CN114295827A - Magnetic particle acridinium ester chemiluminescence detection kit and preparation method and application thereof - Google Patents
Magnetic particle acridinium ester chemiluminescence detection kit and preparation method and application thereof Download PDFInfo
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- CN114295827A CN114295827A CN202111606999.0A CN202111606999A CN114295827A CN 114295827 A CN114295827 A CN 114295827A CN 202111606999 A CN202111606999 A CN 202111606999A CN 114295827 A CN114295827 A CN 114295827A
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- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 title claims abstract description 110
- 239000006249 magnetic particle Substances 0.000 title claims abstract description 79
- 238000002038 chemiluminescence detection Methods 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 102100035194 Placenta growth factor Human genes 0.000 claims abstract description 114
- 238000001514 detection method Methods 0.000 claims abstract description 81
- 101000595923 Homo sapiens Placenta growth factor Proteins 0.000 claims abstract description 76
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- 230000005284 excitation Effects 0.000 claims abstract description 62
- 238000000034 method Methods 0.000 claims abstract description 55
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- 238000002372 labelling Methods 0.000 claims abstract description 25
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- 239000007853 buffer solution Substances 0.000 claims description 34
- 201000011461 pre-eclampsia Diseases 0.000 claims description 33
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 29
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 26
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical group OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 20
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- 239000007987 MES buffer Substances 0.000 claims description 18
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- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 15
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- 108010082093 Placenta Growth Factor Proteins 0.000 claims description 11
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- 229910017604 nitric acid Inorganic materials 0.000 claims description 10
- 108010053096 Vascular Endothelial Growth Factor Receptor-1 Proteins 0.000 claims description 8
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- 238000011002 quantification Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
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- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 239000003513 alkali Substances 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 4
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- YSJGOMATDFSEED-UHFFFAOYSA-M behentrimonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCCCCCCCC[N+](C)(C)C YSJGOMATDFSEED-UHFFFAOYSA-M 0.000 claims description 3
- YIFWXQBNRQNUON-UHFFFAOYSA-M dodecyl(trimethyl)azanium;iodide Chemical compound [I-].CCCCCCCCCCCC[N+](C)(C)C YIFWXQBNRQNUON-UHFFFAOYSA-M 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- LGPJVNLAZILZGQ-UHFFFAOYSA-M hexadecyl(trimethyl)azanium;iodide Chemical compound [I-].CCCCCCCCCCCCCCCC[N+](C)(C)C LGPJVNLAZILZGQ-UHFFFAOYSA-M 0.000 claims description 3
- 230000035484 reaction time Effects 0.000 claims description 3
- CEYYIKYYFSTQRU-UHFFFAOYSA-M trimethyl(tetradecyl)azanium;chloride Chemical compound [Cl-].CCCCCCCCCCCCCC[N+](C)(C)C CEYYIKYYFSTQRU-UHFFFAOYSA-M 0.000 claims description 3
- 235000019270 ammonium chloride Nutrition 0.000 claims description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 2
- 239000004327 boric acid Substances 0.000 claims description 2
- 229940078916 carbamide peroxide Drugs 0.000 claims description 2
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- 238000011049 filling Methods 0.000 claims description 2
- 150000007529 inorganic bases Chemical class 0.000 claims description 2
- 150000007522 mineralic acids Chemical class 0.000 claims description 2
- 150000003242 quaternary ammonium salts Chemical group 0.000 claims description 2
- 239000011541 reaction mixture Substances 0.000 claims description 2
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 11
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- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
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- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 206010061452 Complication of pregnancy Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
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- 208000005107 Premature Birth Diseases 0.000 description 1
- IHDIPBYRHCSTKF-UHFFFAOYSA-N acridine sulfamide Chemical compound NS(N)(=O)=O.C1=CC=CC2=CC3=CC=CC=C3N=C21 IHDIPBYRHCSTKF-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 1
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- DDXLVDQZPFLQMZ-UHFFFAOYSA-M dodecyl(trimethyl)azanium;chloride Chemical compound [Cl-].CCCCCCCCCCCC[N+](C)(C)C DDXLVDQZPFLQMZ-UHFFFAOYSA-M 0.000 description 1
- 230000002222 downregulating effect Effects 0.000 description 1
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Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The embodiment of the invention relates to a magnetic particle acridinium ester chemiluminescence detection kit, a preparation method and a detection method thereof, relating to the field of detection, and comprising an sFlt-1 antibody detection reagent and/or a PIGF antibody detection reagent; also comprises a pre-excitation liquid and an excitation buffer liquid; the sFlt-1 antibody detection reagent comprises a first sFlt-1 antibody marked by acridinium ester and magnetic particles coated with a second sFlt-1 antibody; acridinium ester-labeled sFlt-1 antibodies have a first sFlt-1 antibody to acridinium ester molar ratio of 1: (5-15); the PIGF antibody detection reagent comprises a first PIGF antibody marked by acridinium ester and magnetic particles coated with a second PIGF antibody; the mole ratio of the first PIGF antibody to the acridinium ester in the acridinium ester labeled PIGF antibody is 1: (5-15). The method for labeling the coupling of the acridinium ester and the antibody is simple, has good repeatability, high coupling efficiency and strong luminescent signal, and is convenient for large-scale application. The method has the advantages of higher sensitivity and wide linear range.
