CN114910549A - MALDI-TOF MS-based quantitative detection kit and detection method for insulin-like growth factor I - Google Patents
MALDI-TOF MS-based quantitative detection kit and detection method for insulin-like growth factor I Download PDFInfo
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Abstract
The invention discloses a MALDI-TOF MS-based quantitative detection kit and a detection method for insulin-like growth factor I. The kit comprises: a lysis solution; magnetic beads to which insulin-like growth factor I antibodies are bonded; n15 labeled insulin-like growth factor I internal standard use solution; a calibration product; sinapic acid matrix use solution. The detection method is characterized in that IGF I marked by N15 is used as an internal standard and added into an IGF I-containing standard protein solution and a sample, and the quantitative detection of the IGF I is realized according to the ratio of the IGF I to the internal standard response value obtained on an instrument, so that the detection method has the advantages of good consistency, strong interference resistance, stable methodology, high flux, low cost, small sample dosage, capability of realizing pretreatment automation and the like. The detection method has the capability of detecting IGF I of 1548/day. The LOQ of the detection method is 1ng/mL which is far lower than 20ng/mL of the current clinical detection method, the detection range is expanded from 20-1600 ng/mL of the current method to the range of 5-2000 ng/mL, the method is basically not interfered by IGFBP3, and the CV with good reproducibility is less than 3%.
Description
Technical Field
The invention relates to the technical field of medical detection. More particularly, relates to a MALDI-TOF MS-based kit and a detection method for quantitatively detecting insulin-like growth factor I.
Background
InsuLin-like growth factor I (IGF I), also known as Somatomedin C, is a secreted protein synthesized by the liver and secreted into the peripheral blood, the mature protein of which consists of 70 amino acids, of which 6 cysteines form 3 pairs of disulfide bonds with each other that maintain their spatial structure, and is named because its structure is similar to InsuLin.
The secretion of IGF I is triggered by the binding of Growth Hormone (GH) to its cell surface growth hormone receptor, which is mainly reached by blood circulation, and the secretion of IGF I can directly act on various tissues and organs, and is a performer for the growth promoting action of GH. Therefore, the detecting of GH-IGF I axis can screen out the individual suffering from pituitary growth hormone metabolism disorder in high risk population, diagnose and treat in time to avoid the occurrence of complications, and play an important role in the aspects of tracing the disease cause, guiding the medication after the diagnosis and the like. Since the secretion of GH is influenced by various factors and is in a pulse form (more secretion at night than in the daytime), GH fluctuation in peripheral blood is large, and GH-IGF I axis detection is not suitable as a clinical index. IGF I is a recognized secretory protein with relatively stable content in peripheral blood, and the secretion amount of the IGF I can not only indicate whether GH is combined with a receptor of the GH and trigger the secretion of IGF I, but also regulate the secretion of GH by hypothalamus, and is an ideal index for detecting GH-IGF I axis. At present, the clinical detection method of IGF I is mainly based on a chemiluminescence immunoassay technology, and the technology has many problems in aspects of IGF I quantitative result reproducibility and quantitative accuracy, such as poor quantitative reproducibility, higher result, poor sensitivity, narrow quantitative range, incapability of realizing comparison among laboratories and the like of kits of different brands or different batches of the same brand. The literature reports that the Meito clinic backtracks the IGF1 detection results (Lot No.401-481) of 32 batch ImmuLite 2000 kits used between 10 and 7 of 2007 and finds that the quantitative results of the Lot424-427 kit are low, and the quantitative results of the Lot441-492 are continuously increased. In principle, the chemiluminescence immunoassay technology indirectly realizes the quantification of the substance to be detected by measuring the light intensity emitted by the luminescent substance on the secondary antibody specifically bound with the substance to be detected, so that the stability of the technology is interfered by various factors such as antibody specificity, non-specific adsorption, heterophilic antibody, interferent and the like, and the fluctuation of the quantification result is large, so that the long-term monitoring of the IGF I level of children suffering from growth hormone deficiency disease in clinic cannot be met. In addition, the technology can not distinguish endogenous IGF I and exogenous IGF I in serum, can not realize effective evaluation on treatment effect, and can not prompt a doctor to adjust the treatment direction in time. In recent years, some medical detection laboratories and mass spectrometer companies have developed an IGF I quantification technique based on mass spectrometry (triple quadrupole mass spectrometry), in which after IGF I in a bound state is released by acidic ethanol precipitation, complete IGF I or IGF I digested peptide fragments are quantified in a targeted manner by using LC-MS/MS tandem technology. Compared with the chemiluminescence immunoassay technology, the method has strong anti-interference performance and stable result, but the technology has complex pretreatment, long detection time, low flux and high reagent consumable cost, and is not suitable for being popularized as a clinical IGF I quantitative method.
