WO2016127278A1

WO2016127278A1 - Application of urine complement component 3 (c3) protein

Info

Publication number: WO2016127278A1
Application number: PCT/CN2015/000626
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: CN105988006A

Abstract

An application of a urine complement component 3 (C3) protein to noninvasive detection of urine. Complement component 3 (C3) protein segments can be stably expressed in urine of a normal person. The advantage of acquiring a urine specimen noninvasively is played, the urine complement component (C3) protein is detected by using the urine specimen, the bottleneck problem of urinalysis existing all the time, namely how to eliminate urine volume influences in urine protein/polypeptide detection, is solved, the defects that creatinine serves as an internal reference for related detection of urine are overcome, and a simpler and more convenient detection method is provided for disease diagnosis.

Description

Application of urine complement C3 protein

Technical field

The invention relates to a new use of urine complement C3 protein, in particular to the normal expression of urine complement C3 protein as an internal reference of a urine test method, and abnormal expression as a biomarker for disease diagnosis.

Background technique

Currently, the most widely used type of specimen in clinical routine testing is blood specimens. However, specimens with invasive, lipemia or hemolysis in blood specimens are not detectable for certain items, and collection equipment is often a weapon. This requires not only well-trained and experienced blood collectors, but also blood products themselves that increase occupational exposure. risk. On the other hand, at least 8 hours on an empty stomach increases the risk of hypoglycemia in patients such as pregnant women, the elderly and children. For patients who need frequent and even life-long blood collection, such as diabetes, blood diseases, cancer chemotherapy, etc., it also brings great pain to patients.

Urine is the ultrafiltrate of blood. It is the terminal metabolite produced by glomerular filtration, renal tubular and collecting tube reabsorption, excretion and secretion. It is a window reflecting the changes of the body. Its composition is changed by certain diseases. The specific manifestation of the state, and the collection of urine specimens is simple, non-invasive, multi-volume, repeatable multiple times, convenient to store, high patient compliance, and does not require the help of medical personnel. Compared with blood, urine protein is relatively simple and easy to analyze, and has great advantages in the diagnosis of diseases by body fluids. The composition, quantity and trait changes of urine not only carry various information about the occurrence, development and prognosis of urinary system diseases, but also reflect the overall metabolic state of the body, for studying organ function, evaluating body state, metabolism, and dynamic monitoring. Various information is provided on aspects such as disease progression and judgment of efficacy. In addition to containing a large amount of protein, urine also contains a greater number of polypeptide fragments. Proteins and peptides with a molecular weight of less than 10 kDa can be freely filtered through the glomerulus. The polypeptide fragments in the urine are likely to be products of proteolysis. Analysis of urine may be more suitable for diagnosing systemic diseases and revealing the pathophysiological characteristics of the disease. It is therefore an ideal material for diagnosing and judging disease progression.

Complement C3 is the core protein in the complement system and is mainly produced by hepatocytes and macrophages. The gene encoding complement C3 is located on chromosome 19 and consists of 41 exons. The full length of the cDNA coding sequence is about 5052 bp, which in turn encodes the leader peptide, beta chain and alpha chain. During the synthesis of complement C3, when the single-stranded precursor is transferred to the endoplasmic reticulum, the 22 amino acid guide peptide is cleaved, and the strongly basic linker peptide is discarded, and the cells are secreted in the form of two chains. The complement C3 mature protein contains 1663 amino acids with a relative molecular weight of 180 kDa. In Among the various components of complement, C3 has the highest serum content; in terms of function, C3 is also at the center, which is the intersection of several activation pathways and the basis of C3b-dependent positive feedback loop; meanwhile, C3 is activated and lysed. After that, the series of fragments and their binding proteins produced are complex and diverse, and play an important role in immune defense, immune regulation and immunopathology. In addition, the non-complement function of C3 associated with cell proliferation and differentiation has also been reported, such as the proliferation and establishment of bud base and lens vesicles, involved in the proliferation and growth of B cells in vitro, which may be attributed to the complement component in mitogenic events. Role, C3 may also play a role in early dedifferentiation and even in later differentiation. With the deepening of research, C3 is thought to play a role in tumor formation, signal transduction, and apoptosis. At the same time, it can protect against oxidative stress.

