Quantitative test kit for insulin-like growth factor i variants

Abstract

The present application belongs to the technical field of in-vitro detection, and particularly relates to a quantitative detection kit for insulin-like growth factor I 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 the quantitative detection kit for the variants of insulin-like growth factor I of the present application 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] The present application belongs to the technical field of in vitro detection, and particularly relates to a quantitative detection kit for insulin-like growth factor I variants. BACKGROUND

[0002] Insulin-like growth factor I (IGF-1), also known as Somatomedin C, is a secreted protein synthesized by the liver and secreted into peripheral blood. The mature protein is composed of 70 amino acids, among which 6 cysteines form 3 pairs of disulfide bonds to maintain its spatial structure. It is named because of its structure similar to insulin. IGF-1 is an active protein polypeptide substance, which is the product of autocrine and paracrine of liver cells, kidney cells, spleen cells and other ten kinds of cells in human body. It has the effects of lowering blood sugar, lowering blood lipid, dilating blood vessels, promoting growth, promoting cell differentiation, wound repair, etc. Precise detection of IGF-1 helps disease analysis and medication guidance after diagnosis.

[0003] Although one of the variants of IGF-1, IGF-1LR3, is disclosed in the art, this variant is exogenously synthesized, and 13 amino acids are added at the N-terminal of the original 70 amino acid chain, for a total of 83 amino acids. This IGF-1 variant still shows its original activity to IGF-1 receptors in human tissues. However, there are few reports that IGF-1 can produce variants in vivo, and it is also not realized that the poor linear relationship of individual samples is due to the decomposition of IGF-1 into variants. The existing detection kit can only detect IGF-1, but cannot detect the variants contained in the sample. SUMMARY

[0004] The present application aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, the present application provides a detection kit for variants of insulin-like growth factor I, which can accurately detect des-R IGF-1 variants and des-RR IGF-1 variants, and thus provide fine data for disease analysis of IGF-1, facilitating further clinical diagnosis.

[0005] In a first aspect of the present application, a detection kit for variants of insulin-like growth factor I is provided. According to embodiments of the present application, the kit comprises:

[0006] an IGF-1 specific antibody;

[0007] an internal standard IGF-1 recombinant protein;

[0008] an IGF-1 protein standard;

[0009] an elution solution;

[0010] reaction buffer,

[0011] wherein the elution solution contains a matrix for a MALDI-TOF MS platform and an antigen-antibody complex dissociation reagent,

[0012] The variant of the insulin-like growth factor I includes a des-R IGF-1 variant 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.

[0013] a des-RR IGF-1 variant having a single charge mass-to-charge ratio of 7343-7363 or a double charge mass-to-charge ratio of 3667-3687 or a triple charge mass-to-charge ratio of 2442-2462.

[0014] Currently, the clinical detection method of IGF-1 mainly uses chemiluminescence immunoassay technology. The chemiluminescence immunoassay technology indirectly realizes the quantification of the analyte by measuring the light intensity of the luminescent substance on the second antibody specifically combined with the analyte. However, the stability of this technology is interfered by many factors such as antibody specificity, non-specific adsorption, heterophilic antibody, and interferents, resulting in large fluctuation of the quantitative results.

[0015] The chemiluminescence method for detecting insulin-like growth factor I can only detect the amount of IGF-1 and has no discrimination, and cannot identify variants with different numbers of amino acids from IGF-1. When the inventors detected insulin-like growth factor I by using MALDI-TOF MS, it was found that the detection results of the sample containing insulin-like growth factor I showed peaks of single charge mass-to-charge ratio of 7500-7520 and single charge mass-to-charge ratio of 7343-7363. Through further experimental verification, it was determined that the two peaks correspond to des-R IGF-1 variant and des-RR IGF-1 variant of insulin-like growth factor I, respectively. The inventors found that the detection kit can accurately detect des-R IGF-1 variant and des-RR IGF-1 variant by using the above-mentioned detection kit, especially by bonding IGF-1 antibody to Epoxy magnetic beads, so as to provide refined data for disease analysis of IGF-1. Meanwhile, the inventors also found that bonding IGF-1 internal standard or variant of insulin-like growth factor I to magnetic beads by using other magnetic bead bonding methods, such as biotin-streptavidin system, cannot simultaneously detect des-R IGF-1 variant and des-RR IGF-1 variant, resulting in inaccurate detection results and inability to provide refined data for disease analysis of IGF-1.

[0016] The detection kit of the present application can not only accurately detect the amount of des-R IGF-1 variant and des-RR IGF-1 variant, but also detect the amount of IGF-1, and the total amount of IGF-1 is obtained by adding the amounts of IGF-1, des-R IGF-1 variant and des-RR IGF-1 variant, thereby improving the accuracy of IGF-1 quantification. According to the needs of the hospital, the refined data of IGF-1 disease analysis can be obtained by providing the amount of IGF-1 or the total amount of IGF-1 or the amount of IGF-1 variant.

[0017] According to an embodiment of the present application, the IGF-1 specific antibody is a monoclonal antibody or a polyclonal antibody.

[0018] According to an embodiment of the present application, the affinity constant KD value of the IGF-1 specific antibody is 1x10 -9 -9x10 -12 .