Description
Technical Field
The invention relates to the field of detection, and in particular relates to a magnetic particle acridinium ester chemiluminescence detection kit, a preparation method of the magnetic particle acridinium ester chemiluminescence detection kit, a detection method of the magnetic particle acridinium ester chemiluminescence detection kit, and application of the magnetic particle acridinium ester chemiluminescence detection kit in preparation of a medicament for detecting early epilepsy or preeclampsia.
Background
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Preeclampsia is a common complication of pregnancy and currently has a global incidence of about 3-5%. Meanwhile, preeclampsia is one of the main causes of maternal mortality, and approximately 42% of maternal mortality is caused by preeclampsia. In addition, about 15% of premature births worldwide are also caused by preeclampsia.
The basic pathophysiological changes in preeclampsia are vascular endothelial cell damage and systemic arteriospasm, which may cause damage to various systems and organs of the whole body, resulting in a series of serious complications and serious threat to mother and infant health. At present, the treatment method of preeclampsia is mainly symptomatic treatment, and comprises spasmolysis, blood pressure reduction, diuresis, reasonable dilatation and termination of pregnancy; however, these methods do not fundamentally treat preeclampsia. Only termination of pregnancy can relieve the short-term harm of the disease to the mother. If the risk of preeclampsia can be identified in the early stage of pregnancy and high-risk patients can be prevented, it can be a way to reduce the adverse fate of pregnant and lying-in women.
Disclosure of Invention
Object of the Invention
The invention aims to provide a magnetic particle acridinium ester chemiluminescence detection kit, a preparation method and a detection method thereof, and the detection kit is used for establishing a detection method for quantitatively determining the content of soluble fms-like tyrosine kinase-1 (sFlt-1) and placenta growth factor (PlGF) in serum by utilizing magnetic particles and acridinium ester chemiluminescence immunoassay technology according to the experimental principle of a double-antibody sandwich method. The acridinium ester chemiluminescence immunoassay technology is very accurate quantitative detection, so that the detection accuracy is higher, the accuracy of general immunological detection is obviously lower than that of the acridinium ester chemiluminescence immunoassay technology, and the detection is long in time and inconvenient.
Solution scheme
In order to achieve the purpose of the invention, in a first aspect, the embodiment of the invention provides a magnetic particle acridinium ester chemiluminescence detection kit, which comprises an sFlt-1 antibody detection reagent and/or a PIGF antibody detection reagent; also comprises a pre-excitation liquid and an excitation buffer liquid;
the sFlt-1 antibody detection reagent comprises a first sFlt-1 antibody marked by acridinium ester and magnetic particles coated with a second sFlt-1 antibody; the molar ratio of sFlt-1 antibody to acridinium ester in the acridinium ester-labeled sFlt-1 antibody was 1: (5-15);
the first and second sFlt-1 antibodies recognize different epitopes of soluble fms-like tyrosine kinase-1, respectively;
the PIGF antibody detection reagent comprises a first PIGF antibody marked by acridinium ester and magnetic particles coated with a second PIGF antibody; the molar ratio of the PIGF antibody to the acridinium ester in the acridinium ester labeled PIGF antibody is 1: (5-15);
the first PIGF antibody and the second PIGF antibody respectively recognize different epitopes of the placenta growth factor;
the pre-excitation liquid comprises inorganic acid and an oxidant, and the pH value is less than 2;
the excitation buffer comprises an inorganic base and an enhancer, and the pH of the buffer is > 13;
the reinforcing agent is a quaternary ammonium salt cationic surfactant and is selected from one or more of octaalkyltrimethyl ammonium chloride, dodecyltrimethyl ammonium iodide, tetradecyltrimethyl ammonium chloride, hexadecyltrimethyl ammonium bromide, hexadecyltrimethyl ammonium iodide and docosyltrimethyl ammonium chloride; alternatively cetyltrimethylammonium bromide.
The molar ratio of the first sFlt-1 antibody or the first PIGF antibody to the acridinium ester of the methods of the present invention is 1: (5-15), detection of different antigens is realized, and the conditions of weak luminescent signals or nonspecific combination and the like can occur in improper proportioning, so that the detection sensitivity is low or the result is inaccurate. The molar ratio is an appropriate ratio range screened out through a series of experimental designs.