However, the amount of urine is easily affected by the amount of drinking water and some other physiological factors. Therefore, the concentration of markers related to urine diseases depends not only on the excretion rate of the marker but also on the urine flow. 24h urine is the gold standard for assessing urine markers, but the collection of such specimens is cumbersome and the patient's compliance is poor. At present, the effects of creatinine, urinary permeation or urine specific gravity on the calculation of urine volume are used in the study. The most widely used is urinary creatinine, but these methods are not only affected by age, sex, body fatness, mental state, physiological state, disease state and The impact of diet, etc., and the lack of consistency in different reports and limited cross-over studies, the best way to explore corrected urine output is to address the key to quantitative detection of proteins and peptides in urine.

In order to apply urine testing to clinical work, the present invention first screens for high abundance protein polypeptides that are stably present in urine. The urine protein polypeptide can be stabilized at a certain level when the body is in a normal physiological state, and an increase or decrease in its level is associated with certain diseases.

Summary of the invention

It is an object of the present invention to provide a use of a urine complement C3 protein (Complement Component 3) for the preparation of a urine-related examination.

Preferably, the amino acid sequence of the urine complement C3 protein is as shown in SEQ ID NO: 1:

Preferably, the preparation is a urine complement C3 protein detection kit. The kit is an antibody antigen reaction.

Preferably, the antigen-antibody reaction is coated or labeled with a urine complement C3 protein 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. After mixing 1 μl of the sample with 10 μl of the substrate (0.3% α-cyano-4-hydroxycinnamic acid, HCCA), 1 μl of the spot was taken on an Anchorchip (Autoflex MALDI TOF, Bruker-Dalton) target plate, and the sample was ionized. Mass spectrometry analysis was performed to acquire data in the range of 1000-10000 Da, and a mass spectrometric peptide map composed of protein peaks of different mass-to-charge ratios was obtained. 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 polypeptides by liquid chromatography tandem mass spectrometry and searched for the complement C3 (Complement Component 3) protein in the International Protein Index (IPI human v3.45fasta with 71983entries) database.

The present invention confirmed by studies that the complement C3 protein can stably appear in the urine of a person who is normally examined. Therefore, it is proposed to detect the use of urine complement C3 protein for urine related examination.

The invention exerts the advantage of obtaining non-invasive urine specimens, and uses the random urine specimen to detect the complement C3 protein 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 1726.8 expressed in 30 normal medical specimens.

Figure 3 is a mass spectrum of the complement C3 protein.

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 complement C3 in all urine samples. It can be seen from the figure that m/z 1726.8 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 the International Protein Index (IPI human v3.45fasta 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 complement C3 protein was searched in the database, and the mass spectrum of the complement C3 protein is shown in Fig. 3.

Table 1 Procedure for analytical 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 complement C3 protein 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. Murine anti-human complement C3 monoclonal antibody (Abcam) 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 complement C3 protein 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 complement C3 monoclonal antibody, a labeled secondary antibody, and the like.

Example 7 Detection of sensitivity of the kit

Complement C3 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-complement C3 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 complement C3 was detected. The results showed that the reagent could detect the concentration of 0.01 ng/ml complement C3 protein, indicating a higher detection sensitivity. The above experiments show that the kit of the present invention detects the content of complement C3 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.

Purification of the quantitative complement C3 recombinant protein as a standard, the antigen (centrifuged urine sample) was diluted 1:3, added to the previously coated enzyme plate, incubated at 37 ° C for 2 hours, washed away unbound The antigen, blotting the residual liquid. The mouse anti-human complement C3 protein monoclonal antibody was added and incubated at 37 ° C for 1 hour, 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 the chromogenic substrate, leave it at room temperature for 10 minutes, stop the reaction by adding a stop solution, and read the microplate reader to calculate the content of complement C3 protein in the sample.

By this method, the inventors examined the content of complement C3 protein 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, X is flat). Mean) 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 use of urine complement C3 protein or polypeptide for the preparation of non-invasive detection of urine.
The use according to claim 1, wherein the amino acid sequence of the urine complement C3 protein or polypeptide is as shown in SEQ ID NO: 1.
The use according to claim 1, wherein the preparation is a urine complement C3 protein 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 complement C3 protein or polypeptide and an antibody thereof is coated or labeled in a solid phase or a liquid phase carrier.