CN109738655B - Application of IGFBP3 in preparation of kit for quantitatively detecting liver cancer marker - Google Patents

Abstract

The invention relates to an application of IGF BP3 in preparation of a kit for quantitatively detecting liver cancer markers. The invention discovers that the content change of the IGF BP3 protein has obvious correlation to the state of the liver cancer, and provides a kit capable of accurately detecting the content of the IGF BP3 protein to assist in diagnosing the existence, the stage and the metastasis of the liver cancer.

Description

Application of IGFBP3 in preparation of kit for quantitatively detecting liver cancer marker

Technical Field

The invention belongs to the field of tumor diagnosis, and relates to application of IGFBP3 in preparation of a kit for quantitatively detecting a liver cancer marker.

Background

People talk about cancer discoloration. The threat of malignant tumors to human health has grown in severity since the 21 st century. The fatality rate is second to cardiovascular and cerebrovascular diseases and is the third place. The main reason for the high mortality rate of tumor patients is that early diagnosis cannot be realized, and early diagnosis and early treatment are the most effective methods for preventing and treating tumors and reducing the mortality rate.

As tumor cells develop, certain proteins in tumor patients change, or new abnormal proteins associated with tumors are produced. These chemical species that reflect the presence of tumors are collectively referred to as tumor markers. The tumor markers are antigens and other bioactive substances generated or reduced by the tumor cells in the canceration process due to the change of the expression level of genes, some are not present in normal adult tissues but only in tumor tissues, and the content of some in the tumor tissues is greatly higher than that in the normal tissues. That is, the existence or amount of the tumor marker can indicate the tumor property, so the detection of the tumor marker has great reference significance for the clinical treatment of the tumor, and can be applied to the early diagnosis of the tumor, the staged monitoring of the tumor process and the real-time evaluation of the treatment effect of the drug (ASCO, 1996). In order to meet the requirements of clinical diagnosis and treatment of tumors, research on tumor markers and development of corresponding detection modes are urgently needed to be accelerated.

Currently, tumor markers for clinical diagnosis include 6 major classes, such as carcinoembryonic antigen, enzyme, hormone, glycoprotein, oncogene, and cell surface tumor antigen. The U.S. FDA approved the following serum tumor markers for use in the adjuvant diagnosis of tumors: alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen CA125, cancer antigen CA19-9, cancer antigen CA153, prostate specific antigen (fPSA, tPSA), Thyroglobulin (Thyroglobulin), beta human chorionic gonadotropin (HCGb), and human epididymis secretory protein 4(HE 4). Still other tumor markers commonly used in clinical diagnosis include neuron-specific enolase (NSE), calcitonin (PCT), iron-binding protein (Ferritin), beta 2-microglobulin (beta 2-microglobulin), Pepsinogen (Pepsinogen1,2), Prolactin (Prolactin), and the like. Most of the tumor markers lack sensitivity and specificity, so that the wide application of the tumor markers in tumor diagnosis is limited. For liver cancer, alpha fetoprotein and ultrasonic examination are generally adopted modes for diagnosing high-risk patients, the survival rate of the liver cancer patients is really and obviously improved, but the sensitivity is lower; the tumor antigen CA-125 has higher sensitivity but lacks specificity. Similarly, the blood tumor marker CA15-3 for breast cancer detection is hardly used in early diagnosis due to its low sensitivity. Therefore, early diagnosis of tumors and differentiation between benign and malignant tumors remain a clinical problem, and new technologies and methods are needed to find new tumor markers and to improve the sensitivity and reliability of tumor marker detection.

Insulin Growth Factor Binding Proteins (IGFBPs) are a family of proteins that regulate cell proliferation and apoptosis via insulin-like growth factor-dependent and independent pathways, and have recently been implicated in the development, progression and resistance to chemotherapy of a subset of tumors, particularly IGFBP2, IGFBP3, IGFBP5, which are present in large amounts in vivo. Most IGF in human blood circulation is combined with IGFBP, and there are 6 IGFBPs (IGFBP-1-IGFBP-6) with different properties in human body, wherein IGFBP-3 is closely related to growth hormone, and is a valuable index for diagnosing growth hormone deficiency. IGFBP3 binds to IGF1 and blocks IGF1 signal from being transduced into nucleus via IGF1 receptor, so as to reduce the tumor cell proliferation promoting effect of IGF1 and indirectly inhibit tumor growth. However, in order to apply IGFBP3 to diagnosis and monitoring of tumors, IGFBP3 recombinant protein sold abroad is expensive, and is not beneficial to further production and development. And IGFBP3 recombinant protein developed by domestic manufacturers has low solubility, which is not beneficial to the preparation of the kit.