[0019] The IGF-1 antibody provided by the present application is used for enriching IGF-1 variant, and the sensitivity of detection can be improved.

[0020] According to an embodiment of the present application, the kit further comprises magnetic beads, the magnetic beads are bonded with the IGF-1 specific antibody, and the magnetic beads are Epoxy magnetic beads.

[0021] According to an embodiment of the present application, the particle size of the Epoxy magnetic beads is 0.1-25 μm, preferably 0.5-5 μm.

[0022] According to an embodiment of the present application, the internal standard IGF-1 recombinant protein is an isotope-labeled IGF-1 protein or an IGF-1 protein connected with a tag sequence, or a polypeptide with more than 80% amino acid sequence identity with IGF-1 protein and capable of being enriched by the IGF-1 antibody.

[0023] The internal standard IGF-1 recombinant protein can reduce the influence of sample pretreatment and instrument analysis process on detection reproducibility, and improve the precision of detection.

[0024] According to an embodiment of the present application, the isotope is H2, N15, C13 or O18.

[0025] According to an embodiment of the present application, the tag sequence comprises 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.

[0026] According to an embodiment of the present application, the reaction buffer comprises at least one selected from the group consisting of MES buffer, PBST buffer, Tris-HCl buffer.

[0027] According to an embodiment of the present application, the reaction buffer has a pH value of 6-8.

[0028] The reaction buffer having a pH value of 6-8 can provide an environment for enriching the target having a pH value of 6-8, and improve the enrichment efficiency of the antibody to the IGF-1 variant.

[0029] According to an embodiment of the present application, the reaction buffer is a PBST buffer containing 0.1-0.6% Tween 20.

[0030] According to an embodiment of the present application, the matrix comprises at least one selected from the group consisting of sinapinic acid, a-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, 2,4,6-trihydroxyacetophenone, anthracenetriol, 3-indoleacrylic acid.

[0031] According to an embodiment of the present application, the matrix is a-cyano-4-hydroxycinnamic acid.

[0032] According to an embodiment of the present application, the concentration of the a-cyano-4-hydroxycinnamic acid is 5-20 mg / mL.

[0033] The present application preferably uses a-cyano-4-hydroxycinnamic acid as the matrix of the MALDI-TOF MS platform, which can improve the number and intensity of the mass spectrum peaks.

[0034] According to an embodiment of the present application, the antigen-antibody complex dissociation reagent comprises at least one selected from the group consisting of an organic acid solution, an inorganic acid solution, and an alkali solution,

[0035] The organic acid solution comprises at least one selected from the group consisting of 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;

[0036] The inorganic acid solution comprises at least one selected from the group consisting of hydrochloric acid solution, sulfuric acid solution, and phosphoric acid solution;

[0037] The alkali solution comprises at least one selected from the group consisting of 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.

[0038] According to an embodiment of the present application, the antigen-antibody complex dissociation reagent is trifluoroacetic acid solution.

[0039] According to an embodiment of the present application, the antigen-antibody complex dissociation reagent is a trifluoroacetic acid solution. In this embodiment, the matrix for the MALDI-TOF MS platform in the elution solution does not affect the elution effect of the antigen-antibody complex dissociation reagent.

[0040] According to an embodiment of the present application, the mass concentration of the trifluoroacetic acid solution is 0.05-5%.

[0041] The TFA solution at this concentration as the antigen-antibody complex dissociation reagent can further improve the dissociation efficiency of the variant of insulin-like growth factor I and the IGF-1 antibody, so that the antigen-antibody is completely dissociated, thereby further improving the accuracy of detecting the variant of insulin-like growth factor I.

[0042] According to an embodiment of the present application, the elution solution contains the matrix CHCA (α-cyano-4-hydroxycinnamic acid), acetonitrile, TFA for the MALDI-TOF MS platform.

[0043] According to an embodiment of the present application, the kit further comprises a lysis solution for lysis treatment of the serum or plasma sample to be detected.

[0044] According to an embodiment of the present application, the lysis solution comprises a lysis agent and a first buffer.

[0045] According to an embodiment of the present application, the lysis agent is at least one selected from the group consisting of guanidine hydrochloride, urea, thiourea, sodium dodecyl sulfate, cetyltrimethylammonium bromide, Tween 20, Tween 80, 3-((3-cholamidopropyl)dimethylammonio)propan-1- sulfonate, octyl-β-glucoside, octylthioglucoside, polyethylene glycol octylphenyl ether, and ethylphenyl polyethylene glycol.

[0046] According to an embodiment of the present application, the first buffer is selected from the group consisting of Tris-HCl buffer and PBS buffer.

[0047] According to an embodiment of the present application, the lysis solution contains sodium dodecyl sulfate and PBS buffer.

[0048] According to an embodiment of the present application, the lysis solution is a PBS solution containing 1-5% sodium dodecyl sulfate.

[0049] According to an embodiment of the present application, the kit further comprises a washing solution for washing the antigen-antibody complex before the elution solution elutes the antigen-antibody complex, and the washing solution comprises a first washing solution which is a buffer containing a non-ionic surfactant.

