Quantitative detection method for igf-1 variants

Abstract

The present application belongs to the technical field of in vitro detection, and particularly relates to a quantitative detection method of IGF-1 variants. The present inventors have found that IGF-1 exists in two variant forms, namely a des-R IGF-1 variant with a single charge mass ratio of 7500-7520 and a des-RR IGF-1 variant with a single charge mass ratio of 7343-7363, and by using the detection method of the present application for the variants of insulin-like growth factor I, in particular, the creative finding that when detection is performed, IGF-1 antibodies are bonded to epoxy magnetic beads, so that the detection method can accurately detect the des-R IGF-1 variant and the des-RR IGF-1 variant, thereby providing refined data for disease analysis of IGF-1.

Description

Technical Field

[0001] This invention belongs to the field of in vitro detection technology, specifically relating to a quantitative detection method for IGF-1 variants. Background Technology

[0002] Insulin-like growth factor I (IGF-1), also known as somatomedin C, is a secretory protein synthesized by the liver and secreted into the peripheral blood. Its mature protein consists of 70 amino acids, with six cysteine ​​residues forming three pairs of disulfide bonds to maintain its spatial structure. It is named for its structural similarity to insulin. IGF-1 is an active protein polypeptide, a product of autocrine and paracrine secretion by more than a dozen types of cells in the human body, including hepatocytes, kidney cells, and spleen cells. It has functions such as lowering blood sugar and lipids, dilating blood vessels, promoting growth, promoting cell differentiation, and wound repair. Accurate detection of IGF-1 is helpful for disease analysis and post-diagnosis medication guidance.

[0003] While one variant of IGF-1, IGF-1LR3, has been disclosed in this field, it is exogenously synthesized, consisting of the original 70-amino acid chain with 13 additional amino acids added to its N-terminus, resulting in a total of 83 amino acids. This IGF-1 variant still exhibits its original activity against IGF-1 receptors in human tissues. However, there are very few reports of IGF-1 variants being produced in vivo, and consequently, there is a lack of awareness that poor linearity in individual samples is due to the degradation of IGF-1 into variants. Existing detection methods can only detect IGF-1 and cannot detect variants present in samples. Summary of the Invention

[0004] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, this invention provides a method for detecting variants of insulin-like growth factor I (IGF-1), thereby providing refined data for IGF-1 disease analysis and facilitating further clinical diagnosis.

[0005] In a first aspect, the present invention provides a method for detecting variants of insulin-like growth factor I. According to an embodiment of the present invention, the detection method includes:

[0006] (1) Contact the test sample, IGF-1 internal standard and IGF-1 antibody so that the IGF-1 internal standard and the variant of insulin-like growth factor I contained in the test sample can be specifically bound to the IGF-1 antibody to obtain an antigen-antibody complex enriched with the variant of insulin-like growth factor I and the internal standard IGF-1.

[0007] (2) The antigen-antibody complex is eluted to elute and separate the enriched variant of insulin-like growth factor I and IGF-1 internal standard from the IGF-1 antibody, thereby obtaining a test solution containing the variant of insulin-like growth factor I and IGF-1 internal standard.

[0008] (3) The test solution was analyzed by MALDI ion source mass spectrometry to detect the amount of insulin-like growth factor I variant in the test sample.

[0009] In step (1), the IGF-1 antibody is bonded to Epoxy magnetic beads.

[0010] The variants of insulin-like growth factor I include des-R IGF-1 variants having a single charge mass-to-charge ratio of 7500-7520, a double charge mass-to-charge ratio of 3746-3766, or a triple charge mass-to-charge ratio of 2494-2514.

[0011] The des-RR IGF-1 variants have a mass-to-charge ratio of 7343-7363 for single charge, 3667-3687 for double charge, or 2442-2462 for triple charge.

[0012] Currently, the main clinical detection method for IGF-1 is chemiluminescence immunoassay. Chemiluminescence immunoassay indirectly quantifies the analyte by measuring the light intensity emitted by the luminescent material on the secondary antibody that specifically binds to the analyte. However, the stability of this technique is affected by various factors such as antibody specificity, non-specific adsorption, heterophilic antibodies, and interfering substances, resulting in large fluctuations in the quantitative results.

[0013] Chemiluminescence immunoassay for insulin-like growth factor I (IGF-1) only detects the amount of IGF-1 and lacks discriminative power, failing to identify variants with different amino acid numbers. When using MALDI-TOF MS to detect IGF-1, the inventors discovered peaks with single charge mass-to-charge ratios (MS / MR) of 7500–7520 and 7343–7363 in samples containing IGF-1. Further experimental verification confirmed that these two peaks correspond to the des-R IGF-1 and des-RR IGF-1 variants of IGF-1, respectively. By employing the aforementioned detection method, and particularly by inventively binding IGF-1 antibodies to epoxy magnetic beads, the inventors have enabled the detection method to accurately detect both des-R and des-RR IGF-1 variants, thus providing refined data for IGF-1 disease analysis. The inventors also discovered that using other magnetic bead bonding methods, such as bonding IGF-1 internal standard or insulin-like growth factor I variants to magnetic beads via the biotin-streptavidin system, cannot simultaneously detect both des-R IGF-1 variants and des-RR IGF-1 variants, resulting in inaccurate detection results and an inability to provide refined data for IGF-1 disease analysis.

[0014] The detection method of this invention can accurately detect not only the levels of des-R IGF-1 variants and des-RR IGF-1 variants, but also the level of IGF-1. By adding the detected levels of IGF-1, des-R IGF-1 variants, and des-RR IGF-1 variants, the total IGF-1 level is obtained, thus improving the accuracy of IGF-1 quantification. Based on the hospital's needs, refined data for IGF-1 disease analysis can be obtained by providing the level of IGF-1, the total IGF-1 level, or the level of IGF-1 variants.

[0015] According to an embodiment of the present invention, when the sample to be tested is serum or plasma, the detection method further includes, before step (1), lysing the sample to be tested with a lysis buffer to obtain a lysed solution, and in step (1), contacting the lysed solution, IGF-1 internal standard and IGF-1 antibody.

[0016] According to an embodiment of the present invention, the lysis buffer includes a lysis agent and a first buffer solution.

[0017] According to an embodiment of the present invention, the pyrolysis agent is selected from at least one of guanidine hydrochloride, urea, thiourea, sodium dodecyl sulfate, hexadecyltrimethylammonium bromide, Tween 20, Tween 80, 3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt, octyl-β-glucoside, octyl thioglucoside, polyethylene glycol octylphenyl ether, and ethylphenyl polyethylene glycol.

[0018] According to an embodiment of the present invention, the first buffer solution is selected from Tris-HCl buffer solution and PBS buffer solution.

[0019] According to an embodiment of the present invention, the lysis buffer contains sodium dodecyl sulfate and PBS buffer.

[0020] According to an embodiment of the present invention, the lysis buffer is a PBS solution of sodium dodecyl sulfate with a mass concentration of 1-5%;

[0021] According to an embodiment of the present invention, a test solution containing a variant of the insulin-like growth factor I to be tested and an IGF-1 internal standard is obtained through the following steps:

[0022] (I) Mix the test sample, IGF-1 internal standard and magnetic bead suspension bound to IGF-1 antibody in reaction buffer and react to allow the test variant of insulin-like growth factor I and the IGF-1 internal standard in the test sample to specifically bind to the IGF-1 antibody bound to the magnetic beads, thereby obtaining antigen-antibody complex magnetic beads enriched with the test variant of insulin-like growth factor I and the IGF-1 internal standard.