Further, in the acridinium ester-labeled first sFlt-1 antibody, the molar ratio of the first sFlt-1 antibody to acridinium ester is 1: 10.
further, in the acridinium ester-labeled first PIGF antibody, the molar ratio of the first PIGF antibody to the acridinium ester is 1: 10.
further, the acridinium ester labeled first sFlt-1 antibody also comprises a labeling buffer solution for diluting the acridinium ester labeled first sFlt-1 antibody; optionally the concentration of the acridinium ester-labeled first sFlt-1 antibody is 0.5ug/mL, optionally the labeling buffer is a phosphate buffer or a carbonate buffer, optionally 0.1mol/L carbonate buffer, pH 8.2.
Furthermore, the acridinium ester labeled first PIGF antibody also comprises a labeling buffer solution for fixing volume of the acridinium ester labeled first PIGF antibody; the concentration of the first PIGF antibody, which is optionally acridinium ester labeled, is 0.25ug/mL, the labeling buffer is optionally a phosphate buffer or a carbonate buffer, optionally a 0.1mol/L carbonate buffer, and the pH is 8.2.
Further, in the magnetic particles coated with the second sFlt-1 antibody, the mass ratio of the magnetic particles to the second sFlt-1 antibody is 100: (4-8); optionally, MES buffer solution for fixing the magnetic particles coated with the second sFlt-1 antibody is also included, and optionally, the concentration of the magnetic particles of the second sFlt-1 antibody is 0.5 mg/mL; alternatively, the MES buffer has a concentration of 0.02mol/L and a pH of 4.5 to 5.5.
Further, in the magnetic particles coated with the second PIGF antibody, the mass ratio of the magnetic particles to the second PIGF antibody is 100: (4-8); optionally, the kit further comprises an MES buffer solution for fixing the magnetic particles coated with the second PIGF antibody, and optionally, the concentration of the magnetic particles of the second PIGF antibody is 0.5 mg/mL; alternatively, the MES buffer has a concentration of 0.02mol/L and a pH of 4.5 to 5.5.
Further, in the pre-excitation liquid, the acid is nitric acid or hydrochloric acid or sulfuric acid or boric acid; the oxidant is hydrogen peroxide or carbamide peroxide; optionally, in the pre-excitation liquid, the concentration of the acid is 0.01-0.18M, and the mass concentration of the oxidant is 0.1-2.0%; further optionally, the pre-excitation liquid comprises 0.07M nitric acid and 0.6 wt% hydrogen peroxide, and the pH is 1.1.
Further, in the excitation buffer, the alkali is sodium hydroxide or potassium hydroxide; optionally, in the buffer solution, the concentration of the alkali is 0.2-0.8M, and the mass concentration of the enhancer is 0.2-2.0%;
further optionally, the buffer comprises 0.5M potassium hydroxide and 0.478 wt% cetyltrimethylammonium bromide, pH 13.5.
In a second aspect, a preparation method of the magnetic particle acridinium ester chemiluminescence detection kit is provided, and comprises the following steps:
a method for making an acridinium ester-labeled first sFlt-1 antibody comprises: adding a first sFlt-1 antibody and an acridinium ester solution into a labeling buffer solution, uniformly mixing, carrying out a light-shielding oscillation reaction at room temperature, dialyzing, and adding the labeling buffer solution for quantification to obtain an acridinium ester labeled first sFlt-1 antibody reagent;
a method for preparing magnetic microparticles coated with a second sFlt-1 antibody comprises: adding EDC aqueous solution into the magnetic particle suspension for 30-60min, magnetically separating and removing supernatant, re-suspending, adding a second sFlt-1 antibody for coating, and suspending for 16-20 h; magnetically separating the supernatant, and quantifying to obtain a magnetic particle reagent coated with a second sFlt-1 antibody;
the preparation method of the acridinium ester labeled first PIGF antibody comprises the following steps: adding a first PIGF antibody and an acridinium ester solution into a labeling buffer solution, uniformly mixing, carrying out a photophobic oscillation reaction at room temperature, dialyzing, and adding the labeling buffer solution for quantification to obtain an acridinium ester labeled first PIGF antibody reagent; preferably, the labeling buffer is 0.1mol/L carbonate buffer, pH 8.2;
the preparation method of the magnetic particle coated with the second PIGF antibody comprises the following steps: adding EDC aqueous solution into the magnetic particle suspension for 30-60min, magnetically separating and removing supernatant, re-suspending, adding a second PIGF antibody for coating, and suspending for 16-20 h; magnetically separating the supernatant, and quantifying to obtain a magnetic particle reagent coated with a second PIGF antibody;
further, the method for producing the acridinium ester-labeled first sFlt-1 antibody and the method for producing the acridinium ester-labeled first PIGF antibody independently include: the reaction time is 10-120 minutes under the condition of keeping out of the light and shaking at room temperature.