Therefore, the development of a novel quantitative detection kit for insulin-like growth factor I and a detection method matched with the kit are urgently needed.
Disclosure of Invention
In view of the above problems, a first object of the present invention is to provide a kit for the quantitative detection of insulin-like growth factor I based on MALDI-TOF MS. The kit has high sensitivity, high precision and good accuracy.
The second purpose of the invention is to provide a detection method matched with the kit for use. The detection method is based on MALDI-TOF MS platform to develop IGF I quantitative detection technology which has strong anti-interference, stable methodology, high flux, low cost, less sample consumption and can realize pretreatment automation.
In order to achieve the first object, the invention provides the following technical scheme:
the invention discloses a MALDI-TOF MS-based quantitative detection kit for insulin-like growth factor I, which comprises:
a lysis solution; magnetic beads to which insulin-like growth factor I antibodies are bonded; n15 labeled insulin-like growth factor I internal standard use solution; a calibrator; sinapic acid substrate use solution;
wherein the calibrator is prepared by diluting standard proteins containing insulin-like growth factor I with different concentrations and 1% BSA solution.
The kit provided by the invention uses compounds such as urea, thiourea, SDS, CTAB, Tween 20, CHAPS and the like as a cracking agent for cracking IGF I-IGFBP3-ALS ternary complex in serum/plasma for the first time, so as to avoid interference of IGFBP3 in the detection process, solve the technical problem in the field, and like other protein denaturation reagents or surfactants capable of opening hydrogen bonds between proteins, the kit provided by the invention is also protected by the invention. The kit is combined with MALDI-TOF MS technology to be used for the quantitative detection of the insulin-like growth factor I, and has the advantages of high sensitivity, high precision, good accuracy and the like.
Further, the lysis solution comprises a lysis agent and a buffer. Wherein the cracking agent is selected from one or more of guanidine hydrochloride, urea, thiourea, Sodium Dodecyl Sulfate (SDS), Cetyl Trimethyl Ammonium Bromide (CTAB), tween 20, tween 80 or 3- ((3-cholamidopropyl) dimethylaminopropyl) -1-propanesulfonic acid inner salt (CHAPS), octyl-beta-glucoside, octyl thioglucoside, polyethylene glycol octyl phenyl ether (TritonX-100) and ethyl phenyl polyethylene glycol (NP 40); preferably, the lysis solution contains 4-10M of lysis agent and 0.1M of Tris-HCl buffer solution.
Further, the magnetic beads to which the insulin-like growth factor I antibody is bonded are bonded by utilizing an affinity interaction between biotin and streptavidin.
Further, the Biotin is one of EZ-Link Sulfo-NHS-Biotin, Sulfo-NHS-LC-LC-Biotin or BMCC-Biotin, and can also comprise Biotin containing different modifying groups commonly used in the field; the magnetic beads are streptavidin magnetic beads or carboxyl magnetic beads, and can also comprise magnetic beads modified by other groups matched with the biotin; the diameter of the magnetic bead is 0.1-3.5 μm.
Further, the calibrators may have different concentrations of 1000ng/mL, 750ng/mL, 500ng/mL, 250ng/mL, 100ng/mL, 50ng/mL, 10ng/mL, and 5 ng/mL.
Furthermore, the sinapic acid substrate using solution comprises a solvent, sinapic acid and trifluoroacetic acid.