[0050] According to an embodiment of the present application, the non-ionic surfactant comprises at least one selected from Tween 20, Tween 60, Tween 80, CHAPS, Triton X-100, alkoxypolyethylene hydroxyethanol, octyl glucoside, dodecylmaltoside, n-octyl alpha-D-glucoside, N-octanoyl-N-methylglucamide, N-nonanoyl-N-methylglucamide, N-decanoyl-N-methylglucamide, nonyl-beta-D-glucopyranoside, dodecyl-beta-D-maltoside, N,N-dimethyldodecylamine-N-oxide, decyl glucopyranoside, 1-O-decyl-beta-D-maltoside, Deoxy-Bigchap, saponin, Triton X-114, nonoxynol, tetraethylene glycol monododecyl ether, tetramethylammonium hydroxide pentahydrate, polysorbate-85, tetrabutyl glycol, undecyl-beta-D-maltoside, octyl-beta-D-thioglucopyranoside, n-octyl-beta-D-glucopyranoside, octaethylene glycol monododecyl ether, saponin, saponin, sucrose dodecanoate, glycerol monooleate, 6-O-(N-heptanoyl)-methyl-alpha-D-glucoside, polyethylene glycol monostearate, n-nonyl-beta-d-thiomaltoside, n-dodecyl-beta-d-maltoside.

[0051] According to an embodiment of the present application, the buffer solution comprises at least one selected from PBS buffer solution, MES buffer solution, Tris buffer solution, DPBS buffer solution, CBS buffer solution, BBS buffer solution, HEPES buffer solution, TBS buffer solution, BES buffer solution, TEA buffer solution, MOPS buffer solution, AMPD buffer solution, EPPS buffer solution, MOPSO buffer solution, AMPSO buffer solution, DIPSO buffer solution, TAPSO buffer solution.

[0052] According to an embodiment of the present application, the first washing solution is PBS buffer solution containing at least one of Tween 20, Tween 60, and Tween 80.

[0053] According to an embodiment of the present application, the first washing solution is 0.1% (v / v) Tween 20 PBS buffer solution. In this way, non-specifically adsorbed impurity proteins in the sample can be effectively removed.

[0054] According to an embodiment of the present application, the washing solution further comprises a second washing solution, and the second washing solution is deionized water.

[0055] The washing solution provided by the present application can reduce non-specific adsorption in the antibody enrichment process and improve detection sensitivity. The first washing solution can remove non-specifically adsorbed impurity proteins in the sample, and the second washing solution using deionized water can remove the first washing solution and salt ions.

[0056] In another aspect of the present application, the present application provides a kit for simultaneously detecting insulin-like growth factor I and its variants. According to the embodiments of the present application, the kit is the kit of the first aspect.

[0057] The kit of the first aspect of the present application can be used to simultaneously and accurately detect insulin-like growth factor I and its variants, and according to the detection requirements, the kit of the present application provides the amount of IGF-1 or the total amount of IGF-1 or the amount of IGF-1 variants, to obtain refined data for disease analysis of IGF-1. Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter in the description of the application. BRIEF DESCRIPTION OF DRAWINGS

[0058] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the description of the embodiments, given in conjunction with the accompanying drawings, in which:

[0059] Figure 1 MALDI-TOF MS mass spectrum of IGF-1 standard protein solution of different concentrations detected in Example 1 of the present application is shown;

[0060] Figure 2 IGF-1 standard curve obtained based on IGF-1 standard protein solution of different concentrations in Example 1 of the present application is shown;

[0061] Figure 3 MALDI-TOF MS mass spectrum of serum / plasma sample IGF-1 in Example 1 of the present application is shown;

[0062] Figures 4A-4B MALDI-TOF MS mass spectrum of serum IGF-1 before and after 5 hours of placement in Example 1 of the present application is shown;

[0063] Figures 4C-4D MALDI-TOF MS mass spectrum of IGF-1 before and after adding cocktail protease inhibitors in serum in Example 1 of the present application is shown;

[0064] 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;

[0065] Figure 6 The results of fitting the sum of the quantitative detection results of IGF-1 in Example 1 and des-R IGF-1 variants, des-RR IGF-1 variants as the total amount of IGF-1, and the total amount of IGF-1 with the quantitative detection results of IGF-1 in Example 2 are shown.

[0066] Figure 7 MALDI-TOF MS spectra of IGF-1 and its variants detected using Epoxy magnetic beads in Example 4 are shown;

[0067] Figure 8 MALDI-TOF MS spectra of IGF-1 and its variants detected using Streptavidin magnetic beads in Example 4 are shown. DETAILED DESCRIPTION

[0068] Embodiments of the present application are described in detail herein. The embodiments described herein are merely exemplary and are not to be construed as limiting the present application.

[0069] It should be noted that the terms "first", "second", and the like, are used merely to describe the features and do not imply or connote relative importance or a quantity of the specified technical features. Thus, a feature defined with "first", "second" can explicitly or implicitly include one or more of the feature. Further, in the description of the present application, unless otherwise stated, the meaning of "a plurality" is two or more.

[0070] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The endpoints of the ranges and any values are provided as approximate descriptions of the ranges and are accomplished by the values reasonably related to the stated endpoints. For numeric values, the endpoints of the ranges, the endpoints of the ranges and individual point values, and individual point values can be combined with one another to form one or more new numeric ranges, which are to be considered as specifically disclosed herein.

