CN112379092A - Autoantibody joint detection immunochemiluminescence kit for early lung cancer screening and preparation method thereof - Google Patents
Autoantibody joint detection immunochemiluminescence kit for early lung cancer screening and preparation method thereof Download PDFInfo
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Abstract
The invention relates to an autoantibody joint detection immunochemiluminescence kit for screening early lung cancer and a preparation method thereof, belonging to the technical field of in vitro diagnosis of tumor-related markers. Solves the defect that the prior lung cancer imaging diagnosis technology has false nodules. The kit of the invention comprises: streptavidin magnetic beads; an acridinium ester-labeled antibody or antigen; a biotin-labeled antigen. The kit can be used for supplementing and detecting autoantibodies of P53, MAGEA1, GAGE7, CAGE, GBU4-5, SOX2 and PGP9.5 in blood of a patient to evaluate the risk of lung cancer aiming at the problem that pseudonodules appearing in lung cancer imaging (LDCT) screening cannot be identified. The method can improve the detection sensitivity and specificity of early lung cancer, and is an important auxiliary means for screening lung cancer.
Description
Technical Field
The invention belongs to the technical field of in-vitro diagnosis of tumor-related markers, and particularly relates to an autoantibody joint detection immunochemiluminescence kit for screening early lung cancer and a preparation method thereof.
Background
Lung cancer is the leading place on the leaderboard of cancer incidence. The 5-year survival rate of patients is low because lung cancer spreads early and is usually detected late. The most effective method for improving the survival rate of lung cancer is early discovery and early treatment. Lung cancer screening technologies include imaging and tumor markers, and as chest Computed Tomography (CT), especially low dose thin layer CT (ldct), screening projects are widely developed in china, more and more asymptomatic lung vitreous nodules (GGNs) are discovered. The mass detection of the false nodules is a problem to be solved urgently by LDCT screening, and other auxiliary means are needed for supplement. The blood tumor related markers of lung cancer mainly comprise neuron-specific enolase (NSE), squamous cell carcinoma antigen (SCC), carcinoembryonic antigen (CEA), cytokeratin 19 fragment (CYFRA21-1) and gastrin release precursor (ProGRP), but the markers have increased positive rate in the late stage of lung cancer and are insensitive in the early stage of lung cancer. The research finds that the lung cancer autoantibody can be found 5 years before clinical diagnosis of the lung cancer, and a domestic research on the autoantibody of early lung cancer blood biomarkers comprising P53, melanoma antigen (MAGEA1), G antigen (GAGE7), tumor-associated gene (CAGE), ATP-binding RNA helicase (GBU4-5), sex-determining gene family member 2(SOX2) and protein gene product 9.5(PGP9.5) is included in 818 cases of lung cancer patients in total, and the result shows that the concentration of the antibody in the lung cancer group is higher than that in a benign lung disease group, the total sensitivity reaches 61%, and the specificity reaches 90%.
The use of acridinium esters in immunochemiluminescence assays began in 1979 and can be divided into two main groups: amides and DMAE have similar luminescence properties. The chemical luminescence of the acridinium ester is of a flash type, the intensity of the emitted light reaches the maximum after 0.4s of the starter is added, the half-life period is 0.9s, the luminescence is basically finished within 2s, and the rapid detection can be realized. The acridinium ester labeling process is simple, the labeling reaction is completed in one step, the light-emitting process can directly emit light only by oxidizing hydrogen under an alkaline condition, and other light-emitting catalysts are not needed. Streptavidin is a protein secreted by streptomyces that specifically binds to biotin, with the strongest non-covalent binding known to date. Streptavidin exists in a homotetramer form, and each mole of tetramer molecules can be combined with four moles of biotin molecules, so that the streptavidin has a signal amplification effect in the field of immunoassay.
Disclosure of Invention
Aiming at the defects of pseudonodules in the existing lung cancer imaging diagnosis technology, the invention provides an autoantibody joint detection immunochemiluminescence kit for early lung cancer screening and a preparation method thereof.
In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:
the invention provides an autoantibody joint detection immunochemiluminescence kit for screening early lung cancer, which comprises:
streptavidin magnetic beads;
an acridinium ester-labeled antibody or antigen;
a biotin-labeled antigen;
the antibody is an anti-human IgG antibody, and the antigens are P53 antigen, MAGEA1 antigen, GAGE7 antigen, CAGE antigen, GBU4-5 antigen, SOX2 antigen or PGP9.5 antigen.