[0023] (II) The antigen-antibody complex magnetic beads are eluted and magnetically separated to elute and separate the enriched variant of insulin-like growth factor I and IGF-1 internal standard from the IGF-1 antibody, thereby obtaining a test solution containing the variant of insulin-like growth factor I and IGF-1 internal standard.

[0024] According to an embodiment of the present invention, the particle size of the Epoxy magnetic beads is 0.1 μm to 25 μm, preferably 0.5 μm to 5 μm.

[0025] According to an embodiment of the present invention, the IGF-1 antibody is a monoclonal antibody or a polyclonal antibody.

[0026] According to an embodiment of the present invention, the affinity constant KD value of the IGF-1 antibody is 1×10⁻⁶. -9 -9×10 -12 .

[0027] The IGF-1 antibody provided by this invention is used to enrich IGF-1 variants, which can improve the sensitivity of detection.

[0028] According to an embodiment of the present invention, the IGF-1 internal standard is an isotopically labeled IGF-1 protein or an IGF-1 protein with a linked tag sequence, or a polypeptide with an amino acid sequence similarity of more than 80% to that of the IGF-1 protein that can be enriched by the IGF-1 antibody.

[0029] According to an embodiment of the present invention, the isotope is H2, N15, C13 or O18.

[0030] According to an embodiment of the present invention, the tag sequence includes at least one selected from His tag, Flag tag, GST tag, MBP tag, C-MYC tag, HA tag, FC tag, AVI tag, MBP tag, and DDDDK tag.

[0031] IGF-1 internal standard can reduce the impact of sample pretreatment and instrument analysis on detection reproducibility and improve detection precision.

[0032] According to an embodiment of the present invention, in step (I), the reaction buffer includes at least one selected from MES buffer, PBST buffer, and Tris-HCl buffer.

[0033] According to an embodiment of the present invention, the pH value of the reaction buffer is 6-8.

[0034] A reaction buffer with a pH of 6-8 provides a target enrichment environment with a pH of 6-8, thereby improving the enrichment efficiency of antibodies against IGF-1 variants.

[0035] According to an embodiment of the present invention, the reaction buffer is a PBST buffer containing 0.1-0.6% Tween 20.

[0036] According to an embodiment of the present invention, before step (2) or (II), the product of step (1) or (I) is further subjected to a first wash and a second wash. The first wash uses a buffer solution containing a nonionic surfactant, and the second wash uses deionized water. The washing solution provided by the present invention can reduce nonspecific adsorption during antibody enrichment and improve detection sensitivity. Specifically, the first wash removes nonspecifically adsorbed impurity proteins from the sample, while the second wash, using deionized water, removes the washing solution used in the first wash, as well as the washing solution and salt ions used in the first wash.

[0037] According to an embodiment of the present invention, the nonionic surfactant comprises a selection from Tween 20, Tween 60, Tween 80, CHAPS, Triton X-100, alkoxypolyvinylhydroxoethanol, octyl glucoside, dodecyl maltodextrin, n-octyl α-D-glucoside, N-octanoyl-N-methylglucosamine, N-nonanoyl-N-methylglucosamine, N-decanoyl-N-methylglucosamine, nonyl-β-D-glucopyranoside, dodecyl-β-D-maltodextrin, N,N-dimethyldodecylamine-N-oxide, decyl glucopyranoside, 1-O-decyl-β-D-maltodextrin, Deoxy-Bigchap, and tufted tartaric acid. At least one of the following: flavonoids, Triton X-114, nonylphenol polyoxyethylene ether, tetraethylene glycol monododecyl ether, tetramethylammonium hydroxide pentahydrate, polysorbate-85, tetrabutylphenol aldehyde, undecyl-β-D-maltoside, octyl-β-D-thiopyranoside, n-octyl-β-D-pyranoside, octaethylene glycol monododecyl ether, saponin, span, sucrose dodecanoate, glyceryl monooleate, 6-O-(N-heptaformyl)-methyl-α-D-glucoside, polyethylene glycol monostearate, n-nonyl-β-d-thiomaltoside, and n-dodecyl-β-d-maltoside.

[0038] According to an embodiment of the present invention, the buffer includes a selection from PBS buffer, MES buffer, Tris buffer, DPBS buffer, CBS buffer, BBS buffer, HEPES buffer, TBS buffer, BES buffer, TEA buffer, MOPS buffer, AMPD buffer, EPPS buffer, MOPSO buffer, AMPSO buffer, DIPSO buffer, and TAPSO buffer.

[0039] According to an embodiment of the present invention, the first cleaning solution is a PBS buffer containing at least one of Tween 20, Tween 60, and Tween 80.

[0040] According to an embodiment of the present invention, the first cleaning solution used is a 0.1% (v / v) Tween 20 PBS buffer solution. This effectively removes non-specifically adsorbed impurity proteins from the sample.

[0041] According to an embodiment of the present invention, in step (2) or (II), the elution solution used in the elution treatment contains a matrix and an antigen-antibody complex dissociation reagent, wherein the matrix includes at least one selected from sinapic acid, α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, 2,4,6-trihydroxyacetophenone, anthraquinone, and 3-indoleacrylic acid.

[0042] According to an embodiment of the present invention, the matrix is ​​α-cyano-4-hydroxycinnamic acid.

[0043] According to an embodiment of the present invention, the concentration of α-cyano-4-hydroxycinnamic acid is 5-20 mg / mL.

[0044] The present invention preferably uses α-cyano-4-hydroxycinnamic acid as the matrix of the MALDI-TOF MS platform, which can improve the number and intensity of mass spectra.

[0045] According to an embodiment of the present invention, the antigen-antibody complex dissociation reagent includes at least one selected from organic acid solutions, inorganic acid solutions, and alkaline solutions.

[0046] The organic acid solution includes at least one selected from formic acid solution, acetic acid solution, trifluoroacetic acid solution, propionic acid solution, glycolic acid solution, amino acid solution, butyric acid solution, citric acid, succinic acid, ascorbic acid, lactic acid, malic acid, and tartaric acid;

[0047] The inorganic acid solution includes at least one selected from hydrochloric acid solution, sulfuric acid solution, and phosphoric acid solution;

[0048] The alkaline solution includes at least one selected from sodium hydroxide solution, potassium hydroxide solution, tris(hydroxymethyl)aminomethane solution, tetraethylammonium bromide solution, sodium carbonate solution, sodium bicarbonate solution, ammonium bicarbonate solution, ammonia solution, and urea solution.

[0049] According to an embodiment of the present invention, the antigen-antibody complex dissociation reagent is a trifluoroacetic acid solution. The matrix used for the MALDI-TOF MS platform in the elution solution does not affect the elution effect of the antigen-antibody complex dissociation reagent.

[0050] According to an embodiment of the present invention, the mass concentration of the trifluoroacetic acid solution is 0.05-5%.

[0051] At this concentration, TFA solution, as an antigen-antibody complex dissociation reagent, can further enhance the dissociation efficiency of variants of insulin-like growth factor I and IGF-1 antibody, so that the antigen and antibody are completely dissociated, thereby further improving the accuracy of detecting variants of insulin-like growth factor I.

[0052] According to an embodiment of the present invention, the elution solution contains the matrix CHCA (α-cyano-4-hydroxycinnamic acid), acetonitrile, and TFA for the MALDI-TOF MS platform.