[0071] In order that the present application can be more readily understood, certain technical and scientific terms are specifically defined herein. Unless specifically defined herein, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this application belongs.

[0072] In this document, the terms "comprises" or "comprising" are used in the inclusive, open sense, i.e., meaning "including but not limited to", and not in the exclusive or closed sense, i.e., meaning "including, but not limited to the elements listed".

[0073] In the present context, the terms "identity" and "homology" are used interchangeably to describe the percentage of amino acid residues in an amino acid sequence that are the same when compared to a reference sequence, as determined by a conventional method, e.g., see Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978) Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.). There are a number of algorithms that can be used to align sequences and determine sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the search for similarity method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997); and the BLAST and BLAST 2.0 algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs are available that use these algorithms to determine identity or homology between sequences, and include, but are 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.

[0074] Without substantially affecting the activity of the IGF-1 protein (retaining at least 95% of the activity) and without affecting the binding to the IGF-1 specific antibody, one 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 of the amino acid sequence of the IGF-1 protein of the present application to obtain a polypeptide having more than 80% sequence identity with the amino acid sequence of the IGF-1 protein which is capable of being enriched by the IGF-1 antibody. The polypeptide capable of being enriched by the IGF-1 antibody of the present application can have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (or homology) with the reference sequence (the amino acid sequence of the IGF-1 protein). The sequence identity of the present application can be measured using sequence analysis software. For example, the computer program BLAST, in particular BLASTP, using default parameters.

[0075] According to a specific embodiment of the present application, the present application provides a detection kit of the variant of insulin-like growth factor I, comprising:

[0076] an IGF-1 specific antibody;

[0077] an internal standard IGF-1 recombinant protein;

[0078] an IGF-1 protein standard;

[0079] an elution solution;

[0080] a reaction buffer,

[0081] wherein the elution solution contains a matrix for the MALDI-TOF MS platform and an antigen-antibody complex dissociation reagent,

[0082] the variant of insulin-like growth factor I comprises a des-R IGF-1 variant 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;

[0083] a des-RR IGF-1 variant having a single charge mass-to-charge ratio of 7343-7363 or a double charge mass-to-charge ratio of 3667-3687 or a triple charge mass-to-charge ratio of 2442-2462.

[0084] According to an embodiment of the present application, 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.

[0085] The amino acid sequence shown in SEQ ID NO: 1 is as follows:

[0086] GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDL- LEMYCAPLKPAKSA

[0087] The amino acid sequence shown in SEQ ID NO: 2 is as follows:

[0088] GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDL- LEMYCAPLKPAKSA

[0089] According to a specific embodiment of the present application, the IGF-1 specific antibody is a monoclonal antibody or a polyclonal antibody.

[0090] According to a specific embodiment of the present application, the affinity constant KD value of the IGF-1 specific antibody is 1 x 10 -9 -9 x 10 -12 .

[0091] According to a specific embodiment of the present application, the internal standard IGF-1 recombinant protein is an isotope-labeled IGF-1 protein or an IGF-1 protein connected with a tag sequence, or a polypeptide having more than 80% amino acid sequence identity with the IGF-1 protein and capable of being enriched by the IGF-1 antibody.

[0092] According to a preferred embodiment of the present application, the isotope is H2, N15, C13 or O18.

[0093] According to an embodiment of the present application, the tag sequence comprises at least one selected from a His tag, a Flag tag, a GST tag, a MBP tag, a C-MYC tag, a HA tag, a FC tag, an AVI tag, a MBP tag, a DDDDK tag.

[0094] According to a specific embodiment of the present application, the reaction buffer comprises at least one selected from a MES buffer, a PBST buffer, a Tris-HCl buffer.

[0095] According to an embodiment of the present application, the pH value of the reaction buffer is 6-8.

[0096] The reaction buffer having a pH value of 6-8 can provide an IGF-1 variant-enriched environment having a pH value of 6-8, thereby improving the enrichment efficiency of the antibody for the IGF-1 variant.

[0097] According to a preferred embodiment of the present application, the reaction buffer is a PBST buffer containing 0.1-0.6% Tween 20.

[0098] According to a specific embodiment of the present application, the matrix comprises at least one selected from sinapinic acid, a-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, 2,4,6-trihydroxyacetophenone, anthratrilic acid, 3-indoleacrylic acid. According to a preferred embodiment of the present application, the matrix is a-cyano-4-hydroxycinnamic acid with a concentration of 5-20 mg / mL.

[0099] According to a specific embodiment of the present application, the antigen-antibody complex dissociation reagent comprises at least one selected from an organic acid solution, an inorganic acid solution, an alkali solution, the organic acid solution comprising 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, tartaric acid; the inorganic acid solution comprising at least one selected from hydrochloric acid solution, sulfuric acid solution, phosphoric acid solution; the alkali solution comprising at least one selected from sodium hydroxide solution, potassium hydroxide solution, tris solution, tetraethylammonium bromide solution, sodium carbonate solution, sodium bicarbonate solution, ammonium bicarbonate solution, ammonia solution, urea solution.