Disclosure of Invention

The invention aims to provide application of IGFBP3 in preparation of a kit for quantitatively detecting liver cancer markers, so as to solve at least one technical problem.

According to one aspect of the invention, there is provided a use of an IGFBP in the preparation of a kit for the quantitative detection of a marker for liver cancer.

Preferably, IGFBP3 is a polypeptide comprising SEQ ID NO:2 or the polypeptide fragment of SEQ ID NO:2 into a polypeptide fragment of polar amino acid.

Preferably, IGFBP3 is a polypeptide comprising SEQ ID NO: 3.

According to another aspect of the present invention, there is provided a kit for quantitatively detecting a liver cancer marker, comprising: the reverse protein chip is used for coating broken micro tissues or cells from a sample to be detected; a detection antibody which is a monoclonal antibody of biotinylated anti-IGFBP 3 or a genetically engineered antibody; IGFBP3 standard, which is a polypeptide fragment containing SEQ ID NO. 2 or a polypeptide fragment in which one or two prolines in SEQ ID NO. 2 are mutated to polar amino acids.

Preferably, the IGFBP3 standard peptide is a peptide comprising SEQ ID NO: 3.

Preferably, the purity of the polypeptide fragment is greater than 95%.

Preferably, the detection antibody is a biotinylated monoclonal anti-IGFBP 3 antibody at a concentration of 0.1 mg/L.

Preferably, the detection antibody is a genetically engineered antibody scFv, Fab 'and/or F (ab') 2.

Preferably, the standard is coated on an elisa plate made of polystyrene.

Preferably, the sample to be tested is taken from human blood.

The marker capable of reliably reflecting the liver cancer state is obtained through systematic screening, and based on the fact that the average expression level of IGFBP3 protein in a liver cancer sample is far higher than that of a healthy sample and the correlation between the healthy sample and the malignancy degree of the tumor, IGFBP3 protein is used as a detection target, so that the liver cancer can be predicted, diagnosed, staged, monitored and the like to achieve higher sensitivity, specificity and accuracy. The invention edits the amino acid sequence of IGFBP3, and uses the obtained recombinant protein as a standard substance for detecting IGFBP3 protein, so that the standard substance has good stability, affinity and solubility, is easier to coat on an enzyme label plate, and has stronger binding capacity with a specific antibody. By using the standard substance provided by the invention to calibrate and measure the concentration standard curve of IGFBP3 protein in a sample, the quantitative detection of IGFBP3 protein can be accurately realized.

Drawings

FIG. 1 is a flow chart of screening of liver cancer markers in example 1;

FIG. 2 is a ROC plot of the 17 factors of the training set in example 1;

FIG. 3 is a schematic cross-validation of 17 factors in example 1;

FIG. 4 is a ROC plot of the 6 factors of the training set in example 1;

FIG. 5 is a schematic cross-validation of 6 factors in example 1;

FIG. 6 is a schematic diagram showing the spotting distribution and spotting results of the reversed protein chip in example 2 for detecting β 2-microglobulin, IGFBP3 protein, GP73 protein, GDF15 protein, OPN protein and AFP protein, respectively;

FIG. 7 is a graph comparing the content of IGFBP3 protein in the liver cancer sample and the healthy sample of example 2;

FIG. 8 is a graph showing the effect of IGFBP3 in different IGFBP3 standards on test samples.

Detailed Description

In order to make the technical solutions of the present invention better understood by those skilled in the art, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.

The expression level of IGFBP3 protein can be reflected by direct or indirect means, such as chemiluminescence, time-resolved immunoassay, immunoblotting, etc., and in examples 1-4, the concentration of IGFBP3 protein is determined by the combination of ELISA and absorbance test based on the principle of antigen-antibody reaction.