[0053] According to an embodiment of the present invention, step (3) further includes:

[0054] The solution to be tested is spotted onto a target plate used for MALDI ion source mass spectrometry detection. The target plate after spotting is heat-treated to obtain the crystal to be tested. The target plate with the crystal to be tested is placed on the instrument for data acquisition.

[0055] Optionally, step (3) further includes pre-applying the elution solution to the target plate and subjecting it to heat treatment before spotting, in order to prepare a matrix crystalline layer.

[0056] According to an embodiment of the present invention, two or more layers of the matrix crystalline layer are prepared. Thus, by forming a multilayer crystalline matrix layer as the bottom layer on the target plate, the matrix-assisted laser desorption / ionization effect, i.e., the ionization effect, can be significantly improved, thereby enhancing detection sensitivity.

[0057] According to an embodiment of the present invention, the temperature of the heat treatment is 35-45°C.

[0058] According to an embodiment of the present invention, the MALDI ion source mass spectrometer is a MALDI-TOF MS or a MALDI-TOF-TOF MS.

[0059] A second aspect of the present invention provides a method for simultaneously detecting insulin-like growth factor I and its variants. According to an embodiment of the present invention, the method includes detecting the amounts of insulin-like growth factor I and its variants in a sample using the detection method described in the first aspect.

[0060] Among them, the variants of insulin-like growth factor I include des-R IGF-1 variants having a single charge mass-to-charge ratio of 7500-7520, a double charge mass-to-charge ratio of 3746-3766, or a triple charge mass-to-charge ratio of 2494-2514.

[0061] The des-RR IGF-1 variants have a single charge mass-to-charge ratio of 7343-7363, a double charge mass-to-charge ratio of 3667-3687, or a triple charge mass-to-charge ratio of 2442-2462. The second aspect of the invention provides a method for the simultaneous detection of insulin-like growth factor I and its variants. Utilizing the detection method described in the first aspect, in the method provided in the first aspect, for example, in step (1), an antigen-antibody complex enriched with the target insulin-like growth factor I, its variants, and the IGF-1 internal standard is actually obtained. Step (2) involves elution and separation to obtain a test solution containing the target insulin-like growth factor I and its variants, and the IGF-1 internal standard. During MALDI ion source mass spectrometry detection, the amount of insulin-like growth factor I and its variants can be determined simultaneously.

[0062] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0063] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0064] Figure 1 The MALDI-TOF MS mass spectra of IGF-1 standard protein solutions at different concentrations are shown in Example 1 of this invention.

[0065] Figure 2 The IGF-1 standard curves obtained based on IGF-1 standard protein solutions of different concentrations in Example 1 of the present invention are shown.

[0066] Figure 3 The MALDI-TOF MS mass spectrum of IGF-1 in serum / plasma sample 1 of this invention is shown.

[0067] Figures 4A-4B The MALDI-TOF MS mass spectra of IGF-1 in the serum before and after 5 hours of storage in Example 1 of this invention are shown.

[0068] Figures 4C-4D The MALDI-TOF MS mass spectra of IGF-1 in serum before and after the addition of the cocktail protease inhibitor in Example 1 of this invention are shown.

[0069] Figure 5 The results of fitting the quantitative detection results of IGF-1 in Example 1 with the quantitative detection results of IGF-1 in Example 2 are shown;

[0070] Figure 6 The results show that the sum of the quantitative detection results of IGF-1 in Example 1 and the quantitative detection results of des-R IGF-1 variant and des-RR IGF-1 variant is taken as the total IGF-1, and the total IGF-1 is fitted with the quantitative detection results of IGF-1 in Example 2.

[0071] Figure 7 The MALDI-TOFMS mass spectra of IGF-1 and its variants detected using epoxy magnetic beads in Example 4 are shown.

[0072] Figure 8 The MALDI-TOF MS mass spectra of IGF-1 and its variants detected by streptomycin affinity magnetic beads in Example 4 are shown. Detailed Implementation

[0073] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0074] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0075] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0076] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0077] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0078] In this paper, the terms “identity” and “homology” are used interchangeably to describe the percentage of identical amino acids between two amino acid sequences relative to a reference sequence, determined by conventional methods, such as Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence consistency, including: the homology alignment algorithm by Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm by Smith et al. (1981) Adv. Appl. Math. 2: 482; the similarity search method by Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444; and the Smith-Waterman algorithm (Meth. Mol.). Biol. 70: 173-187 (1997); and the BLASTP and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266: 460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al.

[0079] Without substantially affecting the activity of the IGF-1 protein (retaining at least 95% activity) and without affecting its binding to IGF-1-specific antibodies, those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the amino acid sequence of the IGF-1 protein of the present invention to obtain polypeptides with an amino acid sequence identity of more than 80% that can be enriched by the IGF-1 antibody. The polypeptides enriched by the IGF-1 antibody of the present invention can have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence (amino acid sequence of the IGF-1 protein). The sequence identity described in the present invention can be measured using sequence analysis software, such as the computer program BLAST, especially BLASTP, using default parameters.

[0080] According to a specific embodiment of the present invention, the present invention provides variants of insulin-like growth factor I, including des-RIGF-1 variants having a single charge mass-to-charge ratio of 7500-7520 or a double charge mass-to-charge ratio of 3746-3766 or a triple charge mass-to-charge ratio of 2494-2514.

[0081] The des-RR IGF-1 variants have a mass-to-charge ratio of 7343-7363 for single charge, 3667-3687 for double charge, or 2442-2462 for triple charge.

[0082] According to an embodiment of the present invention, the amino acid sequence of the des-R IGF-1 variant is shown in SEQ ID NO:1, and the amino acid sequence of the des-RR IGF-1 variant is shown in SEQ ID NO:2.

[0083] The amino acid sequence shown in SEQ ID NO:1 is as follows:

[0084] GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLR-LEMYCAPLKPAKSA

[0085] The amino acid sequence shown in SEQ ID NO:2 is as follows:

[0086] GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDL—LEMYCAPLKPAKSA

[0087] According to a specific embodiment of the present invention, the present invention provides a method for detecting a variant of the aforementioned insulin-like growth factor I, comprising:

[0088] (1) Contact the test sample, IGF-1 internal standard and IGF-1 antibody so that the IGF-1 internal standard and the variant of insulin-like growth factor I contained in the test sample can be specifically bound to the IGF-1 antibody to obtain an antigen-antibody complex enriched with the variant of insulin-like growth factor I and the internal standard IGF-1.

[0089] (2) The antigen-antibody complex is eluted to elute and separate the enriched variant of insulin-like growth factor I and IGF-1 internal standard from the IGF-1 antibody, thereby obtaining a test solution containing the variant of insulin-like growth factor I and IGF-1 internal standard.

[0090] (3) The test solution was analyzed by MALDI ion source mass spectrometry to detect the amount of insulin-like growth factor I variant in the test sample.

[0091] In step (1), the IGF-1 antibody is bonded to Epoxy magnetic beads.

[0092] According to a specific embodiment of the present invention, in the process of binding the antibody to the magnetic beads, the particle size of the selected magnetic beads is 0.1μm to 25μm, preferably 0.5μm to 5μm, such as 0.5μm, 1.5μm, 2.7μm, 3.0μm, etc.

[0093] Using the above detection method, not only can the des-R IGF-1 variant and the des-RR IGF-1 variant be detected, but also the amount of IGF-1 contained in the sample. The amount of IGF-1 contained in the sample is actually the sum of the amount of IGF-1 and the des-R IGF-1 variant and the des-RR IGF-1 variant. This detection result is more accurate and provides effective support for further precision medicine.