[0100] According to an embodiment of the present application, the antigen-antibody complex dissociation reagent is a trifluoroacetic acid solution. The mass concentration of the trifluoroacetic acid solution is 0.05-5%.

[0101] According to a specific embodiment of the present application, the method for detecting the variant of insulin-like growth factor I by using the detection kit of the present application comprises:

[0102] (1) contacting the sample to be detected, the internal standard IGF-1 recombinant protein and the IGF-1 specific antibody, so that the internal standard IGF-1 recombinant protein and the variant of insulin-like growth factor I contained in the sample to be detected are respectively specifically combined with the IGF-1 specific antibody, to obtain an antigen-antibody complex enriched with the variant of insulin-like growth factor I and the internal standard IGF-1 recombinant protein;

[0103] (2) performing elution treatment on the antigen-antibody complex, so as to elute and separate the enriched variant of insulin-like growth factor I and the internal standard IGF-1 recombinant protein from the IGF-1 specific antibody, to obtain a detection solution containing the variant of insulin-like growth factor I and the internal standard IGF-1 recombinant protein;

[0104] (3) spotting the detection solution on a target plate for MALDI-TOF MS detection, and performing heat treatment on the target plate after spotting, to obtain a detection crystal;

[0105] (4) placing the target plate with the to-be-tested crystal on a MALDI-TOF MS instrument to collect data, so as to detect the insulin-like growth factor I variant contained in the to-be-tested sample,

[0106] In step (1), the IGF-1 antibody is bonded to the Epoxy magnetic beads. According to a specific embodiment of the present application, the process of bonding the antibody to the magnetic beads is selected, and the particle size of the selected magnetic beads is 0.1 μm to 25 μm, preferably 0.5 μm to 5 μm, such as 0.5 μm, 1.5 μm, 2.7 μm, 3.0 μm, etc.

[0107] Using the above detection method, not only the des-R IGF-1 variant and the des-RR IGF-1 variant can be detected, but also the amount of IGF-1 contained in the sample can be detected, and the 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, and the detection result is more accurate, which provides effective support for further precision medicine.

[0108] It should be noted that the "sum" of the amount of IGF-1 and the des-R IGF-1 variant and the des-RR IGF-1 variant can be the sum of the peak area or the sum of the response intensity, and is preferably the sum of the response intensity.

[0109] The detection kit and the detection method of the present application use Epoxy magnetic beads, and the IGF-1 antibody is bonded to the Epoxy magnetic beads. The Epoxy magnetic beads couple the antibody to the magnetic beads through a chemical bond, which is more firm than the connection through the streptavidin-biotin connection mode. The present application uses Epoxy magnetic beads to bring less background interference to the detection, less impurity peak, reduces non-specific adsorption, and further improves the sensitivity of the detection.

[0110] According to a specific embodiment of the present application, the to-be-tested solution containing the to-be-tested insulin-like growth factor I variant and the internal standard IGF-1 recombinant protein is obtained by the following steps:

[0111] (I) vortexing and mixing the to-be-tested sample, the internal standard IGF-1 recombinant protein and the magnetic bead suspension bonded with the IGF-1 antibody in a reaction buffer to react, so that the to-be-tested insulin-like growth factor I variant in the to-be-tested sample and the internal standard IGF-1 recombinant protein specifically bind to the IGF-1 antibody bonded on the magnetic beads, to obtain an antigen-antibody complex magnetic bead enriched with the to-be-tested insulin-like growth factor I variant and the internal standard IGF-1 recombinant protein;

[0112] (II) performing elution treatment and magnetic separation treatment on the antigen-antibody complex magnetic beads, so as to elute and separate the enriched variant of insulin-like growth factor I and the internal standard IGF-1 recombinant protein from the IGF-1 antibody, and obtain a test solution containing the variant of insulin-like growth factor I and the internal standard IGF-1 recombinant protein.

[0113] According to an embodiment of the present application, before step (3), further comprising: previously spotting the elution solution on the target plate and performing heat treatment, so as to prepare a matrix crystalline layer.

[0114] According to an embodiment of the present application, the matrix crystalline layer is prepared in two or more layers. In this way, by forming a multi-layer crystalline matrix layer as a bottom layer on the target plate, the matrix-assisted laser desorption ionization, i.e. ionization, can be significantly improved, thereby improving the detection sensitivity. The material of the target plate is not particularly limited, for example, it can be a polypropylene 96-hole U-shaped plate, a silicon plate, etc.

[0115] According to a specific embodiment of the present application, the present application provides a method for detecting a variant of insulin-like growth factor I by using MALDI-TOF MS, comprising:

[0116] (1) mixing and reacting the test sample with the internal standard IGF-1 recombinant protein and the magnetic beads to which IGF-1 antibody is bonded, and the variant of insulin-like growth factor I contained in the test sample and the internal standard IGF-1 recombinant protein specifically bind to the IGF-1 antibody bonded on the magnetic beads, to obtain magnetic beads enriched with the variant and the internal standard IGF-1 recombinant protein;

[0117] (2) performing elution treatment and magnetic separation treatment on the magnetic beads enriched with the variant and the internal standard IGF-1 recombinant protein by using an elution solution, to obtain a test solution containing the variant and the internal standard IGF-1 recombinant protein;

[0118] (3) spotting the test solution on a target plate for MALDI-TOF MS detection, and performing heat treatment on the target plate after spotting, so as to crystallize the sample and obtain a crystalline product;

[0119] (4) placing the target plate with the crystalline product in a MALDI-TOF MS instrument to perform data acquisition, so as to detect the variant of insulin-like growth factor I contained in the test sample.