Example 1 screening of liver cancer markers

The experimental volunteers of this example include cancer patients, other cancer patients and healthy people, and the screening process of liver cancer markers is shown in fig. 1. Serum samples from experimental volunteers were well exposed to antibody chips coated with antibodies against 274 serum markers based on ELISA and the antibody chip test results were subjected to artificial neural network analysis. 78 liver cancer samples, 40 other cancer samples and 80 healthy samples are set, and the data provided by the antibody chip is normalized. And (5) obtaining a standard substance curve of the target factor by adopting the related standard substance, and determining the protein concentration according to the standard substance curve. Therefore, the content of the 21 factors in the liver cancer sample is obviously different from that in other cancer samples and healthy samples. According to the requirement that the P value is less than 0.01, 17 factors meeting the requirement are selected for statistical modeling. These 17 factors are AFP, GDF15, CEACAM1, MMP9, GP73, B2M, IGFBP3, ACRP30, Ferritin, Axl, LYVE-1, Fas, DKK-1, HGF, IL8, FGF9, Nidoun 1. And (3) comprehensively screening 17 factors, and evaluating the sensitivity, specificity and accuracy of the selected factors by respectively adopting 4 groups of models of Logistic Regression (LR), Linear Discriminant Analysis (LDA), Random Forest (RF) and Support Vector Machine (SVM).

In this embodiment, a leave-one-out cross-validation screening method (leave-one-out cross-validation approach) is adopted: and dividing the sample N into a training group N-1 and a prediction group 1 in each round to obtain N models, and taking the average of the classification accuracy of the final prediction groups of the N models as the performance index of the classified sample. The advantage of this screening method is that almost all samples in each round are used for training the model, and therefore the distribution of the closest original samples, so the results obtained by evaluation are reliable; no random factors influence the experimental data in the experimental process, and the experimental process is ensured to be reproducible. The model is derived from the training set and verified using the prediction set. It can be seen from fig. 2 that the area under the receiver operating characteristic curve (ROC curve) for 17 factors of the training set collected according to the above 4 models is close to 1, and the accuracy is high. We continued to reduce the number of factors in the model to test, and from 16 factors to 2 factors, observed that the accuracy of 6 factors, β 2 microglobulin, IGFBP3 protein, GP73 protein, GDF15 protein, OPN protein and AFP protein, was comparable to 17 factors, and the area under the ROC curve (shown in fig. 4) for 6 factors was also close to the accuracy and KAPPA value of the cross validation for 1, 17 factors and 6 factors as shown in fig. 3 and 5. Comparing the data provided in tables 1 and 2, the sensitivity, specificity and accuracy in the tables are all close to 1, which shows that the detection results of 17 factors and 6 factors in the tested sample can correctly identify and distinguish patients and non-patients with liver cancer, thus having higher reliability for the experimenter to diagnose the liver cancer state.

Table 117 factor model Performance evaluation

TABLE 26 evaluation of Performance of factor models

Example 2 reverse protein chip to verify the detection effects of 6 factors

The reverse protein chip was operated as follows:

sample treatment: serum samples provided by the experimenter were treated with appropriate buffers and serial dilutions.

Preparing a standard substance: 6 factor recombinant proteins of beta 2-microglobulin, IGFBP3 protein, GP73 protein, GDF15 protein, OPN protein and AFP protein are prepared into 100g/ml (stock solution), and are diluted for 5 times according to different 1/3 times according to the initial concentration of each factor standard curve, and a blank control is set.

Preparing a membrane: diluted serum samples, standards, positive controls and blank controls were spotted onto the membrane. 800cw-Streptavidin was used as a positive control and PBS buffer containing 1% BSA was used as a negative control. After spotting, the membranes were allowed to dry naturally and stored at-80 ℃. Detecting a membrane: after equilibrating the slides to room temperature, incubate for 30 minutes with blocking buffer; adding a biotin labeled antibody solution to incubate for 2 hours; adding 1 × 800cw-conjugated streptavidin (diluted 8000 times with blocking buffer), and incubating at room temperature for 2 h; after washing, the cells were scanned at 532nm by a Genepix 4000B laser scanner.