[0094] It should be noted that the sum of the above-mentioned IGF-1 amount and the des-R IGF-1 variant and the des-RR IGF-1 variant can be the sum of peak areas or the sum of response intensities, preferably the sum of response intensities.

[0095] According to one embodiment of the present invention, the MALDI ion source mass spectrometer is a MALDI-TOF MS or a MALDI-TOF-TOF MS.

[0096] The detection method of this invention uses epoxy magnetic beads. IGF-1 antibodies are bonded to epoxy magnetic beads, and the antibodies are chemically coupled to the beads, resulting in a more robust connection compared to the streptavidin-biotin linkage method. The use of epoxy magnetic beads in this invention reduces background interference, minimizes impurity peaks, and decreases non-specific adsorption, further improving detection sensitivity.

[0097] According to a specific embodiment of the present invention, a test solution containing a variant of the target insulin-like growth factor I and an IGF-1 internal standard is obtained through the following steps:

[0098] (I) The test sample, IGF-1 internal standard and magnetic bead suspension bound to IGF-1 antibody are vortexed and mixed in reaction buffer to allow the test variant of insulin-like growth factor I and IGF-1 internal standard in the test sample to specifically bind to the IGF-1 antibody bound to the magnetic beads, thereby obtaining antigen-antibody complex magnetic beads enriched with test variant of insulin-like growth factor I and IGF-1 internal standard.

[0099] (II) The antigen-antibody complex magnetic beads are eluted and magnetically separated to elute and separate the enriched variant of insulin-like growth factor I and IGF-1 internal standard from the IGF-1 antibody, thereby obtaining a test solution containing the variant of insulin-like growth factor I and IGF-1 internal standard.

[0100] According to an embodiment of the present invention, prior to step (3), the method further includes: pre-applying the elution solution to the target plate and performing heat treatment to prepare a matrix crystalline layer.

[0101] According to an embodiment of the present invention, two or more layers of the matrix crystalline layer are prepared. Thus, by forming multiple layers of crystalline matrix as the bottom layer on the target plate, the matrix-assisted laser desorption / ionization effect, i.e., the ionization effect, can be significantly improved, thereby increasing the detection sensitivity. There are no particular limitations on the material of the target plate; for example, it can be a 96-well polypropylene U-shaped plate, a silicon plate, etc.

[0102] According to a specific embodiment of the present invention, the present invention provides a method for detecting variants of insulin-like growth factor I using MALDI-TOF MS, comprising:

[0103] (1) The sample to be tested is mixed with IGF-1 internal standard and magnetic beads bound to IGF-1 antibody and reacted. The variant of insulin-like growth factor I to be tested and the IGF-1 internal standard contained in the sample to be tested specifically bind to the IGF-1 antibody bound to the magnetic beads to obtain magnetic beads enriched with the variant to be tested and the IGF-1 internal standard.

[0104] (2) The magnetic beads enriched with the target variant and IGF-1 internal standard are eluted and magnetically separated using an elution solution to obtain a test solution containing the target variant and IGF-1 internal standard.

[0105] (3) Spot the solution to be tested onto the target plate for MALDI-TOF MS detection, and heat-treat the target plate after spotting to allow the sample to crystallize and obtain the crystallized product.

[0106] (4) Place the target plate with the crystalline product on the MALDI-TOF MS instrument for data acquisition in order to detect the variant of insulin-like growth factor I contained in the sample to be tested.

[0107] It should be noted that there are no particular restrictions on the temperature and time of the heat treatment, as long as the mixture on the target plate crystallizes to obtain the crystalline product to be detected. For example, the heat treatment temperature can be 35-45℃, preferably 38℃, 39℃, 40℃, etc.

[0108] According to a specific embodiment of the present invention, before spotting the test solution onto the target plate, an elution solution is pre-spotted onto the target plate to pre-form a matrix crystalline layer. This step can be repeated two or more times to form two or more matrix crystalline layers. Therefore, by forming a multi-layered crystalline matrix layer as the bottom layer on the target plate, the matrix-assisted laser desorption / ionization effect, i.e., the ionization effect, can be significantly improved, thereby increasing the detection sensitivity. Furthermore, a hydrophobic target plate can be used during MALDI-TOF MS detection, which can further enhance the detection sensitivity.

[0109] It should be noted that there are no particular restrictions on the data acquisition conditions using a MALDI-TOF MS instrument; the conventional conditions for MALDI-TOF MS in this field can be used. However, the preferred conditions are as follows:

[0110] Laser: Semiconductor laser;

[0111] Laser frequency: 1000-5000Hz;

[0112] Two-dimensional platform moving speed: 0.5-3.0 mm / sec;

[0113] Focus Mass: 4500~9000Da;

[0114] Acquisition quality range: 2kDa~35kDa.

[0115] The MALDI-TOF MS instrument is selected from QuanPro mass spectrometer and QuanTOF type I mass spectrometer.

[0116] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0117] Example 1: Quantitative detection of IGF-1 and IGF-1 variants in 205 serum / plasma samples based on MALDI-TOF MS.

[0118] 1. Reagent preparation:

[0119] (1) Preparation of 10mM PBS solution

[0120] Add 100 mL of 0.1 M PBS solution (2.7 mM potassium chloride, 137 mM sodium chloride, 1.76 mM potassium phosphate) to 900 mL of deionized water and mix by inverting.

[0121] (2) Preparation of 0.1% Tween 20 PBS solution:

[0122] Add 1000 μL of the nonionic surfactant Tween 20 to 1000 mL of 10 mM PBS solution and mix with ultrasonic assistance.

[0123] (3) Preparation of lysis buffer:

[0124] The specific preparation process for a PBS solution containing 1% SDS (sodium dodecyl sulfonate) is as follows: accurately weigh 1.0 g of SDS solid powder into a 100 mL volumetric flask, add about 50 mL of 10 mM PBS solution, and sonicate to dissolve the SDS powder completely. After the liquid returns to room temperature, dilute to the mark with 10 mM PBS solution.

[0125] (4) Preparation of internal standard:

[0126] N15-labeled IGF-1 was prepared into a 250 ng / mL IGF-1 internal standard solution using 0.5% BSA-PBS solution.

[0127] (5) Preparation of the substrate:

[0128] The matrix solution is an acetonitrile aqueous solution containing 10 mg / mL CHCA (α-cyano-4-hydroxycinnamic acid) (acetonitrile:water = 7:3 (v / v)), with a TFA content of 0.5%. The specific preparation process is as follows: Accurately weigh 1 g of CHCA into a 100 mL reagent bottle, add 70 mL of acetonitrile and 30 mL of deionized water, and sonicate for 5 min (ensuring that the pale yellow CHCA is completely dissolved; the sonication time can be appropriately increased). Then add 500 μL of TFA and mix thoroughly by inverting.

[0129] 2. Antibody-magnetic bead bonding

[0130] (1) Add 100ug of IGF-1 antibody (mouse monoclonal antibody) to a 30kDa ultrafiltration tube and centrifuge at 12000×g until all liquid in the tube is removed. Remove the ultrafiltration tube and add 200μL of 0.1M PBS (pH 7.4) buffer solution. Vortex for 30s and centrifuge at 12000×g for 5min (repeat this operation twice). Add 290μL of 0.1M PBS (pH 7.4) solution to the cleaned ultrafiltration tube and vortex to mix before bonding with magnetic beads.