[0120] It should be noted that the temperature and time of heat treatment are not particularly limited, as long as the mixed solution on the target plate is crystallized to obtain a crystalline product for detection. For example, the temperature of heat treatment can be 35-45°C, preferably, such as 38°C, 39°C, 40°C, etc.

[0121] According to a specific embodiment of the present application, before spotting the sample solution on the target plate, an elution solution is first spotted on the target plate to form a matrix crystalline layer, and this step can be repeated twice or more to form two or more layers of matrix crystalline layer. In this way, by forming a multi-layered crystalline matrix layer as a bottom layer on the target plate, the matrix-assisted laser desorption ionization, i.e. ionization, can be significantly improved, and thus the detection sensitivity can be improved. In addition, a hydrophobic target plate can be used in the MALDI-TOF MS detection process, and thus the detection sensitivity can be improved.

[0122] It should be noted that the data acquisition conditions using the MALDI-TOF MS instrument are not particularly limited, and conventional conditions for MALDI-TOF MS in the art can be used, but the preferred conditions are as follows:

[0123] Laser: semiconductor laser;

[0124] Laser frequency: 1000-5000 Hz;

[0125] Two-dimensional platform moving speed: 0.5-3.0 mm / sec;

[0126] Focus Mass: 4500-9000 Da;

[0127] Acquisition mass range: 2 kDa-35 kDa.

[0128] The model of the MALDI-TOF MS instrument is selected from QuanPro mass spectrometer and QuanTOF type I mass spectrometer.

[0129] The schemes of the present application will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be obtained by purchase.

[0130] Example 1: Quantitative detection of IGF-1 and quantitative detection of IGF-1 variants based on MALDI-TOF MS for 205 serum / plasma samples.

[0131] 1. Preparation of reagents:

[0132] (1) Preparation of 10 mM PBS solution

[0133] 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 well by inverting;

[0134] (2) Preparation of 0.1% Tween 20 in PBS:

[0135] Add 1000 μL of non-ionic surfactant Tween 20 to 1000 mL of 10 mM PBS solution, and mix with ultrasonic assistance;

[0136] (3) Preparation of lysis solution:

[0137] Prepare a PBS solution containing 1% SDS (sodium dodecyl sulfate) 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 ultrasonically assist to dissolve the SDS powder completely. After the liquid returns to room temperature, add 10 mM PBS solution to the mark.

[0138] (4) Preparation of internal standard:

[0139] Prepare 250 ng / mL IGF-1 internal standard solution of N15-labeled IGF-1 with 0.5% BSA-PBS solution.

[0140] (5) Preparation of matrix:

[0141] The matrix solution is an aqueous acetonitrile solution (acetonitrile: water = 7:3 (v / v)) containing 10 mg / mL CHCA (α-cyano-4-hydroxycinnamic acid) and 0.5% TFA. Specifically, accurately weigh 1 g of CHCA into a 100 mL reagent bottle, add 70 mL of acetonitrile and 30 mL of deionized water, and ultrasonically assist for 5 min (to ensure that the light yellow CHCA is completely dissolved, the ultrasonic time can be appropriately increased), then add 500 μL of TFA and mix by inversion.

[0142] 2. Antibody-magnetic bead bonding

[0143] (1) Add 100 ug of IGF-1 antibody (mouse monoclonal antibody) to a 30 kDa ultrafiltration tube, and centrifuge at 12000 x g until the liquid in the tube is completely removed. Take out the ultrafiltration tube, add 200 μL of 0.1 M PBS (pH 7.4) buffer solution, vortex for 30 s, and centrifuge at 12000 x g for 5 min (repeat this operation twice). Add 290 μL of 0.1 M PBS (pH 7.4) solution to the washed ultrafiltration tube, mix by vortexing, and bond with magnetic beads.

[0144] (2) Epoxy magnetic beads 100 μL (10 mg) were placed in a 2 mL centrifuge tube and magnetically separated on a magnetic stand. After washing once with 200 μL of 0.1 M PBS (pH 7.4), the magnetic beads were resuspended in 330 μL of 0.1 M PBS (pH 7.4). The 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. After vortex mixing, the mixture was incubated overnight (about 16 hours) at room temperature. After magnetic separation, the liquid was discarded, and the magnetic beads were washed three times with 500 μL of 10 mM PBS solution. After discarding the liquid, the magnetic beads were resuspended in a 1% BSA solution and incubated at room temperature for 30 minutes. After washing once with 500 μL of 0.1% Tween 20-containing PBS solution, the magnetic beads were resuspended in 1 mL of 0.1% Tween 20-containing PBS solution and stored for use.

[0145] 3. Preparation of IGF-1 standard curve

[0146] The IGF-1 standard protein was prepared into an IGF-1 standard protein stock solution at a concentration of 0.1 mg / mL using a PBS solution. Subsequently, the IGF-1 standard protein stock solution was diluted into IGF-1 standard protein solutions at six concentration levels of 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 using a PBS-PA1080 solution.

