CN112611871A

CN112611871A - ELISA kit for detecting human Circ-E-cad protein and application method thereof

Info

Publication number: CN112611871A
Application number: CN202011417417.XA
Authority: CN
Inventors: 张弩
Original assignee: First Affiliated Hospital of Sun Yat Sen University
Current assignee: First Affiliated Hospital of Sun Yat Sen University
Priority date: 2020-12-07
Filing date: 2020-12-07
Publication date: 2021-04-06

Abstract

The invention discloses an ELISA kit for detecting human Circ-E-cad protein, and the specific working solution is prepared as follows: 6 bottles (1 ml/bottle) of standard solution, 7ml of enzyme marker, 7ml of antibody working solution, 7ml of substrate A solution, 7ml of substrate B solution, 7ml of stop solution, 40ml of 20X concentrated washing solution, 50ml of sample diluent and an antigen-coated enzyme label plate. The invention also discloses an application method of the kit. The invention develops an ELISA kit for detecting the human Circ-E-cad protein by preparing the mouse-derived monoclonal antibody for specifically detecting the Circ-E-cad protein, the kit provides all working solutions for target molecule detection, the kit can be used for quantitative detection of the Circ-E-cad protein in human clinical samples, and the kit has the characteristics of strong specificity, good repeatability and high sensitivity, and can quickly realize quantitative detection of the target protein in the samples.

Description

ELISA kit for detecting human Circ-E-cad protein and application method thereof

Technical Field

The invention relates to an ELISA kit and an application method thereof, belongs to the field of protein detection in molecular biology, and particularly relates to an ELISA kit for detecting human Circ-E-cad protein and an application method thereof.

Background

Since 2012, circular RNA (circular RNA) exists in large quantities in living bodies, which is a kind of RNA having a specific function and exists in large quantities objectively. Circular RNA is formed from precursor RNA by cleavage followed by head-to-tail ligation of linear RNA. In the previous research, the objective existence of the circular RNA is not found due to the limitation of the technical level, and researchers really find that a large amount of circular RNA molecules exist in organisms along with the development of deep RNA sequencing and large-scale biological information technology, and the circular RNA is very stable in the organisms due to the formation of closed circles.

There are few reports of studies on the specific functions and molecular mechanisms of action of circular RNAs, and there are currently only a few postulated statements: the circular RNA can be used as a sponge for absorbing miRNA to inhibit the function of the miRNA; the circular RNA directly regulates and controls other RNA levels through base complementary pairing; circular RNAs can bind to proteins, inhibit protein activity, recruit components of a protein complex, or modulate the activity of a protein, and can also serve as templates for translation to direct protein synthesis.

In previous work, a collection of circular RNA candidate molecules that are differentially expressed in glioblastomas and have potential translational capability were discovered by combining ribosomal blotting with Deep Sequencing technologies (Ribosome Profiling and Deep Sequencing) and bioinformatics analysis. The further analysis of the circular RNA database Circbase shows that the circular RNA Circ-E-cad-733nt is most obviously differentially expressed in the normal brain tissues of the tumor and the matched tumor, the expression of the circular RNA Circ-E-cad-733nt in the normal brain tissues of the tumor is very weak or not, the expression is only specifically and highly expressed in the malignant glioma tissues, and a novel protein Circ-E-cad consisting of 254 amino acids is translated by identifying the circular RNA-E-cad-733 through preparing an antibody and protein mass spectrometry. In tumor research and clinical detection, newly discovered proteins are focused, understanding of tumor occurrence and development can be further deepened, and a new detection method is designed aiming at new protein molecular targets, so that the protein has potential clinical application value.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide an ELISA kit for detecting the human Circ-E-cad protein and an application method thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides an ELISA kit for detecting human Circ-E-cad protein, and the specific working solution is prepared as follows:

(1) a 6 bottle (1 ml/bottle) of standard solution, wherein a polypeptide-conjugated human serum albumin BSA molecule for preparing a monoclonal antibody is used as a standard, a prepared sample diluent is 0.01mol/L phosphate solution containing 0.05% of Tween-20, and the pH is finally adjusted to 7.0 by acid and alkali; taking a standard substance, and preparing the standard substance with gradient concentration of 0 mug/ml, 2 mug/ml, 6 mug/ml, 18 mug/ml, 54 mug/ml and 162 mug/ml by using the sample diluent;

(2) enzyme label 7 ml: the enzyme label used in the kit is horseradish peroxidase-labeled goat anti-mouse IgG immunoglobulin, and the IgG is prepared into 4 microgram/ml serving as the enzyme label in the kit by using PBS with the concentration of 0.01M;

(3) antibody working solution 7 ml: the antibody working solution used in the kit is a mouse anti-Circ-E-cad monoclonal antibody, and PBS with the concentration of 0.01M is prepared into the final concentration of 0.5 mu g/ml to be used as the antibody working solution of the kit;