[0131] (2) 100 μL (10 mg) of epoxy resin magnetic beads were placed in a 2 mL centrifuge tube and placed on a magnetic rack for magnetic separation. After washing with 200 μL of 0.1 M PBS (pH 7.4), the solution was resuspended in 330 μL of 0.1 M PBS (pH 7.4). The entire antibody solution in the ultrafiltration tube was transferred to the 2 mL centrifuge tube containing the magnetic beads. 330 μL of 3 M ammonium sulfate solution (dissolved in 0.1 M PBS (pH 7.4)) was added, and the mixture was vortexed and incubated overnight at room temperature (approximately 16 hours) for bonding. After bonding, the magnetic beads were separated by magnetic force and the liquid was discarded. The beads were washed three times with 500 μL of 10 mM PBS solution and the liquid was discarded. The beads were resuspended in 1% BSA solution and inverted at room temperature for 30 min. The beads were washed once with 500 μL of PBS solution containing 0.1% Tween 20 and then resuspended in 1 mL of PBS solution containing 0.1% Tween 20 for later use.

[0132] 3. Preparation of IGF-1 standard curve

[0133] IGF-1 standard protein was prepared into a 0.1 mg / mL stock solution using PBS. The stock solution was then serially diluted with PBS-PA1080 to obtain six concentration levels of IGF-1 standard protein solutions: 1256.02 ng / mL, 981.56 ng / mL, 670.99 ng / mL, 351.85 ng / mL, 65.01 ng / mL, and 6.73 ng / mL.

[0134] Accurately transfer 40 μL of IGF-1 standard protein solution samples of different concentrations into 0.6 mL centrifuge tubes. Add 130 μL of 0.1% Tween 20 PBS solution and 30 μL of IGF-1 internal standard, respectively. Vortex to mix. Then add 10 μL of magnetic bead suspension containing the bound antibody and vortex to mix. Invert and mix on a centrifuge for 10 min. Remove and centrifuge briefly for 10 s. Immediately place on a magnetic rack for magnetic separation for 1 min. Discard the liquid in the tube and add 200 μL of 0.1% Tween 20 PBS solution. Vortex for about 30 s to ensure that the magnetic beads are completely suspended. Place on a magnetic rack for magnetic separation for 1 min (repeat the operation 3 times). After discarding the supernatant, add 200 μL of deionized water to a 0.6 mL centrifuge tube, vortex for 30 s, then place on a magnetic rack for magnetic separation for 1 min. Discard the supernatant again, and repeat the operation once more with 100 μL of deionized water. Centrifuge the empty centrifuge tube in a mini centrifuge for 30 s, then place on a magnetic rack for magnetic separation for 30 s. Carefully aspirate the trace amount of liquid at the bottom of the centrifuge tube with a 10 μL pipette tip, then accurately add 10 μL of the matrix working solution, vortex for 30 s, and place on a magnetic rack for magnetic separation for 30 s. Take 2 μL of colorless and transparent eluent (matrix solution containing the target protein) and spot it on a target plate preheated at 39 °C. After the sample on the target plate has completely crystallized, acquire data using MALDI-TOF MS. The results are shown in [Figure number missing]. Figure 1 .

[0135] The MALDI-TOF MS acquisition conditions are as follows:

[0136] Laser: Semiconductor laser; Laser frequency: 1000Hz; 2D platform movement speed: 1.5mm / sec; Focus Mass: 4500~9000Da; Acquisition quality range: 2kDa~35kDa.

[0137] The ratio of the Y-values ​​of IGF-1 in the standard solution to that of the internal standard (N15-labeled IGF-1) obtained on MALDI-TOF MS was used as x, and the concentration of IGF-1 was used as y to fit a quantitative standard curve. A power function was chosen for standard curve fitting; see [link to documentation]. Figure 2 Y = A * X B

[0138] Where A = 0.867, B = 1.0699, R 2 =0.9968.

[0139] 4. Quantification of IGF-1 in serum / plasma samples

[0140] Accurately transfer 20 μL of serum / plasma into a 0.6 mL centrifuge tube, add 20 μL of lysis buffer, vortex, and shake at 1500 rpm for 10 min at room temperature. Then add 130 μL of 0.6% Tween 20 PBS solution and 30 μL of IGF-1 internal standard solution, vortex to mix, add 10 μL of antibody-bound magnetic bead suspension, vortex to mix, and incubate at room temperature on a centrifuge for 10 min. Remove and centrifuge for 10 s, then immediately place on a magnetic rack for magnetic separation for 1 min. Discard the liquid in the tube, add 200 μL of 0.1% Tween 20 PBS solution, vortex for about 30 s to ensure complete suspension of the magnetic beads, and then place on a magnetic rack for magnetic separation for 1 min (repeat the operation 3 times). After discarding the supernatant, add 200 μL of deionized water to a 0.6 mL centrifuge tube, vortex for 30 s, then place on a magnetic rack for magnetic separation for 1 min. Discard the supernatant again, and repeat the operation once more with 100 μL of deionized water. Centrifuge the liquid-free centrifuge tube in a mini centrifuge for 30 s, then place on a magnetic rack for magnetic separation for 30 s. Carefully aspirate the trace amount of liquid at the bottom of the centrifuge tube using a 10 μL pipette tip. Then accurately add 10 μL of matrix, vortex for 30 s, and place on a magnetic rack for magnetic separation for 30 s. Take 2 μL of colorless and transparent eluent and spot it onto a target plate preheated on a 39°C hot plate. After the sample on the target plate has completely crystallized, acquire data using MALDI-TOF MS.

[0141] The ratio of IGF-1 / IGF-1 variant to the internal standard (N15-labeled IGF-1) for each sample is used as the X value and input into the standard curve Y = A*X. B In the process, the concentration of IGF-1 / variant in the sample tube is calculated. The IGF-1 content in the sample is calculated according to the following formula:

[0142]

[0143] C IGFI —IGF-1 concentration in the reaction tube (ng / mL)

[0144] V — Sample volume (μL)

[0145] The inventors used MALDI TOF MS to quantitatively detect IGF-1 in 205 serum / plasma samples. During the detection process, they discovered two additional peaks besides the IGF-1 peak in each mass spectrometry result, suggesting the possible existence of two variants of IGF-1. The variant with a mass-to-charge ratio (MMR) of 7500–7520 was named des-R IGF-1, and the variant with a MMR of 7343–7363 was named des-RR IGF-1. This invention provides an example of the mass spectrometry result of one sample, such as... Figure 3 As shown.

[0146] The MALDI TOF MS quantification results of IGF-1 in 205 serum / plasma samples are shown in Table 1 below.

[0147] Table 1:

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154] Example 2: IGF-1 was quantitatively detected in 205 serum / plasma samples from the same cases as in Example 1 using a chemiluminescence platform (Huayin).

[0155] The detection data (total IGF-1) of chemiluminescent immunoassay in 205 serum / plasma samples are shown in Table 2 below.

[0156] Table 2:

[0157]

[0158]

[0159] Example 3: Verification that the des-R IGF-1 variant and the des-RR IGF-1 variant are IGF-1 variants

[0160] Using the same detection method as in Example 1, the following experimental and control groups were set up:

[0161] Experimental group: After adding IGF-1 standard to serum, the serum was left at room temperature for about 5 hours. Samples were tested at 0 hours and 5 hours after being left at room temperature. Figure 4A and 4B The mass spectrometry results of serum before and after 5 hours of storage are shown. The results indicate that after 5 hours at room temperature, the quantitative result of IGF-1 decreased from 524 ng / mL to 321 ng / mL, and the mass spectrometry... Figure 4B A new substance 1 with a mass-to-charge ratio of 7508 and a new substance 2 with a mass-to-charge ratio of 7351 were found.

