with 2mM methionine-2% acetic acid to prepare low, medium and high concentration "standard samples" of 300, 5000 and 7000 ng / mL. The mixed human serum sample was used as the serum sample for the recovery experiment. 900 μL of serum sample was taken and 100 μL of 2mM methionine-2% acetic acid, low, medium and high concentration "standard samples" were added and mixed evenly to obtain the basic sample and the experimental samples of low, medium and high concentrations. The above samples were tested using the detection method of this invention. Each sample was tested in 6 replicates. The measured values ​​were recorded and the recovery rate of the experimental samples was calculated. The results are shown in Table 9.

[0040] Table 9 ; Correct verification results: The single concentration recovery rate and average recovery rate of the reagent kit in the spike recovery experiment are both within the range of [85%, 115%], which meets the requirements, indicating that the correctness of the reagent kit has been verified.

[0041] Example 2 The Influence of Magnetic Bead Types Used for Specific Antibody Conjugation Magnetic bead preparation: (1) Carboxyl magnetic beads, (2) Tosyl magnetic beads Antibody preparation: Prepare 5 mg to 6 mg of IGF-1 antibody and allow it to return to room temperature. Add 1 mg of antibody in batches to the prepared ultrafiltration centrifuge tube and wash three times with 400 μL phosphate buffer (0.1 M PBS, pH 7.4).

[0042] Preparation of carboxyl magnetic bead enrichment solution: Weigh 25 mg of carboxyl magnetic beads, resuspend and wash once with 1 mL of magnetic bead washing buffer (0.05% Tween-20, 0.05 M MES buffer, pH 6.0, MES is 2-(N-morpholino)-ethanesulfonic acid), and wash twice with 1 mL of magnetic bead buffer (0.05 M MES buffer, pH 6.0). Then, magnetically separate the beads, discard the supernatant, add 200 μL of coupling buffer (10 mg / mL EDC, 0.05 M MES buffer) and 1800 μL of magnetic bead buffer, mix at 200 rpm at room temperature for 30 min, discard the supernatant, add 1 mg of antibody, and replenish the magnetic bead buffer to 2 mL, mix at 200 rpm at room temperature for 3 h for the coupling reaction. After the reaction, resuspend the magnetic beads once with 1 mL of magnetic bead buffer and wash twice to remove uncoupled antibodies. Then, the beads were magnetically separated, the supernatant was discarded, and 1 mL of blocking buffer (polymer blocking agent CE510) was added. The mixture was stirred at 200 rpm at room temperature for 2 hours to block unreacted active sites. After 2 hours of blocking, the antibody beads were washed three times with washing buffer (3% bovine serum albumin, 0.2% Tween-20, 0.05 M MES buffer, pH 6.0), resuspended in 5 mL of washing buffer containing 0.05% ProClin 300 to achieve an antibody concentration of 0.2 mg / mL, and stored at 2–8 °C.

[0043] Tosyl magnetic bead protein enrichment solution preparation: Refer to the protein enrichment solution preparation kit.

[0044] Comparison of protein enrichment solutions extraction effects: Five mixed serum samples were tested using the two protein enrichment solutions described above according to the kit detection method, and the peak area of ​​IGF-1 after extraction was compared. The enrichment effects of the protein enrichment solutions prepared with carboxyl groups and Tosyl magnetic beads are shown in Table 10.

[0045] Table 10 ; Comparison of protein enrichment solution extraction effects: The peak area of ​​Tosyl magnetic beads is higher than that of carboxyl magnetic beads, indicating that the protein enrichment solution prepared with Tosyl magnetic beads has a better enrichment effect.

[0046] Example 3 Effect of eluent components Eluent-acid system-30% acetonitrile: (1) 0.5% trifluoroacetic acid (TFA) as control; (2) 0.1% formic acid; (3) 0.1% formic acid-0.008% hydrochloric acid; (4) 5% acetic acid; (5) 5% acetic acid-0.008% hydrochloric acid.

[0047] Elution efficiency comparison: Five mixed serum samples were taken and tested according to the kit method. The above-mentioned elution solutions were used, with each solution applied in triplicate, and the average peak area was recorded. The elution efficiency comparison results of different elution solutions are shown in Table 11.