Preferably, the kit comprises a magnetic particle reaction solution R1, an acridinium ester marker reaction solution R2 and a biotin marker reaction solution R3;
the magnetic particle reaction solution R1 comprises streptavidin magnetic beads, a first surfactant, sodium chloride, a first buffer solution, a first preservative and a first protein stabilizer;
the acridinium ester marker reaction liquid R2 comprises an acridinium ester marked antigen, sodium chloride, a second buffer solution, a second preservative, a second surfactant and a second protein stabilizer;
alternatively, the acridinium ester label reaction liquid R2 comprises an acridinium ester-labeled anti-human IgG antibody, sodium chloride, a second buffer, a second preservative, a second surfactant, and a second protein stabilizer;
the biotin-labeled reaction solution R3 contains a biotin-labeled antigen, sodium chloride, a third buffer solution, a third preservative, a third surfactant, and a third protein stabilizer.
More preferably, in the magnetic particle reaction solution R1, the concentration of streptavidin magnetic beads is 0.01 wt% to 0.2 wt%; the particle size of the streptavidin magnetic bead is 0.1-5 μm.
More preferably, the acridinium ester label reaction liquid R2 contains the acridinium ester-labeled antigen or the acridinium ester-labeled anti-human IgG antibody at a concentration of 0.01 to 2. mu.g/mL, respectively; labeling antigen or anti-human IgG antibody and acridinium ester according to the molar concentration ratio of 1 (1-20); the acridine ester is N-hydroxysuccinimide (NHS) -acridine ester, and the structural formula is shown as the formula I:
in the formula I, R1Is composed of
R2And R3Independently selected from: H.
1-12, n ═ 1;
R4is composed of
n＝1-12；
R5Is composed of
More preferably, the concentration of the biotin-labeled antigen in the biotin-labeled reaction solution R3 is 0.05 to 5. mu.g/mL; labeling the antigen and biotin according to the molar concentration of 1 (1-50); the structural formula of biotin is shown as formula II or formula III:
in the formulas II and III, n is 1-13.
More preferably, the first, second and third buffers are independently selected from 2- (N-morpholino) ethanesulfonic acid (MES), piperazine-NN-bis (2-ethanesulfonic acid) (PIPES), sodium 3- (N-enkephalyl) -2-hydroxypropanesulfonate (MOPSO), Tris-hydrochloric acid, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bistris), Phosphate (PB) or 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), at respective pH's of 5.0 to 8.5 and respective concentrations of 10 to 600 mmoL/L; the pH is 5.0-8.5, the concentration of the first buffer solution in the magnetic particle reaction solution R1 is 10-600mmoL/L, the concentration of the second buffer solution in the acridinium ester marker reaction solution R2 is 10-600mmoL/L, and the concentration of the third buffer solution in the biotin marker reaction solution R3 is 10-600 mmoL/L.
More preferably, the first preservative, the second preservative and the third preservative are independently selected from the group consisting of NaN3And Proclin 300 or Proclin 950, wherein the mass concentration of the first preservative in the magnetic particle reaction liquid R1 is 0.01-1%, the mass concentration of the second preservative in the acridine ester marker reaction liquid R2 is 0.01-1%, and the mass concentration of the third preservative in the biotin marker reaction liquid R3 is 0.01-1%.
More preferably, the first surfactant, the second surfactant and the third surfactant are independently selected from tween, polyethylene glycol, poloxamer, triton or choline chloride, the mass concentration of the first surfactant in the magnetic particle reaction solution R1 is 0.01% -2%, the mass concentration of the second surfactant in the acridinium ester marker reaction solution R2 is 0.01% -2%, and the mass concentration of the third surfactant in the biotin marker reaction solution R3 is 0.01% -2%.
More preferably, the first protein stabilizer, the second protein stabilizer and the third protein stabilizer are independently selected from one or more of Bovine Serum Albumin (BSA), casein, bovine serum Gamma globulin (BGG), egg protein and fish protein, and the mass concentration is 0.1-5%; the mass concentration of the first protein stabilizer in the magnetic particle reaction liquid R1 is 0.1-5%, the mass concentration of the second protein stabilizer in the acridine ester marker reaction liquid R2 is 0.1-5%, and the mass concentration of the third protein stabilizer in the biotin marker reaction liquid R3 is 0.1-5%.