Further, the solvent is a mixed solution of acetonitrile and water, the volume ratio of the acetonitrile to the water is 1:2, the concentration of sinapic acid is 5-15 mg/mL, and the volume content of trifluoroacetic acid is 0.7%.
Further, the kit also comprises 0.1% Tween 20 in PBS buffer.
In order to achieve the second object, the invention provides the following technical scheme:
the invention provides a MALDI-TOF MS-based quantitative detection method for insulin-like growth factor I, which comprises the following steps:
(1) placing a sample to be detected or a calibrator into a centrifuge tube, adding 0.5-1 times of volume of lysis solution for vortex mixing, and then adding N15-marked insulin-like growth factor I internal standard use solution and 3-10 times of volume of PBS buffer solution containing 0.1% Tween 20 for vortex mixing;
(2) adding the magnetic beads bonded with the insulin-like growth factor I antibody into the centrifugal tube, uniformly mixing, and reacting at room temperature for 1-60 min;
(3) after instantaneous centrifugation for 10s, carrying out magnetic separation to remove the supernatant;
(4) sequentially washing by using PBS buffer solution containing 0.1 percent Tween-20 and deionized water;
(5) eluting with sinapic acid matrix solution, and magnetically separating to obtain eluate containing insulin-like growth factor I;
(6) spotting the eluent containing the insulin-like growth factor I on a hydrophobic target plate, drying and crystallizing, and detecting on MALDI-TOF MS;
(7) and inputting the ratio of the obtained insulin-like growth factor I in the sample to be detected to the internal standard response value into a standard curve to calculate the concentration of the insulin-like growth factor I in the sample to be detected.
In order to solve the problems of poor anti-interference performance, generally higher result, large fluctuation of results among different batches and brands of reagents, low flux, high cost and the like in IGF I quantitative detection by the traditional detection method, the invention develops a detection method based on MALDI-TOF MS technology, which can effectively solve the problems, the detection method takes IGF I marked by N15 as an internal standard to be added into standard protein solution and samples containing IGF I, realizes the quantitative detection of IGF I according to the ratio of IGF I (7649Da) and internal standard (7742Da) response values obtained on an instrument, has the advantages of good consistency, strong anti-interference, stable methodology, high flux, low cost, less sample consumption, realization of pretreatment automation and the like, the clinical popularization of the detection technology can better diagnose GH-IGF I axis related diseases, and can promote the mutual identification of IGF I quantitative projects among different hospitals, promote the integration and configuration optimization of medical resources.
Furthermore, the standard curve is fit by taking the ratio of the insulin-like growth factor I in the calibrator solution to the response value of the internal standard on MALDI-TOF MS as x and the concentration of the insulin-like growth factor I as y.
Further, the mass ratio of the antibody to the magnetic beads is 1: 50-200.
The invention has the following beneficial effects:
the invention develops a MALDI-TOF MS-based quantitative detection kit for insulin-like growth factor I. The kit comprises: a lysis solution; magnetic beads to which insulin-like growth factor I antibodies are bonded; n15 labeled insulin-like growth factor I internal standard use solution; a calibrator; sinapic acid matrix use solution. The kit adopts compounds such as urea, thiourea, SDS, CTAB, Tween 20, CHAPS and the like as a cracking agent for cracking IGF I-IGFBP3-ALS ternary complex in serum/plasma for the first time, so as to avoid interference of IGFBP3 in the detection process, and has the advantages of high sensitivity, high precision, good accuracy and the like.
The invention also develops a detection method matched with the kit, the detection method takes IGF I marked by N15 as an internal standard to be added into a standard protein solution containing IGF I and a sample, and realizes the quantitative detection of the IGF I according to the ratio of IGF I (7649Da) to internal standard (7742Da) response values obtained on an instrument, and the detection method has the advantages of good consistency, strong anti-interference, stable methodology, high flux, low cost, less sample consumption, capability of realizing pretreatment automation and the like.
The pretreatment time of the detection method to each sample is 1.1min, the mass spectrum detection time of each sample is 0.93min, and the IGF I detection capacity is 1548/day. The LOQ of the detection method is 1ng/mL which is far lower than 20ng/mL of the current clinical detection method, the detection range is expanded from 20-1600 ng/mL of the current method to 5-2000 ng/mL, and CV (constant Current) with good reproducibility is less than 3% (n is 96).
