CN113735958A - CHI3L1 antigen, antibody and kit for predicting internal arteriovenous fistula loss of self body - Google Patents
CHI3L1 antigen, antibody and kit for predicting internal arteriovenous fistula loss of self body Download PDFInfo
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Abstract
The invention discloses a CHI3L1 epitope peptide, a CHI3L1 antigen prepared by using the CHI3L1 epitope peptide, an antibody prepared by using a corresponding antigen, and an in-vitro test box with specificity based on the CHI3L1 antigen antibody, which can quickly and simply predict the loss of work of an autokinetic-venous fistula (AVF); the kit mainly comprises a CHI3L1 microporous enzyme label plate, a CHI3L1 antibody micropore enzyme label plate, a CHI3L1 calibrator and a quality control product, a CHI3L1 detection antibody, avidin-labeled Horse Radish Peroxidase (HRP), substrate solutions A and B and stop solution; it predicts early failure of autologous arteriovenous fistula by detecting the concentration of chitinase 3-like protein 1(CHI3L1) in the serum of ERSD patients.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a CHI3L1 antigen, an antibody and a kit for predicting the internal arteriovenous fistula loss of an autologous body.
Background
ESRD (renal disease) imposes a heavy burden on the whole society, families and individuals. The data show that the total number of uremia patients in China is continuously increased in recent years, and the number of new onset patients and total dialysis patients in China in 2011 is 15.4 and 237.3 people respectively per million population. Taking the sea above as an example, new uremic patients have risen from 89.4 per million population in 2007 to 128.1 per million population in 2014, and total dialysis patients have risen from 409.8 to 898 per million population. The national hemodialysis registration system (CNDRS) showed that by 2018, about 58 million patients are on national hemodialysis and about 9.5 million patients are on peritoneal dialysis. Because of the limited source and number of kidney transplant donors, hemodialysis is currently a key treatment to remove uremic toxins, correct complications, alter quality of life, and prolong the life of uremic patients.
Autologous arteriovenous fistulas (AVFs) have the advantages of long service life, low infection rate, few complications and the like compared to other vascular access (intra-graft fistulas, subcutaneous tunnel catheters with cuff), are the first dialysis access for hemodialysis patients, and are called "life lines" for hemodialysis patients. However, loss of work of AVF severely limits its clinical utility, and recent studies have shown that AVF1 has primary and secondary patency rates of 64% and 79%, respectively, with about 21% of AVF being discarded after establishment and not used. With the age of dialysis, the loss of AVF is always a problem that disturbs the expansion of hemodialysis patients and medical staff.
At present, no product for simply and conveniently predicting early dysfunction of the internal arteriovenous fistula exists in clinic, and studies prove that the level of a post-dysfunction serous inflammatory marker CHI3L1 of AVF is increased in ESRD patients. In addition, no product for simply and conveniently predicting early failure of the internal arteriovenous fistula in the body exists clinically. Therefore, the invention provides the CHI3L1 antigen, the antibody and a kit for predicting the loss of function of the autoarteriovenous fistula.
Disclosure of Invention
The invention aims to provide a CHI3L1 epitope peptide, a CHI3L1 antigen prepared from the CHI3L1 epitope peptide, an antibody prepared from a corresponding antigen, and a kit for specifically predicting the loss of work of an autologous arteriovenous internal fistula based on the CHI3L1 antigen antibody, wherein the loss of work of the autologous arteriovenous internal fistula (AVF) can be rapidly and simply predicted.
The invention discloses a CHI3L1 epitope peptide, which comprises a CHI3L1 epitope peptide I and a CHI3L1 epitope peptide II,
the amino acid chain fragment I of the CHI3L1 epitope peptide I is as follows:
Cys-Cys-Cys-Thr-Cys-Thr-Cys-Ala-Cys-Cys-Ala，
the amino acid chain fragment II of the CHI3L1 epitope peptide II is as follows:
Ala-Thr-Gln-Cys-Cys-Ala-Thr-Cys-Ala-Ala。
the CHI3L1 epitope peptide is prepared by synthesizing the CHI3L1 epitope peptide I by the amino acid chain fragment I through a solid phase method and synthesizing the CHI3L1 epitope peptide II by the amino acid chain fragment II through a solid phase method.
