CN116087386A

CN116087386A - Human insulin-like growth factor detection method and kit

Info

Publication number: CN116087386A
Application number: CN202310378354.9A
Authority: CN
Inventors: 郑坚; 司慧娟; 姜宽
Original assignee: West Lake Omi Hangzhou Biotechnology Co ltd
Current assignee: West Lake Omi Hangzhou Biotechnology Co ltd
Priority date: 2023-04-11
Filing date: 2023-04-11
Publication date: 2023-05-09

Abstract

The invention mainly relates to a method and a kit for detecting human insulin-like growth factors, wherein the method comprises the following steps: dissociation of proteins; precipitating protein; and (3) solid phase extraction and purification: mass spectrometry detection: loading LC-MS/MS detection to obtain the Area Ratio value of the sample to be detected; and (3) calculating: and replacing the sample to be detected with the standard working solution, repeating the pretreatment step and the mass spectrum detection step to obtain a standard curve, substituting the Area Ratio value of the sample to be detected into the standard curve, and calculating the content of IGF-I and IGF-II. The kit comprises: a dissociating agent; a precipitant; standard working solution; a quality control product working solution; the standard internal standard fluid comprises an isotopically labeled diluent of IGF-I. The complexity of the pretreatment process is obviously reduced, IGF-I and IGF-II can be detected simultaneously, the method has high sensitivity, strong specificity, good accuracy, high precision, short detection time and high detection flux.

Description

Human insulin-like growth factor detection method and kit

Technical Field

The invention mainly relates to the technical field of medical detection, in particular to a method and a kit for detecting human insulin-like growth factors.

Background

Growth Hormone Deficiency (GHD) is a disease causing short stature of children, is mostly caused by insufficient secretion of Growth Hormone (GH) by pituitary gland, clinically manifests as symptoms of short stature, normal intelligence, slow bone growth and the like, and has bone age behind normal children of the same age, and can also cause changes of bone metabolism and sugar metabolism to seriously influence the growth of children.

GHD diagnosis has no gold standard at present, laboratory examination uses GH excitation test as main diagnosis basis, but GH is unstable, takes pulse and has circadian rhythm, and needs injection medicine excitation to have higher level score. GH excitation test requires 5 times of blood drawing within 2 hours, and has poor repeatability and accuracy and more influencing factors. Serum IGF-I (human insulin-like growth factor-I) is relatively stable without obvious pulse secretion and circadian rhythm, and can better reflect the endogenous GH secretion state, so that the serum IGF-I is once considered as a screening index of GHD. Clinical examination of primary patients with GHD or of individuals with height monitoring generally preceded IGF-I and IGFBP-3 (human insulin-like growth factor binding protein 3), combined with these two results and clinical manifestations, the GH detection was performed in the suspected case of GHD.

Human insulin-like growth factor (IGF) is a group of polypeptide substances with growth promoting effect, secretion cells of the IGF are widely distributed in tissues such as human liver, kidney, lung, heart and intestine, the IGF family comprises IGF-I and IGF-II (human insulin-like growth factor-II), IGF-I is an alkaline peptide containing 70 amino acids and coded by a gene of chromosome 12, has a structure similar to insulin, can mediate the growth promoting effect of GH on organisms, has strong growth promoting effect and is an important growth factor in childhood, and IGF-I synthesized in each tissue plays the growth promoting effect in a self-secretion or paracrine mode, so researches show that the change of IGF-I, IGFBP-3 level is closely related to GHD; insulin-like action of IGF-II is stronger, is a single-chain peptide containing 67 amino acids and plays an important role in fetal growth, and early papers indicate that IGF-I/IGF-II performance is superior to IGF-I single-test or IGF-I+IGFBP-3 dual-test performance in judging whether children suffer from GHD.