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 MALDI-TOF MS mass spectrum overlay of 8 concentration gradients of IGF I standard curve.
Figure 2 five parameter Logistic fitted IGF I quantitative standard curve.
FIG. 3 is a MALDI-TOF MS mass spectrum of sample number 2020122307.
FIG. 4 is a graph showing the correlation between the detection technique of the present invention and the chemiluminescence immunoassay technique.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
In the present invention, the preparation method is a conventional method unless otherwise specified. The starting materials used are commercially available from published sources unless otherwise specified.
Experimental example 1
1. Serum/plasma samples
Randomly selecting 18 serum/plasma samples, and quantitatively testing IGF I in the samples by using a chemiluminescence immunoassay technology, wherein the specific information and detection results of the samples are shown in the following table:
TABLE 1 detection data table of 18 serum/plasma samples by chemiluminescence immunoassay
2. Reagent preparation
(1) Preparation of 10mM PBS solution:
0.1M PBS (2.7mM potassium chloride, 137mM sodium chloride, 1.76mM potassium phosphate) was diluted 10-fold with deionized water.
(2) Preparation of 0.1% Tween 20 in PBS:
500uL of nonionic surfactant Tween 20 is added into 500mL of 10mM PBS solution, and the mixture is mixed evenly by ultrasonic assistance.
(3) Preparation of 20mM Biotin aqueous solution:
1mg of EZ-Link Sulfo-NHS-Biotin was added to 224uL of deionized water and vortexed to dissolve the Biotin completely.
(4) Preparation of 1% BSA solution:
accurately weighing 0.5mg bovine serum albumin in a 50mL centrifuge tube, adding 50mL 10mM PBS solution, and dissolving the protein solid completely under the assistance of ultrasound.
(5) Preparation of lysate
The lysis solution contains 8M urea and 0.1M Tris-HCl with the pH value of 8.5, and the specific preparation process comprises the following steps: 24.024g of urea and 0.6057g of Tris are added with 30mL of water, the pH value is adjusted to 8.5 by 1M hydrochloric acid, and the volume is adjusted to 50mL by water.
(6) Preparation of 250ng/mL IGF I internal standard solution
N15 labeled IGF I was formulated in 250ng/mL IGF I internal standard solution with 1% BSA solution.
(7) Preparation of matrix stock solution of Sinapic Acid (SA)
60mg of sinapic acid solid powder is accurately weighed into a 10mL centrifuge tube, 2mL of acetonitrile (HPLC grade) and 4mL of absolute ethyl alcohol (HPLC grade) are added for vortex mixing, 70uL of trifluoroacetic acid solution is added for mixing to serve as an SA matrix stock solution, and the SA matrix stock solution is stored in a dark place.
(8) Preparation of matrix use solution of Sinapic Acid (SA)
Taking a proper amount of matrix stock solution of Sinapic Acid (SA), and mixing the matrix stock solution and deionized water in a vortex mode according to the volume ratio of 3:2 of the matrix stock solution to the deionized water.
