CN113533747A - Simoa kit of biomarker CRP and use method thereof - Google Patents
Simoa kit of biomarker CRP and use method thereof Download PDFInfo
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- CN113533747A CN113533747A CN202110841139.9A CN202110841139A CN113533747A CN 113533747 A CN113533747 A CN 113533747A CN 202110841139 A CN202110841139 A CN 202110841139A CN 113533747 A CN113533747 A CN 113533747A
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Abstract
The invention relates to a single-molecule array detection (Simoa) kit of a biomarker CRP in an immunoassay technology and a using method thereof. The Simoa kit is used for detecting the content of acute-phase reactive protein (CRP) in human body fluid based on a Simoa platform monomolecular array technology and a double-antibody sandwich method, and comprises magnetic beads coated with capture antibodies, CRP standard products, biotinylated detection antibodies, SBG solution, fluorogenic substrate solution and sample diluent. The sensitivity of the Simoa kit reaches 0.06pg/mL, the sample loading amount only needs 1 mu L, the repeatability is good (CV is less than 10%), the detection range is 0.1-1000pg/mL, and the CRP content in human body fluid can be accurately detected.
Description
Technical Field
The invention relates to the technical field of immunodetection, in particular to a Simoa kit of a biomarker CRP and a using method thereof.
Background
The acute phase reaction Protein (CRP) is synthesized by the induction of interleukin 6(IL-6) in the liver, the CRP content is very low under the physiological state, but the IL-6 is actively expressed and greatly secreted in the process of forming and infiltrating tumors, and under the stimulation of IL-6 which is an inflammatory factor, the CRP content in normal serum is 0-5 mug/mL, the CRP serum level of a lung cancer patient is far higher than that of a healthy person, and the CRP level is rapidly reduced along with the improvement of the disease condition after chemotherapy or other treatment, so the CRP can be used as an effective biomarker for clinical diagnosis and treatment guidance.
The CRP detection method used clinically at present mainly comprises immunodiffusion, latex agglutination, immunoturbidimetry and immunochromatography, and the immunodiffusion, latex agglutination, immunoturbidimetry and immunochromatography can only be qualitative and not quantitative, and can only be used as an auxiliary means for diagnosis. The enzyme-linked immunosorbent assay has low sensitivity, poor repeatability, poor quantification accuracy, long operation time and low automation degree.
Single molecule array (Simoa) technology, also known as digital ELISA, is a magnetic bead-based ultrasensitive detection method for proteins. In a traditional double antibody sandwich ELISA, the fluorescent product of the enzyme-substrate reaction diffuses to a large volume of about 50-100 μ L, relying on millions of enzyme-labeled immune complexes to generate a measurable fluorescent signal above background. In digital ELISA, the fluorescent product of the enzyme-substrate reaction is confined in 46-femtoliter (fL) sized microwells, which are referred to as single molecule arrays (Simoa). Using this method, the presence of a single enzyme molecule can be detected. In Simoa immunoassays, antibody-coated capture beads are added in excess to a sample containing a low concentration of target protein molecules. Based on the poisson distribution principle, one or zero target protein molecules will bind to each magnetic bead. Magnetic beads that bind more than one protein molecule are negligible. The magnetic beads are then incubated with biotinylated detection antibody and streptavidin-beta-galactosidase to form an enzyme-labeled immunocomplex. Next, the beads were loaded onto a 46fL microwell array, where each well was capable of holding only one bead. The microwells were filled with fluorogenic substrate and sealed with perfluorooil. A bead containing a single immunocomplex will result in a large number of substrate molecules being enzymatically catalyzed to produce a large number of fluorescent products. Since the fluorescent product is limited to a very small volume of about 46fL, the fluorescence imaging of a single micropore can be realized by combining the fluorescence microscopic imaging technology, so that the detection of a single protein molecule is realized. In this way, Simoa can quantify protein concentration using a digital readout mode and achieve a detection range of over 4 orders of magnitude.
Therefore, the development of the CRP detection method which is high in sensitivity, high in automation degree, rapid, good in accuracy and suitable for popularization based on the Simoa technology has great significance for rapidly detecting the biomarker CRP.
Disclosure of Invention
A Simoa kit for a biomarker CRP, comprising a capture antibody coated magnetic bead, a CRP standard, a biotinylated detection antibody, a SBG solution, a fluorogenic substrate solution, and a sample diluent.
The technical scheme of the invention is realized as follows:
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 monoclonal antibody L30601; the buffer used for ultrafiltration was 50-60mmol/mL morpholine ethanesulfonic acid buffer at pH 6.2-6.4.
