CN111579788A - Simoa kit of high-sensitivity tumor marker EPHA2 and application thereof - Google Patents
Simoa kit of high-sensitivity tumor marker EPHA2 and application thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
Abstract
The invention belongs to the fields of immunodetection technology and tumor detection, and particularly relates to a Simoa kit of a high-sensitivity tumor marker EPHA2 and application thereof. The Simoa kit comprises capture antibody coated magnetic beads, EPHA2 standard, biotinylated detection antibody, SBG solution, fluorogenic substrate solution, and sample diluent. The Simoa kit for detecting the content of the EPHA2 protein in human serum based on the Simoa platform and the double antibody sandwich method provided by the invention can qualitatively and quantitatively detect the content of the EPHA2 in the human serum; the method has the characteristics of high sensitivity, high flux, less sample amount, automation, short time, simple and convenient operation, wide detection range and the like.
Description
Technical Field
The invention belongs to the fields of immunodetection technology and tumor detection, and particularly relates to a Simoa kit of a high-sensitivity tumor marker EPHA2 and application thereof.
Background
The erythropoietin-producing hepatocyte (EPH) a2 receptor (EPHA 2) belongs to the EPH Receptor Tyrosine Kinase (RTK) family, which is divided into two subfamilies (EPHA and EPHB) on the basis of sequence homology and binding to the ligand of "ephrins". Ephrin-based signals play a role in the growth, migration, and invasion of cancer cells through a variety of pathways, including RAS and AKT, integrin-mediated adhesion, and epithelial-to-mesenchymal transition. In many malignancies, different ephrins and their receptors (including EPHA 2) are activated, possibly leading to increased malignancy and poor prognosis. In colorectal cancer, EPHA2 and ephrin a1 were found to be significantly higher in stages I to II than in stages III to IV, suggesting a potential role in the early stages of disease progression. Furthermore, EPHA2 is also associated with poor prognosis in stages II and III. In 2009, Brannan et al analyzed EphA2 expression in non-small cell lung cancer (NSCLC) tissue samples using immunohistochemical techniques. As a result, EphA2 overexpression was detected in greater than 90% of NSCLC tumor samples, and EphA2 expression was positively correlated with activated Epidermal Growth Factor Receptor (EGFR).
The currently clinically used detection method of EPHA2 is mainly a tissue biopsy method-immunohistochemistry. However, biopsy is an invasive method of detection and can cause a lot of pain to the patient. In contrast, liquid biopsy is a well-accepted non-invasive detection method. Thus, the detection of EPHA2 in blood is of greater importance for the diagnosis and prognostic assessment of cancer. However, because of the relatively low level of EPHA2 in blood, no commercial kit is currently available for the detection of EPHA2 in blood. Simoa is a digital ultra-sensitive detection technology platform for protein markers based on Poisson distribution principle and single molecule technology. The platform adopts a classic ELISA double-antibody sandwich method, and immune magnetic beads with single protein to be detected can be loaded into a micropore with about 46 femtoliters for fluorescence imaging. By counting the luminescent microwells, the platform allows qualitative and quantitative detection of proteins.
Simoa is a digital ultra-sensitive detection technology platform for protein markers based on Poisson distribution principle and single molecule technology. The platform adopts a classic ELISA double-antibody sandwich method, and immune magnetic beads with single protein to be detected can be loaded into a micropore with about 46 femtoliters for fluorescence imaging. By counting the luminescent microwells, the platform allows for ultrasensitive quantitative detection of proteins.
Therefore, the method for detecting the EPHA2, which is high in sensitivity, high in automation degree, rapid, good in accuracy and suitable for popularization, is developed based on the Simoa technology, and has great significance for rapidly detecting the tumor marker EPHA 2.
Disclosure of Invention
The invention provides a Simoa kit of a high-sensitivity tumor marker EPHA2 and application thereof, so as to solve the technical problems of complex operation, large sample quantity requirement, insufficient sensitivity and the like in the prior art. The method is simple and convenient to operate, high in flux, high in sensitivity, and less in sample amount, and is suitable for large-scale screening.
The technical scheme of the invention is realized as follows:
a Simoa kit of a high-sensitivity tumor marker EPHA2 comprises magnetic beads coated with capture antibodies, an EPHA2 standard, biotinylated detection antibodies, an SBG solution, a fluorogenic substrate solution and a sample diluent.