(4) substrate a solution 7 ml: weighing 0.1g of TMB (tetramethylbenzidine), adding 100ml of absolute ethyl alcohol, adding sterilized deionized water to 1L, fully and uniformly mixing, and subpackaging 7ml of the mixture to be used as a substrate A solution of the kit;

(5) substrate B solution 7 ml: 1.5g of sodium hydrogen phosphate, 0.95g of citric acid and 0.65ml of 0.75% urea hydrogen peroxide, adding sterilized deionized water to 100ml, adjusting the pH value to 5.2-5.5, and subpackaging 7ml as a substrate B solution of the kit;

(6) 7ml of stop solution: the stop solution is sulfuric acid with the concentration of 1 mol/L;

(7)20X concentrated Wash 40 ml: contains 1% of tween-20 phosphate buffer solution with the concentration of 0.2 mol/L;

(8) sample diluent 50 ml: the sample diluent is 0.01mol/L phosphate solution containing 0.05% Tween-20, and the pH is finally adjusted to 7.0 by acid and alkali;

(9) antigen-coated elisa plate: using carbonate buffer solution with the concentration of 0.1M as diluent, diluting the BSA-coupled polypeptide antigen to the concentration of 10 mu g/ml, adding 100 mu l of the diluted polypeptide antigen into a hole of an enzyme-labeled plate, coating the solution at 4 ℃ overnight, drying the solution by drying, adding 1% gelatin into the solution according to the concentration of 100 mu l/hole, incubating the solution for 1 hour at 37 ℃ by using phosphate buffer solution, drying the solution at low temperature in vacuum, vacuumizing and packaging the solution in a tin foil paper tape, sealing the solution, and storing the solution in a refrigerator at 2-8 ℃.

Further, a coupled BSA polypeptide antigen is pre-coated on the hole of the micropore strip by adopting an indirect competition ELISA method, the Circ-E-cad protein in the sample is detected to compete with the antibody of the coupled antigen pre-coated on the micropore strip, the color is developed by using a tetramethyl benzidine substrate, and the light absorption value of the sample is inversely related to the content of the Circ-E-cad protein contained in the sample.

Further, the mouse anti-Circ-E-cad monoclonal antibody described in (3) specifically recognizes and binds to amino acid sequence TNLCDGGHSHRRGR at the C-terminal end of the Circ-E-cad protein; the Circ-E-Cad protein is transcribed by circular RNA Circ-E-Cad-733 n; the nucleotide sequence of the circular RNA Circ-E-Cad-733n is shown as SEQ ID No. 1; the amino acid sequence of the Circ-E-cad protein is shown in SEQ ID No. 2.

Further, the mouse anti-Circ-E-cad monoclonal antibody described in (3) is prepared by the following preparation method:

3.1) predicting and translating the amino acid sequence of the Circ-E-Cad protein according to the nucleotide sequence SEQ ID No.1 of the circular RNA Circ-E-Cad-733n, wherein the amino acid sequence of the Circ-E-Cad protein is shown as SEQ ID No. 2;

3.2) synthesizing a polypeptide TNLCDGGHSHRRGR amino acid sequence specific to the Circ-E-Cad protein by a method of chemically synthesizing polypeptide according to the amino acid sequence of the Circ-E-Cad protein, wherein the polypeptide is coupled with keyhole limpet hemocyanin as immunogen;

3.3) polypeptide coupling keyhole limpet hemocyanin immune mice to prepare a hybridoma cell line;

3.4) carrying out expanded cell culture on the hybridoma cells with positive detection to produce the mouse anti-Circ-E-cad monoclonal antibody.

Further, in step 3.3), the step of immunizing the mouse and preparing the hybridoma cell line comprises the following steps:

3.3.1) immunizing animals: BALB/c female mice, 8-10 weeks old; immunizing mice with 50 μ g of keyhole limpet hemocyanin-coupled polypeptide TNLCDGGHSHRRGR and 50 μ l of immunoadjuvant 5 times at time points d0, d21, d42, d63, d 8; taking serum after 103 days to carry out ELISA to detect the antibody titer;

3.3.2) cell fusion: the mice are sacrificed, the spleen is taken out through aseptic operation, and spleen cell suspension is prepared; spleen cells and myeloma cells were mixed according to 1: 10, and adding polyethylene glycol, and fusing the cells to form hybridoma cells;

3.3.3) monoclonal testing: after 10-14 days, when cell clone is visible to naked eyes and part of the culture medium of the wells begins to turn yellow, all the wells are detected by enzyme linked immunosorbent assay, after 3 days, ELISA detection is carried out on the positive clone identified by the first ELISA, after 3 days, ELISA detection is carried out on the positive clone identified by the second ELISA, and the positive cells are subjected to amplification cell culture for mass production of monoclonal antibodies.

Further, in step 3.3.2), the polyethylene glycol is added at every 3 × 10⁸1.5ml of polyethylene glycol was slowly added to the mixed cells.

Further, the specific preparation method of the 20X concentrated washing solution in (7) comprises weighing 160g of sodium chloride, 4g of potassium hydrogen phosphate, 58g of sodium hydrogen phosphate dodecahydrate and 4g of potassium chloride, adding sterilized deionized water to 990ml of water, adding 10ml of Tween-20, and adjusting the pH to 7.0.