Further, the labeling buffer is phosphate buffer or carbonate buffer, optionally 0.1mol/L carbonate buffer, pH 8.2.
Further, the dialysis method comprises: filling the reaction mixture into a dialysis bag, dialyzing in the dark at the temperature of 2-8 ℃, and replacing the dialysate for 2 h/time, wherein the dialysate is phosphate buffer solution, and the concentration of the dialysate is 0.02 mol/L.
Further, the method for preparing magnetic microparticles coated with a second sFlt-1 antibody and the method for preparing magnetic microparticles coated with a second PIGF antibody independently include: adding 0.5-1mL of EDC aqueous solution with the concentration of 10mg/mL into each 100g of magnetic particles; optionally, the magnetic particle suspension is obtained by resuspending the magnetic particles with MES buffer; optionally, using MES buffer for resuspension or quantification; optionally, the concentration of MES buffer is 0.02mol/L, and the pH is 4.5-5.5; alternatively, the quantitative concentration is 0.5 mg/mL; optionally, in the magnetic particle suspension or the resuspension solution, 10mL of MES buffer solution is used for every 100g of magnetic particles; alternatively, the suspension is room temperature suspension.
The labeled buffer solution is phosphate buffer solution or carbonate buffer solution, and different buffer solution systems and the concentration and pH thereof have different influences on the luminous intensity of the acridinium ester.
Further, the time for the reaction of the antibody and the acridinium ester with shaking at room temperature and in the dark is 10-120 minutes. The length of the reaction time generally affects the degree of antibody coupling and the intensity of the luminescent signal, but as the time is prolonged, a higher false positive phenomenon occurs, and therefore, the optimal coupling time needs to be experimentally verified.
Further, the preparation method of the pre-excitation liquid comprises the following steps: mixing purified water, concentrated nitric acid and 30% hydrogen peroxide, fixing the volume, uniformly stirring, and filtering to obtain pre-excitation liquid; the pH of the pre-excitation liquid is 1.10, wherein the concentration of each component is as follows: 0.07M nitric acid and 0.6 wt% hydrogen peroxide.
Further, the preparation method of the excitation buffer comprises the following steps: dissolving purified water and a reinforcing agent, adding potassium hydroxide, dissolving, fixing the volume, and filtering to obtain an excitation buffer solution; optionally, the pH of the excitation buffer is 13.5, optionally, the concentration of potassium hydroxide in the excitation buffer is 0.5M; optionally, the enhancer is cetyltrimethylammonium bromide, optionally with a mass fraction of 0.478 wt%.
In a third aspect, a chemiluminescence detection method for magnetic particle acridinium ester is provided, which comprises the following steps:
s1, adding 20 parts by volume of calibrator, quality control material or sample to be tested into the detection tube;
s2, adding 50 parts by volume of magnetic particles coated with the antibody into a detection tube;
s3, adding 50 parts by volume of acridinium ester labeled antibody into a detection tube;
s4, mixing evenly, and incubating for 15 minutes at 37 +/-0.5 ℃;
s5, adding 300 parts by volume of cleaning solution into a detection tube, and uniformly mixing;
s6, magnetically separating out the supernatant;
s7, repeating the steps S5 and S6 for at least two times;
s8, adding pre-excitation liquid and excitation buffer solution into the detection tube, wherein the addition amount of the pre-excitation liquid and the excitation buffer solution is 200 parts by volume;
detecting the luminous intensity after S9 and 1S;
wherein, when quantitatively detecting soluble fms-like tyrosine kinase-1 in the sample, the antibody in step S2 is a second sFlt-1 antibody, and the antibody in step S3 is a first sFlt-1 antibody; when the placental growth factor in the sample is quantitatively assayed, the antibody in step S2 is a second PIGF antibody, and the antibody in step S3 is a first PIGF antibody;
the volume ratio of the pre-excitation liquid to the excitation buffer liquid is 2: 5-5: 2; alternatively 1: 1;
optionally, the method further comprises: and calculating the ratio of the soluble fms-like tyrosine kinase-1 to the placenta growth factor to be used as a basis for judging early epilepsy.
In a fourth aspect, the invention provides an application of a magnetic particle acridinium ester chemiluminescence detection kit in preparation of a drug for detecting early epilepsy or preeclampsia, and the magnetic particle acridinium ester chemiluminescence detection kit prepared by the magnetic particle acridinium ester chemiluminescence detection kit in the first aspect or the preparation method in the second aspect is adopted.
The invention also provides a preparation method of the acridinium ester marker obtained by the method for two quantitative kits of sFlt-1/PIGF. The detection specificity and the luminescence signal are both strong, and the sensitivity is high.