[0162] Control group: Cocktail protease inhibitor (manufacturer Roche, catalog number 0589279100) was added to serum, followed by IGF-1 standard protein. Samples were tested at 0 hours and 5 hours after being incubated at room temperature. Figure 4C and 4D The mass spectrometry results of serum before and after 5 hours of storage are shown. The results indicate that after 5 hours at room temperature, the IGF-1 quantification of the sample did not change significantly, and no new substance 1 (mass-to-charge ratio 7508) or substance 2 (mass-to-charge ratio 7351) was observed. It can be inferred that the cocktail protease inhibitor inhibited the degradation of IGF-1.

[0163] Based on the experimental results of the experimental group and the control group, it can be determined that the new substance 1 with a mass-to-charge ratio of 7508 and the new substance 2 with a mass-to-charge ratio of 7351 are produced by the decomposition of IGF-1. Therefore, it can be determined that the new substance 1 with a mass-to-charge ratio of 7508 and the new substance 2 with a mass-to-charge ratio of 7351 are variants of IGF-1.

[0164] By further comparing the mass-to-charge ratios of novel substances 1 and 2 with those of IGF-1 and their amino acid sequences, the amino acid sequence of novel substance 1 is shown in SEQ ID NO:1, and it is named the des-R IGF-1 variant:

[0165] GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLR-LEMYCAPLKPAKSA(SEQ ID NO:1)

[0166] The amino acid sequence of the new substance 2 is shown in SEQ ID NO:2, and it is named the des-RR IGF-1 variant:

[0167] GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDL—LEMYCAPLKPAKSA(SEQ ID NO:2)

[0168] Results analysis:

[0169] (1) The quantitative detection results of IGF-1 in Example 1 were fitted with the quantitative detection results of IGF-1 in Example 2, and the results are as follows: Figure 5 As shown, the fitting method is a power function, y = 0.4796x 1.0765 The goodness of fit was 0.7905, and the correlation coefficient r between the two sets of data was 0.8891.

[0170] The fitting results show that the quantitative results of IGF-1 in this invention are well correlated with the quantitative results of chemiluminescent immunoassay, and IGF-1 variants can be detected.

[0171] (2) The sum of the quantitative detection results of IGF-1 in Example 1 and the quantitative detection results of IGF-1 variants (des-R IGF-1 variant + des-RRIGF-1 variant) is taken as the total IGF-1 amount. The total IGF-1 amount is then fitted with the quantitative detection results of IGF-1 in Example 2 using the same fitting method as above. Figure 6 As shown, y = 0.3926x 1.1604 The goodness of fit was 0.912, and the correlation coefficient r between the two sets of data was 0.9550.

[0172] The comparison shows that the correlation coefficient r after fitting the total IGF-1 amount with the quantitative IGF-1 detection results in Example 2 is 0.0659 higher than the correlation coefficient r after fitting only the quantitative IGF-1 detection results with the quantitative IGF-1 detection results in Example 2. Moreover, the correlation coefficient r of fitting the total IGF-1 amount is almost close to 1, indicating that the analysis of IGF-1 using the total IGF-1 amount (IGF-1 + des-R IGF-1 variant + des-RR IGF-1 variant) can significantly improve the quantitative accuracy.

[0173] At the same time, it can also output quantitative results for IGF-1, des-R IGF-1 variants, and des-RR IGF-1 variants, providing more indicators to support the clinical diagnosis of the relationship between IGF-1 and disease.

[0174] In summary, the results of Examples 1-3 of this invention indicate that the quantitative detection results of IGF-1 variants affect the accuracy of IGF-1 quantitative results; using the total amount of IGF-1 and IGF-1 variants as the detection result of IGF-1 can improve the accuracy of IGF-1 detection results; combining Examples 1, 2, and 3 and the IGF-1 protein structure, it can be determined that des-R IGF-1 and des-RR IGF-1 are IGF-1 variants.

[0175] Example 4: Selection of magnetic beads suitable for simultaneous detection of IGF-1 and IGF-1 variants

[0176] 1. Preparation of reagents required for testing:

[0177] The preparation of 10 mM PBS solution, 0.1% Tween 20 PBS solution, lysis buffer, internal standard, and matrix is ​​as described in Example 1.

[0178] Calibrators: Calibrator R1 is available in 6 concentrations: 1000 ng / mL, 750 ng / mL, 500 ng / mL, 250 ng / mL, 50 ng / mL and 5 ng / mL. Multiple concentrations of calibrators can be used to determine a wide range of IGF-1 content samples.

[0179] 2. Antibody-magnetic bead bonding:

[0180] (1) The preparation of IGF-1 antibody-epoxy (epoxy resin) magnetic beads is as follows:

[0181] 100 μg of IGF-1 antibody (mouse monoclonal antibody) was added to a 30 kDa ultrafiltration tube and centrifuged at 12000 × g until all liquid in the tube was removed. The ultrafiltration tube was then removed and 200 μL of 0.1 M PBS (pH 7.4) buffer solution was added. The tube was vortexed for 30 seconds and then centrifuged at 12000 × g for 5 minutes (this operation was repeated twice). 290 μL of 0.1 M PBS (pH 7.4) solution was added to the cleaned ultrafiltration tube and vortexed to mix before bonding with magnetic beads.

[0182] 100 μL (10 mg) of epoxy resin magnetic beads were placed in a 2 mL centrifuge tube and magnetically separated on a magnetic rack. The solution was washed once with 200 μL of 0.1 M PBS (pH 7.4) and then resuspended in 330 μL of 0.1 M PBS (pH 7.4). The entire antibody solution in the ultrafiltration tube was transferred to the 2 mL centrifuge tube containing the magnetic beads, and 330 μL of 3 M ammonium sulfate solution (dissolved in 0.1 M PBS (pH 7.4)) was added. The mixture was vortexed and incubated overnight at room temperature (approximately 16 hours) for bonding. After bonding, the magnetic beads were separated by magnetic force and the liquid was discarded. The beads were washed three times with 500 μL of 10 mM PBS solution and the liquid was discarded. The beads were resuspended in 1% BSA solution and inverted at room temperature for 30 min. The beads were washed once with 500 μL of PBS solution containing 0.1% Tween 20 and then resuspended in 1 mL of PBS solution containing 0.1% Tween 20 for later use.

[0183] (2) The preparation of IGF-1 antibody-biotin-streptavidin-magnetic beads is as follows:

[0184] 100 μg of IGF-1 antibody was added to a 30 kDa ultrafiltration tube and centrifuged at 12000 × g until all liquid in the tube was removed. The tube was then removed and 200 μL of 10 mM PBS buffer was added. The tube was vortexed for 30 seconds and then centrifuged at 12000 × g for 5 minutes (this operation was repeated twice).