Further, the specific preparation method of the sample diluent in (8) is to weigh 8g of sodium chloride, 0.2g of potassium hydrogen phosphate, 2.9g of sodium hydrogen phosphate dodecahydrate and 0.2g of potassium chloride, add sterilized deionized water to 999.5ml, add 0.5ml of tween-20, and adjust the PH to 7.0.

Further, the BSA coupled polypeptide antigen in (9) is a polypeptide of chemically synthesized keyhole limpet hemocyanin KLH, and the sequence of the polypeptide is TNLCDGGHSH RRGR.

Further, the application method of the ELISA kit for detecting the human Circ-E-cad protein comprises the following steps:

preparing a 1X washing solution: 40ml of 20 Xconcentrated washings were diluted with deionized water in a 1: 19 diluting or preparing a washing solution according to the required dosage;

a step of processing a sample to be detected:

a. centrifuging a sample to be detected at 10 ℃ at 4000r/min for 10min to remove impurities in the sample;

b. taking 50 mul of sample to be detected, adding 200 mul of sample diluent, mixing (dilution multiple is 5 times), and mixing for 30 s;

c. 100 μ l of liquid was taken for analysis;

the experimental operation steps are as follows:

a. taking out the required reagent from the refrigeration environment, balancing at room temperature (20-25 deg.C) for more than 30min, and shaking each liquid reagent before use;

b. taking out the microporous strips according to the plate frame and the requirement, putting the unused micropores into a self-sealing bag, and storing at 2-8 ℃;

c. numbering: numbering the corresponding micropores of the samples and the standard products in sequence, making 2 holes for each sample and each standard product in parallel, and recording the positions of the standard holes and the positions of the sample holes;

d. add standard/sample 100. mu.l/well, then add enzyme marker 25. mu.l/well, then add antibody working solution 25. mu.l/well. Lightly shaking and mixing, covering with a cover plate film, and reacting at 25 deg.C for 10 min;

e. taking out, washing the plate with 250 μ l of washing solution for 4-5 times, soaking for 15-30 s each time, and patting to dry (bubbles which are not removed after patting to dry can be punctured by a clean gun head);

f. color development: adding 50 μ l of substrate solution A and 50 μ l of solution B into each well, mixing by gentle shaking, and developing at 25 deg.C in dark for 10 min;

g. and (3) determination: adding 50 μ l of stop solution into each well, gently oscillating and mixing, setting an enzyme-labeling instrument at 450nm (it is recommended to use double-wavelength 450/630nm to detect and read data within 5 min), and determining absorbance value (OD value);

h. and (4) judging a result:

h1, rough decision: comparing the average absorbance value of the sample with a standard value to obtain the concentration range (ng/ml);

h2, quantitative analysis:

calculation of percent absorbance: the percent absorbance of a standard or sample is equal to the average of the percent absorbance values of the standard or sample (double well) divided by the absorbance value of the first standard (0 standard) and multiplied by 100%, i.e.:

b-average absorbance value of Standard solution or sample solution

B₀Average absorbance value of 0ng/ml standard solution

Drawing and calculating a standard curve:

the standard curve is plotted with the percent absorbance of the standard as the ordinate and the semilog of the conjugated antigen (ng/ml) as the abscissa. And substituting the percent absorbance of the sample into the standard curve, reading out the concentration corresponding to the sample from the standard curve, and multiplying the concentration by the corresponding dilution multiple to obtain the actual content of the Circ-E-cad protein in the sample.

Compared with the prior art, the invention has the following beneficial effects:

the invention develops an ELISA kit for detecting the human Circ-E-cad protein by preparing the mouse-derived monoclonal antibody for specifically detecting the Circ-E-cad protein, the kit provides all working solutions for target molecule detection, the kit can be used for quantitative detection of the Circ-E-cad protein in human clinical samples, and the kit has the characteristics of strong specificity, good repeatability and high sensitivity, and can quickly realize quantitative detection of the target protein in the samples.

Drawings

FIG. 1 is a diagram showing the circular RNA formation pattern of Circ-E-Cad-733 nt;

FIG. 2 shows the result of sequencing and identification of Circ-E-Cad-733nt circular RNA;

FIG. 3 is a standard curve of the effect test example 1 for the detection of Circ-E-cad ELISA;

FIG. 4 is a precision curve of the Circ-E-cad protein assay kit in effect test example 2;

FIG. 5 shows the results of testing human cerebrospinal fluid with the Circ-E-cad ELISA kit in Experimental example 3.

Detailed Description

The invention will now be further described with reference to the accompanying drawings and specific embodiments.