The invention also provides a preparation method of the autoantibody joint detection immunochemiluminescence kit for screening early lung cancer, which comprises the following steps:
step one, preparation of magnetic particle reaction liquid R1
Mixing a first surfactant, sodium chloride, a first buffer solution, a first preservative and a first protein stabilizer to prepare an R1 basic buffer solution;
washing streptavidin magnetic beads with PBS (phosphate buffer solution) with pH7.2, and then resuspending the magnetic beads into R1 basic buffer solution to prepare magnetic particle reaction solution R1;
step two, preparation of acridinium ester marker reaction liquid R2
Mixing sodium chloride, a second buffer solution, a second preservative, a second surfactant and a second protein stabilizer to prepare an R2 basic buffer solution;
placing an acridinium ester labeled antigen or an acridinium ester labeled anti-human IgG antibody in an R2 basic buffer solution to prepare an acridinium ester labeled reactant reaction solution R2;
step three, preparation of biotin marker reaction liquid R3
Mixing sodium chloride, a third buffer solution, a third preservative, a third surfactant and a third protein stabilizer to prepare an R3 basic buffer solution;
and placing the biotin-labeled antigen in an R3 basic buffer solution to prepare a biotin-labeled reactant R3.
The detection principle of the autoantibody joint detection immunochemiluminescence kit for screening early lung cancer of the invention is as follows:
(1) principle of double antigen sandwich method
The kit comprises streptavidin magnetic particles, acridinium ester labeled antigen and biotin labeled antigen; as shown in FIG. 1, the reaction epitopes of the acridinium ester labeled antigen and the biotin labeled antigen are different from those of the autoantibody, the acridinium ester labeled antigen and the biotin labeled antigen are immunoreactive with the autoantibody in the sample, and are further fixed on the surface of magnetic beads through a biotin-streptavidin connecting bridge, the magnetic beads are fixed in a specific area of a reaction cup under the magnetic field of a chemiluminescence analyzer, after the interfering substances are cleaned, the acridinium ester is excited by acid and alkali to emit light, and the content of the autoantibody in the sample is in direct proportion to the Relative Light Unit (RLU) detected by the system.
(2) Principle of indirect method
The kit comprises streptavidin magnetic particles, an acridinium ester labeled anti-human IgG antibody and a biotin labeled antigen; as shown in FIG. 2, acridinium ester labeled anti-human IgG antibody and biotin labeled antigen are immunoreactive with autoantibodies in a sample, and are further immobilized on the surface of magnetic beads through a biotin streptavidin connecting bridge, the magnetic beads are immobilized in a specific area of a reaction cup under the magnetic field of a chemiluminescence analyzer, after interfering substances are washed away, the acridinium ester is excited by acid and alkali to emit light, and the content of the autoantibodies in the sample is in direct proportion to Relative Light Units (RLU) detected by the system.
Compared with the prior art, the invention has the beneficial effects that:
the autoantibody joint detection immunochemiluminescence kit for screening early lung cancer is based on acridinium ester chemiluminescence and a streptavidin magnetic bead-biotin amplification reaction system, the test principle can be a double-source sandwich method or an indirect method, a chemiluminescence analyzer is used for sample detection, and qualitative analysis of a detected object can be realized according to a standard curve of acridinium ester luminescence intensity (RLU) and the concentration of the detected object.
The autoantibody joint detection immunochemiluminescence kit for screening early lung cancer aims at the problem that false nodules appearing in lung cancer imaging (LDCT) screening cannot be identified, and is used for supplementing and detecting autoantibodies of P53, MAGEA1, GAGE7, CAGE, GBU4-5, SOX2 and PGP9.5 in blood of a patient to carry out risk assessment on lung cancer. The method can improve the detection sensitivity and specificity of early lung cancer, and is an important auxiliary means for screening lung cancer.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of the detection principle of the combined detection of autoantibodies and immunochemiluminescence kit for early stage lung cancer screening according to the present invention by a double antigen sandwich method;
FIG. 2 is a schematic diagram of the detection principle of the detection by indirect kit of the autoantibody joint detection immunochemiluminescence kit for early lung cancer screening of the present invention;
in the figure, the position of the upper end of the main shaft,
which represents a magnetic particle of streptavidin and is,
an antigen that represents a biotin label, and a biotin label,
indicates an autoantibody (analyte),
represents an acridinium ester-labeled antigen,
an acridinium ester-labeled anti-human IgG antibody is shown.
Detailed Description
For a further understanding of the invention, preferred embodiments of the invention are described below in conjunction with the detailed description, but it is to be understood that the description is intended to further illustrate the features and advantages of the invention and not to limit the claims to the invention.