The preparation method of the magnetic bead coated with the capture antibody comprises the following steps:
(1) carrying out liquid change treatment on the capture antibody through an ultrafiltration tube;
(2) 2.7 μm magnetic beads were activated with EDC;
(3) mixing the capture antibody processed in the step (1) with the activated magnetic beads processed in the step (2) and incubating for 2-3h at 4 ℃;
(4) and (4) washing the product incubated in the step (3) twice by using a PBST solution, then sealing by using a sealing solution at 37 ℃, finally washing twice by using a magnetic bead diluent to obtain magnetic beads coated with the capture antibody, and storing in the magnetic bead diluent.
The capture antibody in the step (1) is a mouse monoclonal antibody; the buffer used in the ultrafiltration tube was 50 mmol/L morpholine ethanesulfonic acid buffer, pH = 6.2.
The reaction concentration of the capture antibody in the step (3) is 0.05-0.5 mg/mL, and the reaction concentration of the activated magnetic beads is 1.2 × 109/mL。
The PBST solution in the step (4) is 10mmol/L PBS +1% Tween20, and the pH = 7.4; blocking solution is 10mmol/L PBS +1% BSA, pH = 7.4; the magnetic bead dilution was 50 mmol/L Tris-HCl + 10mmol/L EDTA +0.1% Tween20 +1% BSA, pH = 7.4.
The preparation method of the biotinylated detection antibody comprises the following steps: and (2) carrying out liquid exchange treatment on the detection antibody by using a PBS solution through an ultrafiltration tube, adding NHS-Biotin or a derivative thereof at room temperature, reacting for 30 min to obtain a biotinylated detection antibody, and purifying by using the ultrafiltration tube to obtain the biotinylated detection antibody, wherein the detection antibody is a rabbit polyclonal antibody.
The quantity ratio of the detection antibody to the reaction substance of the NHS-Biotin or the derivative thereof is 1: 40; PBS solution was 10mmol/L phosphate buffer, pH = 7.4.
The SBG solution was diluted from the original concentration to a concentration of 150 pmol/L by an enzyme diluent of 10mmol/L PBS + 0.5% fetal bovine serum +1 mmol/L MgCl2The kit comprises a sample, a standard EPHA2, a fluorogenic substrate, a fluorescent substrate solution and a pH =7.4, wherein the standard EPHA2 is prepared into 1 mL by sample diluent, and other standards can be automatically diluted and prepared according to requirements, the sample diluent is 10mmol/L PBS + 5mmol/L EDTA +0.1% Tween20 + 2% BSA, the fluorogenic substrate is resorufin- β -galactoside, and the concentration of the fluorogenic substrate solution is 100 mu mol/L;
the tumor marker EPHA2 is used in the preparation of cancer diagnostic reagent.
The tumor marker EPHA2 Simoa kit is applied to the detection of the content of EPHA2 in human serum.
The invention has the following beneficial effects:
the high-sensitivity Simoa kit for detecting the content of the EPHA2 protein in human serum based on the Simoa platform and the double-antibody sandwich method, provided by the invention, is a human EPHA2 double-antibody sandwich digital ELISA detection kit which uses a mouse monoclonal antibody as a capture antibody and uses a rabbit polyclonal antibody for detecting the antibody, and can qualitatively and quantitatively detect the content of the EPHA2 in the human serum; the method has the characteristics of high sensitivity, high flux, low sample size of 4 mu L, automation, short time consumption, simple and convenient operation, wide detection range and the like, and the lowest detection limit of the method can reach 0.12 pg/mL as can be seen from the attached figure 1. The invention can be used as a non-invasive method for diagnosing and monitoring the course of cancer (especially non-small cell lung cancer and pancreatic cancer).
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a standard graph of the EPHA2 Simoa kit of the present invention.
FIG. 2 is a serum dilution curve obtained using the EPHA2 Simoa kit according to the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
Examples
The high-sensitivity Simoa kit for detecting the content of EPHA2 in human serum provided by the invention comprises 2.7 mu m magnetic beads coated by capture antibodies, an EPHA2 standard, biotinylated detection antibodies, a streptavidin-beta-galactosidase (SBG) solution, a fluorescent substrate solution, a sample diluent and an electronic file of a test method.
In the present invention, raw materials of magnetic beads, capture antibodies, standards, detection antibodies, biotinylation reagents, SBG, and fluorescent substrates are obtained by purchase; wherein the capture antibody is a mouse monoclonal antibody, and the detection antibody is a rabbit polyclonal antibody.