The detection method commonly used in IGF-I detection in the current market is a chemiluminescence method, wherein the detection of IGFBP-3 is mainly due to the fact that 95% of IGF-I in blood is combined with the IGFBP-3, and the influence of IGFBP-3 is still avoided even if the IGFBP-3 is treated before detection. Meanwhile, the detection limit of the current detection method is 25ng/mL, and the IGF-I content in the blood of the low-age children is generally below 10ng/mL, so the detection limit defect exists in the method. The mass spectrometry detection of IGF-I can avoid the influence of IGFBP-3, realize the accurate quantification of IGF-I, and is hopeful to omit IGFBP-3 detection; on the other hand, the detection limit can reach below 10ng/mL, thereby realizing the detection of low-concentration IGF-I. IGF-II is a loop in the GH-IGF axis and is theoretically possible to influence the GH-IGF axis and thus the GH secretion, and studies have shown that IGF-I/IGF-II performance is superior to IGF-I screening or IGF-I+IGFBP-3 performance in determining whether children are GHD. Therefore, the simultaneous quantitative detection of IGF-I and IGF-II can help to screen out GHD patients better clinically, thereby obtaining effective treatment.

In the prior art, there are few quantitative detection patents for IGF-I and/or IGF-II, and only the application number of the intelligent organism is 202210059159, the invention name is a quantitative detection kit and a detection method for insulin sample growth factor-I based on MALDI-TOF+MS, and the invention patent applications of the Nyquist diagnosis investment company with the grant publication numbers of CN105067698B and CN 102596230B. The invention of the intelligent organism is mainly based on detection and quantification of IGF-I by MALDI-TOF technology, and the quantification adopts a nonlinear fitting mode according to the characteristics of the mass spectrum; in addition, in the selection of lysate (dissociating agent), protein denaturant and surfactant are considered, the protection scope of the lysate does not relate to the reagent such as trifluoroethanol, and the antibody enrichment is needed, and the difference from the established methodology of the research is large; from the flow, the experiment is complicated, and the steps of combining an antibody with a magnetic bead, enriching target protein, washing, resuspension and the like are involved, so that materials such as a ultrafilter tube and the like and materials such as an antibody and a target plate are also needed when the magnetic bead bond of the antibody is prepared. The nyquist diagnosis investment company patent is mainly based on MALDI-TOF technology quantification, the quantification adopts a nonlinear fitting mode according to the characteristics of the mass spectrum, in addition, in the selection of lysate (dissociating agent), protein denaturant and surfactant are considered, and antibody enrichment is needed, and the main technical points are concentrated on online SPE separation and purification and IGF-I quantification by high-resolution mass spectrum.

The foregoing background knowledge is intended to assist those of ordinary skill in the art in understanding the prior art that is closer to the present invention and to facilitate an understanding of the inventive concepts and aspects of the present application, and it should be understood that the foregoing background art should not be used to assess the novelty of the technical aspects of the present application without explicit evidence that such matter is disclosed prior to the filing date of the present application.

Disclosure of Invention

In order to solve at least one technical problem mentioned in the background art, the invention aims to provide a method and a kit for simultaneously detecting IGF-I and IGF-II based on LC-MS/MS, wherein the method remarkably reduces the complexity of a pretreatment process, can simultaneously detect IGF-I and IGF-II, and has the advantages of high sensitivity, strong specificity, good accuracy, high precision, short detection time and high detection flux.

Provided is a method for detecting human insulin-like growth factor, comprising:

protein dissociation: adding a dissociation agent and a standard internal standard solution into a sample to be detected to carry out protein dissociation;

protein precipitation: adding a precipitant into the dissociated sample solution, and taking a supernatant for later use;

and (3) solid phase extraction and purification: loading samples to the SPE plate, and eluting sequentially;

mass spectrometry detection: loading LC-MS/MS detection to obtain the Area Ratio value of the sample to be detected;

and (3) calculating: the steps of protein dissociation, protein precipitation, solid phase extraction and purification and mass spectrum detection are repeatedly carried out by replacing a sample to be detected with a standard working solution, a standard curve is obtained, and the Area Ratio value of the sample to be detected is substituted into the standard curve to calculate the content of IGF-I and IGF-II;

wherein, the liquid crystal display device comprises a liquid crystal display device,

the standard internal standard solution comprises an isotope labeling diluent of IGF-I;

the standard working fluid comprises dilutions of IGF-I and IGF-II with different concentrations.