3. Antibody-magnetic bead bonding
300ug of IGF I antibody was added to a 30kDa ultrafiltration tube, centrifuged at 12000 Xg centrifugal force until all the liquid in the tube was removed, 200uL of 10mM PBS buffer was added, vortexed for 30 seconds, and centrifuged at 12000 Xg centrifugal force for 5min (this operation was repeated 2 times). After removing the ultrafiltration tube, 400. mu.L of 10mM PBS buffer solution and 12. mu.L of 20mM Biotin solution (EZ-Link SuLfo-NHS-Biotin) were added, vortexed for 30 seconds, and then allowed to react at room temperature for 30 min. After the Biotin and the antibody had reacted sufficiently, the tube was centrifuged at 12000 Xg for 10min in a centrifuge, and 200. mu.L of 10mM PBS buffer was added and vortexed for 30s, and then centrifuged at 12000 Xg for 5min (this operation was repeated 2 times). Adding 30mg of streptavidin magnetic beads (the diameter of the magnetic beads is 1.5um) into a 5mL centrifugal tube, placing the centrifugal tube containing the magnetic beads on a magnetic frame for magnetic separation for 5min, removing all supernatant, taking down the centrifugal tube, re-dissolving the magnetic beads by using 2.5mL of 10mM PBS buffer solution, then completely transferring antibodies bonded with the Biotin into magnetic bead suspension, washing the magnetic bead suspension for multiple times by using 10mM PBS, transferring the antibodies in the ultrafiltration tube into the magnetic bead suspension, uniformly vortexing, and then overturning at room temperature for 3 hours to ensure that the antibodies bonded with the Biotin are fully bonded with the streptavidin magnetic beads. After the antibody is bonded with the magnetic beads, 5mL of the test tube is placed on a magnetic frame for magnetic separation for 5min, then the magnetic beads are washed with 2mL of 10mM PBS for 2 times, and finally the magnetic beads are resuspended with 3mL of 0.1% Tween 20 PBS solution for standby, wherein the concentration of the antibody is 100 ug/mL.
4. Preparation of IGF I Standard Curve
IGF I standard protein was formulated as a 0.1mg/mL stock of IGF I standard protein with 1% BSA solution. Then, the IGF I standard protein stock solution was diluted stepwise with 1% BSA solution to IGF I standard protein solutions at 8 concentration levels of 1000ng/mL, 750ng/mL, 500ng/mL, 250ng/mL, 100ng/mL, 50ng/mL, 10ng/mL and 5 ng/mL. Accurately transferring 40uL of IGF I standard protein solution samples with different concentrations into a 0.6mL centrifuge tube, adding 20uL of lysis solution, vortexing at room temperature at 1500 rpm for 10min, adding 130uL of 0.1% Tween 20 PBS solution and 30uL of IGF1 internal standard solution, vortexing and uniformly mixing, adding 10uL of magnetic bead suspension of bonded antibodies (using a front vortex for 30s, and vortexing every 5 samples once for 10 s), vortexing, placing on a turnover instrument at room temperature for reaction for 10min, taking off and placing on a magnetic frame for magnetic separation for 1min immediately after being rotated for 10s by an instant centrifuge, discarding the liquid in the tube, adding 200uL of 0.1% Tween 20 PBS solution, vortexing for about 30s to ensure that the magnetic beads are completely suspended, and placing on the magnetic frame for magnetic separation for 1min (repeating the operation for 3 times). After discarding the supernatant, 200uL of deionized water was added to a 0.6mL centrifuge tube, vortexed for 30 seconds, placed on a magnetic rack, magnetically separated for 1min, and the supernatant was discarded, followed by repeating this operation once with 100uL of deionized water. Placing a centrifuge tube without liquid in a mini centrifuge for 30s, placing the centrifuge tube on a magnetic frame for magnetic separation for 30s, carefully absorbing the trace liquid at the bottom of the centrifuge tube by using a 10uL gun head, then accurately adding 10uL SA matrix, using liquid for 30s by using vortex, placing the centrifuge tube on the magnetic frame for magnetic separation for 30s, taking 2uL colorless transparent eluent to point on a preheated target plate on an electric heating plate at 39 ℃ for heating, and collecting data by using MALDI-TOF MS after all samples on the target plate are crystallized. The results are shown in FIG. 1.
Wherein the acquisition conditions of MALDI-TOF MS are as follows:
a laser: a semiconductor laser; laser frequency: 1000 Hz; moving speed of the two-dimensional platform: 1.5 mm/sec; focus Mass: 4500-7500 Da; collection quality range: 2kDa to 35 kDa.