Advantageous effects
(1) On the basis of evaluating blood pressure and proteinuria, two joint inspection project kits under a magnetic particle-acridinium ester system platform are developed, two projects of sFlt-1/PlGF can be detected simultaneously, the ratio of the two projects is calculated to judge the result, early and rapid clinical diagnosis of preeclampsia and prediction of poor pregnancy outcome can be assisted, and doctors are helped to identify and treat high-risk groups, so that the safety of pregnant mothers and infants is guaranteed. The method for labeling the coupling of the acridinium ester and the antibody is simple, has good repeatability, high coupling efficiency and strong luminescent signal, and is convenient for large-scale application. The method has the advantages of higher sensitivity and wide linear range.
(2) The main biological functions of PIGF include inducing the proliferation, migration and activation of vascular endothelial cells and promoting the formation of placental blood vessels, while sFlt-1 has the biological activity of down-regulating and inhibiting PIGF to promote the growth of placental blood vessels. Studies have found that changes in sFlt-1 and PIGF concentration occur significantly earlier than in preeclampsia, and that the sFlt-1/PIGF ratio better reflects placental vascular growth. On the basis of evaluating proteinuria and blood pressure, the combined detection of the sFlt-1/PIGF ratio has good prediction value and diagnosis guiding significance on preeclampsia.
(3) The invention provides a magnetic particle-based acridinium ester chemiluminescence immunoassay method for early detection of preeclampsia and development of an sFlt-1/PIGF magnetic particle acridinium ester chemiluminescence kit. The adopted chemiluminescence analysis method has the advantages of high sensitivity, simple equipment, convenient operation, wide linear range, fast analysis, convenient realization of automation and the like. In addition, the preferable acridinium ester system is a luminescent agent commonly used in a chemiluminescence method, and the process of labeling the antibody directly influences the luminous efficiency, namely influences the sensitivity and accuracy of a detection result. The invention aims to explore a quantitative detection immunodiagnosis method and a reagent with good stability and excellent performance, assist in clinical early and rapid preeclampsia diagnosis and poor pregnancy outcome prediction, and help doctors identify and treat high-risk people, thereby ensuring the safety of mothers and infants in pregnancy.
(4) Preparation of acridinium ester marker of the present invention: the acridinium ester marker has a special group generating luminescence on a chemical structure, can directly participate in a luminescence reaction after an excitation liquid is added in a luminescence immunoassay process, does not need a substrate liquid, generally has no background luminescence of the substances, and is a luminescent agent with high luminescence efficiency. Both acridinium ester and acridine sulfamide can be combined with antibody (or antigen) to produce a marker with strong chemiluminescent activity and high immunoreaction specificity, the acridinium ester is usually marked on the amino group of the antibody or antigen, and the antibody is preferably directionally coupled on the fixed area of the antibody when being marked, so that the antibody can be marked with high efficiency and the activity of the antibody cannot be damaged. The magnetic particle is a microsphere or particle polymerized by high molecular monomer, the diameter is mostly micron-sized or millimeter-sized, and the surface of the magnetic particle has functional groups capable of being combined with antibody or antigen, such as amino, carboxyl, hydroxyl and the like, so that the magnetic particle can form chemical coupling by a specific coupling method, and has the advantages of strong binding force and large capacity. In the immune reaction, the magnetic particles can be uniformly dispersed in the reaction solution, the specific surface area is large, the reaction is accelerated, and the reaction rate is improved.
(5) The coupling method of directly coating the antibody on the magnetic particles and directly labeling the antibody with acridinium ester is adopted, a biotin-streptavidin system is not required to be introduced, the operation is simple, the introduced uncertain factors are few, and the production risk is small.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.
Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In some embodiments, materials, elements, methods, means, and the like that are well known to those skilled in the art are not described in detail in order to not unnecessarily obscure the present invention.
Example 1
1. The first sFlt-1 antibody labeled with acridinium ester was prepared by the following method:
1) measuring a marking buffer solution in a centrifuge tube;
2) adding the first sFlt-1 antibody, and mixing well;
3) adding the acridinium ester solution, fully and uniformly mixing, and carrying out vibration reaction at room temperature in a dark place; the molar ratio of acridinium ester to first sFlt-1 antibody is 1: 10; the time of the first sFlt-1 antibody and acridinium ester in light-shielding oscillation reaction at room temperature is 10-120 minutes;
4) putting the above mixture into dialysis bag, dialyzing at 2-8 deg.C in dark place, and replacing dialysate for 2 hr per time; the dialyzate was 0.02mol/L phosphate buffer solution.
5) Adding a proper amount of a labeling buffer solution for quantification, and sealing and storing at 2-8 ℃.