The invention discloses an autoantibody joint detection immunochemiluminescence kit for screening early lung cancer, which comprises: streptavidin magnetic beads, acridinium ester labeled antibodies or antigens and biotin labeled antigens. Wherein the antibody is anti-human IgG antibody, and the antigens are P53 antigen, MAGEA1 antigen, GAGE7 antigen, CAGE antigen, GBU4-5 antigen, SOX2 antigen or PGP9.5 antigen. Preferably, the reagent comprises magnetic particle reaction liquid R1, acridinium ester marker reaction liquid R2 and biotin marker reaction liquid R3; the magnetic particle reaction solution R1 comprises streptavidin magnetic beads, a first surfactant, sodium chloride, a first buffer solution, a first preservative and a first protein stabilizer; the acridinium ester marker reaction liquid R2 comprises an acridinium ester labeled antigen, sodium chloride, a second buffer solution, a second preservative, a second surfactant and a second protein stabilizer; alternatively, the acridinium ester label reaction solution R2 comprises an acridinium ester labeled anti-human IgG antibody, sodium chloride, a second buffer solution, a second preservative, a second surfactant and a second protein stabilizer; the biotin-labeled reaction solution R3 contains a biotin-labeled antigen, sodium chloride, a third buffer solution, a third preservative, a third surfactant, and a third protein stabilizer.
In the technical scheme, the concentration of streptavidin magnetic beads in the magnetic particle reaction solution R1 is preferably 0.01-0.2 wt%; the preferred particle size of the streptavidin magnetic beads is 0.1-5 μm, and the particle size is 1-3 μm.
In the above technical scheme, the concentrations of the acridinium ester-labeled antigen or acridinium ester-labeled anti-human IgG antibody in the acridinium ester-labeled reaction liquid R2 are 0.01-2 μ g/mL, respectively; labeling antigen or anti-human IgG antibody and acridinium ester according to the molar concentration ratio of 1 (1-20); the acridine ester is N-hydroxysuccinimide (NHS) -acridine ester, and the structural formula is shown as the formula I:
in the formula I, R1Is composed of
R2And R3Independently selected from: H.
1-12, n ═ 1;
R4is composed of
n＝1-12；
R5Is composed of
In the technical scheme, the concentration of the biotin-labeled antigen in the biotin-labeled reaction solution R3 is 0.05-5 mug/mL; labeling the antigen and biotin according to the molar concentration of 1 (1-50); the structural formula of biotin is shown as formula II or formula III:
formula II or formula III, n is 1-13.
In the technical scheme, the first buffer solution, the second buffer solution and the third buffer solution are independently selected from 2- (N-morpholine) ethanesulfonic acid (MES), piperazine-NN-bis (2-ethanesulfonic acid) (PIPES), 3- (N-enkephalyl) -2-hydroxypropanesulfonic acid sodium salt (MOPSO), Tris-hydrochloric acid, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bistris), Phosphate (PB) and 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES), the pH values are respectively 5.0-8.5, and the concentrations in the respective reaction solutions are respectively 10-600 mmoL/L.
In the above technical solution, the first preservative, the second preservative and the third preservative are independently selected from NaN3Proclin 300 or Proclin 950, concentrated in massThe degrees are respectively 0.01-1%.
In the technical scheme, the first surfactant, the second surfactant and the third surfactant are independently selected from tween, polyethylene glycol, poloxamer, triton and choline chloride, and the mass concentration of the first surfactant, the second surfactant and the third surfactant in respective reaction liquid is 0.01-2%.
In the above technical scheme, the first protein stabilizer, the second protein stabilizer and the third protein stabilizer are independently selected from one or more of Bovine Serum Albumin (BSA), casein, bovine serum Gamma globulin (BGG), egg protein and fish protein, and the mass concentration of each of the first protein stabilizer, the second protein stabilizer and the third protein stabilizer in the respective reaction solution is 0.1% -5%.
According to the technical scheme, the molar concentration of sodium chloride in the magnetic particle reaction liquid R1, the acridinium ester marker reaction liquid R2 and the biotin marker reaction liquid R3 is 0.15 mol/L;
in the above technical solution, the kit may further include a calibrator, which is a reference substance used as an independent variable value in the calibration function, and includes two to more concentration levels for calibrating the detection items during the quantitative detection.
The preparation method of the autoantibody joint detection immunochemiluminescence kit for screening early lung cancer comprises the following steps:
step one, preparation of magnetic particle reaction liquid R1
Mixing a first surfactant, sodium chloride, a first buffer solution, a first preservative and a first protein stabilizer to prepare an R1 basic buffer solution;
washing streptavidin magnetic beads with PBS (phosphate buffer solution) with pH7.2, then resuspending the magnetic beads into R1 basic buffer solution to prepare R1 reaction solution with mass concentration of 0.01-0.2%, wherein the PBS buffer solution comprises 20mmol/L PB and 150mmol/L sodium chloride;
step two, preparation of acridinium ester marker reaction liquid R2
Mixing sodium chloride, a second buffer solution, a second preservative, a second surfactant and a second protein stabilizer to prepare an R2 basic buffer solution;
placing an acridinium ester labeled antigen or an acridinium ester labeled anti-human IgG antibody in an R2 basic buffer solution to prepare an acridinium ester labeled reactant reaction solution R2 with the concentration of 0.01-2 mu g/mL;
step three, preparation of biotin marker reaction liquid R3
And mixing sodium chloride, a third buffer solution, a third preservative, a third surfactant and a third protein stabilizer to prepare an R3 basic buffer solution.