The invention also provides a human insulin-like growth factor detection kit which can quantitatively detect IGF-I and IGF-II simultaneously; the kit comprises:

a dissociating agent;

and (3) a precipitant:

standard working solution;

standard internal standard fluids, including isotopically labeled dilutions of IGF-I;

a quality control product working solution;

wherein, the standard working solution and the quality control working solution comprise diluted solutions with different concentrations of IGF-I and IGF-II, and the concentrations of IGF-I and IGF-II are different.

In some preferred embodiments, the standard working fluid comprises:

IGF-I solution at a concentration of 8, 24, 80, 200, 600, 1200ng/mL; and

IGF-II solution at a concentration of 12, 36, 120, 300, 900, 1800 ng/mL.

In some preferred embodiments, the quality control product working fluid comprises:

IGF-I solution at a concentration of 600, 200, 50 ng/mL; and

IGF-II solution at concentrations of 900, 300, 75 ng/mL.

The invention also provides application of the kit in simultaneous detection of serum or plasma IGF-I and IGF-II.

The beneficial effects of this application are:

the prior art is mainly based on MALDI-TOF technology, has high detection cost and complex pretreatment steps, and the inventor provides a method and a kit based on LC-MS/MS technology and capable of simultaneously detecting IGF-I and IGF-II in serum or plasma in consideration of enriching IGF-I detection technology and synchronously detecting IGF-I and IGF-II, wherein the pretreatment process is simple and only comprises three treatment processes: the method has the advantages that the dissociation of the analyte and the binding protein, the protein precipitation and the solid phase extraction purification can be carried out after the pretreatment is finished, the quantitative detection result can be obtained, the complexity of the pretreatment process is obviously reduced, the IGF-I and IGF-II are detected simultaneously based on the LC-MS/MS technology, the method has high sensitivity, strong specificity, good accuracy, high precision, short detection time and high detection flux, and the detection result can assist a clinician in diagnosing diseases related to abnormal IGF-I and IGF-II content in adults and/or minors.

Drawings

To make the above and/or other objects, features, advantages and examples of the present invention more comprehensible, the accompanying drawings which are needed in the detailed description of the present invention are simply illustrative of the present invention and other drawings can be obtained without inventive effort for those skilled in the art.

FIG. 1 is a flow chart of a method for simultaneous detection of IGF-I and IGF-II;

FIG. 2 is an IGF-I standard curve;

FIG. 3 is IGF-II standard curve;

FIG. 4 is a chromatogram of an IGF-I internal standard isotope;

FIG. 5 is an IGF-I chromatogram;

FIG. 6 is an IGF-II chromatogram.

Detailed Description

Suitable substitutions and/or modifications of the process parameters will be apparent to those skilled in the art from the disclosure herein, however, it is to be expressly pointed out that all such substitutions and/or modifications are intended to be encompassed by the present invention. While the products and methods of preparation of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the products and methods of preparation described herein without departing from the spirit and scope of the invention.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The present invention uses the methods and materials described herein; other suitable methods and materials known in the art may be used. The materials, methods, and examples described herein are illustrative only and not intended to be limiting. All publications, patent applications, patents, provisional applications, database entries, and other references mentioned herein, and the like, are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Unless specifically stated otherwise, the materials, methods, and examples described herein are illustrative only and not intended to be limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

Unless specifically indicated, the weight ratios, percentages, and the like described herein are by volume weight ratios and volume percentages.

In order to facilitate an understanding of the embodiments of the present invention, abbreviations and key terms that may be involved in the embodiments of the present invention are first explained or defined.