2. The preparation method of the acridinium ester labeled first PIGF antibody comprises the following steps:
1) measuring a marking buffer solution in a centrifuge tube;
2) adding a first PIGF antibody, and fully and uniformly mixing;
3) adding the acridinium ester solution, fully and uniformly mixing, and carrying out vibration reaction at room temperature in a dark place; the molar ratio of the acridinium ester to the first PIGF antibody is 1: 10; the time of the first PIGF antibody and the acridinium ester in the dark shock reaction at room temperature is 10-120 minutes;
4) putting the above mixture into dialysis bag, dialyzing at 2-8 deg.C in dark place, and replacing dialysate for 2 hr per time; the dialyzate was 0.02mol/L phosphate buffer solution.
5) Adding a proper amount of a labeling buffer solution for quantification, and sealing and storing at 2-8 ℃.
3. The magnetic microparticles coated with the second sFlt-1 antibody are prepared by the method comprising:
1) taking 100mg of magnetic particles, magnetically separating out supernatant, and resuspending with 10mL of 0.02mol/L MES buffer solution with pH 4.5-5.5;
2) adding 0.5-1mL of freshly prepared EDC aqueous solution with the concentration of 10mg/mL, and suspending at room temperature for 30-60 min;
3) magnetic separation, supernatant removal, 0.02mol/L, pH4.5-5.5MES buffer solution 10mL heavy suspension;
4) adding 4-8mg of a second sFlt-1 antibody, and suspending at room temperature for 16-20 h;
5) and (4) carrying out magnetic separation, removing supernatant, diluting with a magnetic particle buffer solution, and suspending to 0.5mg/mL to complete the preparation of the magnetic separation reagent.
4. The preparation method of the magnetic particle coated with the second PIGF antibody comprises the following steps:
1) taking 100mg of magnetic particles, magnetically separating out supernatant, and resuspending with 10mL of 0.02mol/L MES buffer solution with pH 4.5-5.5;
2) adding 0.5-1mL of freshly prepared EDC aqueous solution with the concentration of 10mg/mL, and suspending at room temperature for 30-60 min;
3) magnetic separation, supernatant removal, 0.02mol/L, pH4.5-5.5MES buffer solution 10mL heavy suspension;
4) adding 4-8mg of second PIGF antibody, and suspending at room temperature for 16-20 h;
5) and (4) carrying out magnetic separation, removing supernatant, diluting with a magnetic particle buffer solution, and suspending to 0.5mg/mL to complete the preparation of the magnetic separation reagent.
The preparation method of the pre-excitation liquid in the embodiment comprises the following steps: sequentially adding 0.8L of purified water, 4.862mL of concentrated nitric acid and 5.46mL of 30% hydrogen peroxide into a 1L of light-proof wide-mouth glass container, adding the purified water to a constant volume of 1L, stirring and mixing uniformly, and filtering to obtain pre-excitation liquid; the pH value is 1.10, wherein the concentration of each component is as follows: nitric acid: 0.07M; hydrogen peroxide: 0.6 percent;
the method for preparing the excitation buffer in this example was as follows: sequentially adding 0.8L of purified water and 4.82g of hexadecyl trimethyl ammonium bromide into a 1L wide-mouth glass container, stirring until the solid is completely dissolved, adding 28.056g of potassium hydroxide, stirring until the solid is completely dissolved, adding the purified water to a constant volume of 1L, and filtering to obtain an excitation buffer solution; buffer B was prepared as above with pH 13.5, with the following concentrations of components: potassium hydroxide: 0.5M; cetyl trimethylammonium bromide: 0.478 wt%.
Example 2
The difference from example 1 is that cetyltrimethylammonium bromide (CTAB) in the excitation buffer was replaced by cetyltrimethylammonium chloride (CTAC).
Example 3
The difference from example 1 is that cetyltrimethylammonium bromide (CTAB) in the excitation buffer was replaced by cetyltrimethylammonium iodide.
Example 4
The difference from example 1 is that cetyltrimethylammonium bromide (CTAB) in the excitation buffer was replaced by behenyltrimethylammonium chloride.
Example 5
The difference from example 1 is that cetyltrimethylammonium bromide (CTAB) in the excitation buffer was replaced by tetradecyltrimethylammonium chloride.
Example 6
The difference from example 1 is that cetyltrimethylammonium bromide (CTAB) in the excitation buffer was replaced by dodecyltrimethylammonium iodide.
Example 7
The difference from example 1 is that cetyltrimethylammonium bromide (CTAB) in the excitation buffer was replaced by dodecyltrimethylammonium chloride.
Example 8
The difference from example 1 is that cetyltrimethylammonium bromide (CTAB) in the excitation buffer was replaced by octaalkyltrimethylammonium chloride.