A CHI3L1 antigen comprising: the CHI3L1 antigen I is prepared by coupling the CHI3L1 antigen epitope peptide I and carrier protein KLH; CHI3L1 antigen II was prepared by coupling the epitope peptide II of CHI3L1 described above with the carrier protein KLH.
A CHI3L1 antibody comprising: monoclonal antibody I prepared from CHI3L1 antigen I described above and monoclonal antibody II prepared from CHI3L1 antigen II described above.
A kit for predicting the loss of function of an autologous arteriovenous internal fistula comprises the monoclonal antibody I and the monoclonal antibody II.
A kit for predicting autologous arteriovenous internal fistula loss of work comprises a CHI3L1 antibody micropore ELISA plate prepared from a monoclonal antibody I or a monoclonal antibody II, wherein the preparation method of the CHI3L1 antibody micropore ELISA plate comprises the following steps:
firstly, selecting any one of a monoclonal antibody I or a monoclonal antibody II, and preparing the monoclonal antibody I or the monoclonal antibody II into a solution A with the concentration of 0.5-1.5 ng/ml by using a PBS (phosphate buffer solution) with the concentration of 1XPH being 7.4;
dripping the solution A into a micropore enzyme label plate, and placing the micropore enzyme label plate in a 37 ℃ incubator for culturing for 1.5-2 h;
and then taking out, blocking with 2% BSA, taking out after blocking, washing with PBS buffer solution with 1XPH being 7.4, and drying for 0.5-1 h for later use.
A kit for predicting autologous arteriovenous internal fistula loss of work further comprises a CHI3L1 detection antibody prepared by a monoclonal antibody I or a monoclonal antibody II, wherein the preparation method of the CHI3L1 detection antibody comprises the following steps:
firstly, selecting one of a monoclonal antibody I or a monoclonal antibody II, ensuring that the selected antibody is different from a CHI3L1 antibody selected when the CHI3L1 antibody micropore ELISA plate is prepared, then diluting the selected antibody, coupling the diluted antibody with biotin, mixing and rotating for 2 hours;
then pouring the coupled solution into a dialysis bag, dialyzing in PBS buffer solution with the concentration of 1XPH being 7.4 at the constant temperature of 4 ℃ for 24h, changing the dialyzate for 4 times, and collecting the solution in the dialysis bag to obtain the high-concentration CHI3L1 detection antibody;
finally preparing a detection antibody preservative solution; and then diluting the obtained high-concentration detection antibody with a detection antibody preservation solution to prepare a solution of 0.2-1.0 ng/ml for later use.
Preferably, the volume of the biotin is 26 ul-60 ul, and the concentration of the biotin is 1 umol/L-10 mmol/L;
the volume of the diluted antibody coupled with biotin is 1ml, and the concentration of the diluted antibody is 150 ug/ml-2 mg/ml;
the formula of the preservation solution for detecting the antibody is as follows: 2.2-5.4 g of NaH2PO422.15-31.08 g of Na2HPO4·12H2O, 3-10 g of casein, 0.15-0.35 g of tween20 and 1L of distilled water;
a kit for predicting the loss of work of an autologous arteriovenous internal fistula further comprises a CHI3L1 calibrator, a quality control material, an avidin-labeled HRP, a substrate solution A, a substrate solution B and a stop solution;
diluting the avidin-labeled HRP by using an enzyme-labeled preservation solution; the formula of the enzyme-labeled preservation solution is as follows: 1-1.36 mol/L of glycerol, 8.5-10 mmol/L of sodium phosphate, 26-30 umol/L of bovine serum albumin, and 14-20 umol/L of cytochrome C;
the formula of the substrate liquid A is as follows: 10.3-13.6 g of sodium acetate, 1.2-1.6 g of citric acid, 0.28-0.35 ml of 30% hydrogen peroxide and 1L of distilled water;
the formula of the substrate liquid B is as follows: 0.15-0.24 g of disodium ethylene diamine tetraacetate, 0.14-0.18 g of citric acid, 36-52 ml of glycerol, 0.12-0.16 g of TMB and 1L of distilled water;
the stop solution is sulfuric acid, and the concentration of the sulfuric acid is 1.6-2 mol/L.