Scanning with ImageQuant LAS4000 chemiluminescent imaging analysis System

1) Scanning the instrument: ImageQuant LAS4000Scanner

2) Brand name: GE corporation, USA (GE Healthcare corporation)

3) The producing area: USA

4) Scanning parameters are as follows: high resolution

Data was extracted using the instrumental self-contained analysis software and analyzed using IBM SPSS analysis software.

As shown in fig. 6, the data analysis revealed that 6 factors had significant differences (P < 0.05).

Serum samples of hepatocellular carcinoma patients and healthy persons were tested for IGFBP3 protein. As shown in FIG. 7, the mean expression level of IGFBP3 was much higher in serum samples from hepatocellular carcinoma patients than in the healthy group. By data analysis, significant differences were found for IGFBP3 (P < 0.05).

Reagent source information:

name (R)	Company(s)	Detection of antibodies	Initial concentration of antigen
				IGFBP3	raybiotech	102-17505	200000pg/ml
GDF15	raybiotech	144-00185	2000pg/ml
				β2M	raybiotech	DS-MB-00113	10000pg/ml
OPN	raybiotech	119-15527	1000000pg/ml
				GP73	raybiotech	130-10311	10000pg/ml
AFP	raybiotech	DS-MB-00070	1000pg/ml

EXAMPLE 3 preparation of Stable IGFBP3 Standard

IGFBP3 protein is a protein composed of 291 amino acids, and its full-length amino acid sequence is shown in SEQ ID NO. 1. The standard curve for determining IGFBP3 concentration is calibrated by IGFBP3 standard, the IGFBP3 standard is obtained from blood sample of tumor patient, or from gene recombinant expression, and IGFBP3 standard is selected from the group consisting of those with purity of more than 95%: IGFBP3 protein, recombinant IGFBP3 protein, full-length or fragments comprising the amino acid sequence of SEQ ID NO. 1 or complexes thereof coupled to other groups, and other derivatives. And the IGFBP3 standard substance with high solubility is selected, so that the acquisition of experimental data is facilitated. Standard curve for IGFBP3 concentration a corresponding curve of concentration and absorbance measurements measured by ELISA method was prepared using a known concentration of IGFBP3 standard.

A great deal of research finds that the prokaryotic protein expressed by recombination is lack of glycosylation, and the stability of the prokaryotic protein is poorer than that of the natural protein. Furthermore, insoluble inclusion bodies are obtained during the purification of recombinant proteins and are not easily bound to specific antibodies. Currently, the IGFBP3 calibration products on the market generally have poor stability. IGFBP3 recombinant protein purchased from abroad is expensive and is not beneficial to further production and development. However, recombinant proteins developed by domestic manufacturers have low solubility, which is not favorable for preparation of kits. However, the inventor finds out through antigen epitope design experiments that the peptide chain structure of the polypeptide fragment obtained by mutating one or two amino acids in the SEQ ID NO. 2 sequence is relatively stable, which is beneficial to the combination of the polypeptide fragment and a specific antibody. In addition, the mutated polypeptide fragment has better affinity and is easier to coat on an enzyme label plate than the recombinant protein.

In this example, the sequence from amino acid 267 to amino acid 283 of SEQ ID NO. 1 (SEQ ID NO:2) was selected, in which proline 5 (Pro) of SEQ ID NO:2 was mutated to the polar amino acid lysine (Lys), and the resulting amino acid sequence was KYGQKLPGYTTKGKEDV, labeled SEQ ID NO:3, and biochemically synthesized by Shanghai Gill. The amino acid sequences of SEQ ID NO 2 and the mutated SEQ ID NO 3 selected in this example all contain > 25% of charged amino acids (E, D, K, R and H) and < 25% of hydrophobic residues, all of which are hydrophilic amino acid polypeptides.

The prior art considers that polypeptide chains rich in proline can firmly bind two proteins when performing biological functions, while polypeptide chains lacking proline often have poor binding capacity. ELISA was performed with the recombinant protein of SEQ ID NO. 1, the polypeptide of SEQ ID NO. 2, and the polypeptide of SEQ ID NO. 3 at different concentrations, respectively, and IGFBP3 antibody at a concentration of 1.5. mu.g/mL. The results are shown in table 3, with increasing antigen concentration, titers: SEQ ID NO 3> SEQ ID NO 2 > SEQ ID NO 1, which shows that the polypeptide of SEQ ID NO 3 has the strongest binding ability with the IGFBP3 antibody.