LC-MS/MS: liquid chromatography-tandem mass spectrometry;

IGF-I: human insulin-like growth factor-I;

IGF-II: human insulin-like growth factor-II;

GH: growth hormone;

GHD: growth hormone deficiency;

IGFBP-3: human insulin-like growth factor binding protein 3;

calibration material: the method is used for calibrating detection items during quantitative detection;

MALDI-TOF: time-of-flight mass spectrometry;

TFE: tetrafluoroethylene;

SDS: sodium dodecyl sulfate;

CHAPS:3- [3- (cholestamidopropyl) dimethylamino ] propane sulfonic acid inner salt;

BSA: bovine serum albumin;

HQC: high-concentration quality control product working solution;

MQC: medium-concentration quality control product working solution;

LQC: low-concentration quality control product working solution;

area Ratio: area ratio.

The present invention is described in detail below.

A method for detecting human insulin-like growth factor, comprising:

In some preferred embodiments, the protein dissociation specifically comprises: 100 mu L of sample is taken in a centrifuge tube with the volume of 1.5mL by a pipette, then a dissociating agent and an internal standard solution are added, and the sample is incubated at 37 ℃ and 500-2500 rpm.

In some preferred embodiments, the dissociating agent is at least one of TFE, SDS, or CHAPS.

In a partially preferred embodiment, the TFE is present in a volume percentage of 20 to 80%.

In a partially preferred embodiment, the volume percent of SDS is 0.2 to 0.8%.

In a partially preferred embodiment, the CHAPS is present in an amount of 0.1 to 1.0% by volume.

In some preferred embodiments, the precipitation specifically comprises: taking 50-500 mu L of precipitant by using a pipette, centrifuging for 10-40 min, taking supernatant, adding 1mL of 5% ammonia water, and uniformly mixing for later use.

In some preferred embodiments, the precipitating agent is at least one of acetonitrile, methanol, ethanol, or acetonitrile.

In a part of the preferred embodiments, the volume ratio of acetic acid and acetonitrile in the acetonitrile acetate is 5:95.

In some preferred embodiments, the volume percentages of methanol, ethanol, acetonitrile are all 100%.

In some preferred embodiments, the SPE plates are also equilibrated prior to rinsing, specifically, 500 μl of 5% ammonia is removed to the activated SPE plates and plate equilibration is performed using a biological sample pretreatment instrument.

In some preferred embodiments, the SPE plate is activated prior to equilibration, specifically, 500 μl methanol is removed into the SPE plate well and plate activation is performed using a biological sample pretreatment instrument.

In some preferred embodiments, the sample solution after the protein precipitation step is transferred to an SPE plate after the activation equilibration, and sample loading is performed using a biological sample pretreatment instrument.

In some preferred embodiments, the rinsing specifically comprises:

leaching 1: transferring 300 mu L of 5% ammonia water into the SPE plate holes, and leaching for one time by using a biological sample pretreatment instrument;

leaching 2: and transferring 300 mu L of acetic acid methanol aqueous solution into the SPE plate holes, and performing secondary leaching by using a biological sample pretreatment instrument.

In some preferred embodiments, the eluting specifically comprises:

elution 1: transferring 50 mu L of acetonitrile formate solution (0.2% formic acid and 80% acetonitrile) into a 96-well plate, eluting by using a biological sample pretreatment instrument, and repeating for 2 times;

elution 2: move and get 50 mu LddH ₂ And (3) eluting in the O-96 well plate by using a biological sample pretreatment instrument, and transferring the eluent to a sample injection bottle or the 96 well plate.

In some preferred embodiments, the standard internal standard solution is at a concentration of 10 μg/mL.

In some preferred embodiments, the standard working fluid comprises:

IGF-I solution at a concentration of 8, 24, 80, 200, 600, 1200ng/mL; and

IGF-II solution at a concentration of 12, 36, 120, 300, 900, 1800 ng/mL.

The liquid chromatography-tandem mass spectrometry (liquid chromatography-tandem mass spectrometry, LC-MS/MS) technology is widely used for quantitative detection of specific protein markers due to its advantages of high sensitivity and good specificity, but unfortunately, there is no commercialized and mature detection procedure for detecting IGF-I and IGF-II in blood/plasma, and it is not possible to achieve the detection requirements of high accuracy and high precision in the process of evaluating the aforementioned factors. Based on the method, the invention provides a method for simultaneously detecting IGF-I and IGF-II based on an LC-MS/MS technology, the pretreatment flow only comprises dissociation by a dissociation agent, precipitation by a precipitator and solid-phase extraction by an SPE96 pore plate, and then mass spectrometry detection can be carried out.