A kit for predicting the loss of work of an autologous arteriovenous internal fistula is used for an in-vitro test for predicting the early loss of work of the autologous arteriovenous internal fistula.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
1. the kit for predicting the dysfunction of the autoarteriovenous internal fistula based on the original CHI3L1 antigen and antibody provided by the technical scheme of the invention provides a reliable in-vitro test method for predicting the early dysfunction of the autoarteriovenous internal fistula for clinic, and the kit is simple and convenient to operate and does not need complex instruments for cooperation, so that the kit can be widely popularized and used.
2. The CHI3L1 epitope peptide provided by the technical scheme of the invention has immunogenicity and immunoreactivity after being connected with a carrier protein, and can generate a specific antibody combined with CHI3L 1.
Drawings
Fig. 1 is a flow chart of a preparation process of a kit for predicting the loss of work of an autologous arteriovenous internal fistula in the technical scheme;
FIG. 2 is a schematic diagram of Fmoc synthesis of solid phase peptides according to the invention;
FIG. 3 is a schematic diagram of the coupling of a monoclonal antibody of the present invention to biotin;
FIG. 4 is a line graph of the kit of the present invention;
FIG. 5 is a graph showing the correlation between the results of the kit of the present invention and the Hangzhou Puqiao kit.
Detailed Description
In order to facilitate the understanding of the technical solutions of the present invention for those skilled in the art, the technical solutions of the present invention will be further described with reference to the drawings attached to the specification.
The invention provides a CHI3L1 epitope peptide
The CHI3L1 protein, chitinase 3-like protein 1, described in the present invention is known in the art, and the amino acid sequence thereof is known in the art and can be found in the national center for biological information or professional databases such as NCBI.
Through a large amount of theoretical researches and experimental groping, the inventor finally screens two CHI3L1 epitope peptides I and CHI3L1 epitope peptides II with good antigenicity. The molecular weights are 1254.55 and 1104.24, respectively, and the amino acid chain fragments are as follows:
Cys-Cys-Cys-Thr-Cys-Thr-Cys-Ala-Cys-Cys-Ala；
Ala-Thr-Gln-Cys-Cys-Ala-Thr-Cys-Ala-Ala。
namely, the CHI3L1 epitope peptide comprises CHI3L1 epitope peptide I and CHI3L1 epitope peptide II,
the amino acid chain fragment I of the CHI3L1 epitope peptide I is as follows:
Cys-Cys-Cys-Thr-Cys-Thr-Cys-Ala-Cys-Cys-Ala。
the amino acid chain fragment II of the CHI3L1 epitope peptide II is as follows:
Ala-Thr-Gln-Cys-Cys-Ala-Thr-Cys-Ala-Ala。
the research of the invention finds that the epitope peptide I and the epitope peptide II have immunogenicity and immunoreactivity after being connected with carrier protein, and can generate specific antibodies combined with CHI3L 1.
The amino acid chain fragments are respectively synthesized into epitope peptide. Namely, the CHI3L1 epitope peptide comprises the CHI3L1 epitope peptide I synthesized by an amino acid chain fragment I through a solid phase method and the CHI3L1 epitope peptide II synthesized by an amino acid chain fragment II through a solid phase method.
The solid phase method is a solid phase peptide synthesis method, and the main idea of the solid phase peptide synthesis method is as follows: the carboxyl group of the carboxyl terminal amino acid of the peptide chain to be synthesized is firstly connected with an insoluble macromolecular compound (resin) in a covalent bond mode, then the amino acid combined on the solid phase carrier is taken as an amino component, and the peptide chain is lengthened by removing the amino protecting group and reacting with an excessive activated carboxyl component. Repeating the above operations until the desired length of the synthesized peptide chain is reached.
FIG. 2 shows a schematic diagram of epitope peptide synthesis by solid phase peptide synthesis (schematic diagram of solid phase peptide synthesis by Fmoc method) according to the present invention. The method mainly comprises the following steps: step (1) activation of the Fmoc-protected amino acid (HOBt/DCC method) → step (2) attachment of the amino acid to the resin → step (3) removal of the Fmoc protecting group of the amino acid → step (4) activation of another amino acid → step (5) coupling.