Table 3 results of ELISA with IGFBP3 antibody

Example 4 kit for quantitative determination of liver cancer markers

The kit for quantitatively detecting the liver cancer marker comprises the following components:

ELISA plate: the capture antibody is coated by a polystyrene plate with good adsorption performance, low blank value and stable batch, and is treated by confining liquid in advance.

2. Detecting an antibody: specific antibodies directed against β 2-microglobulin, IGFBP3 protein, GP73 protein, GDF15 protein, OPN protein and AFP protein may be selected from monoclonal antibodies or antigen binding fragments thereof, such as scFv, Fab 'and F (ab') 2. The concentration of the diluted detection antibody was 0.1 mg/L.

3. Washing liquid: 20 Xconcentrated wash containing 0.1% Tween 20.

4. A standard, comprising: the protein standard comprises a beta 2-microglobulin standard, an IGFBP3 protein standard, a GP73 protein standard, a GDF15 protein standard, an OPN protein standard and an AFP protein standard, wherein the IGFBP3 protein standard is a protein standard containing 95% purity of SEQ ID NO:3, polypeptide fragment standard antigen dry powder.

5. Diluent A15 ml of 5 Xconcentrated diluent (0.02mol/LpH7.4 PBS, 0.05% Tween-20) for sample dilution

6 Diluent B15 ml of 5 Xconcentrated Diluent 7.200 μ l of 300X concentrated HRP-streptavidin solution used to dilute the antibodies and HRP-streptavidin.

8. Substrate: 12ml of TMB solution.

9. Stopping liquid: 8ml of a 0.2M strength sulfuric acid solution.

10. Target detection protein: the beta 2-microglobulin, IGFBP3 protein, GP73 protein, GDF15 protein, OPN protein and AFP protein in the blood plasma or serum refer to the above 6 proteins existing in the blood, which are not intracellular and on the cell surface, and can exist alone or exist in combination with other extracellular proteins in the blood.

The kit for quantitatively detecting the liver cancer marker provided by the embodiment is used in the following way:

(1) adding a standard substance which is diluted by a diluent in a gradient manner and a serum sample to be detected, repeating each sample for two times, adding 100 mu l of the standard substance and the serum sample to be detected in each hole, and reacting for 40 minutes at 37 ℃;

(2) preparing 1 Xwashing liquid to wash the plate for 5 times for 10 minutes on a plate washing machine;

(3) adding the diluent B into a biotinylated detection antibody and HRP-streptavidin, uniformly mixing, and adding into a microporous plate for incubation for 40 minutes;

(4) and washing again, adding a substrate for reaction for 10 minutes, adding a stop solution for color development, reading on an enzyme label plate, calculating a standard curve according to the reading to obtain a linear relation between the reading and a standard substance, and substituting the OD value of the sample into a linear formula to obtain the content of the sample.

The whole process does not exceed 2 hours.

Comparative examples

As a control to example 4, a kit was set up with a purity of 95% of SEQ ID NO:1 recombinant protein Standard antigen Dry powder as IGFBP3 Standard, the remaining setup and procedure were identical to example 2. Serum samples from healthy group experimental volunteers and from liver cancer group experimental volunteers were taken and tested using the kits of example 4 and this comparative example. The results indicate that both kits can significantly distinguish between the healthy and liver cancer groups by the detection of β 2-microglobulin, IGFBP3 protein, GP73 protein, GDF15 protein, OPN protein and AFP protein, and specifically, the results of the detection of IGFBP3 protein are shown in fig. 8.

The invention takes IGFBP3 as a liver cancer marker, designs an antibody chip kit capable of accurately and quantitatively detecting IGFBP3, and provides a reliable and convenient method for the prediction, diagnosis, staging and monitoring of liver cancer. In addition to being a liver cancer marker, IGFBP3 can be optimized for the detection of tumors such as lung cancer, stomach cancer, esophageal cancer, osteosarcoma, pancreatic cancer, lymph cancer, colon cancer, breast cancer, prostate cancer, oral cancer, nasopharyngeal cancer, cervical cancer, leukemia, malignant melanoma, sarcoma, renal cancer, biliary cancer, and the like.