The reaction concentration of the capture antibody in the step (3) is 0.1-0.2 mg/mL; the reaction concentration of the activated magnetic beads is 0.5-2.1X 109/mL。
The PBST solution in the step (4) is 10-12mmol/L PBS + 1-1.5% Tween20, and the pH value is 7.4-7.9; the blocking solution is 10-16mmol/L PBS + 1-1.2% BSA, and the pH value is 7.4-8.2; the magnetic bead diluent is 50-60mmol/L Tris-HCl +10-13mmol/L EDTA + 0.1-0.3% Tween20+ 1-1.9% BSA, and the pH value is 7.4-9.0.
The preparation method of the biotinylated detection antibody comprises the following steps: and (2) carrying out liquid change 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 30-45min, and carrying out ultrafiltration purification to obtain a biotinylated detection antibody, wherein the detection antibody is a monoclonal antibody L30602.
The quantitative ratio of the detection antibody to the reaction substance of the NHS-Biotin or the derivative thereof is 1:30-1:50, the PBS solution is 10-30mmol/L phosphate buffer solution, and the pH value is 7.4-9.0.
The concentration of the SBG solution is 50-400pmol/L, and the SBG buffer solution is 10-13mmol/L PBS + 0.5% BSA +1-2mmol/L MgCl2The pH value is 7.4-8; the fluorogenic substrate is resorufin-beta-galactoside, and the concentration of the fluorogenic substrate solution is 100-120 mu mol/L.
A method for using the Simoa kit of the biomarker CRP in the preparation of diagnostic reagents.
The use method of the Simoa kit of the biomarker CRP in detecting CRP content in human body fluid.
The invention has the following beneficial effects:
the Simoa kit for detecting CRP content in human body fluid based on the Simoa monomolecular array technology platform and the double-antibody sandwich method provided by the invention can quantitatively detect CRP content in human body fluid by taking the monoclonal antibody L30601 as a capture antibody and the monoclonal antibody L30602 as a detection antibody;
the complete on-machine detection reagent is provided, only a sample needs to be processed or an instrument needs to be automatically diluted, the operation is simple and convenient, the manual error is reduced, the repeatability is high, and the accuracy of clinical diagnosis is improved;
the detection can be simultaneously carried out on 288 samples at most at one time, and the detection time is shortened by two times compared with the detection time of similar products, namely, one sample is detected every 2 min. A great deal of time is saved;
the minimum detection limit is 0.06pg/mL, and the low-level CRP content can be accurately detected;
the sample amount is low, and 1 mu L of sample can be detected;
the detection range is 0.1-1000pg/mL, the detection range is wider, and the application range is wide;
as can be seen from FIG. 1, the minimum detection limit of the present application can reach 0.06 pg/mL.
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 CRP Simoa kit of 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 Simoa kit for detecting CRP content in human body fluid provided by the invention comprises 2.7 mu m magnetic beads coated by capture antibodies, CRP standard substances, biotinylated detection antibodies, SBG solution, fluorogenic substrate solution, sample diluent and test method electronic files.
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 monoclonal antibody L30601, and the detection antibody is monoclonal antibody L30602.
The preparation process of the capture antibody coated magnetic bead comprises the following steps: first, the stored antibody was subjected to a solution exchange treatment through an ultrafiltration tube using MES as a buffer solution of 50 to 60mmol/mL morpholine ethanesulfonic acid at pH 6.2 to 6.4.
Then, the magnetic beads were activated with EDC and the capture antibody and activated magnetic beads were incubated for 2h at 4 ℃. And then washing with PBST twice, blocking with blocking solution at 37 ℃, finally washing with magnetic bead diluent twice, and storing in the magnetic bead diluent to obtain the magnetic beads coated with the capture antibody. It is composed ofThe reaction concentration of the middle capture antibody is 0.1-0.2 mg/mL; the reaction concentration of the activated magnetic beads is 0.5-2.1X 109/mL。
The PBST formula comprises: 10-12mmol/L PBS + 1-1.5% Tween20, pH 7.4-7.9; the formula of the sealing liquid is as follows: 10-16mmol/L PBS + 1-1.2% BSA, pH 7.4-8.2. The magnetic bead diluent is 50-60mmol/L Tris-HCl +10-13mmol/LEDTA + 0.1-0.3% Tween20+ 1-1.9% BSA, and the pH value is 7.4-9.0.