Placing the biotin-labeled antigen in an R3 basic buffer solution to prepare a biotin-labeled reactant R3 with the concentration of 0.05-5 mug/mL.
Step four, preparation of calibration product
The calibrator is prepared according to the conventional method of those skilled in the art according to the kind of the analyte (autoantibody), such as P53, MAGEA1, GAGE7, CAGE, GBU4-5, SOX2 or PGP 9.5.
The detection method of the autoantibody joint detection immunochemiluminescence kit for early lung cancer screening, disclosed by the invention, specifically comprises the following steps of:
double antigen sandwich detection method 1
Firstly, adding a sample, biotin marker reaction liquid R3 and acridinium ester marker reaction liquid R2 (containing acridinium ester labeled antigen) into a reaction cup, and incubating for 0-25 min; secondly, adding magnetic particle reaction liquid R1 and incubating for 5-25 min; thirdly, adding cleaning fluid for cleaning; and fourthly, adding pre-excitation liquid (hydrogen peroxide and nitric acid solution) and excitation liquid (sodium hydroxide solution) into the reaction mixture to excite the luminescence reaction. The generated light quantum number is measured by a chemiluminescence analyzer, and the content of the measured object in the sample is in direct proportion to the light quantum number.
Double antigen sandwich detection method 2
Firstly, adding a sample, biotin marker reaction liquid R3 and magnetic particle reaction liquid R1 into a reaction cup, and incubating for 0-25 min; secondly, adding acridinium ester marker reaction liquid R2 (containing acridinium ester marker antigen) for incubation for 5-25 min; thirdly, adding cleaning fluid for cleaning; and fourthly, adding pre-excitation liquid (hydrogen peroxide and nitric acid solution) and excitation liquid (sodium hydroxide solution) into the reaction mixture to excite the luminescence reaction. The generated light quantum number is measured by a chemiluminescence analyzer, and the content of the measured object in the sample is in direct proportion to the light quantum number.
Indirect detection method
Firstly, adding a sample, a biotin marker reaction solution R3 and a magnetic particle reaction solution R1 into a reaction cup, and incubating for 0-25 min; secondly, adding cleaning fluid for cleaning; thirdly, adding acridinium ester marker reaction liquid R2 (acridinium ester marked anti-human IgG antibody) for incubation for 5-25 min; fourthly, adding cleaning fluid for cleaning; and fifthly, adding pre-excitation liquid (hydrogen peroxide and nitric acid solution) and excitation liquid (sodium hydroxide solution) into the reaction mixture to excite the luminescence reaction. The generated light quantum number is measured by a chemiluminescence analyzer, and the content of the measured object in the sample is in direct proportion to the light quantum number.
The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.
In order to make those skilled in the art better understand the technical solution of the present invention, the present invention will be further described in detail with reference to the following embodiments.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art. Materials, reagents, devices, instruments, apparatuses and the like used in the following examples are commercially available unless otherwise specified.
Example 1
1.1 composition of autoantibody joint detection immunochemiluminescence kit for early lung cancer screening
1.2 preparation method of autoantibody joint detection immunochemiluminescence kit for early lung cancer screening
Step one, preparation of magnetic particle reaction liquid R1
Mixing a first surfactant, sodium chloride, a first buffer solution, a first preservative and a first protein stabilizer to prepare an R1 basic buffer solution;
and (3) taking streptavidin magnetic beads with the particle size of 3 mu m, washing the streptavidin magnetic beads with PBS (phosphate buffer solution) with the pH value of 7.2 for three times, and then re-suspending the magnetic beads into R1 basic buffer solution to prepare magnetic particle reaction solution R1 with the mass concentration of the magnetic beads of 0.2%.
Step two, preparation of acridinium ester marker reaction liquid R2
Mixing sodium chloride, a second buffer solution, a second preservative, a second surfactant and a second protein stabilizer to prepare an R2 basic buffer solution;
taking the acridinium ester labeled antigen to prepare an acridinium ester labeled reactant reaction solution R2 with the antigen concentration of 0.2 mu g/mL in an R2 basic buffer solution.