The specific process is as follows:
fmoc protected amino acids
Step (1): activation of amino acid (HOBt/DCC method)
Step (2): attaching amino acids to resins
And (3): fmoc protecting group for removing amino acid
And (4): activation of another amino acid
And (5): coupling of
In the above-mentioned procedure or as shown in FIG. 2, the steps (3) and (5) were repeated several times until 142mg of the peptide resin of CHI3L1 epitope peptide I and 158mg of the peptide resin of CHI3L1 epitope peptide II were obtained.
Then, the two peptide resins were cleaved separately, in the following procedure: cutting the peptide chain by TFA (trifluoroacetic acid), reacting for 4.0h at room temperature by using EDT (dimercaptoethane, 3.0% volume fraction) and thioanisole (2.0% volume fraction) as a scavenging agent, removing the cutting reagent, and extracting by using ether to respectively obtain crude products of the CHI3L1 epitope peptide I and the CHI3L1 epitope peptide II.
Finally, crude products of CHI3L1 epitope peptide I and CHI3L1 epitope peptide II are purified.
CHI3L1 epitope peptide I and CHI3L1 epitope peptide II were purified by the existing technique and identified by the existing technique.
Purifying the obtained CHI3L1 epitope peptide I and CHI3L1 epitope peptide II in corresponding steps, and identifying to obtain synthetic peptide as the target product.
Secondly, the invention provides a CHI3L1 antigen
The CHI3L1 antigen comprises the following components: the CHI3L1 antigen I is prepared by coupling the CHI3L1 antigen epitope peptide I with carrier protein KLH (keyhole limpet hemocyanin); CHI3L1 antigen II was prepared by coupling the above-described CHI3L1 epitope peptide II with the carrier protein KLH (keyhole limpet hemocyanin).
The CHI3L1 antigen I and the CHI3L1 antigen II have immunogenicity and immunoreactivity, and can generate specific antibodies combined with the CHI3L 1.
Thirdly, the invention provides a CHI3L1 antibody
A CHI3L1 antibody of the invention, comprising: monoclonal antibody I prepared from CHI3L1 antigen I described above and monoclonal antibody II prepared from CHI3L1 antigen II described above.
Fourthly, the invention provides a CHI3L1 antigen kit
The CHI3L1 antigen kit, namely the kit for predicting the dysfunction of the autologous arteriovenous internal fistula, comprises the monoclonal antibody I and the monoclonal antibody II.
Based on the technical scheme of the CHI3L1 antigen kit, specific examples of the CHI3L1 antigen kit are provided below.
A kit for predicting the loss of work of autologous arteriovenous internal fistula is a kit of CHI3L1 antigen prepared based on CHI3L1 epitope peptide and antibody prepared by corresponding antigen (CHI3L1 antigen kit for short). The test kit is provided with a CHI3L1 antibody micropore ELISA plate, a CHI3L1 calibrator and quality control material, a CHI3L1 detection antibody, avidin labeled horse radish peroxidase, a substrate solution A, a substrate solution B and a stop solution.
The principle of the CHI3L1 antigen kit is as follows: coating an antibody prepared by a monoclonal antibody I or a monoclonal antibody II on a microporous enzyme label plate to form a solid-phase antibody, sequentially adding a proper amount of a sample to be detected and a CHI3L1 detection antibody into the microporous plate to form a solid-phase antibody-antigen-detection antibody complex, washing the enzyme label plate, then adding avidin to mark HRP, finally forming the solid-phase antibody-antigen-detection antibody-avidin to mark HRP complex, completely washing the enzyme label plate, sequentially adding substrates A and B and a stop solution, and then measuring the OD value in the microporous plate by using an enzyme label instrument at the wavelength of 450nm to obtain the antigen content of CHI3L1 in the sample.