The invention also provides a human insulin-like growth factor detection kit,

the kit can quantitatively detect IGF-I and IGF-II simultaneously;

the kit comprises:

a dissociating agent;

and (3) a precipitant:

standard working solution;

a quality control product working solution;

In some preferred embodiments, the precipitating agent is at least one of acetonitrile, methanol, or ethanol acetate.

In some preferred embodiments, the standard working fluid comprises:

IGF-I solution at a concentration of 8, 24, 80, 200, 600, 1200ng/mL; and

IGF-II solution at a concentration of 12, 36, 120, 300, 900, 1800 ng/mL.

IGF-I solution at a concentration of 600, 200, 50 ng/mL; and

IGF-II solution at concentrations of 900, 300, 75 ng/mL.

The kit provided by the invention obtains pure peptide fragments by using a dissociating agent and a precipitating agent, is free from the interference of other proteins such as IGFBP-3 and the like, is used for quantitative detection of human insulin-like growth factor-I and human insulin-like growth factor-II by combining an LC-MS/MS technology, wherein the single application of an isotope labeling diluent of IGF-I as a standard internal standard liquid saves detection time and detection cost, has high sensitivity, strong specificity, good accuracy, high precision, short detection time and high detection flux, and can be used for quantitative detection of IGF-I and IGF-II content in adults and/or minors.

Example 1:

there is provided a kit for simultaneous detection of IGF-I and IGF-II based on an LC-MS/MS method, comprising:

a dissociating agent;

and (3) a precipitant:

standard working solution;

a quality control product working solution;

The dissociating agent is 0.5% SDS.

The precipitant was acetonitrile acetate (volume ratio 5:95).

The standard working solution is prepared by the following steps:

accurately weighing IGF-I and IGF-II standard substances, respectively dissolving with 0.1% BSA solution to prepare 200 mug/mL standard stock solution, and respectively diluting the stock solutions into 6 standard substance working solutions with different concentrations, wherein the specific working solutions are shown in Table 1.

TABLE 1 working fluid for Standard substance

ng/mL	STD1	STD2	STD3	STD4	STD5	STD6
							IGF-I	8	24	80	200	600	1200
IGF-II	12	36	120	300	900	1800

The standard internal standard solution is prepared by the following steps:

accurately weighing IGF-I-N15 isotope internal standard substance, and preparing 10 mug/mL working solution by using 0.1% BSA solution.

The quality control product working solution is prepared by the following steps:

accurately weighing IGF-I and IGF-II standard substances, respectively dissolving with 0.1% BSA solution to prepare 200 mug/mL standard storage liquid, and respectively diluting the standard storage liquid with three concentration gradients of high, medium and low to obtain quality control product working liquids, wherein the working liquids are shown in Table 2.

Table 2, quality control product working solution

ng/mL	HQC	MQC	LQC
				IGF-I	600	200	50
IGF-II	900	300	75

Example 2:

a method for simultaneously detecting IGF-I and IGF-II in a sample is provided, and the flow chart of the detection method is shown in FIG. 1.

The sample pretreatment flow comprises dissociation, precipitation and solid phase extraction.

Dissociation: 100 μl of the sample was removed by pipetting into a 1.5mL centrifuge tube, then adding the dissociating agent 40% tfe and internal standard solution and incubating at 37 ℃ at 1500 rpm.

Precipitation: taking 100 mu L of precipitator acetonitrile by using a pipette, centrifuging for 30min, taking the supernatant, adding 1mL of 5% ammonia water, and uniformly mixing for later use.

