WO2016127276A1

WO2016127276A1 - Application of urine alpha-fetoprotein

Info

Publication number: WO2016127276A1
Application number: PCT/CN2015/000624
Authority: WO
Inventors: 张曼
Original assignee: 张曼
Priority date: 2015-02-10
Filing date: 2015-09-01
Publication date: 2016-08-18
Also published as: CN105988005A

Abstract

An application of a urine alpha-fetoprotein (AFP), particularly an expression of the urine alpha-fetoprotein in urine and an application thereof to urine content detection. A research verifies that an alpha-fetoprotein in urine has a relatively high abundance expression, and therefore information about disease diagnosis is obtained by detecting the expression quantity of the alpha-fetoprotein in the urine. The advantages that a urine specimen is acquired noninvasively and is saved conveniently are played, and the alpha-fetoprotein is detected by using the urine specimen.

Description

Application of urine alpha-fetoprotein

Technical field

The invention relates to a new use of urinary alpha fetoprotein, in particular to the expression of alpha fetoprotein in urine and its application in the detection of urine content.

Background technique

Alpha-fetoprotein is the main component of embryonic plasma protein, and its gene encoding human albumin and vitamin D binding protein belongs to the albumin gene family, which are located on chromosome 4. It is produced by yolk sac and fetal liver. It is mainly composed of fetal liver synthesis after 12 weeks of gestation. The serum concentration during fetal period is very high, and it decreases after birth. From February to March, alpha-fetoprotein is basically replaced by albumin. More difficult to check out.

Alpha-fetoprotein has many important physiological functions, the most basic of which is the transport function. Alpha-fetoprotein can bind estrogen, fatty acid, bilirubin, Cu2+ and Ni2+. One of the most important functions is to transport fatty acids. Both fetal and embryonic alpha-fetoprotein can bind 2 to 3 fatty acid molecules. Part is unsaturated fatty acids. In addition, alpha-fetoprotein is involved in cell proliferation, regulation of metabolism, and interaction between macrophages and T lymphocytes. The role of alpha-fetoprotein in the immune response is immunosuppression, mainly manifested by inhibition of the maternal immune response to embryonic development and tumor patients' immune response to tumors. Alpha-fetoprotein can also be synthesized in hepatocellular carcinoma, embryonic tumors, and some extrahepatic tumors. Therefore, alpha-fetoprotein is associated with primary liver cancer, gastric cancer, lung cancer, pancreatic cancer, cholangiocarcinoma, and testicular tumor. It can also be elevated in patients with partial hepatitis (15% to 58%) and cirrhosis (11% to 47%).

Due to the special source of alpha-fetoprotein, it is difficult to detect it in the serum of normal adults. This only indicates that the serum concentration is too low, exceeding the minimum detection limit of the general test, so that it cannot be detected. However, our research has confirmed that a part of alpha-fetoprotein is found in the urine of normal people. Therefore, normal people can still synthesize a certain amount of alpha-fetoprotein, which plays a certain role in normal physiological metabolism, and can also be used in urine. A certain amount of a fragment of alpha-fetoprotein is stably present.

Urine is the ultrafiltrate of blood, which is the terminal metabolite produced by the body. The composition change is a specific manifestation of certain disease states, and the collection of urine specimens is non-invasive, multi-volume, patient compliance, and not The advantage of requiring the help of medical personnel, in summary, the present invention aims to apply the advantages of urine specimens, and provides a simple kit for detecting alpha-fetoprotein fragments in urine.

Summary of the invention

It is an object of the present invention to provide an expression of urine alpha-fetoprotein in urine and its use in the detection of urine content.

Preferably, the amino acid sequence of the urine alpha-fetoprotein is as shown in SEQ ID NO: 1:

Preferably, the preparation is a urine alpha-fetoprotein detection kit. The kit is an antibody antigen reaction.

Preferably, the antigen-antibody reaction is coated or labeled with a urine alpha-fetoprotein or polypeptide and an antibody thereof in a solid phase or liquid phase carrier.