[0048] Table 11 ; Elution buffer vs. acid elution efficiency comparison results: Under 30% acetonitrile conditions, the elution efficiency of the combined system of formic acid / acetic acid and trace amounts of hydrochloric acid is superior to that of a single acid system. The eluent containing 0.1% formic acid and 0.008% hydrochloric acid can maximize the signal response of the IGF-1 target peak, with a peak area 30% higher than that of TFA.

[0049] Furthermore, in practical application, the chromatographic column used in this invention maintained excellent performance even after 6000 consecutive injections into the target elution system. The chromatographic system pressure remained consistently within the normal range without abnormal fluctuations; the peak shapes were regular, with stable symmetry and resolution, and no peak broadening or tailing was observed; moreover, the linearity fully met the experimental method requirements, satisfying the standard usage for column life in routine analysis. In addition, the chromatographic column used in this invention is a domestically produced product, with a single column costing only 1500-2000 yuan. While ensuring performance and lifespan, the cost of experimental analysis is also relatively low, demonstrating a significant cost-performance advantage.

[0050] Example 4 Comparison of conventional methods and automated detection: Standard method: Take 100 μL of serum sample, add 45 μL of internal standard solution and 100 μL of dissociation agent, vortex mix for 10 s, add 400 μL of acetonitrile, shake at 900 rpm for 10 min, centrifuge at 12000 g for 7 min, take 600 μL of supernatant to a new low-adsorption centrifuge tube, freeze dry, and reconstitute with 150 μL of 1% acetic acid.

[0051] Automated detection: Refer to Specific Implementation Example 1.

[0052] Method comparison: Eight serum samples were collected and pretreated and detected using both conventional and automated methods. Each serum sample was replicated in quadruplicate, and the mean peak area of ​​IGF-1 was recorded. The mean peak area results of serum samples detected by different methods are shown in Table 12.

[0053] Table 12 ; Method comparison results: Compared with conventional methods, the automated detection method improved the peak area of ​​IGF-1 by 15%~33%, while the interference peaks were smaller, such as... Figure 3As shown, this helps improve the accuracy and sensitivity of low-concentration sample analysis. Furthermore, in the conventional method's pretreatment process, the 600 μL supernatant used contains 38% aqueous phase, and freeze-drying takes 2 hours, severely reducing pretreatment efficiency. In contrast, the automated detection method shortens the entire pretreatment process to 1 hour, significantly improving detection efficiency.

[0054] Example 5 Comparison of matrix solution pretreatment methods Pretreatment methods: (1) Macroporous resin adsorption; (2) Acetic acidification-macroporous resin adsorption-ammonia water neutralization pretreatment; wherein, the macroporous resin is a non-polar hydrophobic interaction type resin, preferably a polystyrene skeleton structure resin, and HPD-100 type resin is used in this embodiment.

[0055] Macroporous resin adsorption: 500 g of macroporous resin was placed in a beaker, soaked in ethanol, and then filtered to remove the solvent using a Buchner funnel and medical absorbent gauze. The resin was then washed twice with ethanol and purified water to obtain activated macroporous adsorption resin. 1 L of human serum was added to 500 g of activated macroporous resin at a ratio of 0.5 g / mL. The mixture was stirred at 75 rpm at room temperature for 1 h, filtered once using a vacuum filtration device, and centrifuged to obtain a blank matrix solution.

[0056] Acetic acidification-macroporous resin adsorption-ammonia water neutralization pretreatment: Refer to Specific Example 1 for pretreatment.

[0057] Comparison of pretreatment methods: The serum samples after the two pretreatment methods were tested according to the kit detection method. Each serum sample was tested in quadruplicate, and the average peak area of ​​IGF-1 was recorded. The removal rate of IGF-1 by different pretreatment methods is shown in Table 13.

[0058] Table 13 ; Pretreatment method comparison results: After acidification with acetic acid, adsorption with macroporous resin, and neutralization with ammonia, the removal rate of IGF-1 in serum can reach 98%, which is significantly higher than the effect of adsorption with macroporous resin alone.