Solid phase extraction:

SPE plate activation: remove 500 μl methanol into SPE plate wells, perform plate activation using biological sample pretreatment instrument:

SPE plate balancing: transferring 500 mu L of 5% ammonia water into an SPE pore plate, and balancing the plate by using a biological sample pretreatment instrument;

loading: the sample solution after the precipitation step is moved to an SPE plate after the activation balance, and a biological sample pretreatment instrument is used for sample loading;

leaching 2: transferring 300 mu L of acetic acid methanol aqueous solution into SPE plate holes, and performing secondary leaching by using a biological sample pretreatment instrument;

elution 1: transferring 50 mu L of acetonitrile acetate solution (0.2% formic acid, 80% acetonitrile and the balance water) into a 96-well plate, eluting by using a biological sample pretreatment instrument, and repeating for 2 times;

The liquid chromatography parameters are as follows.

Sample injection amount: 10-20 mu L;

mobile phase information: fluidity a:0.1% formic acid in water, mobile phase B:0.1% acetonitrile formate solution.

The elution procedure is shown in table 3.

TABLE 3 elution procedure

Time(min)	Flow(mL/min)	B.Conc(%)
			1.0	0.3	5
3.0	0.3	45
			4.5	0.3	50
4.6	0.3	95
			5.6	0.3	95
5.7	0.3	5
			7.0	0.3	5

In electrospray ionization mode (ESI), positive ion scanning mass spectrometry parameters were performed using the MRM multiple reaction monitoring mode as shown in table 4.

Table 4, positive ion scanning mass spectrometry parameters

Parameter name	Numerical value
		Air curtain gas (CUR)	30
Collision gas (CAD)	9
		Spray voltage (IS)	5500
Ionization Temperature (TEM)	550
		Ion source GaS1 (GS 1)	55
Ion source GaS2 (GS 2)	55
		Injection voltage (EP)	10

The mass spectrum test compound parameters are shown in table 5.

Table 5, mass spectrum detection parameters

Compounds of formula (I)	Q1	Q3	De-cluster voltage (DP)	Collision voltage (CE)
					IGF-I	957	1175.61	80	45
IGF-II	1068	1065.4	80	46
					IGF-I-IS	968.5	1189.7	100	45

Example 3:

on the basis of the previous examples, the detection of IGF-I and IGF-II in the samples is continued, the mixed standard working solution shown in the table 1 is used for replacing the samples, the pretreatment flow is carried out under the same experimental conditions, the IGF-I concentration is 8-1200 ng/mL, the IGF-II concentration is 12-1800ng/mL, the detection is carried out according to the mass spectrometry conditions of the previous examples from low to high after the sample injection, the quantitative peak area-concentration is plotted to obtain a standard curve, and the result shows that the linear range and the quantitative limit of IGF-I and IGF-II are as follows:

linear range: IGF-I: 8-1200 ng/mL; IGF-II: 12-1800 ng/mL;

quantitative limit: IGF-I:8ng/mL; IGF-II:12ng/mL;

linear coefficient: IGF-I: r= 0.99963; IGF-II: r= 0.99889;

the standard curves are shown in fig. 2 and 3, respectively.

The chromatograms of IGF-I and its internal standard isotopes IGF-I-IS and IGF-II are shown in figures 4-6, the retention time of IGF-I IS 3.72min, the retention time of IGF-II IS 3.66min, and according to figures 4-6, the detection of two compounds of IGF-I and IGF-II has high accuracy, small interference and strong specificity.

The 6 repeated test results are shown in Table 6.

TABLE 6 repeated test results

Example 4:

for the dissociation agents in the foregoing examples, 40% TFE, 0.8% SDS and 0.6% CHAPS were prepared, and these three agents were used as dissociation agents, and were treated according to the pretreatment procedure, information was collected by mass spectrometry, and the Area Ratio of IGF1 and IGF2 was calculated, respectively, and from the results, SDS performance response was the best, TFE was the worst, and all 3 agents were used as dissociation agents, and specific data are shown in table 7.

TABLE 7 Area Ratio values and precision Using different dissociating agents

For the precipitants in the foregoing examples, acetonitrile acetate (volume ratio 5:95), acetonitrile, methanol and acetone reagents were prepared and used as precipitants, respectively, pretreatment of the samples was performed according to the sample pretreatment flow, collection of mass spectra, screening of the precipitants was performed by adopting a calculation recovery method, and the results showed that acetonitrile acetate, acetonitrile and methanol could be used as precipitants, acetone could not be used as precipitants, and specific data are shown in table 8.