The inventors first collected random urine specimens of normal physical examination, took the supernatant after centrifugation, and purified and separated urine specimens using weak cation exchange magnetic beads. 1 μl of sample with 10 μl of substrate (0.3% α-cyano-4- After mixing hydroxycinnamic acid and HCCA), 1 μl was spotted on an Anchorchip (Autoflex MALDI TOF, Bruker-Dalton) target plate, and the sample was ionized and subjected to mass spectrometry. Data in the range of 1000-10000 Da was collected to obtain different mass loads. A mass spectrometric peptide map composed of protein peaks. All mass spectra were analyzed using ClinProTools 2.1 analysis software to screen for highly expressed high-abundance protein peptides. The inventors then identified these screened protein peptides by liquid chromatography tandem mass spectrometry and searched for the alpha-fetoprotein (Alpha-fetoprotein) protein in the International Protein Index (IPI human v3.45fasta with 71983entries) database. .

The present invention confirmed by studies that alpha fetoprotein can stably appear in the urine of a person who is normally examined. Therefore, it is proposed to detect the application of urine protein alpha fetoprotein in urine related examination.

The invention exerts the advantage of non-invasiveness of urine specimens, and uses random urine specimens to detect alpha-fetoprotein or polypeptide.

The above and other objects, features, and advantages of the present invention will become more apparent and understood by the appended claims appended claims

DRAWINGS

Figure 1 is the average value of all points in the normal physical examination specimens between 1000 and 10000.

Figure 2 is a scatter plot of the point of mass-to-charge ratio of 1893.6 expressed in 30 normal medical specimens.

Figure 3 is a mass spectrum of alpha-fetoprotein.

detailed description

Example 1 Collection and processing of urine specimens

30 normal physical examinations (Beijing Metropolitan University Hospital, Beijing Medical University Hospital) were collected to randomly clean the middle urine samples, centrifuged within 1500 rpm (1500 rpm, 5 min), and the supernatant was retained. After storage, the refrigerator was stored at -80 °C.

Example 2 Purification and Separation of Peptides from Urine Specimens by Magnetic Beads

The urine sample was taken out from the -80 ° C refrigerator, recombined at 4 ° C, centrifuged (3000 rpm, 10 min), and the supernatant was taken for use. The weak cation magnetic beads (MB-WCX) were equilibrated at room temperature and the magnetic bead suspension was manually mixed. Add 10 ul of MB-WCX and 10 ul of magnetic bead binding buffer to the sample tube. Mix the sample gun up and down to avoid foaming. Add 5 ul of urine supernatant to the sample tube, mix well and let stand for 1 minute on the magnetic stand. The magnetic beads are separated from the suspended liquid. Use a sample gun to remove the suspended clear liquid. The tip should avoid contact with the magnetic beads and avoid picking up the magnetic beads. Add 100 ul of magnetic bead washing buffer to the sample tube, mix well, and then leave the sample tube on the magnetic stand for 1 minute. The magnetic beads are attached to the wall and separated from the suspended liquid. The suspended liquid is removed by a sample gun. Repeat 3 times and discard the suspension. Add 5 ul of magnetic bead elution buffer to the sample tube and repeatedly pipette 10 times or more to mix the magnetic beads and the elution buffer to avoid foaming. The sample tube was placed on a magnetic stand and allowed to stand for 2 min to separate the magnetic beads from the suspension. The supernatant (eluent) was transferred to a new 0.5 ml sample tube labeled. Add 5 ul of Stabilization Buffer and carefully pipette and mix.