[0059] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A kit for the quantitative determination of IGF-1, characterized in that: The kit comprises a calibrator, a quality control, an internal standard, an immunological enrichment solution, a dissociation solution, a washing solution, an elution solution, mobile phase 1 and mobile phase 2. The calibrator comprises multiple concentrations of IGF-1. The quality control comprises one or more concentrations of IGF-1. The internal standard solution comprises IGF-1-N 15 ; The immunological enrichment solution comprises IGF-1 specific antibody coupled Tosyl magnetic beads, which are obtained by coupling IGF-1 specific antibody with Tosyl magnetic beads. The dissociation solution comprises acetic acid. The washing solution comprises PBS buffer. The elution solution comprises formic acid, hydrochloric acid and acetonitrile. The mobile phase 1 comprises aqueous formic acid. The mobile phase 2 comprises formic acid acetonitrile solution.

2. The kit for quantitative determination of IGF-1 according to claim 1, characterized by: The concentration of the calibrator is 10-1000 ng / mL, and the concentration of the quality control is 50-500 ng / mL.

3. The kit for quantitative determination of IGF-1 according to claim 1, characterized by: The internal standard solution is IGF-1-N at 80-300 ng / mL 15 The immunological enrichment solution is Tosyl magnetic beads coupled with IGF-1 specific antibodies at 0.2-0.5 mg / mL, the dissociation solution is 0.8%-1.2% acetic acid, the elution solution is PBS buffer at pH 6.9-7.5, the elution solution comprises 0.05%-0.15% formic acid, 0.005%-0.01% hydrochloric acid and 25%-35% acetonitrile, the mobile phase 1 is 5%-15% formic acid aqueous solution, and the mobile phase 2 is 5%-15% formic acid acetonitrile solution.

4. A method for preparing the kit for quantitative determination of IGF-1 according to claim 1, characterized in that The kit comprises the following steps: (1) Matrix solution pretreatment: human serum is acidified with acetic acid, adsorbed with macroporous resin, and neutralized with ammonia water for pretreatment to obtain a blank matrix solution; (2) Preparation of calibrator and quality control: standard is dissolved and diluted with methionine-acetic acid to obtain a calibrator and quality control intermediate solution, which is diluted with a blank matrix solution to obtain a calibrator and quality control solution; (3) Preparation of internal standard: internal standard is dissolved and diluted with methionine-acetic acid to obtain an internal standard intermediate solution, which is diluted with a blank matrix solution to obtain an internal standard solution; (4) Preparation of protein enrichment solution: IGF-1 specific antibody is coupled with Tosyl magnetic beads to obtain IGF-1 specific antibody coupled Tosyl magnetic beads; (5) Preparation of dissociation solution: acetic acid is taken, water is added, and mixed to obtain an acetic acid solution; (6) Preparation of washing solution: PBS buffer powder is placed in a clean beaker, and purified water is added to dissolve thoroughly to obtain a washing solution; (7) Preparation of elution solution: formic acid, hydrochloric acid, acetonitrile and purified water are taken into a beaker, and mixed thoroughly to obtain an elution solution; (8) Preparation of mobile phase 1: purified water and formic acid are taken into a centrifuge tube, and mixed thoroughly to obtain a mobile phase 1 solution; 5. The method for preparing a kit for quantitative determination of IGF-1 according to claim 4, characterized in that (9) Preparation of mobile phase 2: acetonitrile and formic acid are taken into a centrifuge tube, and mixed thoroughly to obtain a mobile phase 2 solution.

6. The method for preparing a kit for quantitative determination of IGF-1 according to claim 5, characterized in that Step (1) is specifically: human serum is taken, a magnetic stirrer is used, the temperature is set to 32-40℃, the rotation speed is set to 600-1000 rpm, acetic acid is added during liquid mixing, stirring for 3-8 min, then activated macroporous resin is added, an electric stirrer is used, the rotation speed is set to 60-90 rpm, stirring at room temperature for 0.5-2 h, and then filtering; the filtrate is transferred to a beaker, a magnetic stirrer is used, the rotation speed is set to 600-1000 rpm, ammonia water is added during liquid mixing, stirring for 3-8 min, and then centrifuging to obtain a blank matrix solution.