The invention provides a preparation method and an application method of an ELISA kit for detecting human Circ-E-cad protein. The detailed contents of the invention are summarized as follows:

in the invention, E-Cadherin in a cyclization mode is identified and found in a human brain glial cell line U251 at the early stage, secondary circular RNA is circular RNA with the size of 733nt formed by circularizing the 7 th-10 th exons of the E-Cadherin gene and is named as circular-E-Cad-733 nt (see figure 1, sequence SEQ ID. No.1), and the accurate cyclization interface of the circular-E-Cad-733 nt is identified by a sanger DNA sequencing method (the Junction site of the circular RNA is shown in figure 2); translation potential analysis shows that the open reading frame in the Circ-E-Cad starts from ATG, translates the new protein with the length of 254aa across the cyclization interface position, and the protein is named as the Circ-E-Cad (sequence SEQ ID No. 2); a mouse monoclonal antibody is prepared aiming at a section of specific amino acid sequence (TNLCDGGHSHRRGR) at the C terminal of the Circ-E-cad and is used as a main raw material for preparing an ELISA kit in the later stage.

According to the circular RNA database, circular base suggests that the four exons 7 to 10 of the human E-Cadherin gene form a circular RNA molecule, namely, circular-E-cad-733 nt, and further analysis shows that the circular-E-cad-733 nt can translate a circular-E-cad protein consisting of 254 amino acids. The invention develops an ELISA kit for detecting the human Circ-E-cad protein by preparing the mouse-derived monoclonal antibody for specifically detecting the Circ-E-cad protein, the kit provides all working solutions for target molecule detection, the kit can be used for quantitative detection of the Circ-E-cad protein in human clinical samples, and the kit has the characteristics of strong specificity, good repeatability and high sensitivity, and can quickly realize quantitative detection of the target protein in the samples.

Example 1

The specific implementation process of the preparation of the human Circ-E-cad protein ELISA kit is as follows:

1. the E-Cadherin gene is located in the region of the human chromosome 16 long arm chr16(q11.2) through the analysis of UCSC (http:// genome. UCSC. edu /) online database software, the genome spans 98324bp, and the variant for coding the longest protein 882 amino acids has 16 exon compositions; according to circular RNA information recorded by circular RNA authority database circBase (http:// circrna. org /), the 7 th to 10 th exons of E-Cadherin gene are found to form closed circular RNA molecules through head-to-tail connection, the length of the circular RNA molecules is 733nt, and the circular RNA molecules are named as Circ-E-Cad-733 n; PCR amplification primers are designed on two sides of the circular RNA connecting site, sequences at two wings of the circular RNA connecting site are amplified, and the accurate circular site of the Circ-E-Cad-733n circular RNA is obtained by a sanger DNA sequencing method. The reaction system and conditions for amplifying the target fragment by PCR using the cDNA of the brain glioma U251 cell as a template are described as follows, wherein the PCR is a 30. mu.l total system, specifically, 15. mu.l of 2 XPCR MIX (Vazyme company), 1.5. mu.l of each of upstream and downstream primers (10mM), and 1. mu.l of cDNA template, and 30. mu.l of sterile water is used for supplementing the system. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 15s in a cycle, annealing at 60 ℃ for 30s, extension at 72 ℃ for 25s for 40 cycles, continuous extension at 72 ℃ for 5min after PCR reaction cycle, and then storage at 16 ℃. The PCR product was purified and subjected to sanger DNA sequencing.

2. Prediction and recognition of Circ-E-Cad-733nt circular RNA translation protein and mouse monoclonal antibody preparation

Predicting the translated protein product sequence (see sequence SEQ ID No.2) by using online software ORF finder (https:// indera. mullins. microbial. washington. edu/sms2/ORF _ find. html) to perform circular RNA translation potential prediction according to the nucleic acid sequence of Circ-E-Cad-733 n; nucleotide sequence analysis of the Circ-E-Cad-733nt circular RNA molecule shows that RNA can form an open reading frame consisting of ATG-TGA after cyclization, a novel protein of the Circ-E-Cad consisting of 254 amino acids is generated by translation, and the molecular weight of the protein is predicted to be about 28KD by protein molecular weight prediction software (http:// www.bio-soft.

According to the composition of the amino acid sequence of the Circ-E-Cad, a specific antigen is designed, and a monoclonal antibody is prepared by using a mouse as a host by the following method: synthesizing a polypeptide TNLCDGGHSHRRGR amino acid sequence which is specifically aimed at the Circ-E-Cad protein by a method of chemically synthesizing the polypeptide as an immunogen; chemically synthesized polypeptide is coupled with Keyhole Limpet Hemocyanin (KLH) KLH molecules, then a mouse is immunized, a hybridoma cell line is prepared, and an antibody effect is tested; chemical synthesis of peptides and peptide-coupled keyhole limpet hemocyanin was performed by Shanghai Potta scientific Co.

The mouse monoclonal antibody preparation entrusts Nanjing Kinshire company to adopt a standardized preparation and purification process. The detailed preparation process of monoclonal antibody is shown in the following methodology:

1) immunizing animals

5 BALB/c female mice, 8-10 weeks old, were used; mice were immunized 5 times (time points d0, d21, d42, d63, d84) with 50 μ g of Keyhole Limpet Hemocyanin (KLH) conjugated polypeptide TNLCDGGHSHRRGR and 50 μ l of immunoadjuvant (total volume 100 μ l); mice were bled before each immunization and sera (100 μ l) were collected; serum was taken 103 days later for ELISA to detect antibody titer.