Example 3 Generation of Point Targets and Peptide Spectra of Urine Specimens

After calibrating the instrument with the standard, 1 μl of the eluate was mixed with 10 μl of the substrate (0.3% α-cyano-4-hydroxycinnamic acid, HCCA), and 1 μl of the spot was taken at the Anchorchip (Autoflex MALDI TOF, Bruker-Dalton) target. Dry on the plate at room temperature. The sample was ionized by irradiation with a nitrogen laser, mass spectrometry was performed, and data in the range of 1000-10000 Da was collected to obtain a mass spectrum composed of protein peaks of different mass-to-charge ratios. For each MALDI crystallization point, a total of 400 lasers were irradiated (each of the 8 different positions of each crystallization point was irradiated 50 times), and the average value represents one specimen, thereby obtaining a polypeptide map of all samples. The ClinProTools2.1 analysis software was used to analyze the mass spectra of normal control group, type 2 diabetes without complications and comorbidity group and type 2 diabetes mellitus with early kidney injury group, and to screen differential polypeptides. Screening conditions: mass range 1000-10000 Da, signal to noise ratio (S/N) greater than 5, mass drift no more than 0.1%, all mass spectra normalized according to total ion current. Specifically, please refer to FIG. 1 , which shows the average value of all the mass-to-charge ratio points between 1000-10000 Da in 30 urine samples; the peak area as a quantitative standard, and FIG. 2 shows the expression of alpha fetoprotein in all urine samples. It can be seen from the figure that m/z 1893.6 has a peak area greater than 600 in all specimens.

Example 4 Identification of Protein Polypeptides

The magnetic bead eluate in the sample tube was evaporated to dryness, dissolved in 20 ul of mobile phase A (5% acetonitrile, 0.1% formic acid in water), and transferred to a sample bottle. The injection volume was 18 ul, firstly desalted into the trap column at a rate of 15 μl/min, and the capture time was 3 min. Then enter the analytical column at a flow rate of 400 nl / min for gradient elution, the elution gradient is 5% B-50% B-80% B-80% B-50% B-5% B (mobile phase B: 95% acetonitrile) , 0.1% aqueous solution of formic acid, see Table 1). The analysis time was 60 min, the column temperature was 35 ° C, and all eluted components were analyzed by mass spectrometry. Nano ion source, spray voltage 1.8kV; mass spectrometry mode for data dependence and dynamic exclusion, scan range 400-2000m/z; first-order scan (MS) using Obitrap, resolution set to 100000; CID and secondary scan using LTQ; A single isotope of the strongest 10 ions was selected as the parent ion in the MS spectrum for MS/MS (single charge exclusion, not as parent ion). The mass spectrometry scan time was 60 min. Sequest ^TM search was performed using the data analysis software Bioworks Browser 3.3.1 SP1. The search database is International Protein Index (IPI human v3.45 fasta with 71983entries). The mother ion error was set to 100 ppm, the fragment ion error was set to 1 Da, the digestion method was non-enzymatically cut, and the variable modification was methionine oxidation. The search result parameters are set to deltacn ≥ 0.10, two charges Xcorr 2.6, three charges Xcorr 3.1, and three charges above Xcorr 3.5. The protein alpha-fetoprotein is retrieved in the database, and the mass spectrum of the alpha-fetoprotein is shown in Figure 3.

Table 1 Procedure for analyzing column gradient elution

Example 5 Preparation of the kit

1. Selection of working concentration of coated antibody and enzyme labeled antibody

The antibody and antigen concentrations were determined according to Pierce's BCA Protein Concentration Kit instructions, and then the rabbit anti-human alpha-fetoprotein polyclonal antibody (Abcam) was diluted to a concentration using a standard checkerboard assay. 10.0 ng/ml, 1.0 ng/ml, and 0.1 ng/ml were coated on solid phase ELISA plates and liquid phase magnetic beads, respectively, each concentration consisting of three wales, overnight at 4 ° C, and washed 3 times. A strong positive antigen solution was added to one of the transverse coated wells, a weak positive antigen solution was added to the other row, and a negative control was added to the third row. Incubate for 2 hours at 37 ° C and wash 3 times. A murine anti-human alpha fetoprotein monoclonal antibody (Abeam) was added, incubated at 37 ° C for 1 hour, and washed 3 times. The labeled secondary antibody was added, incubated at 37 ° C for 30 minutes, washed 4 times, the substrate was added, and the mixture was allowed to stand at room temperature for 20 minutes in the dark, and the stop solution was added for reading. Choose the optimal concentration of coated antibody.

2. Preparation of the kit

The rabbit anti-human alpha-fetoprotein polyclonal antibody was diluted with a coating buffer, added to a solid phase microplate and liquid phase magnetic beads, and gently shaken overnight at 4 °C. The uncoated liquid was poured out, washed 3 times, and a blocking solution was added to prevent non-specific binding sites, incubated at 37 ° C for 1 hour, and washed 3 times. Store at 4 ° C for later use. The kit is divided into a mouse anti-human alpha fetoprotein monoclonal antibody, a labeled secondary antibody, and the like.