As shown in fig. 1, the preparation method of the CHI3L1 antigen kit is shown in the flow chart, the CHI3L1 antigen kit contains a CHI3L1 antibody microplate, a CHI3L1 calibrator and quality control material, a CHI3L1 detection antibody, avidin-labeled horseradish peroxidase, a substrate solution a, a substrate solution B and a stop solution, and the CHI3L1 antibody microplate, the CHI3L1 detection antibody, the avidin-labeled horseradish peroxidase, the substrate solution a, the substrate solution B and the stop solution are respectively prepared, and the preparation steps are described in detail later. CHI3L1 calibrator and quality control were purchased directly from the manufacturer. After all parts are prepared, all parts are detected to be qualified and then combined, accommodated and placed in a reagent box.
CHI3L1 antibody micropore enzyme label plate
The CHI3L1 antibody micropore ELISA plate is prepared by monoclonal antibody I or monoclonal antibody II. The preparation method of the CHI3L1 antibody micropore ELISA plate comprises the following steps:
first, either monoclonal antibody i or monoclonal antibody ii was selected and prepared into 0.5 to 1.5ng/ml solution a using 1 XPH-7.4 PBS buffer (phosphate buffered saline).
And dripping the solution A into a micropore enzyme label plate, and then placing the micropore enzyme label plate in a 37 ℃ incubator for culturing for 1.5-2 h.
And then, the sample was taken out, blocked with 2% BSA (bovine serum albumin), and taken out after blocking, washed with 1 XPH-7.4 PBS buffer (phosphate buffered saline), and then dried for 0.5 to 1h for use.
(II) CHI3L1 detection antibody
The CHI3L1 detection antibody was prepared from monoclonal antibody I or monoclonal antibody II. It is noted that the detection antibody prepared by CHI3L1 is different from the monoclonal antibody prepared by CHI3L1 antibody microporous enzyme label plate. When the coating (CHI3L1 antibody microplate) antibody was derived from one of monoclonal antibody I or monoclonal antibody II, the antibody for preparing the detection antibody of CHI3L1 was derived from the other of monoclonal antibody I or monoclonal antibody II.
The preparation method of the CHI3L1 detection antibody comprises the following steps:
firstly, diluting the selected antibody (monoclonal antibody I or monoclonal antibody II) to the concentration of 150 ug/ml-2 mg/ml, coupling 1ml of the diluted antibody with 26 ul-60 ul of biotin with the concentration of 1 umol/L-10 mmol/L, mixing and rotating for 2 h.
Then the solution after coupling was poured into a dialysis bag, and dialyzed in PBS buffer solution of 1XPH ═ 7.4 at a constant temperature of 4 ℃ for 24 hours, during which time the dialysate was changed 4 times, and then the solution in the dialysis bag was collected, to obtain a high concentration of CHI3L1 detection antibody.
Finally preparing a detection antibody preservative solution; the formula of the preservation solution for detecting the antibody is as follows: 2.2-5.4 g of NaH2PO422.15-31.08 g of Na2HPO4·12H2O, 3-10 g of casein, 0.15-0.35 g of tween20 (Jushan mountain)Pyrister), distilled water 1L. And diluting the obtained high-concentration detection antibody with a detection antibody preservation solution to prepare a solution of 0.2-1.0 ng/ml for later use.
(III) avidin-labeled HRP
The avidin-labeled HRP in the kit was purchased from Biotechnology, Inc. at a concentration of 500U/ml. When in use, the enzyme-labeled preservation solution is diluted by a certain amount of enzyme-labeled preservation solution, and the dilution ratio is 1: 10000-1: 30000. The formula of the enzyme-labeled preservation solution is as follows: 1-1.36 mol/L of glycerol, 8.5-10 mmol/L of sodium phosphate, 26-30 umol/L of bovine serum albumin, and 14-20 umol/L of cytochrome C.
CHI3L1 calibrator and quality control product
And (3) directly purchasing the CHI3L1 calibrator and the quality control product as standard products.
(V) substrate solution A, substrate solution B and stop solution
The formula of the substrate liquid A is as follows: 10.3-13.6 g of sodium acetate, 1.2-1.6 g of citric acid, 0.28-0.35 ml of 30% hydrogen peroxide and 1L of distilled water.
The formula of the substrate liquid B is as follows: 0.15-0.24 g of disodium ethylene diamine tetraacetate, 0.14-0.18 g of citric acid, 36-52 ml of glycerin, 0.12-0.16 g of TMB and 1L of distilled water.