Detection example 1
The detection process is as follows:
the method of using the quantitative detection kit for soluble fms-like tyrosine kinase-1 (sFlt-1) is as follows:
1. adding 20 mu L of calibrator, quality control material or sample to be tested into the detection tube;
2. adding 50. mu.L of a second sFlt-1 antibody-magnetic microparticle to the detection tube;
3. add 50. mu.L of the first sFlt-1 antibody-acridinium ester to the detection tube;
4. after mixing evenly, incubating for 15 minutes at 37 plus or minus 0.5 ℃;
5. adding 300 mu L of cleaning solution into the detection tube, and uniformly mixing;
6. magnetically separating the supernatant;
7. repeating the steps 5 and 6 twice;
8. adding 100 mu L of pre-excitation liquid A and 100 mu L of excitation liquid B into a detection tube;
9. the luminescence intensity was measured after 1 s.
The usage method of the placenta growth factor (PlGF) quantitative detection kit is as follows:
1. adding 50 mu L of calibrator, quality control material or sample to be tested into the detection tube;
2. adding 50 μ L of a second PlGF antibody-magnetic microparticle to the detection tube;
3. adding 50 μ L of the first PlGF antibody-acridinium ester to the detection tube;
4. after mixing evenly, incubating for 10 minutes at 37 plus or minus 0.5 ℃;
5. adding 300 mu L of cleaning solution into the detection tube, and uniformly mixing;
6. magnetically separating the supernatant;
7. repeating the steps 5 and 6 twice;
8. adding 100 mu L of pre-excitation liquid A and 100 mu L of excitation liquid B into a detection tube;
9. the luminescence intensity was measured after 1 s.
The kit of examples 1 and 2 was used to test samples according to the method of the test examples.
Firstly, the signal-to-noise ratio of the reinforcing agents of the embodiments 1 to 8 is detected, and the results are shown in table 1, and it is obvious that the signal-to-noise ratio of the reinforcing agent added with CTAB is higher, that is, the sensitivity of the reinforcing agent is higher, and the method is an ideal choice.
The signal-to-noise ratio detection method comprises the following steps: the luminous values of the acridinium ester sample and the blank background are detected by using a full-automatic chemiluminescence immunoassay analyzer, the ratio of the acridinium ester sample to the blank background is used for obtaining the signal-to-noise ratio, and the performance is better if the signal-to-noise ratio is larger.
TABLE 1-1 SNR data for surfactants of example 2(CTAC) and example 1(CTAB)
TABLE 1-2 SNR data for surfactants of examples 3-5
TABLE 1-3 SNR data for surfactants of examples 6-8
Detection example 2
sFlt-1 kit principle:
the kit adopts a double-antibody one-step sandwich method, and is a detection method combining a chemiluminescence immunoassay method with a magnetic particle separation technology. The detection principle is as follows: and sequentially adding the sample to be detected, the magnetic bead coated sFlt-1 antibody and the acridinium ester marked sFlt-1 antibody, and combining a pair of sFlt-1 monoclonal antibodies with sFlt-1 antigen molecules in the sample to form an immune complex. After washing to remove unbound antibodies and impurities, excitation solutions a and B were added in order to measure the relative luminescence intensity (RLU). In a certain range, the RLU is in positive correlation with the concentration of the sFlt-1 antigen, and the content of the sFlt-1 in the sample to be detected can be calculated from the standard curve through the RLU.
Principle of PLGF kit:
the kit adopts a double-antibody one-step sandwich method, and is a detection method combining a chemiluminescence immunoassay method with a magnetic particle separation technology. The detection principle is as follows: and sequentially adding a sample to be detected, the PLGF antibody coated by the magnetic beads and the PLGF antibody marked by acridine ester, and combining a pair of PLGF monoclonal antibodies with PLGF antigen molecules in the sample to form an immune complex. After washing to remove unbound antibodies and impurities, excitation solutions a and B were added in order to measure the relative luminescence intensity (RLU). The RLU is in positive correlation with the concentration of the PLGF antigen in a certain range, and the content of the PLGF in the sample to be detected can be calculated from the standard curve through the RLU.
In order to better verify the detection effect of the kit of the invention, the kit of examples 1 and 2 is used for detecting samples according to the method of the detection example.
Primary analytical Performance of the first, sFlt-1 kits
1. Minimum limit of detection
The method comprises the following steps: the lowest detection limit is not more than 1ng/mL, and the detection results are shown in Table 2.
TABLE 2 minimum detection limit of sFlt-1 kit for detection of sFlt-1
2. Linearity
The method comprises the following steps: in the measuring range of [0, 400] ng/mL, the correlation coefficient r of the kit should be more than or equal to 0.9900, and the detection results are shown in Table 3.