Example 7 Detection of sensitivity of the kit

The alpha-fetoprotein recombinant protein (OriGene, Germany) was diluted with PBS to 200 ng/ml, 100 ng/ml, 50 ng/ml, 25 ng/ml, 10 ng/ml, 2 ng/ml, 0.5 ng/ml, 0.05 ng/ml, 0.01. Ng/ml, 0 ng/ml, 100 ul per well was added to the above coated ELISA plate and the liquid magnetic beads, incubated at 37 ° C for 2 hours, and washed 3 times. The mouse anti-alphafetoprotein monoclonal antibody was diluted 1:2000, 100 ul was added per well, incubated at 37 ° C for 1 hour, and washed 3 times. The labeled secondary antibody was added, incubated at 37 ° C for 30 minutes, and washed 3 times. The substrate was added to room temperature for 15 minutes, and the stop solution was added for reading. The lowest amount of alpha-fetoprotein was detected. The results showed that the reagent could detect the concentration of 0.01 ng/ml alpha-fetoprotein, indicating a higher detection sensitivity. The above experiments show that the kit of the present invention detects the content of alpha-fetoprotein in the urine sample, and has high sensitivity. The minimum detection limit of the sample is 0.01 ng/ml, and the recovery rate is 90%±13%. The kit requires fewer instruments and requires only a microplate reader, an oscillator, a centrifuge, a pipette, etc., and the cost is low.

Example 8 Detection of specificity and stability of the kit

Take a normal medical examination (Beijing Shijitan Hospital affiliated to Capital Medical University) to measure 30-50ml of random urine in the middle of clean, put into a cleaned urinary catheter, women should avoid the menstrual period when taking urine specimens, and prevent vaginal secretions from mixing into the urine. Centrifuge at 1500 rpm for 5 minutes at room temperature, and take the supernatant for examination.

Purify and quantify the recombinant alpha-fetoprotein recombinant protein as a standard. Dilute the antigen (centrifuged urine sample) 1:3, add it to the previously coated enzyme plate, incubate at 37 °C for 2 hours, wash away The bound antigen is used to blot dry residual liquid. The mouse anti-human alpha fetoprotein monoclonal antibody was added to incubate for 1 hour at 37 ° C, the unbound antibody was washed away, and the residual liquid was blotted. The labeled secondary antibody was added, incubated at 37 ° C for 30 minutes, washed 4 times, and the residual liquid was blotted dry. Add chromogenic substrate, leave it at room temperature for 10 minutes, stop the reaction by adding stop solution, read the microplate reader, and calculate the content of alpha-fetoprotein in the sample.

By this method, the inventors examined the content of alpha-fetoprotein in 100 normal urine urine samples with an accuracy of more than 97%, and had good specificity.

Two random urines of normal physical examination were taken, and ELISA was performed by the above method. The measurement was performed once a day for 10 times. The coefficient of variation (CV)=S/X×100% (S is the standard deviation and X is the average). Value) Calculate the inter-batch and intra-assay coefficient of variation. Finally, the intra- and inter-assay coefficients of variation were 2.81% and 3.26%, respectively, indicating good stability.

While the invention has been described above in terms of a preferred embodiment, it is not intended to limit the invention, and those skilled in the art can make some modifications and improvements without departing from the spirit and scope of the invention. Therefore, the scope of the invention is defined by the claims.

Claims

The expression of alpha-fetoprotein and its polypeptide in urine and its application in the detection of urine content.
The use according to claim 1, wherein the amino acid sequence of the urine alpha-fetoprotein is as shown in SEQ ID NO: 1.
The use according to claim 1, wherein the preparation is a urine alpha-fetoprotein or polypeptide detection kit.
The use according to claim 3, wherein the kit is an antigen-antibody reaction.
The use according to claim 4, wherein the reaction is a urine alpha-fetoprotein or polypeptide and an antibody thereof is coated or labeled in a solid phase or a liquid phase carrier.