2) Cell fusion

Preheating the used cell culture solution and PEG 1500-37 deg.C, counting myeloma cells, and culturing the cells in RPMI1640+ 10% FCS; splenocytes collection is a mouse immunized by euthanasia of cervical vertebra, the whole mouse is placed in a beaker containing 200ml of 80% povidone-iodine, 20% of 70% ethanol at a ratio, the spleen of the mouse is excised in a sterile environment, the spleen is transferred to a culture dish containing 5ml of RPMI1640+ 10% FCS cell culture solution, fat and connective tissue are trimmed, then the spleen is cut into small pieces, and the cut spleen is transferred to a new culture dish containing 1ml of RPMI1640+ 10% FCS; using two sterile forceps, fixing the spleen by one forceps, and stripping the spleen cells into the culture medium by the other forceps; transferring the cells into a 50ml test tube, washing 4 times with 2ml of medium, pipetting the cell suspension 10 times, and then standing for 1 minute until larger tissue pieces fall to the bottom of the test tube; collecting the upper cell suspension, placing into a new 50ml test tube, centrifuging at 1000rpm for 5 minutes, pouring out the supernatant, and taking the splenocytes into 20ml RPMI1640+ 10% FBS culture medium; spleen cells and myeloma cells were mixed according to 1: 10, the cells were mixed by suspending the mixed cells in 25ml of RPMI1640 medium (without additives) and then centrifuged at 1000rpm for 5 minutes every 3X 10⁸Slowly adding 1.5ml PEG into mixed cells, incubating at 37 deg.C for 3 min, and slowly adding20ml of RPMI1640 medium was slowly added, followed by centrifugation at 1000rpm for 5 minutes, and the cells were directly seeded into a 96-well plate.

3) Monoclonal testing:

after 10-14 days, when cell clone is visible to naked eyes and part of the culture medium of the wells begins to turn yellow, all the wells are detected by enzyme-linked immunosorbent assay (ELISA), after 3 days, ELISA detection is carried out on positive clone identified by the first ELISA, after 3 days, ELISA detection is carried out on positive clone identified by the second ELISA, and the positive cells are subjected to amplification cell culture for mass production of monoclonal antibodies.

3. Preparation of human Circ-E-cad enzyme-linked immunosorbent assay (ELISA) detection kit

The Circ-E-cad kit adopts an indirect competition ELISA method, conjugate BSA polypeptide antigen is pre-coated on a micropore, the Circ-E-cad protein in a sample and the conjugate antigen competition antibody pre-coated on the micropore are detected, after an enzyme marker is added, a tetramethyl benzidine (TMB) substrate is used for developing color, the light absorption value of the sample is negatively correlated with the content of the Circ-E-cad protein contained in the sample, and the content of the Circ-E-cad in the sample to be detected can be obtained by comparing the light absorption value with a standard curve.

Preparing a working solution in the kit:

(1) a 6 bottle (1 ml/bottle) of standard solution, wherein a polypeptide-conjugated human serum albumin BSA molecule for preparing a monoclonal antibody is used as a standard, a prepared sample diluent is 0.01mol/L phosphate solution containing 0.05% of Tween-20, and the pH is finally adjusted to 7.0 by acid and alkali; taking the standard substance, and then preparing the standard substance with gradient concentration of 0. mu.g/ml, 2. mu.g/ml, 6. mu.g/ml, 18. mu.g/ml, 54. mu.g/ml and 162. mu.g/ml by using the sample diluent.

(2) Enzyme label 7 ml: the enzyme label used in the kit is goat anti-mouse IgG immunoglobulin labeled with Horseradish Peroxidase (HRP), and the IgG is prepared to have the maximum concentration of 4 mu g/ml by using PBS with the concentration of 0.01M and is used as the enzyme label in the kit.

(3) Antibody working solution 7 ml: the antibody working solution used in the kit is a mouse anti-Circ-E-cad monoclonal antibody, and PBS with the concentration of 0.01M is prepared into the final concentration of 0.5 mu g/ml to be used as the antibody working solution of the kit.

(4) Substrate a solution 7 ml: weighing 0.1g of TMB (tetramethylbenzidine), adding 100ml of absolute ethyl alcohol, adding sterilized deionized water to 1L, fully and uniformly mixing, and subpackaging 7ml of the mixture to be used as a substrate A liquid of the kit.

(5) Substrate B solution 7 ml: 1.5g of sodium hydrogen phosphate, 0.95g of citric acid and 0.65ml of 0.75% urea hydrogen peroxide, adding sterilized deionized water to 100ml, adjusting the pH to 5.2-5.5, and subpackaging 7ml to obtain a substrate B solution of the kit.