7. The method for preparing a kit for quantitative determination of IGF-1 according to claim 4, characterized by The activated macroporous resin is prepared by the following steps: macroporous resin is taken into a beaker, soaked with ethanol, and then the solvent is filtered out; then washed with ethanol and purified water respectively to obtain. Step (4) is specifically: (4-1) Prepare ultrafiltration centrifuge tube: take the ultrafiltration centrifuge tube, add water, and centrifuge; add magnetic bead buffer solution for replacement; wherein the magnetic bead buffer solution is sodium bicarbonate-sodium carbonate buffer salt, pH 9.0-9.5; (4-2) Antibody preparation: Take the IGF-1 specific antibody, room temperature, add the antibody to the prepared ultrafiltration centrifuge tube in batches, centrifuge, then add magnetic bead buffer solution, centrifuge; Transfer the antibody in the ultrafiltration centrifuge tube to a 1.5 mL centrifuge tube, wash the ultrafiltration centrifuge tube with magnetic bead buffer solution, and the washing solution is also transferred to a 1.5 mL centrifuge tube, and the antibody concentration is detected; (4-3) Prepare magnetic bead solution: Take the Tosyl magnetic bead solution, mix well, and add it to a 5 mL centrifuge tube. Magnetic separation is performed on the magnetic stand to retain the magnetic beads and discard the supernatant. Then wash the magnetic beads with magnetic bead washing solution. Magnetic separation is performed to retain the magnetic beads and discard the supernatant. Then wash the magnetic beads with magnetic bead buffer solution for more than 2 times. Finally, resuspend the magnetic beads with magnetic bead buffer solution. The composition of the magnetic bead washing solution includes Tween-20 and sodium bicarbonate-sodium carbonate buffer salt, with a pH of 9.0-9.5; (4-4) Antibody magnetic bead coupling: Mix the antibody and magnetic bead solution, add coupling buffer, and rotate in a 35-40°C constant temperature incubator for 6-16h. The coupling buffer is ammonium sulfate; (4-5) Blocking reaction: Add blocking buffer, rotate at room temperature for 2-6h; (4-6) Washing: Magnetic separation is performed to retain the magnetic beads and discard the supernatant. Wash with washing buffer for several times, and resuspend in the washing buffer to make the concentration of the antibody in it 0.2-0.5 mg / mL. The composition of the washing buffer includes Tween-20, bovine serum albumin, proclin300, and PBS buffer, with a pH of 7.0-7.

4.

8. A method for the quantitative determination of IGF-1 for non-diagnostic purposes, characterized in that: The kit of claim 1 comprises the following steps: (I) Reagent preparation; (II) Automatic pretreatment using a full-automatic magnetic solid phase extraction instrument: Place several 96-well deep well plates in the designated position of the magnetic solid phase extraction instrument. According to the preset pretreatment program, automatically add different components to different columns. According to the set time sequence, extract the sample. After the operation is completed, automatically transfer the eluent to the 96-well sample plate for mass spectrometry analysis.

9. The detection method of claim 8, wherein Step (I) specifically includes: (I-1) Take out each component of the kit and let it stand at room temperature; (I-2) Reconstitute the calibrators, quality controls, and internal standard to obtain calibrators J1-J6, quality controls Z7-Z8, and internal standard solution; (I-3) Dilute mobile phase 1 with purified water, ultrasonic, and mix well to obtain the aqueous phase. Dilute mobile phase 2 with acetonitrile, ultrasonic, and mix well to obtain the organic phase.

10. The detection method of claim 8, wherein In step (II), the chromatographic parameters are controlled as follows: Chromatographic column: Sepax Bio C4 + online filter; Flow rate: 0.4 mL / min; Column temperature: 55°C; Injection volume: 20 μL; Liquid chromatography elution gradient: 0-2.0 min, mobile phase A ratio 95%, mobile phase B ratio 5%; 2.0-3.1 min, mobile phase A ratio 40-95%, mobile phase B ratio 5-60%; 3.1-3.2 min, mobile phase A ratio 40-95%, mobile phase B ratio 5-60%; 3.2-4.3 min, mobile phase A ratio 40-95%, mobile phase B ratio 5-60%; 4.3-4.4 min, mobile phase A 40-95%, mobile phase B 5-60%; 4.4-5.0 min, mobile phase A 95%, mobile phase B 5%; Mass spectrometry parameters were controlled as follows: The ion source was an electrospray ion source in positive ion mode; The capillary voltage was 2.0 kv, the desolvation temperature was 600 °C, the desolvation gas flow rate was 1000 L / hr, and the cone gas flow rate was 150 L / hr; The scan mode was multiple reaction monitoring mode; the cone voltage was 20-50 V, and the collision voltage was 20-50 V.

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