The stop solution is sulfuric acid, and the concentration of the sulfuric acid is 1.6-2 mol/L.
The CHI3L1 antigen kit provided by the technical scheme of the invention is used for in-vitro test for predicting early failure of autologous arteriovenous internal fistula. It predicts early failure of autologous arteriovenous fistula by detecting the concentration of chitinase 3-like protein 1(CHI3L1) in the serum of ERSD patients. When the serum of a patient is detected, a sample to be detected of the patient is only required to be added into the micropore, then the detection antibody is added, then after incubation, the avidin-labeled horse radish peroxidase is added, secondary incubation is carried out, and finally, the substrate solutions A and B and the stop solution are added for detection. The kit has the advantages of simple and convenient operation and capability of providing a reliable method for predicting the early failure of the arteriovenous internal fistula for clinic.
In order to verify the quality of the CHI3L1 antigen kit provided by the technical scheme of the invention, various performances of the kit are detected, and the main performances of the kit comprise: the kit comprises determination of detection critical value, relative sensitivity, relative specificity, coincidence rate, linear range, accuracy, repeatability, stability and batch-to-batch difference, and the detection method of each performance of the kit correlation is as follows.
1. Determination of a threshold value
50 negative sera were tested by the established ELISA method and the detailed test results are shown in tables 1.1-1.
Tables 1.1-1: ELISA test results of 50 negative sera
Determining the critical value of negative and positive according to the statistical principle
That is, the detection value of the sample to be detected is not less than 0.2943 and is positive, the detection value of the sample to be detected is not more than 0.2590 and is negative, and the sample to be detected is suspicious between the two.
2. Relative sensitivity, relative specificity and total coincidence rate
The established ELISA detection method is used for detecting 50 serum samples with different antibody titers, and compared with the detection result of CHI3L1ELISA kit of Hangzhou prestige biotechnology limited (in the process of kit development, the developed kit needs to be compared with the existing kit on the market so as to obtain the effectiveness of the developed kit as an important item in kit detection), the relative sensitivity, the relative specificity and the total coincidence rate of the method are analyzed, and the calculation formulas are as follows (2.1), (2.2) and (2.3).
Relative sensitivity (%) { positive number/(positive number + false negative number) } × 100% … … … (2.1)
Relative specificity (%) { negative number/(negative number + false positive number) } × 100% … … … (2.2)
Percent (%) coincidence { (number of positive + number of negative)/total number of detections } × 100% … … … … … (2.3)
50 serum samples with different antibody titers are detected by the established ELISA method, the detection result is positive 28 parts and negative 22 parts, the detection result of CHI3L1ELISA kit of Hangzhou Puqiang biotechnology limited company is positive 29 parts and negative 21 parts, the specific data are shown in a table 2.1-1, and the relative sensitivity, the relative specificity and the total coincidence rate are respectively as follows: 96.54 percent, 91.67 percent and 94 percent, and meets the requirements of the kit.
Tables 2.1-1: results of 50 serum tests
3. Linear range
Diluting a high-value sample with the concentration of CHI3L1 protein of 1.2ng/mL into 5 concentrations according to a multiple ratio, repeatedly detecting each concentration sample for 3 times, calculating the average value of the concentration, performing straight line fitting on the result average value and the dilution ratio, and calculating a linear correlation coefficient r, wherein the result is not lower than 0.9950, the detection result is shown in tables 3.1-1 and a linear curve graph 4, and the kit is known to be linear and meet the requirements according to the information of the graph.
Tables 3.1-1: linear form
Correlation of theoretical values with measured values: r is 0.9998
4. Accuracy of
Different concentrations of CHI3L1 protein are respectively added into a blank distilled water sample, the adding recovery rate of the kit is measured according to the operation steps of the kit, the measurement result is shown in table 4.1-1, and as can be seen from table 4.1-1, the recovery rate of the CHI3L1 protein double antibody sandwich method kit is 86.11-104.61%, the variation coefficient is 3.98-7.21%, and is less than 15%, which indicates that the kit has high accuracy.