Step three, preparation of biotin marker reaction liquid R3
Mixing sodium chloride, a third buffer solution, a third preservative, a third surfactant and a third protein stabilizer to prepare an R3 basic buffer solution;
the biotin-labeled antigen was taken out and placed in R3 base buffer to prepare a biotin-labeled reaction solution R3 with a concentration of 0.7. mu.g/mL.
1.3 detection method of autoantibody joint detection immunochemiluminescence kit for early lung cancer screening
Double antigen sandwich method 1: firstly, adding 50 mu L of sample, 60 mu L of biotin marker reaction solution R3 and 60 mu L of acridinium ester marker reaction solution R2 into a reaction cup, and incubating for 5 min; secondly, adding 50 mu L of magnetic particle reaction solution R1 and incubating for 10 min; thirdly, adding cleaning fluid for cleaning; and a fourth step of adding a pre-excitation liquid (hydrogen peroxide and nitric acid solution) and an excitation liquid (sodium hydroxide solution) to the reaction mixture to excite a luminescence reaction, and measuring the number of generated light quanta using a chemiluminescence analyzer.
Double antigen sandwich detection method 2
Double antigen sandwich method 2: firstly, adding 50 mu L of sample, 60 mu L of biotin marker reaction solution R3 and 60 mu L of magnetic particle reaction solution R1 into a reaction cup, and incubating for 15 min; secondly, adding acridinium ester marker reaction liquid R2 and incubating for 5 min; thirdly, adding cleaning fluid for cleaning; and a fourth step of adding a pre-excitation liquid (hydrogen peroxide and nitric acid solution) and an excitation liquid (sodium hydroxide solution) to the reaction mixture to excite a luminescence reaction, and measuring the number of generated light quanta using a chemiluminescence analyzer.
The assay of the kit of example 1 was >0.99 linear, > 5% reproducible and > 85% recovered.
Note: the antigens used in the kit are classified into P53, MAGEA1, GAGE7, CAGE, GBU4-5, SOX2 and PGP9.5 according to the detection of autoantibodies.
Example 2
1.1 composition of autoantibody joint detection immunochemiluminescence kit for early lung cancer screening
1.2 preparation method of autoantibody joint detection immunochemiluminescence kit for early lung cancer screening
Step one, preparation of magnetic particle reaction liquid R1
Mixing a first surfactant, sodium chloride, a first buffer solution, a first preservative and a first protein stabilizer to prepare an R1 basic buffer solution;
streptavidin magnetic beads with the particle size of 1 mu m are taken, washed three times by PBS buffer solution with the pH value of 7.2, and then the magnetic beads are resuspended in R1 basic buffer solution to prepare magnetic particle reaction solution R1 with the mass concentration of 0.01 percent.
Step two, preparation of acridinium ester marker reaction liquid R2
Mixing sodium chloride, a second buffer solution, a second preservative, a second surfactant and a second protein stabilizer to prepare an R2 basic buffer solution;
acridinium ester labeled anti-human IgG antibody is taken to be placed in R2 basic buffer solution to prepare acridinium ester labeled reactant reaction solution R2 with the antibody concentration of 1 mu g/mL.
Step three, preparation of biotin marker reaction liquid R3
Mixing sodium chloride, a third buffer solution, a third preservative, a third surfactant and a third protein stabilizer to prepare an R3 basic buffer solution;
the biotin-labeled antigen R3 basic buffer solution is taken to prepare biotin-labeled reactant R3 with the concentration of 4 mug/mL.
1.3 detection method of autoantibody joint detection immunochemiluminescence kit for early lung cancer screening double-antigen sandwich detection method 2
Firstly, adding 50 mu L of sample, 90 mu L of magnetic particle reaction solution R1 and 90 mu L of biotin marker reaction solution R3 into a reaction cup, and incubating for 20 min; secondly, adding 70 mu L of acridinium ester marker reaction liquid R2 into the reaction cup and incubating for 5 min; thirdly, adding cleaning solution and cleaning for five times; and fourthly, adding pre-excitation liquid (hydrogen peroxide and nitric acid solution) and excitation liquid (sodium hydroxide solution) into the reaction mixture to excite the luminescence reaction. And measuring the generated light quantum number, wherein the content of the measured object in the sample is in direct proportion to the light quantum number.
Indirect detection method
Firstly, adding a sample, biotin marker reaction liquid R3 and magnetic particle reaction liquid R1 into a reaction cup, and incubating for 0-25 min; secondly, adding cleaning fluid for cleaning; thirdly, adding acridinium ester marker reaction liquid R2 to incubate for 5-25 min; fourthly, adding cleaning fluid for cleaning; and fifthly, adding pre-excitation liquid (hydrogen peroxide and nitric acid solution) and excitation liquid (sodium hydroxide solution) into the reaction mixture to excite the luminescence reaction. The generated light quantum number is measured by a chemiluminescence analyzer, and the content of the measured object in the sample is in direct proportion to the light quantum number.