Fitting of a quantitative standard curve was performed with the ratio of IGF I in the standard solution to the Y-value of an internal standard (IGF I labeled N15) on MALDI-TOF MS as x and the concentration of IGF I as Y. According to the method, the square root of the ratio of the x variable to the y variable is used as the y variable, the IGF I concentration is used as the x variable to be input, five-parameter Logistic is selected to fit an IGF I quantitative standard curve, the equation for fitting the five-parameter Logistic curve is shown in FIG. 2, and the equation for fitting the five-parameter Logistic curve is as follows:
y＝(A-D)/[(1+(x/C) B )] n + D (correlation coefficient r) 2 ＝0.9996)，
Wherein: a is 63.95659;
B＝-0.24037；
C＝3411.95918；
D＝0.13802；
n＝3.63501。
5. quantification of IGF I in serum/plasma samples
Accurately transferring 20uL of serum/plasma into a 0.6mL centrifuge tube, adding 20uL of lysis solution, vortexing at room temperature at 1500 rpm for 10min, adding 130uL of 0.1% Tween 20 PBS solution and 30uL of IGF1 internal standard use solution, vortexing and mixing, adding 10uL of magnetic bead suspension of bonded antibody (using a front vortex for 30s, and using the mixture after vortexing for 10s once every 5 samples), vortexing and mixing, placing on a turnover instrument at room temperature for reaction for 10min, taking off, placing on a magnetic rack immediately after being vortexed for 10s by an instant centrifuge for magnetic separation for 1min, discarding the liquid in the tube, adding 200uL of 0.1% Tween 20 PBS solution, vortexing for about 30s to ensure that the magnetic beads are completely suspended, and placing on the magnetic rack for magnetic separation for 1min (repeating the operation for 4 times). After discarding the supernatant, 200uL of deionized water was added to a 0.6mL centrifuge tube, vortexed for 30 seconds, placed on a magnetic rack, magnetically separated for 1min, and the supernatant was discarded, followed by repeating this operation once with 100uL of deionized water. The centrifuge tube without liquid is placed in a mini centrifuge for 30s, then placed on a magnetic rack for magnetic separation for 30s, and a tip of 10uL is used to carefully suck out the trace liquid at the bottom of the centrifuge tube. Then 10uL of SA matrix is accurately added, the SA matrix is placed on a magnetic frame for magnetic separation for 30s after being vortexed for 30s, 2uL of colorless transparent eluent is spotted on a preheated target plate on an electric hot plate at 39 ℃ for heating, and after all samples on the target plate are crystallized, MALDI-TOF MS is used for collecting data.
The IGF I concentration in the sample tube was calculated by inputting the obtained ratio of IGF I to internal standard (IGF I labeled N15) for each sample as y-value into the fitting curve, and the spectra were processed as: baseline correction: a DefauLt mode; and (3) smoothing a mass spectrum: setting 9 windows by adopting a Moving-Average mode; MiniSNR is 1; % Height to use: 1; and (3) calibrating the mass-to-charge ratio: calibration was performed with m/z7649 (peak of IGF I) mass spectrum peak; the calibration method comprises the following steps: single-point calibration; automatic internal calibration mass to charge ratio: m/z7742 (mass spectral peak of internal standard); automatic internal calibration limits: 2000in PPM. The calculation is performed according to the following calculation formula:
the IGF I content in the sample was calculated according to the following formula:
C IGFI concentration of IGF I in reaction tube (ng/mL)
V-sample volume (μ L)
The results of the quantification of the 18 sera are shown in the following table:
TABLE 2 Table of data of 18 serum/plasma samples tested by the test method of the present invention
FIG. 3 is a MALDI-TOF MS mass spectrum of sample number 2020122307.
As can be seen from comparing Table 1 and Table 2, the detection method of the present invention has lower determination results than the conventional IGF I detection platform (chemiluminescence immunoassay), and the correlation analysis is shown in FIG. 4, and the correlation coefficient of the two methods is 0.9883, which illustrates that the difference between the quantitative results of the two methods is the systematic deviation caused by the methodology itself.
The method has the advantages of stable methodology, high flux, low cost, small sample demand and the like, and has good correlation with the existing method, so that the method can meet the clinical requirement on IGF I quantification.
It should be understood that the above-described embodiments of the present invention are examples for clearly illustrating the invention, and are not to be construed as limiting the embodiments of the present invention, and it will be obvious to those skilled in the art that various changes and modifications can be made on the basis of the above description, and it is not intended to exhaust all embodiments, and obvious changes and modifications can be made on the basis of the technical solutions of the present invention.