(6) 7ml of stop solution: the stop solution is 1mol/L sulfuric acid (2 ml of sulfuric acid is added into 34ml of sterilized deionized water and mixed gently).

(7)20X concentrated Wash 40 ml: the phosphate buffer solution containing 1% of Tween-20 and having a concentration of 0.2mol/L is prepared by weighing 160g of sodium chloride, 4g of potassium hydrogen phosphate, 58g of sodium hydrogen phosphate dodecahydrate and 4g of potassium chloride, adding sterilized deionized water to 990ml, adding 10ml of Tween-20, and adjusting the pH to 7.0.

(8) Sample diluent 50 ml: the sample diluent is 0.01mol/L phosphate solution containing 0.05% of Tween-20, and the pH is finally adjusted to 7.0 by acid and alkali, wherein the specific preparation method comprises the steps of weighing 8g of sodium chloride, 0.2g of potassium hydrogen phosphate, 2.9g of sodium hydrogen phosphate dodecahydrate and 0.2g of potassium chloride, adding sterilized deionized water to the volume of 999.5ml, adding 0.5ml of Tween-20, and adjusting the pH to 7.0.

(9) Preparing an antigen-coated ELISA plate: using carbonate buffer solution with the concentration of 0.1M as diluent, diluting the BSA-coupled polypeptide antigen to the concentration of 10 mu g/ml, adding 100 mu l of the diluted polypeptide antigen into a hole of an enzyme-labeled plate, coating the solution at 4 ℃ overnight, drying the solution by drying, adding 1% gelatin into the solution according to the concentration of 100 mu l/hole, incubating the solution for 1 hour at 37 ℃ by using phosphate buffer solution, drying the solution at low temperature in vacuum, vacuumizing and packaging the solution in a tin foil paper tape, sealing the solution, and storing the solution in a refrigerator at 2-8 ℃. The BSA-coupled polypeptide antigen is specifically a polypeptide chemically synthesized with keyhole limpet hemocyanin KLH, the polypeptide sequence is TNLCDGGHSHRRGR, and the specific coupling method is a standard sodium periodate method.

4. Operating method of human Circ-E-cad enzyme-linked immunosorbent assay (ELISA) detection kit

Preparation of 1 × washing solution 40ml of concentrated washing solution (20-fold concentration) was washed with deionized water according to a 1: 19 dilution (1 part of concentrated washing solution +19 parts of deionized water), or the washing solution is prepared according to the required dosage. Sample pre-processing care must be taken when processing any sample:

A. disposable tips must be used in the experiments, and the tips must be changed when different reagents are to be aspirated.

B. Before the experiment, whether various experimental instruments are clean or not needs to be checked, and the experimental instruments can be cleaned if necessary so as to avoid pollution and interference on the experimental result.

A step of processing a sample to be detected:

c. 100 μ l of the liquid was taken for analysis.

The experimental operation steps are as follows:

a. taking out the required reagent from the refrigeration environment, and balancing at room temperature (20-25 deg.C) for more than 30min, wherein each liquid reagent should be shaken well before use.

b. Taking out the microporous strip according to the plate frame and the requirement, putting the unused micropores into a self-sealing bag, and storing at 2-8 ℃.

c. Numbering: and numbering the corresponding micropores of the samples and the standard products in sequence, making 2 holes in parallel for each sample and standard product, and recording the positions of the standard holes and the sample holes.

d. Add standard/sample 100. mu.l/well, then add enzyme marker 25. mu.l/well, then add antibody working solution 25. mu.l/well. The mixture is gently shaken and evenly mixed, and then the mixture is covered by a cover plate film and reacted for 10min at the temperature of 25 ℃.

e. Taking out, washing the plate with 250 μ l of washing solution for 4-5 times, soaking for 15-30 s each time, and drying (the bubbles which are not removed after drying can be punctured with clean gun head).

f. Color development: adding 50 μ l of substrate solution A and 50 μ l of solution B into each well, mixing by gentle shaking, and developing at 25 deg.C in dark for 10 min.

g. And (3) determination: add 50 μ l of stop solution into each well, mix by gentle shaking, set the microplate reader at 450nm (it is recommended to read the data within 5min with the double wavelength 450/630nm detection), determine the absorbance value (OD value).

And (4) judging a result:

there are two methods for determining the result, the rough determination method can be used in the 1 st method, and the quantitative determination method can be used in the 2 nd method. Note that the absorbance of the sample is inversely related to the content of the Circ-E-cad protein.

Rough judgment:

the concentration range (ng/ml) can be obtained by comparing the average absorbance value of the sample with the standard value. Assuming that the absorbance value of sample 1 is 0.221 and the absorbance value of sample 2 is 0.795, the absorbance values of the standard solutions are: 0ppm is 1.81; 2ppm is 1.50; 6ppm is 1.114; 18ppm is 0.660; 54ppm is 0.318; 162ppm was 0.145. The concentration range of sample 1 is 54ppm-162 ppm; the concentration range of sample 2 is 6ppm to 18 ppm. Multiplying the dilution times by the corresponding dilution times to obtain the actual content of the Circ-E-cad protein in the sample.