The assay of the kit of example 2 was >0.99 linear, > 5% reproducible and > 85% recovered.
Note: the antigens used in the kit are classified into P53, MAGEA1, GAGE7, CAGE, GBU4-5, SOX2 and PGP9.5 according to the detection of autoantibodies.
It should be understood that the above embodiments are only examples for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither necessary nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.
Claims (10)
1. An autoantibody combined detection immunochemiluminescence kit for early lung cancer screening, which is characterized by comprising:
streptavidin magnetic beads;
an acridinium ester-labeled antibody or antigen;
a biotin-labeled antigen;
the antibody is an anti-human IgG antibody, and the antigens are P53 antigen, MAGEA1 antigen, GAGE7 antigen, CAGE antigen, GBU4-5 antigen, SOX2 antigen or PGP9.5 antigen.
2. The autoantibody combined detection immunochemiluminescence kit for screening early lung cancer according to claim 1, wherein the kit comprises a magnetic particle reaction solution R1, an acridinium ester marker reaction solution R2 and a biotin marker reaction solution R3;
the magnetic particle reaction solution R1 comprises streptavidin magnetic beads, a first surfactant, sodium chloride, a first buffer solution, a first preservative and a first protein stabilizer;
the acridinium ester marker reaction liquid R2 comprises an acridinium ester marked antigen, sodium chloride, a second buffer solution, a second preservative, a second surfactant and a second protein stabilizer;
alternatively, the acridinium ester label reaction liquid R2 comprises an acridinium ester-labeled anti-human IgG antibody, sodium chloride, a second buffer, a second preservative, a second surfactant, and a second protein stabilizer;
the biotin-labeled reaction solution R3 contains a biotin-labeled antigen, sodium chloride, a third buffer solution, a third preservative, a third surfactant, and a third protein stabilizer.
3. The autoantibody joint detection immunochemiluminescence kit for screening early lung cancer according to claim 2, wherein in the magnetic particle reaction solution R1, the concentration of streptavidin magnetic beads is 0.01 wt% to 0.2 wt%; the particle size of the streptavidin magnetic bead is 0.1-5 μm.
4. The kit for combined detection of immune chemiluminescence for screening early lung cancer according to claim 2, wherein the concentration of the acridinium ester labeled antigen or acridinium ester labeled anti-human IgG antibody in the acridinium ester label reaction liquid R2 is 0.01-2 μ g/mL; labeling antigen or anti-human IgG antibody and acridinium ester according to the molar concentration ratio of 1 (1-20); the acridine ester is N-hydroxysuccinimide (NHS) -acridine ester, and the structural formula is shown as the formula I:
in the formula I, R1Is composed of
R2And R3Independently selected from: H.
1-12, n ═ 1;
R4is composed of
n＝1-12；
R5Is composed of
5. The kit for combined detection of immune chemiluminescence for screening early lung cancer according to claim 2, wherein the concentration of biotin-labeled antigen in the biotin-label reaction solution R3 is 0.05-5 μ g/mL; labeling the antigen and biotin according to the molar concentration of 1 (1-50); the structural formula of biotin is shown as formula II or formula III:
in the formulas II and III, n is 1-13.
6. The kit for detecting combined autoantibody and immunochemiluminescence for screening early lung cancer according to claim 2, the method is characterized in that the first buffer solution, the second buffer solution and the third buffer solution are independently selected from 2- (N-morpholine) ethanesulfonic acid, piperazine-NN-bis (2-ethanesulfonic acid), 3- (N-enkephaline) -2-sodium hydroxypropanesulfonate, Tris-hydrochloric acid, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane, phosphate or 4-hydroxyethyl piperazine ethanesulfonic acid, the pH values are 5.0-8.5 respectively, the concentration of the first buffer solution in the magnetic particle reaction solution R1 is 10-600mmoL/L, the concentration of the second buffer solution in the acridine ester marker reaction solution R2 is 10-600mmoL/L, and the concentration of the third buffer solution in the biotin marker reaction solution R3 is 10-600 mmoL/L.
7. The kit for combined detection of immune chemiluminescence for screening early lung cancer according to claim 2, wherein the first preservative, the second preservative and the third preservative are independently selected from NaN3And Proclin 300 or Proclin 950, wherein the mass concentration of the first preservative in the magnetic particle reaction liquid R1 is 0.01-1%, the mass concentration of the second preservative in the acridine ester marker reaction liquid R2 is 0.01-1%, and the mass concentration of the third preservative in the biotin marker reaction liquid R3 is 0.01-1%.