Claims (10)
1. A kit for the quantitative detection of insulin-like growth factor I based on MALDI-TOF MS, the kit comprising:
a lysis solution; magnetic beads to which insulin-like growth factor I antibodies are bonded; n15 labeled insulin-like growth factor I internal standard use solution; a calibrator; sinapic acid substrate use solution;
wherein the calibrator is prepared by diluting insulin-like growth factor I standard protein with different concentrations and 1% BSA solution.
2. The kit of claim 1, wherein the lysis solution comprises a lysis agent and a buffer; preferably, the cracking agent is selected from one or more of guanidine hydrochloride, urea, thiourea, sodium dodecyl sulfate, hexadecyl trimethyl ammonium bromide, tween 20, tween 80 or 3- ((3-cholamidopropyl) dimethylaminopropyl) -1-propanesulfonic acid inner salt, octyl-beta-glucoside, octyl thioglucoside, polyethylene glycol octyl phenyl ether and ethyl phenyl polyethylene glycol; preferably, the lysis solution contains 4-10M of lysis agent and 0.1M of Tris-HCl buffer solution.
3. The kit according to claim 1, wherein the magnetic beads having the insulin-like growth factor I antibodies bonded thereto are bonded by an affinity interaction between biotin and streptavidin.
4. The kit of claim 3, wherein the Biotin is one of EZ-Link Sulfo-NHS-Biotin, Sulfo-NHS-LC-LC-Biotin, or BMCC-Biotin; the magnetic beads are streptavidin magnetic beads or carboxyl magnetic beads; the diameter of the magnetic bead is 0.1-3.5 μm.
5. The kit of claim 1, wherein the calibrant has a different concentration of 1000ng/mL, 750ng/mL, 500ng/mL, 250ng/mL, 100ng/mL, 50ng/mL, 10ng/mL, and 5 ng/mL.
6. The kit of claim 1, wherein the sinapic acid substrate use solution comprises a solvent, sinapic acid, and trifluoroacetic acid.
7. The kit according to claim 6, wherein the solvent is a mixed solution of acetonitrile and water, the volume ratio of the acetonitrile to the water is 1:2, the sinapic acid concentration is 5-15 mg/mL, and the trifluoroacetic acid content is 0.7% by volume.
8. A method for the quantitative detection of insulin-like growth factor I based on MALDI-TOF MS, characterized in that it comprises the following steps:
(1) placing a sample to be detected or a calibrator into a centrifuge tube, adding 0.5-1 times of volume of lysis solution for vortex mixing, and then adding N15-marked insulin-like growth factor I internal standard use solution and 3-10 times of volume of PBS buffer solution containing 0.1% Tween 20 for vortex mixing;
(2) adding the magnetic beads bonded with the insulin-like growth factor I antibody into the centrifugal tube, uniformly mixing, and reacting at room temperature for 1-60 min;
(3) after instantaneous centrifugation for 10s, carrying out magnetic separation to remove the supernatant;
(4) sequentially washing the mixture by using PBS buffer solution containing 0.1% Tween-20 and deionized water;
(5) eluting with sinapic acid matrix solution, and magnetically separating to obtain eluate containing insulin-like growth factor I;
(6) spotting the eluent containing the insulin-like growth factor I on a hydrophobic target plate, drying and crystallizing, and detecting on MALDI-TOF MS;
(7) and inputting the ratio of the obtained insulin-like growth factor I in the sample to be detected to the internal standard response value into a standard curve to calculate the concentration of the insulin-like growth factor I in the sample to be detected.
9. The quantitative determination method according to claim 8, wherein the standard curve is fit by taking the ratio of the insulin-like growth factor I in the calibrator solution to the response value of the internal standard on MALDI-TOF MS as x and the concentration of the insulin-like growth factor I as y.
10. The quantitative detection method of claim 8, wherein the mass ratio of the antibody to the magnetic beads is 1:50 to 200.
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|---|---|---|---|---|
| CN116087386A (en) * | 2023-04-11 | 2023-05-09 | 西湖欧米(杭州)生物科技有限公司 | Human insulin-like growth factor detection method and kit |
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