[0185] After removing the ultrafiltration tube, add 400 μL of 10 mM PBS buffer and 12 μL of 20 mM Biotin solution (EZ-Link SuLfo-NHS-Biotin), vortex for 30 s, and then incubate at room temperature for 30 min. After the Biotin has fully reacted with the antibody, centrifuge the ultrafiltration tube at 12000 × g for 10 min, add 200 μL of 10 mM PBS buffer, vortex for 30 s, and then centrifuge at 12000 × g for 5 min (repeat this operation twice). Add 30 mg of streptavidin magnetic beads (1.5 μm in diameter) to a 5 mL centrifuge tube. Place the centrifuge tube containing the magnetic beads on a magnetic rack for magnetic separation for 5 min, discarding all supernatant. Remove the centrifuge tube and resuspend the magnetic beads in 2.5 mL of 10 mM PBS buffer. Transfer all the biotin-bonded antibody to the magnetic bead suspension. Wash the tube multiple times with 10 mM PBS. Transfer the antibody from the ultrafiltration tube to the magnetic bead suspension, vortex until homogeneous, and incubate at room temperature for 3 hours to ensure complete bonding between the biotin-bonded antibody and the streptavidin magnetic beads. After the antibody and magnetic beads are bonded, place the 5 mL tube on a magnetic rack for magnetic separation for 5 min, wash the magnetic beads twice with 2 mL of 10 mM PBS, and finally resuspend the magnetic beads in 3 mL of 0.1% Tween 20 PBS solution for later use.

[0186] 3. Preparation of IGF-1 standard curve

[0187] For calibrators, take 40 μL directly into a 0.6 mL EP tube; for serum / plasma samples, take 20 μL into an EP tube and add 20 μL of lysis buffer, vortex to mix, and shake at 1500 rpm for 10 min at room temperature; add 130 μL of PBST to the sample, vortex to mix, then add 10 μL of antibody (vortex thoroughly before use to ensure the magnetic bead solution is homogeneous (about 30 s), and vortex once for every 5 samples), vortex to mix, and react on a vortex mixer at room temperature for 10 min. Remove and centrifuge for 10 s, then immediately place on a magnetic rack for magnetic separation for 1 min. Discard the liquid in the tube, add 200 μL of PBST, vortex for about 30 s to ensure all magnetic beads are suspended, and then place on a magnetic rack for magnetic separation for 1 min (repeat the operation 3 times). After discarding the supernatant, add 200 μL of deionized water to a 0.6 mL centrifuge tube, vortex for 30 s, and then place it on a magnetic rack for magnetic separation for 1 min. Discard the supernatant again, and repeat the operation once more with 100 μL of deionized water. Centrifuge the liquid-free centrifuge tube in a mini centrifuge for 30 s, then place it on a magnetic rack for magnetic separation for 30 s. Carefully aspirate the trace amount of liquid at the bottom of the centrifuge tube with a 10 μL pipette (small tip), then accurately add 10 μL of matrix solution, vortex for 30 s, and place it on a magnetic rack for magnetic separation for 30 s. Take 2 μL of colorless and transparent eluent and spot it onto a preheated target plate well (2.4 mm, hydrophobic target plate) at 39 °C. Heat the target plate until all the sample on the target plate has crystallized, then remove the target plate and load it into the QuanPRO protein spectrometer. Once the vacuum level meets the requirements, begin data acquisition.

[0188] QuanPRO protein spectrometer detection parameters:

[0189] Laser: Semiconductor laser; Laser frequency: 1000Hz; 2D platform movement speed: 1.5mm / sec; Focus Mass: 4500~9000Da; Acquisition quality range: 2kDa~35kDa.

[0190] The instrument response values ​​of six calibrators at different concentrations were fitted with calibration curves (power function) to their corresponding labeled concentrations. The x-axis represents the instrument response value, and the y-axis represents the calibrator concentration indicated on the "Human Insulin-like Growth Factor-I Assay Kit Data Information Card". The quantitative results of IGF-1 and IGF-1 variants can be directly calculated using the human insulin-like growth factor-1 quantification system.

[0191] Antibody magnetic beads bonded using the two methods described above were used to test six concentrations of standards and the same samples. Quantitative analysis of the samples was performed, and the results are shown in Table 3. Figure 7 , 8 As shown.

[0192] Table 3:

[0193]

[0194] Mass spectrometry results using epoxy magnetic beads are as follows: Figure 7 As shown, the variant samples des-R IGF-1 (m / z = 7510) and des-RR IGF-1 (m / z = 7353) can be clearly detected. The mass spectrometry results of the antibody magnetic beads bonded using streptomycin are shown below. Figure 8 As shown, only the variant sample des-R IGF-1 (m / z = 7510) was detected, and the des-RR IGF-1 (m / z = 7353) concentration was only 0.43 ng / mL. This detection value is below the detection limit of the QuanPRO protein spectrometer, and the result is inaccurate and should be considered as undetectable. In summary, using the epoxy resin magnetic beads of this invention to detect IGF-1 and IGF-1 variants yields more accurate results and can accurately quantify both IGF-1 and IGF-1 variants. Other methods of binding magnetic beads to antibodies cannot achieve accurate detection of IGF-1 and IGF-1 variants.

[0195] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0196] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for detecting variants of insulin-like growth factor I, characterized in that, include: (1) Contact the test sample, IGF-1 internal standard and IGF-1 antibody so that the IGF-1 internal standard and the variant of insulin-like growth factor I contained in the test sample can be specifically bound to the IGF-1 antibody, respectively, to obtain an antigen-antibody complex enriched with the variant of insulin-like growth factor I and IGF-1 internal standard. (2) The antigen-antibody complex is eluted to elute and separate the enriched variant of insulin-like growth factor I and IGF-1 internal standard from the IGF-1 antibody, so as to obtain a test solution containing the variant of insulin-like growth factor I and IGF-1 internal standard. (3) The test solution was analyzed by MALDI ion source mass spectrometry to detect the amount of insulin-like growth factor I variant in the test sample. In step (1), the IGF-1 antibody is bonded to epoxy magnetic beads. The variants of insulin-like growth factor I include des-R IGF-1 variants having a single charge mass-to-charge ratio of 7500-7520, a double charge mass-to-charge ratio of 3746-3766, or a triple charge mass-to-charge ratio of 2494-2514. The des-RR IGF-1 variants have a mass-to-charge ratio of 7343-7363 for single-charged cells, 3667-3687 for double-charged cells, or 2442-2462 for triple-charged cells. The amino acid sequence of the des-R IGF-1 variant is shown in SEQ ID NO:

1. The amino acid sequence of the des-RR IGF-1 variant is shown in SEQ ID NO:

2. The detection method is used for non-diagnostic purposes.

2. The detection method according to claim 1, characterized in that, When the sample to be tested is serum or plasma, the detection method further includes, before step (1), lysing the sample to be tested with a lysis buffer to obtain a lysed solution, and in step (1), contacting the lysed solution, IGF-1 internal standard and IGF-1 antibody.

3. The detection method according to claim 2, characterized in that, The lysis buffer includes a lysis agent and a first buffer solution.

4. The detection method according to claim 3, characterized in that, The pyrolysis agent is selected from at least one of guanidine hydrochloride, urea, thiourea, sodium dodecyl sulfate, hexadecyltrimethylammonium bromide, Tween 20, Tween 80, 3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt, octyl-β-glucoside, octyl thioglucoside, polyethylene glycol octylphenyl ether, and ethylphenyl polyethylene glycol.

5. The detection method according to claim 3, characterized in that, The first buffer solution is selected from Tris-HCl buffer and PBS buffer.

6. The detection method according to claim 3, characterized in that, The lysis buffer contains sodium dodecyl sulfate and PBS buffer.

7. The detection method according to claim 3, characterized in that, The lysis buffer is a PBS solution with a mass concentration of 1-5% sodium dodecyl sulfate.