Finally, it should be noted that the above-mentioned embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the modifications and equivalents of the specific embodiments of the present invention can be made by those skilled in the art after reading the present specification, but these modifications and variations do not depart from the scope of the claims of the present application.

Sequence listing

<110> Guangzhou Riboao Biotechnology Ltd

Application of <120> IGFBP3 in preparation of kit for quantitatively detecting liver cancer marker

<130>

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 291

<212> PRT

<213> Artificial sequence

<400> 1

Met Gln Arg Ala Arg Pro Thr Leu Trp Ala Ala Ala Leu Thr Leu Leu

1 5 10 15

Val Leu Leu Arg Gly Pro Pro Val Ala Arg Ala Gly Ala Ser Ser Ala

20 25 30

Gly Leu Gly Pro Val Val Arg Cys Glu Pro Cys Asp Ala Arg Ala Leu

35 40 45

Ala Gln Cys Ala Pro Pro Pro Ala Val Cys Ala Glu Leu Val Arg Glu

50 55 60

Pro Gly Cys Gly Cys Cys Leu Thr Cys Ala Leu Ser Glu Gly Gln Pro

65 70 75 80

Cys Gly Ile Tyr Thr Glu Arg Cys Gly Ser Gly Leu Arg Cys Gln Pro

85 90 95

Ser Pro Asp Glu Ala Arg Pro Leu Gln Ala Leu Leu Asp Gly Arg Gly

100 105 110

Leu Cys Val Asn Ala Ser Ala Val Ser Arg Leu Arg Ala Tyr Leu Leu

115 120 125

Pro Ala Pro Pro Ala Pro Gly Asn Ala Ser Glu Ser Glu Glu Asp Arg

130 135 140

Ser Ala Gly Ser Val Glu Ser Pro Ser Val Ser Ser Thr His Arg Val

145 150 155 160

Ser Asp Pro Lys Phe His Pro Leu His Ser Lys Ile Ile Ile Ile Lys

165 170 175

Lys Gly His Ala Lys Asp Ser Gln Arg Tyr Lys Val Asp Tyr Glu Ser

180 185 190

Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser Glu Ser Lys Arg Glu Thr

195 200 205

Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu Asp Thr Leu Asn His Leu

210 215 220

Lys Phe Leu Asn Val Leu Ser Pro Arg Gly Val His Ile Pro Asn Cys

225 230 235 240

Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly

245 250 255

Arg Lys Arg Gly Phe Cys Trp Cys Val Asp Lys Tyr Gly Gln Pro Leu

260 265 270

Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp Val His Cys Tyr Ser Met

275 280 285

Gln Ser Lys

290

<210> 2

<211> 17

<212> PRT

<213> Artificial sequence

<400> 2

Lys Tyr Gly Gln Pro Leu Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp

1 5 10 15

Val

<210> 3

<211> 17

<212> PRT

<213> Artificial sequence

<400> 3

Lys Tyr Gly Gln Lys Leu Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp

1 5 10 15

Val

Claims (6)

1. The application of IGFBP3 in preparing a kit for quantitatively detecting liver cancer markers is characterized in that: the IGFBP3 is a polypeptide with a sequence of SEQ ID NO:3, and the sample suitable for detection by using the kit is human blood.

2. A kit for quantitatively detecting a liver cancer marker, comprising:

the reverse protein chip is used for coating a sample to be detected, and the sample to be detected is human blood;

a detection antibody which is a monoclonal antibody of biotinylated anti-IGFBP 3 or a genetically engineered antibody;

IGFBP3 standard, which is a polypeptide fragment with sequence SEQ ID NO. 3.

3. The kit for quantitatively detecting a marker for liver cancer according to claim 2, wherein: the purity of the polypeptide fragment is more than 95%.

4. The kit for quantitatively detecting a marker for liver cancer according to claim 3, wherein: the detection antibody is a monoclonal antibody of biotinylated anti-IGFBP 3, and the concentration is 0.1 mg/L.

5. The kit for quantitatively detecting a marker for liver cancer according to claim 2, wherein: the detection antibody is at least one of genetically engineered antibodies scFv, Fab 'and F (ab') 2.

6. The kit for quantitatively detecting a marker for liver cancer according to claim 2, wherein: the standard substance is coated on an ELISA plate made of polystyrene.

Images