Quantitative analysis:

a. calculation of percent absorbance, the percent absorbance of a standard or sample is equal to the average of the percent absorbance values of the standard or sample (double well) divided by the absorbance value of the first standard (0 standard) and multiplied by 100%, i.e.:

b-average absorbance value of Standard solution or sample solution

B₀Average absorbance value of 0ng/ml standard solution

b. Drawing and calculating standard curve

Effect test example 1 drawing of Standard Curve

Drawing a standard curve as shown in figure 3 strictly according to the effect of the human Circ-E-cad enzyme-linked immunosorbent assay (ELISA) kit on testing the kit by using a standard substance carried by the kit by using an operation method; the standard curve is drawn to show that the kit can effectively detect the content of the target protein as low as 2 mu g/ml.

Effect test example 2 and test of kit precision

The precision of the test kit with the standard substance is determined by repeating the test twice, a plurality of different experimental results show that the precision of the Circ-E-cad protein test kit is determined by the results in FIG. 4, the variation coefficient (% CV) of the obtained standard absorption value is plotted against the concentration of the corresponding Circ-E-cad protein, the variation coefficient in all ranges is less than 5 percent, the obtained kit has good reproducibility, and the results show that the variation coefficient of the test data in the prepared kit plate is less than 5 percent, the result stability is good, and the precision is high.

Effect test example 3, human Circ-E-cad Clinical cerebrospinal fluid specimen detection of ELISA detection kit

Collecting cerebrospinal fluid of a human from clinic to perform a kit detection experiment, collecting 10 cases of cerebrospinal fluid of patients accompanied with cerebral edema but without brain glioma as a control, collecting 100 cases of patients suffering from brain glioma as an experimental group, performing the detection experiment strictly according to the kit operation instructions, and referring to a detection result shown in fig. 5; by detecting clinical human cerebrospinal fluid samples, the Circ-E-cad ELISA detection kit detects that the expression of the Circ-E-cad is high in abundance in cerebrospinal fluid of patients with brain glioma, and the expression of the Circ-E-cad protein of patients with non-brain tumors is very low.

The present invention is not limited to the above-described embodiments, and various changes and modifications of the present invention are intended to be included within the scope of the claims and the equivalent technology of the present invention if they do not depart from the spirit and scope of the present invention.

Sequence listing

<110> secondary first hospital of Zhongshan university

<120> ELISA kit for detecting human Circ-E-cad protein and application method thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 733

<212> RNA

<213> Circ-E-Cad-733nt(Homo sapiens)

<400> 1

gaaccucugu gauggagguc acagccacag acgcggacga ugaugugaac accuacaaug 60

ccgccaucgc uuacaccauc cucagccaag auccugagcu cccugacaaa aauauguuca 120

ccauuaacag gaacacagga gucaucagug uggucaccac ugggcuggac cgagagaguu 180

ucccuacgua uacccuggug guucaagcug cugaccuuca aggugagggg uuaagcacaa 240

cagcaacagc ugugaucaca gucacugaca ccaacgauaa uccuccgauc uucaauccca 300

ccacguacaa gggucaggug ccugagaacg aggcuaacgu cguaaucacc acacugaaag 360

ugacugaugc ugaugccccc aauaccccag cgugggaggc uguauacacc auauugaaug 420

augauggugg acaauuuguc gucaccacaa auccagugaa caacgauggc auuuugaaaa 480

cagcaaaggg cuuggauuuu gaggccaagc agcaguacau ucuacacgua gcagugacga 540

augugguacc uuuugagguc ucucucacca ccuccacagc caccgucacc guggaugugc 600

uggaugugaa ugaagccccc aucuuugugc cuccugaaaa gagaguggaa guguccgagg 660

acuuuggcgu gggccaggaa aucacauccu acacugccca ggagccagac acauuuaugg 720

aacagaaaau aac 733

<210> 2

<211> 254

<212> PRT

<213> Circ-E-Cad(Homo sapiens)