In the scheme of the invention, the capture antibody coats the magnetic beads, and the preparation process comprises the following steps: first, the stored antibody was subjected to a fluid change treatment through an ultrafiltration tube using MES buffer of 50 mmol/L morpholinoethanesulfonic acid buffer, pH = 6.2.
Then, the magnetic beads are activated by EDC, the capture antibody and the activated magnetic beads are incubated for 2 h at the temperature of 4 ℃, then, the magnetic beads are washed twice by PBST, then, the PBST is sealed by sealing liquid at the temperature of 37 ℃, finally, the magnetic beads are washed twice by magnetic bead diluent and stored in the magnetic bead diluent, and the magnetic beads coated by the capture antibody are obtained, wherein the reaction concentration of the capture antibody is 0.1-0.2 mg/mL, and the reaction concentration of the activated magnetic beads is 1.2 × 109/mL。
The PBST formula comprises: 10mmol/L PBS +1% Tween20, pH = 7.4; the formula of the sealing liquid is as follows: 10 mmol/LPBS +1% BSA, pH = 7.4. The magnetic bead diluent formula comprises: 50 mmol/L Tris-HCl + 10mmol/L EDTA +0.1% Tween20 +1% BSA, pH = 7.4.
In the biotinylated detection antibody scheme of the invention, the preparation process comprises the following steps: first, the originally stored detection antibody was subjected to a fluid change treatment with PBS through an ultrafiltration tube. Then, NHS-Biotin or its derivative was added thereto at room temperature and reacted for 30 min. And finally, purifying the biotinylated detection antibody by using an ultrafiltration tube to obtain the biotinylated detection antibody. PBS formulation: 10mmol/L Phosphate Buffered Saline (PBS), pH = 7.4. Wherein the quantity ratio of the detection antibody to the reaction substance of NHS-Biotin or the derivative thereof is 1: 40.
Preparation of enzyme and substrate solutions: SBG was diluted from the original concentration to 150 pmol/L by enzyme dilution and substrate was diluted to 100. mu. mol/L with PBS as described above; wherein the enzyme diluent is: 10mmol/L PBS + 0.5% fetal bovine serum +1 mmol/LMgCl2，pH=7.4。
The final concentrations are all working concentrations for final imaging.
The substrate is: resorufin-beta-galactoside (RGP)
Preparing a standard solution: 100 ng/mL of LEPHA2 standard was formulated in 1 mL of sample diluent. Other standard products can be prepared according to the needs, wherein the sample diluent is as follows: 10mmol/L PBS + 5mmol/L EDTA +0.1% Tween20 + 2% BSA.
Method of use of the EPHA2 Simoa kit:
firstly, an electronic document of a test method is imported to an HD-1/HD-X Simoa analyzer produced by Quanterix, and the electronic document of the test method comprises a test running mode. The method on the electronic document of the test method can be changed according to the requirements so as to be suitable for the reagent application of the kit. The present invention utilizes serum dilution curves obtained with the EPHA2 Simoa kit.
The electronic document of the test method comprises the following steps: reaction time of magnetic beads, sample and detection antibody (35 min), amount of magnetic beads (25. mu.L), amount of detection antibody (20. mu.L), amount of standard and sample (100. mu.L), amount of SBG (100. mu.L) and reaction time (5 min). The default reaction time is set to be 35 min-5 min, and the default method is a Simoa 2.0 two-step method. The standard concentration can be custom set, with a default setting of (1000, 333, 111, 37, 12.3, 4.11, 1.37, 0 pg/mL).
Then, the concentration was 2 × 107Magnetic beads/mL, detection antibody at a concentration of 1. mu.g/mL, SBG at a concentration of 150 pmol/L, fluorogenic substrate RGP at a concentration of 100. mu. mol/L and 96-well plate with standards and samples added were loaded into the analyzer at the indicated positions while scanning the label and setting the positional parameters.
Finally, the test was run. After the test is finished, a test result is derived, the instrument automatically fits a curve and calculates a sample result, as shown in fig. 1, the graph 1 shows that the concentration of EPHA2 and AEB have a good linear relation, and the lower detection limit (0.12 pg/mL) can be calculated according to a fitting equation, so that a foundation is laid for quantitatively detecting EPHA2 in blood. In addition, the serum dilution curve obtained by using the EPHA2 Simoa kit is shown in FIG. 2, and it can be seen from FIG. 2 that the kit can quantitatively detect serum EPHA2 with different dilution degrees, and can also be realized when only a small amount of sample (4 μ L) is available.