TABLE 8 recovery rates with different precipitants

Example 5:

standard working solutions of IGF-I and IGF-II were prepared to have 3 concentrations of high, medium and low as shown in table 9, and recovery experiments and precision verification were performed, and recovery rates and precision were measured according to the methods of the foregoing examples, as shown in tables 10 and 11, respectively.

TABLE 9 concentration by adding standard

Watch 10, precision

TABLE 11 recovery rate

By integrating the verification experiments, the quantitative limit, the linearity, the recovery rate, the precision and other technical indexes of the scheme meet the requirements, the method can simultaneously detect the IGF-I and IGF-II contents in the blood plasma/serum, the repeatability is good, the labeling recovery rate is high, and the accuracy of the detection result is improved. Meanwhile, the substrate interference can be greatly eliminated by adopting the isotope internal standard method for quantification, and the method is not influenced by the conditions of pretreatment process, sample loading volume, flow and the like, so that accurate quantification can be achieved.

The conventional technology in the above embodiments is known to those skilled in the art, and thus is not described in detail herein.

The specific embodiments described herein are offered by way of example only to illustrate the spirit of the invention. Various modifications or additions to the described embodiments may be made by those skilled in the art to which the invention pertains or may be substituted in a similar manner without departing from the spirit of the invention or beyond the scope of the appended claims.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

While the above detailed description has shown, described, and pointed out novel features as applied to various embodiments, it will be understood that various omissions, substitutions, and changes in the form and details of the device or method illustrated may be made without departing from the spirit of the disclosure. In addition, the various features and methods described above may be used independently of one another, or may be combined in various ways. All possible combinations and subcombinations are intended to fall within the scope of this disclosure. Many of the embodiments described above include similar components, and thus, these similar components are interchangeable in different embodiments. While the invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the invention extends beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and obvious modifications and equivalents thereof. Therefore, the present invention is not intended to be limited by the specific disclosure of the preferred embodiments herein.

The invention is a well-known technique.

Claims

1. A method for detecting human insulin-like growth factor, comprising:

the standard working fluid comprises serial dilutions of IGF-I and IGF-II, and the concentrations of IGF-I and IGF-II are different.

2. The method according to claim 1, characterized in that: the dissociating agent is at least one of TFE, SDS, or CHAPS.

3. The method according to claim 1, characterized in that: the precipitant is at least one of acetonitrile, methanol, ethanol or acetonitrile.

4. A method according to any one of claims 1 to 3, characterized in that: the standard working solution comprises the following components:

IGF-I solution at a concentration of 8, 24, 80, 200, 600, 1200ng/mL; and

IGF-II solution at a concentration of 12, 36, 120, 300, 900, 1800 ng/mL.

5. A human insulin-like growth factor detection kit, characterized in that:

the kit can quantitatively detect IGF-I and IGF-II simultaneously;

the kit comprises:

a dissociating agent;

and (3) a precipitant:

standard working solution;

a quality control product working solution;

6. The kit of claim 5, wherein: the dissociating agent is at least one of TFE, SDS, or CHAPS.

7. The kit of claim 5, wherein: the precipitant is at least one of acetonitrile, methanol, ethanol or acetonitrile.

8. The kit according to any one of claims 5 to 7, wherein: the standard working solution comprises the following components:

IGF-I solution at a concentration of 8, 24, 80, 200, 600, 1200ng/mL; and

IGF-II solution at a concentration of 12, 36, 120, 300, 900, 1800 ng/mL.

9. The kit according to any one of claims 5 to 7, wherein: the quality control product working solution comprises:

IGF-I solution at a concentration of 600, 200, 50 ng/mL; and

IGF-II solution at concentrations of 900, 300, 75 ng/mL.

10. Use of a kit according to any one of claims 5 to 9 for simultaneous detection of serum or plasma IGF-I and IGF-II.