The preparation process of the biotinylation detection antibody 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: 10-30mmol/L Phosphate Buffered Saline (PBS), pH 7.4-9.0. Wherein the quantity ratio of the detection antibody to the reaction substance of the NHS-Biotin or the derivative thereof is 1:30-1: 50.
Preparation of enzyme and substrate solutions: the concentration of the SBG solution is 50-400pmol/L, and the SBG buffer solution is: 10-13mmol/LPBS + 0.5% BSA +1-2mmol/L MgCl2，pH＝7.4-8.0；
The final concentrations are all working concentrations for final imaging.
The substrate is: resorufin-beta-galactoside (RGP)
Preparing a standard solution: the 100ng/mL CRP standard was formulated to 1mL with sample dilutions. The other standard products can be prepared according to the needs.
The use method of the CRP Simoa kit comprises the following steps:
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 electronic document of the test method comprises the following steps: reaction time of magnetic beads, sample and detection antibody (35min), 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 35min-5min, 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, 37.0, 12.3, 4.12, 1.37, 0 pg/mL).
Then, the concentration was 2X 107Magnetic beads/mL, detection antibody at a concentration of 1-2. mu.g/mL, SBG at a concentration of 50-400pmol/L, fluorogenic substrate RGP at a concentration of 100-120. mu. mol/L and 96-well plate with standard and sample added were loaded into the analyzer at the indicated positions while scanning the label and setting positional parameters.
Finally, the test was run. After the experimental test is finished, the test result is derived, the instrument automatically fits the curve and calculates the sample result, as shown in fig. 1.
The fitting equation is: a four parameter Logistic curve fitting equation: y ═ a-D/[ 1+ (x/C)^B]+D。
The application method of the Simoa kit in detecting CRP content in human body fluid 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 for the biomarker CRP, characterized in that: the Simoa kit comprises capture antibody coated magnetic beads, CRP standards, biotinylated detection antibody, streptavidin-beta-galactosidase (SBG) solution, fluorogenic substrate solution, and sample diluent.
2. The Simoa kit for biomarker CRP according to claim 1, wherein the magnetic beads coated with capture antibodies are prepared by the method comprising:
(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 the temperature of 2-8 ℃;
(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 for the biomarker CRP according to claim 2, characterized in that: the capture antibody in the step (1) is a monoclonal antibody L30601; the buffer used for ultrafiltration was 50-60mmol/L morpholine ethanesulfonic acid buffer at pH 6.0-6.4.
4. The Simoa kit for the biomarker CRP according to claim 2, characterized in that: the reaction concentration of the capture antibody in the step (3) is 0.1-0.2 mg/mL; the reaction concentration of the activated magnetic beads is 0.5-2.1X 109/mL。
5. The Simoa kit for the biomarker CRP according to claim 2, characterized in that: the PBST solution in the step (4) is 10-12mmol/L PBS + 1-1.5% Tween20, and the pH value is 7.4-7.9; the blocking solution is 10-16mmol/L PBS + 1-1.2% BSA, and the pH value is 7.4-8.2; the magnetic bead diluent is 50-60mmol/L Tris-HCl +10-13mmol/L EDTA + 0.1-0.3% Tween20+ 1-1.9% BSA, and the pH value is 7.4-9.0.
6. The Simoa kit for the biomarker CRP of claim 1, wherein the biotinylated detection antibody is prepared by: and (2) carrying out liquid change 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 30-45min, and carrying out ultrafiltration purification to obtain a biotinylated detection antibody, wherein the detection antibody is a monoclonal antibody L30602.
7. The Simoa kit for the biomarker CRP according to claim 6, characterized in that: the quantitative ratio of the detection antibody to the reaction substance of the NHS-Biotin or the derivative thereof is 1:30-1: 50; the PBS solution is 10-30mmol/L phosphate buffer solution, and the pH value is 7.4-9.0.
8. The Simoa kit for the biomarker CRP according to claim 1, characterized in that: preparation of enzyme and substrate solutions: the concentration of the SBG solution is 50-400pmol/L, and the SBG buffer solution is 10-13mmol/L PBS + 0.5% BSA +1-2mmol/L MgCl2The pH value is 7.4-8.0; the fluorogenic substrate is resorufin-beta-galactoside, and the concentration of the fluorogenic substrate solution is 100-120 mu mol/L.
9. A method of using the Simoa kit for the biomarker CRP of any one of claims 1 to 8 in the preparation of a cancer diagnostic reagent.
10. Use of the Simoa kit for the biomarker CRP of any one of claims 1 to 8 for the detection of CRP levels in human body fluids.
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