Table 4.1-1: determination result of recovery rate of CHI3L1 protein double-antibody sandwich method kit
5. Repeatability of
5.1 in-batch repeatability
30 parts of known background serum are respectively detected by using CHI3L1ELISA detection kits of the same batch, which are manufactured by a laboratory, on days 1, 2, 3, 5 and 7, wherein 10 parts of negative serum, 10 parts of strong positive serum and 10 parts of weak positive serum are respectively detected, each part of serum is subjected to 3 repetitions, a standard deviation and a coefficient of variation are calculated, the batch repeatability is analyzed, the determination results are shown in tables 5.1-1, 5.1-2 and 5.1-3, and the table results show that the coefficient of variation of 30 parts of serum is less than 15 percent, which indicates that the batch repeatability of the kit of the same batch is better.
Tables 5.1-1: in-batch repeatability test results
Tables 5.1-2: in-batch repeatability test results
Tables 5.1-3: in-batch repeatability test results
5.2 repeatability between batches
30 parts of known background serum are respectively detected by using CHI3L1ELISA detection kits manufactured in laboratories of 3 different batches (the batch numbers are 20200307, 20200415 and 20200416 respectively), wherein 10 parts of negative serum, 10 parts of strong positive serum and 10 parts of weak positive serum are repeated for 3 times, the results are shown in tables 5.2-1, 5.2-2 and 5.2-3, and the table results show that the variation coefficients of the 30 parts of serum are less than 15%, which indicates that the batch repeatability of the kits of 3 batches is better.
Table 5.2-1: results of batch-to-batch repeatability tests
TABLE 5.2-2 results of the inter-batch reproducibility measurements
TABLE 5.2-3 results of the inter-batch reproducibility measurements
6. Stability test
3 different batches of CHI3L1ELISA detection kits (batch numbers are 20200307, 20200415 and 20200416 respectively) are stored at 2-8 ℃, are taken out at 0 month, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months and 12 months respectively, are subjected to ELISA detection by using the same 50 parts of serum, and are compared with the Hangzhou prestige ELISA detection kit in sensitivity, specificity and coincidence rate, and the results are shown in tables 6.1-1. The results show that the sensitivity, specificity and coincidence rate of the kit are stable, which indicates that the kit can be stored for 8 months or more.
TABLE 6.1-1 test results on stability of the kit
7. Correlation test
The CHI3L1 calibrator of the invention was used for calibration, 100 human serum samples were randomly tested, and the test results were compared with those of CHI3L1ELISA kit of Hangzhou Puwang Biotechnology, Inc., and the comparison results are shown in FIG. 5. As can be seen from fig. 5, the correlation R of the test sample was 0.9942, indicating that the kit has good correlation.
By testing the critical value, relative sensitivity, relative specificity, coincidence rate, linear range, accuracy, repeatability, batch difference and stability of the kit, all performances of the product can be known to meet the requirements, and therefore the quality of the kit is qualified.
Technical solution of the invention is described above with reference to the accompanying drawings, it is obvious that the specific implementation of the invention is not limited by the above-mentioned manner, and it is within the scope of the invention to adopt various insubstantial modifications of the inventive method concept and technical solution, or to apply the inventive concept and technical solution to other occasions without modification.
Claims (10)
1. The CHI3L1 epitope peptide is characterized by comprising a CHI3L1 epitope peptide I and a CHI3L1 epitope peptide II,
the amino acid chain fragment I of the CHI3L1 epitope peptide I is as follows:
Cys-Cys-Cys-Thr-Cys-Thr-Cys-Ala-Cys-Cys-Ala，
the amino acid chain fragment II of the CHI3L1 epitope peptide II is as follows:
Ala-Thr-Gln-Cys-Cys-Ala-Thr-Cys-Ala-Ala。
2. the CHI3L1 epitope peptide according to claim 1, wherein the amino acid chain fragment I is synthesized into the CHI3L1 epitope peptide I by a solid phase method, and the amino acid chain fragment II is synthesized into the CHI3L1 epitope peptide II by a solid phase method.
3. A CHI3L1 antigen comprising: preparing CHI3L1 antigen i by coupling CHI3L1 epitope peptide i according to claim 2 with carrier protein KLH; CHI3L1 antigen II was prepared by coupling CHI3L1 epitope peptide II of claim 2 to a carrier protein KLH.