The fitting equation is: a four parameter Logistic curve fitting equation: y = (A-D)/[1+ (x/C)^B]+ D, wherein a =0.02, B =1.3, C =21997.5, D = 493.0.
The application of the Simoa kit in detecting the content of EPHA2 in human serum also belongs to the protection scope of the invention.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A Simoa kit of high sensitivity tumor marker EPHA2, characterized in that: the Simoa kit comprises capture antibody coated magnetic beads, EPHA2 standard, biotinylated detection antibody, SBG solution, fluorogenic substrate solution, and sample diluent.
2. The Simoa kit of the highly sensitive tumor marker EPHA2 according to claim 1, wherein the magnetic beads coated with the capture antibodies are prepared by the following steps:
(1) carrying out liquid change treatment on the capture antibody through an ultrafiltration tube;
(2) 2.7 μm magnetic beads were activated with EDC;
(3) mixing the capture antibody processed in the step (1) with the activated magnetic beads processed in the step (2) and incubating for 2-3h at 4 ℃;
(4) and (4) washing the product incubated in the step (3) twice by using a PBST solution, then sealing by using a sealing solution at 37 ℃, finally washing twice by using a magnetic bead diluent to obtain magnetic beads coated with the capture antibody, and storing in the magnetic bead diluent.
3. The Simoa kit of the highly sensitive tumor marker EPHA2 according to claim 2, wherein: the capture antibody in the step (1) is a mouse monoclonal antibody; the buffer used in the ultrafiltration tube was 50 mmol/L morpholine ethanesulfonic acid buffer, pH = 6.2.
4. The Simoa kit of the highly sensitive tumor marker EPHA2 according to claim 2, wherein the reaction concentration of the capture antibody in step (3) is 0.1-0.2 mg/mL, and the reaction concentration of the activated magnetic beads is 1.2 × 109/mL。
5. The Simoa kit of the high sensitivity tumor marker EPHA2 according to claim 2, wherein: the PBST solution in the step (4) is 10mmol/L PBS +1% Tween20, and the pH = 7.4; blocking solution is 10mmol/L PBS +1% BSA, pH = 7.4; the magnetic bead dilution was 50 mmol/L Tris-HCl + 10mmol/L EDTA +0.1% Tween20 +1% BSA, pH = 7.4.
6. The Simoa kit of the highly sensitive tumor marker EPHA2 according to claim 1, wherein the biotinylated detection antibody is prepared by: and (2) carrying out liquid exchange treatment on the detection antibody by using a PBS solution through an ultrafiltration tube, adding NHS-Biotin or a derivative thereof at room temperature, reacting for 30 min to obtain a biotinylated detection antibody, and purifying by using the ultrafiltration tube to obtain the biotinylated detection antibody, wherein the detection antibody is a rabbit polyclonal antibody.
7. The Simoa kit of highly sensitive tumor marker EPHA2 according to claim 6, wherein: the quantity ratio of the detection antibody to the reaction substance of the NHS-Biotin or the derivative thereof is 1: 40; PBS solution was 10mmol/L phosphate buffer, pH = 7.4.
8. The Simoa kit of the highly sensitive tumor marker EPHA2 according to claim 1, wherein: the SBG solution is diluted from the original concentration to the concentration of 150 pmol/L by enzyme diluent, wherein the enzyme diluent is 10 mmol/LPBS + 0.5% fetal bovine serum +1 mmol/L MgCl2And the pH =7.4, the EPHA2 standard is prepared into 1 mL by a sample diluent, and the other standards can be prepared by self-dilution according to requirements, wherein the sample diluent is 10mmol/L PBS + 5mmol/L EDTA +0.1% Tween20 + 2% BSA, the fluorescent substrate is resorufin- β -galactoside, and the concentration of the fluorescent substrate solution is 100 mu mol/L.
9. Use of the high sensitivity Simoa kit of the tumor marker EPHA2 according to any one of claims 1 to 8 for the preparation of a cancer diagnostic reagent.
10. The use of the high sensitivity Simoa kit of the tumor marker EPHA2 according to any one of claims 1 to 8 as a kit for detecting the EPHA2 content in human serum, characterized in that: the detection limit of the kit is 0.12 pg/mL.
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