8. The detection method according to any one of claims 1-7, characterized in that, The test solution containing the variant of insulin-like growth factor I to be tested and the internal standard IGF-1 is obtained through the following steps: (I) The test sample, IGF-1 internal standard and magnetic bead suspension bound to IGF-1 antibody are mixed in reaction buffer and reacted so that the test variant of insulin-like growth factor I and the IGF-1 internal standard in the test sample specifically bind to the IGF-1 antibody bound to the magnetic beads, thereby obtaining antigen-antibody complex magnetic beads enriched with the test variant of insulin-like growth factor I and the IGF-1 internal standard; (II) The antigen-antibody complex magnetic beads are eluted and magnetically separated to elute and separate the enriched variant of insulin-like growth factor I and IGF-1 internal standard from the IGF-1 antibody, thereby obtaining a test solution containing the variant of insulin-like growth factor I and IGF-1 internal standard.

9. The detection method according to claim 1, characterized in that, The Epoxy magnetic beads have a particle size of 0.1μm to 25μm.

10. The detection method according to claim 1, characterized in that, The Epoxy magnetic beads have a particle size of 0.5μm to 5μm.

11. The detection method according to claim 1, characterized in that, The IGF-1 antibody is a monoclonal antibody or a polyclonal antibody.

12. The detection method according to claim 11, characterized in that, The affinity constant KD value of the IGF-1 antibody is 1×10⁻⁶. -9 - 9×10 -12 .

13. The detection method according to claim 1, characterized in that, The IGF-1 internal standard is an isotopically labeled IGF-1 protein or an IGF-1 protein with a linked tag sequence, or a polypeptide with an amino acid sequence identity of more than 80% that can be enriched by the IGF-1 antibody.

14. The detection method according to claim 13, characterized in that, The isotopes are H2, N15, C13 or O18.

15. The detection method according to claim 13, characterized in that, The tag sequence includes at least one selected from His tag, Flag tag, GST tag, MBP tag, C-MYC tag, HA tag, FC tag, AVI tag, MBP tag, and DDDDK tag.

16. The detection method according to claim 8, characterized in that, In step (I), the reaction buffer includes at least one selected from MES buffer, PBST buffer, and Tris-HCl buffer.

17. The detection method according to claim 8, characterized in that, The pH value of the reaction buffer is 6-8.

18. The detection method according to claim 8, characterized in that, The reaction buffer is PBST buffer, which contains 0.1-0.6% Tween 20.

19. The detection method according to claim 8, characterized in that, Before step (2) or (II), the product of step (1) or (I) is further subjected to a first cleaning and a second cleaning, wherein the cleaning solution used for the first cleaning is a buffer solution containing a nonionic surfactant, and the second cleaning uses deionized water.

20. The detection method according to claim 19, characterized in that, The nonionic surfactants include those selected from Tween 20, Tween 60, Tween 80, CHAPS, Triton X-100, alkoxypolyvinylhydroxoethanol, octyl glucoside, dodecyl maltodextrin, n-octyl α-D-glucoside, N-octanoyl-N-methylglucosamine, N-nonanoyl-N-methylglucosamine, N-decanoyl-N-methylglucosamine, nonyl-β-D-glucopyranoside, N,N-dimethyldodecylamine-N-oxide, decyl glucopyranoside, 1-O-decyl-β-D-maltodextrin, deoxy-Bigcha p, at least one of the following: digitoxin, triaton X-114, nonylphenol polyoxyethylene ether, tetraethylene glycol monododecyl ether, tetramethylammonium hydroxide pentahydrate, polysorbate-85, tetrabutylphenol aldehyde, undecyl-β-D-maltodextrin, octyl-β-D-thiopyranoside, n-octyl-β-D-pyranoside, octaethylene glycol monododecyl ether, saponin, span, sucrose dodecanoate, glyceryl monooleate, 6-O-(N-heptaformyl)-methyl-α-D-glucoside, polyethylene glycol monostearate, and n-nonyl-β-D-thiomaltodextrin.

21. The detection method according to claim 19, characterized in that, The buffers include those selected from PBS buffer, MES buffer, Tris buffer, DPBS buffer, CBS buffer, BBS buffer, HEPES buffer, TBS buffer, BES buffer, TEA buffer, MOPS buffer, AMPD buffer, EPPS buffer, MOPSO buffer, AMPSO buffer, DIPSO buffer, and TAPSO buffer.

22. The detection method according to claim 19, characterized in that, The first cleaning solution used is a PBS buffer containing at least one of Tween 20, Tween 60, and Tween 80.

23. The detection method according to claim 8, characterized in that, In step (2) or (II), the elution solution used in the elution treatment contains a matrix and an antigen-antibody complex dissociation reagent, wherein the matrix includes at least one selected from sinapic acid, α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, 2,4,6-trihydroxyacetophenone, anthraquinone, and 3-indoleacrylic acid.

24. The detection method according to claim 23, characterized in that, The matrix is ​​α-cyano-4-hydroxycinnamic acid.

25. The detection method according to claim 24, characterized in that, The concentration of the α-cyano-4-hydroxycinnamic acid is 5-20 mg / mL.

26. The detection method according to claim 23, characterized in that, The antigen-antibody complex dissociation reagent includes at least one selected from organic acid solutions, inorganic acid solutions, and alkaline solutions; The organic acid solution includes at least one selected from formic acid solution, acetic acid solution, trifluoroacetic acid solution, propionic acid solution, glycolic acid solution, amino acid solution, butyric acid solution, citric acid, succinic acid, ascorbic acid, lactic acid, malic acid, and tartaric acid; The inorganic acid solution includes at least one selected from hydrochloric acid solution, sulfuric acid solution, and phosphoric acid solution; The alkaline solution includes at least one selected from sodium hydroxide solution, potassium hydroxide solution, tris(hydroxymethyl)aminomethane solution, tetraethylammonium bromide solution, sodium carbonate solution, sodium bicarbonate solution, ammonium bicarbonate solution, ammonia solution, and urea solution.

27. The detection method according to claim 23, characterized in that, The antigen-antibody complex dissociation reagent is a trifluoroacetic acid solution.

28. The detection method according to claim 27, characterized in that, The mass concentration of the trifluoroacetic acid solution is 0.05-5%.

29. The detection method according to claim 23, characterized in that, Step (3) further includes: The solution to be tested is spotted onto a target plate used for MALDI ion source mass spectrometry detection. The target plate after spotting is heat-treated to obtain the crystal to be tested. The target plate with the crystal to be tested is placed on the instrument for data acquisition.

30. The detection method according to claim 23, characterized in that, Step (3) further includes pre-dipling the elution solution onto the target plate and performing heat treatment before targeting, in order to prepare a matrix crystalline layer.

31. The detection method according to claim 30, characterized in that, Prepare to form two or more layers of the matrix crystalline layer.

32. The detection method according to claim 30, characterized in that, The heat treatment temperature is 35-45℃.

33. The detection method according to claim 1, characterized in that, The MALDI ion source mass spectrometer is either MALDI-TOFMS or MALDI-TOF-TOF MS.

34. A method for simultaneously detecting insulin-like growth factor I and its variants, characterized in that, include: Using the detection method according to any one of claims 1-33, the amount of insulin-like growth factor I and its variants in the sample to be tested is detected. Among them, the variants of insulin-like growth factor I include des-R IGF-1 variants having a single charge mass-to-charge ratio of 7500-7520, a double charge mass-to-charge ratio of 3746-3766, or a triple charge mass-to-charge ratio of 2494-2514. The des-RR IGF-1 variants have a mass-to-charge ratio of 7343-7363 for single-charged cells, 3667-3687 for double-charged cells, or 2442-2462 for triple-charged cells. The method is used for non-diagnostic purposes.

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