4. A CHI3L1 antibody, comprising: monoclonal antibody i prepared from CHI3L1 antigen i of claim 3; monoclonal antibody II prepared from CHI3L1 antigen II of claim 3.
5. A kit for predicting autologous arteriovenous fistula loss, comprising the monoclonal antibody I and the monoclonal antibody II of claim 4.
6. The kit for predicting autologous arteriovenous fistula loss of function of claim 5, which comprises a CHI3L1 antibody micropore ELISA plate prepared from a monoclonal antibody I or a monoclonal antibody II, wherein the CHI3L1 antibody micropore ELISA plate is prepared by the following steps:
firstly, selecting any one of a monoclonal antibody I or a monoclonal antibody II, and preparing the monoclonal antibody I or the monoclonal antibody II into a solution A with the concentration of 0.5-1.5 ng/ml by using a PBS (phosphate buffer solution) with the concentration of 1XPH being 7.4;
dripping the solution A into a micropore enzyme label plate, and placing the micropore enzyme label plate in a 37 ℃ incubator for culturing for 1.5-2 h;
and then taking out, blocking with 2% BSA, taking out after blocking, washing with PBS buffer solution with 1XPH being 7.4, and drying for 0.5-1 h for later use.
7. The kit for predicting autologous arteriovenous fistula loss of function of claim 5, which comprises a CHI3L1 detection antibody prepared from monoclonal antibody I or monoclonal antibody II, wherein the CHI3L1 detection antibody is prepared by the following steps:
firstly, selecting one of a monoclonal antibody I or a monoclonal antibody II, ensuring that the selected antibody is different from a CHI3L1 antibody selected when the CHI3L1 antibody micropore ELISA plate is prepared, then diluting the selected antibody, coupling the diluted antibody with biotin, mixing and rotating for 2 hours;
then pouring the coupled solution into a dialysis bag, dialyzing in PBS buffer solution with the concentration of 1XPH being 7.4 at the constant temperature of 4 ℃ for 24h, changing the dialyzate for 4 times, and collecting the solution in the dialysis bag to obtain the high-concentration CHI3L1 detection antibody;
finally preparing a detection antibody preservative solution; and then diluting the obtained high-concentration detection antibody with a detection antibody preservation solution to prepare a solution of 0.2-1.0 ng/ml for later use.
8. The kit for predicting autologous arteriovenous fistula loss according to claim 7, wherein the volume of biotin is 26 ul-60 ul, and the concentration of biotin is 1 umol/L-10 mmol/L;
the volume of the diluted antibody coupled with biotin is 1ml, and the concentration of the diluted antibody is 150 ug/ml-2 mg/ml;
the formula of the preservation solution for detecting the antibody is as follows: 2.2-5.4 g of NaH2PO422.15-31.08 g of Na2HPO4·12H2O, 3-10 g of casein, 0.15-0.35 g of tween20 and 1L of distilled water.
9. The kit for predicting autologous arteriovenous fistula loss of function of claim 5, which is characterized by further comprising CHI3L1 calibrator and quality control, avidin-labeled HRP, substrate solution A and B and stop solution;
diluting the avidin-labeled HRP by using an enzyme-labeled preservation solution; the formula of the enzyme-labeled preservation solution is as follows: 1-1.36 mol/L of glycerol, 8.5-10 mmol/L of sodium phosphate, 26-30 umol/L of bovine serum albumin, and 14-20 umol/L of cytochrome C;
the formula of the substrate liquid A is as follows: 10.3-13.6 g of sodium acetate, 1.2-1.6 g of citric acid, 0.28-0.35 ml of 30% hydrogen peroxide and 1L of distilled water;
the formula of the substrate liquid B is as follows: 0.15-0.24 g of disodium ethylene diamine tetraacetate, 0.14-0.18 g of citric acid, 36-52 ml of glycerol, 0.12-0.16 g of TMB and 1L of distilled water;
the stop solution is sulfuric acid, and the concentration of the sulfuric acid is 1.6-2 mol/L.
10. The kit of claim 5 for predicting the loss of function of an autologous arteriovenous internal fistula in an in vitro test for predicting early loss of function of an autologous arteriovenous internal fistula.
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