[0147] Accurately pipette 40 uL of IGF-1 standard protein solution sample with different concentrations into 0.6 mL centrifuge tube, add 130 uL of 0.1% Tween 20 PBS solution and 30 uL of IGF-1 internal standard, vortex mix, then add 10 uL of magnetic bead suspension of bonded antibody, vortex mix. Mix on a tumbler for 10 min, remove and centrifuge for 10 s, then place on a magnetic stand for magnetic separation for 1 min, discard the liquid in the tube, then add 200 uL of 0.1% Tween 20 PBS solution, vortex for about 30 s, ensure that the magnetic beads are completely suspended, then place on a magnetic stand for magnetic separation for 1 min (repeat 3 times). Discard the supernatant, add 200 uL of deionized water to the 0.6 mL centrifuge tube, vortex for 30 s, then place on a magnetic stand for magnetic separation for 1 min, then discard the supernatant, repeat the operation once more with 100 uL of deionized water. Place the centrifuge tube without liquid in a mini centrifuge and centrifuge for 30 s, then place on a magnetic stand for magnetic separation for 30 s, carefully aspirate the trace amount of liquid at the bottom of the centrifuge tube with a 10 uL gun tip, then accurately add 10 uL of matrix solution, vortex for 30 s, then place on a magnetic stand for magnetic separation for 30 s, take 2 uL of colorless transparent eluent (matrix solution containing target protein) and spot on a target plate preheated on an electric hot plate at 39°C for heating, when the sample on the target plate is completely crystallized, collect data with MALDI-TOF MS. The results are shown in Figure 1 .

[0148] The MALDI-TOF MS collection conditions are as follows:

[0149] Laser: semiconductor laser; laser frequency: 1000 Hz; two-dimensional platform moving speed: 1.5 mm / sec; FocusMass: 4500-9000 Da; collection mass range: 2 kDa-35 kDa.

[0150] The ratio of Y-value of IGF-1 to internal standard (N15-labeled IGF-1) obtained by MALDI-TOF MS in the standard solution is x, and the concentration of IGF-1 is y. The power function is selected for standard curve fitting, see Figure 2 , Y = A*X B

[0151] A = 0.867, B = 1.0699, R 2 = 0.9968.

[0152] 4. Quantification of IGF-1 in serum / plasma samples

[0153] Accurately pipette 20 μL of serum / plasma into a 0.6 mL centrifuge tube, add 20 μL of lysis solution, vortex, 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, add 10 μL of bonded antibody magnetic bead suspension, vortex, place on a rotator for 10 min at room temperature, remove and centrifuge for 10 s, then immediately place on a magnetic stand 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, ensure that the magnetic beads are completely suspended, then place on a magnetic stand for magnetic separation for 1 min (repeat 3 times). Discard the supernatant, add 200 μL of deionized water to the 0.6 mL centrifuge tube, vortex for 30 s, then place on a magnetic stand for magnetic separation for 1 min, discard the supernatant, and repeat the operation once more with 100 μL of deionized water. Place the centrifuge tube without liquid in a mini centrifuge and centrifuge for 30 s, then place on a magnetic stand for magnetic separation for 30 s, carefully aspirate the trace amount of liquid at the bottom of the centrifuge tube with a 10 μL gun tip. Then accurately add 10 μL of matrix solution, vortex for 30 s, then place on a magnetic stand for magnetic separation for 30 s, take 2 μL of colorless transparent eluent and spot it on a target plate preheated on an electric hot plate at 39°C for heating. When the sample on the target plate is completely crystallized, collect data using MALDI-TOF MS.

[0154] The ratio of IGF-1 / IGF-1 variant to internal standard (N15-labeled IGF-1) of each sample obtained is taken as X value and input into the standard curve Y=A*X B to calculate the concentration of IGF-1 / variant in the sample tube. The content of IGF-1 in the sample is calculated according to the following formula:

[0155]

[0156] C IGFI — concentration of IGF-1 in the reaction tube (ng / mL)

[0157] V— sample volume (μL)

[0158] The inventors quantitatively detected IGF-1 in 205 serum / plasma samples using MALDI TOF MS. During the detection process, it was found that in each mass spectrum result, in addition to the peak of IGF-1, there were two peaks, which were speculated to be two variants of IGF-1. The mass-to-charge ratio range of 7500-7520 was named des-R IGF-1, and the mass-to-charge ratio range of 7343-7363 was named des-RR IGF-1. The mass spectrum result of one of the samples is shown in Figure 3 .

[0159] The quantitative results of IGF-1 MALDI TOF MS in 205 serum / plasma samples are shown in Table 1 below.

[0160] Table 1:

[0161]

[0162]

[0163]

[0164]

[0165]

[0166] 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).

[0167] The detection data (total IGF-1) of chemiluminescent immunoassay in 205 serum / plasma samples are shown in Table 2 below.