8. The kit of claim 2, wherein the first surfactant, the second surfactant and the third surfactant are independently selected from tween, polyethylene glycol, poloxamer, triton or choline chloride, the mass concentration of the first surfactant in the magnetic particle reaction solution R1 is 0.01% -2%, the mass concentration of the second surfactant in the acridinium ester marker reaction solution R2 is 0.01% -2%, and the mass concentration of the third surfactant in the biotin marker reaction solution R3 is 0.01% -2%.
9. The kit for combined detection of immunochemiluminometry of autoantibodies for screening early lung cancer according to claim 2, wherein the first protein stabilizer, the second protein stabilizer and the third protein stabilizer are independently selected from one or more of bovine serum albumin, casein, bovine serum Gamma globulin, egg protein, fish protein; the mass concentration of the first protein stabilizer in the magnetic particle reaction liquid R1 is 0.1-5%, the mass concentration of the second protein stabilizer in the acridine ester marker reaction liquid R2 is 0.1-5%, and the mass concentration of the third protein stabilizer in the biotin marker reaction liquid R3 is 0.1-5%.
10. The method for preparing the kit for detecting the combination of the autoantibody and the immunochemiluminescence for screening the early lung cancer according to any one of claims 2 to 9, comprising:
step one, preparation of magnetic particle reaction liquid R1
Mixing a first surfactant, sodium chloride, a first buffer solution, a first preservative and a first protein stabilizer to prepare an R1 basic buffer solution;
washing streptavidin magnetic beads with PBS (phosphate buffer solution) with pH7.2, and then resuspending the magnetic beads into R1 basic buffer solution to prepare magnetic particle reaction solution R1;
step two, preparation of acridinium ester marker reaction liquid R2
Mixing sodium chloride, a second buffer solution, a second preservative, a second surfactant and a second protein stabilizer to prepare an R2 basic buffer solution;
placing an acridinium ester labeled antigen or an acridinium ester labeled anti-human IgG antibody in an R2 basic buffer solution to prepare an acridinium ester labeled reactant reaction solution R2;
step three, preparation of biotin marker reaction liquid R3
Mixing sodium chloride, a third buffer solution, a third preservative, a third surfactant and a third protein stabilizer to prepare an R3 basic buffer solution;
and placing the biotin-labeled antigen in an R3 basic buffer solution to prepare a biotin-labeled reactant R3.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113702638A (en) * | 2021-08-24 | 2021-11-26 | 山东大学 | Based on CuInS2Chemiluminescence immunoassay kit for quantitatively detecting CEA antigen by adopting @ ZnS nanocrystalline |
| WO2024001044A1 (en) * | 2022-07-01 | 2024-01-04 | 广州市丹蓝生物科技有限公司 | Biomarker combination related to lung cancer, kit containing same, and use thereof |
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| CN103869086A (en) * | 2014-04-14 | 2014-06-18 | 杭州凯保罗生物科技有限公司 | Serum autoantibody detection kit |
| CN105548565A (en) * | 2015-12-30 | 2016-05-04 | 深圳市新产业生物医学工程股份有限公司 | Kit for detecting trypanosoma cruzi antibody as well as preparation and application thereof |
| CN110146692A (en) * | 2019-05-28 | 2019-08-20 | 迪瑞医疗科技股份有限公司 | One kind being based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin iodine system and detection kit |
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| CN103869086A (en) * | 2014-04-14 | 2014-06-18 | 杭州凯保罗生物科技有限公司 | Serum autoantibody detection kit |
| CN105548565A (en) * | 2015-12-30 | 2016-05-04 | 深圳市新产业生物医学工程股份有限公司 | Kit for detecting trypanosoma cruzi antibody as well as preparation and application thereof |
| CN110146692A (en) * | 2019-05-28 | 2019-08-20 | 迪瑞医疗科技股份有限公司 | One kind being based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin iodine system and detection kit |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113702638A (en) * | 2021-08-24 | 2021-11-26 | 山东大学 | Based on CuInS2Chemiluminescence immunoassay kit for quantitatively detecting CEA antigen by adopting @ ZnS nanocrystalline |
| CN113702638B (en) * | 2021-08-24 | 2024-02-27 | 山东大学 | CuInS-based 2 Chemiluminescent immunoassay kit for quantitatively detecting CEA antigen by adopting @ ZnS nanocrystals |
| WO2024001044A1 (en) * | 2022-07-01 | 2024-01-04 | 广州市丹蓝生物科技有限公司 | Biomarker combination related to lung cancer, kit containing same, and use thereof |
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