<400> 2

Met Glu Val Thr Ala Thr Asp Ala Asp Asp Asp Val Asn Thr Tyr Asn

1 5 10 15

Ala Ala Ile Ala Tyr Thr Ile Leu Ser Gln Asp Pro Glu Leu Pro Asp

20 25 30

Lys Asn Met Phe Thr Ile Asn Arg Asn Thr Gly Val Ile Ser Val Val

35 40 45

Thr Thr Gly Leu Asp Arg Glu Ser Phe Pro Thr Tyr Thr Leu Val Val

50 55 60

Gln Ala Ala Asp Leu Gln Gly Glu Gly Leu Ser Thr Thr Ala Thr Ala

65 70 75 80

Val Ile Thr Val Thr Asp Thr Asn Asp Asn Pro Pro Ile Phe Asn Pro

85 90 95

Thr Thr Tyr Lys Gly Gln Val Pro Glu Asn Glu Ala Asn Val Val Ile

100 105 110

Thr Thr Leu Lys Val Thr Asp Ala Asp Ala Pro Asn Thr Pro Ala Trp

115 120 125

Glu Ala Val Tyr Thr Ile Leu Asn Asp Asp Gly Gly Gln Phe Val Val

130 135 140

Thr Thr Asn Pro Val Asn Asn Asp Gly Ile Leu Lys Thr Ala Lys Gly

145 150 155 160

Leu Asp Phe Glu Ala Lys Gln Gln Tyr Ile Leu His Val Ala Val Thr

165 170 175

Asn Val Val Pro Phe Glu Val Ser Leu Thr Thr Ser Thr Ala Thr Val

180 185 190

Thr Val Asp Val Leu Asp Val Asn Glu Ala Pro Ile Phe Val Pro Pro

195 200 205

Glu Lys Arg Val Glu Val Ser Glu Asp Phe Gly Val Gly Gln Glu Ile

210 215 220

Thr Ser Tyr Thr Ala Gln Glu Pro Asp Thr Phe Met Glu Gln Lys Ile

225 230 235 240

Thr Asn Leu Cys Asp Gly Gly His Ser His Arg Arg Gly Arg

245 250

Claims

1. An ELISA kit for detecting human Circ-E-cad protein is characterized in that the specific working solution is prepared as follows:

2. The ELISA kit for detecting human Circ-E-cad protein of claim 1, characterized in that: adopting an indirect competition ELISA method to pre-coat coupled BSA polypeptide antigen on the micropore, detecting the competitive antibody of the Circ-E-cad protein in the sample and the pre-coated coupled antigen on the micropore, developing by using a tetramethyl benzidine substrate, and the light absorption value of the sample is inversely related to the content of the Circ-E-cad protein contained in the sample.

3. The ELISA kit for detecting human Circ-E-cad protein of claim 1, characterized in that:

(3) the mouse anti-Circ-E-cad monoclonal antibody specifically recognizes and binds to an amino acid sequence TNLCDGGHSHRRGR at the C-terminal end of the Circ-E-cad protein; the Circ-E-Cad protein is transcribed by circular RNA Circ-E-Cad-733 n; the nucleotide sequence of the circular RNA Circ-E-Cad-733n is shown as SEQ ID No. 1; the amino acid sequence of the Circ-E-cad protein is shown in SEQ ID No. 2.

4. The ELISA kit for detecting human Circ-E-cad protein of claim 1, wherein the mouse anti-Circ-E-cad monoclonal antibody described in (3) is prepared by the following preparation method:

5. The ELISA kit for detecting human Circ-E-cad protein as claimed in claim 4, wherein in step 3.3), the step of immunizing mouse and preparing hybridoma cell line comprises:

6. The ELISA kit for detecting human Circ-E-cad protein of claim 5, wherein: in step 3.3.2), the addition of polyethylene glycol is carried out at a rate of every 3X 10⁸1.5ml of polyethylene glycol was slowly added to the mixed cells.

7. The ELISA kit for detecting human Circ-E-cad protein of claim 1, characterized in that: (7) the specific preparation method of the medium 20X concentrated washing solution comprises the steps of weighing 160g of sodium chloride, 4g of potassium hydrogen phosphate, 58g of sodium hydrogen phosphate dodecahydrate and 4g of potassium chloride, adding sterilized deionized water to 990ml of constant volume, adding 10ml of Tween-20, and adjusting the pH to 7.0.

8. The ELISA kit for detecting human Circ-E-cad protein of claim 1, characterized in that: (8) the specific preparation method of the medium sample diluent comprises the steps of weighing 8g of sodium chloride, 0.2g of potassium hydrogen phosphate, 2.9g of sodium hydrogen phosphate dodecahydrate and 0.2g of potassium chloride, adding sterilized deionized water to the volume of 999.5ml, adding 0.5ml of Tween-20, and adjusting the pH value to 7.0.

9. The ELISA kit for detecting human Circ-E-cad protein of claim 1, characterized in that: (9) the polypeptide antigen of the coupled BSA is a polypeptide of chemically synthesized coupled keyhole limpet hemocyanin KLH, and the sequence of the polypeptide is TNLCDGGHSHRRGR.

10. The method of using the ELISA kit for detecting human Circ-E-cad protein of claim 1, comprising the steps of:

a step of processing a sample to be detected:

c. 100 μ l of liquid was taken for analysis;

the experimental operation steps are as follows:

h. and (4) judging a result:

h2, quantitative analysis:

b-average absorbance value of Standard solution or sample solution

B₀Average absorbance value of 0ng/ml standard solution

Drawing and calculating a standard curve:

drawing a standard curve graph by taking the percent absorbance of the standard substance as a vertical coordinate and taking the semilogarithm of the coupled antigen (ng/ml) as a horizontal coordinate; and substituting the percent absorbance of the sample into the standard curve, reading out the concentration corresponding to the sample from the standard curve, and multiplying the concentration by the corresponding dilution multiple to obtain the actual content of the Circ-E-cad protein in the sample.