[0168] Table 2:

[0169]

[0170]

[0171]

[0172] Example 3: Verification that the des-R IGF-1 variant and the des-RR IGF-1 variant are IGF-1 variants

[0173] Using the same detection method as in Example 1, the following experimental and control groups were set up:

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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:

[0178] GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLR-LEMYCAPLKPAKSA(SEQ ID NO:1)

[0179] 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:

[0180] GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDL—LEMYCAPLKPAKSA(SEQ ID NO:2)

[0181] Results analysis:

[0182] (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.

[0183] 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.

[0184] (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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] Example 4: Selection of magnetic beads suitable for simultaneous detection of IGF-1 and IGF-1 variants

[0189] 1. Preparation of reagents required for testing:

[0190] 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.

[0191] 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.

[0192] 2. Antibody-magnetic bead bonding:

[0193] (1) The preparation of IGF-1 antibody-epoxy (epoxy resin) magnetic beads is as follows:

[0194] 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.

[0195] 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.

[0196] (2) The preparation of IGF-1 antibody-biotin-streptavidin-magnetic beads is as follows:

[0197] 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).

[0198] 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.

[0199] 3. Preparation of IGF-1 standard curve

[0200] 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 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 a QuanPRO protein spectrometer (MALDI-TOF MS). Once the vacuum level meets the requirements, begin data acquisition.

[0201] QuanPRO protein spectrometer detection parameters:

[0202] Laser: Semiconductor laser; Laser frequency: 1000Hz; 2D platform movement speed: 1.5mm / sec; Focus Mass: 4500~9000Da; Acquisition quality range: 2kDa~35kDa.

[0203] 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.

[0204] 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.

[0205] Table 3

[0206]

[0207] 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.

[0208] 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.

[0209] 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 kit for detecting variants of insulin-like growth factor I, characterized in that, include: IGF-1 specific antibody; Internal standard IGF-1 recombinant protein; IGF-1 protein standard; Elution solution; reaction buffer, The elution solution contains a matrix and an antigen-antibody complex dissociation reagent for the MALDI-TOF MS platform. 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 kit further includes magnetic beads that are bonded to the IGF-1 specific antibody, and the magnetic beads are epoxy magnetic beads.

2. The reagent kit according to claim 1, characterized in that, The IGF-1 specific antibody is a monoclonal antibody or a polyclonal antibody.

3. The reagent kit according to claim 2, characterized in that, The affinity constant KD value of the IGF-1 specific antibody is 1×10⁻⁶. -9 - 9×10 -12 .

4. The reagent kit according to claim 1, characterized in that, The Epoxy magnetic beads have a particle size of 0.1μm to 25μm.

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

6. The reagent kit according to claim 1, characterized in that, The internal standard IGF-1 recombinant protein 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% with that of the IGF-1 protein that can be enriched by the IGF-1 specific antibody.

7. The reagent kit according to claim 6, characterized in that, The isotopes are H2, N15, C13 or O18.

8. The reagent kit according to claim 6, 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.

9. The reagent kit according to claim 1, characterized in that, The reaction buffer includes at least one selected from MES buffer, PBST buffer, and Tris-HCl buffer.

10. The kit according to claim 1, characterized in that, The pH value of the reaction buffer is 6-8.

11. The reagent kit according to claim 1, characterized in that, The reaction buffer is PBST buffer, which contains 0.1-0.6% Tween 20.

12. The kit according to claim 1, characterized in that, The matrix comprises 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.

13. The reagent kit according to claim 1, characterized in that, The matrix is ​​α-cyano-4-hydroxycinnamic acid.

14. The kit according to claim 13, characterized in that, The concentration of the α-cyano-4-hydroxycinnamic acid is 5-20 mg / mL.

15. The kit according to claim 1, 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.

16. The reagent kit according to claim 1, characterized in that, The antigen-antibody complex dissociation reagent is a trifluoroacetic acid solution.

17. The kit according to claim 16, characterized in that, The mass concentration of the trifluoroacetic acid solution is 0.05-5%.

18. The reagent kit according to claim 1, characterized in that, The kit further includes a lysis buffer for lysing serum or plasma samples to be tested.

19. The reagent kit according to claim 18, characterized in that, The lysis buffer includes a lysis agent and a first buffer solution.

20. The kit according to claim 19, 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.

21. The reagent kit according to claim 19, characterized in that, The first buffer solution is selected from Tris-HCl buffer and PBS buffer.

22. The reagent kit according to claim 19, characterized in that, The lysis buffer contains sodium dodecyl sulfate and PBS buffer.

23. The reagent kit according to claim 19, characterized in that, The lysis buffer is a PBS solution with a mass concentration of 1-5% sodium dodecyl sulfate.

24. The kit according to claim 1, characterized in that, The kit further includes a washing solution for washing the antigen-antibody complex before elution by the elution solution. The washing solution includes a first washing solution, which is a buffer solution containing a nonionic surfactant.

25. The kit according to claim 24, 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.

26. The kit according to claim 24, 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.

27. The kit according to claim 24, characterized in that, The first washing solution is a PBS buffer containing at least one of Tween 20, Tween 60, and Tween 80.

28. The kit according to claim 24, characterized in that, The cleaning solution further includes a second cleaning solution, which is deionized water.

29. A kit for the simultaneous detection of insulin-like growth factor I and its variants, characterized in that, The kit is the kit according to any one of claims 1-28.

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