TABLE 3 Linear correlation of sFlt-1 detection by the sFlt-1 kit
3. Repeatability of
The method comprises the following steps: the Coefficient of Variation (CV) should be no greater than 10% with the results shown in Table 4.
TABLE 4 reproducibility of sFlt-1 kit for detection of sFlt-1
Second, major analytical Properties of PLGF kit
1. Minimum limit of detection
The method comprises the following steps: the lowest detection should be not more than 1.0pg/mL, and the detection results are shown in Table 5.
TABLE 5 minimum detection limit for PLGF detection by PLGF kit
2. Linearity
The method comprises the following steps: within the measuring range of [0, 1000] ng/mL, the correlation coefficient r of the kit should be more than or equal to 0.9900, and the detection results are shown in Table 6.
TABLE 6 detection of linear correlation of PLGF by the PLGF kit
3. Repeatability of
The method comprises the following steps: the Coefficient of Variation (CV) should be not more than 10%, and the results are shown in Table 7.
TABLE 7 reproducibility of PLGF detection by PLGF kit
Clinical detection data of three, sFlt-1, PLGF kit
The detection method comprises the following steps:
the method comprises the steps of respectively detecting the contents of two serum markers, namely sFlt-1 and PLGF, in the serum of a preeclampsia group and a normal pregnancy group by adopting two sFlt-1 and PLGF kits of the patent, calculating sFlt-1/PLGF, comparing data difference between the two groups, and carrying out data analysis and comparison to obtain an sFlt-1/PLGF ratio so as to verify the specificity and sensitivity of the sFlt-1/PLGF ratio to the prediction of the incidence rate of the preeclampsia, wherein the detection results are shown in tables 8-9.
TABLE 8 serum sFlt-1, PLGF levels and ratios of the two in the normal pregnancy group (group A)
TABLE 9 Pre-eclampsia group (group B) serum sFlt-1, PLGF levels and ratios of the two
And (4) analyzing results: the serum sFlt-1 level of the normal pregnancy group is obviously lower than that of the pre-eclampsia group, the serum PLGF level of the normal pregnancy group is obviously higher than that of the pre-eclampsia group, the sFlt-1/PLGF of the pre-eclampsia group is obviously higher than that of the normal pregnancy group, and related studies prove that the sFlt-1/PLGF is more than 85, so that the pre-eclampsia can be judged. sFlt-1 alone was tested to be 79.2% sensitive and 73.6 specific; PLGF sensitivity was 74.3% and specificity was 77.2%; the sensitivity of sFlt-1/PLGF is 85.8 percent, and the specificity is 88.6 percent, so that the ratio of the sensitivity and the specificity for detecting the preeclampsia is larger than that of a single index for detecting the preeclampsia alone.
Preeclampsia is usually diagnosed based on the triple signs of hypertension, proteinuria and edema. Given that edema is a common manifestation of normal pregnancy, it has not been considered as one of the criteria in recent diagnostic criteria. The treatment of preeclampsia and eclampsia is based on prevention, and when the preeclampsia symptoms are found to be detected by the conventional method and are detected for emergency examination, the prevention period is generally missed, and the preeclampsia symptoms need to be admitted for treatment, and once eclampsia occurs, emergency rescue measures are taken immediately.
For preeclampsia, especially early onset preeclampsia that occurs as early as gestation between 20 and 34 weeks, early, reliable diagnosis is critical for clinical control of this disease. In particular, early onset preeclampsia is troublesome to clinicians due to its serious side effects and adverse consequences commonly associated therewith. Furthermore, the early, reliable diagnosis and prognosis of preeclampsia are critical for planning both preventive and therapeutic intervention studies.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A magnetic particle acridinium ester chemiluminescence detection kit is characterized by comprising an sFlt-1 antibody detection reagent and/or a PIGF antibody detection reagent; also comprises a pre-excitation liquid and an excitation buffer liquid;
the sFlt-1 antibody detection reagent comprises a first sFlt-1 antibody marked by acridinium ester and magnetic particles coated with a second sFlt-1 antibody; the molar ratio of sFlt-1 antibody to acridinium ester in the acridinium ester-labeled sFlt-1 antibody was 1: (5-15);
the first and second sFlt-1 antibodies recognize different epitopes of soluble fms-like tyrosine kinase-1, respectively;
the PIGF antibody detection reagent comprises a first PIGF antibody marked by acridinium ester and magnetic particles coated with a second PIGF antibody; the molar ratio of the PIGF antibody to the acridinium ester in the acridinium ester labeled PIGF antibody is 1: (5-15);
the first PIGF antibody and the second PIGF antibody respectively recognize different epitopes of the placenta growth factor;
the pre-excitation liquid comprises inorganic acid and an oxidant, and the pH value is less than 2;
the excitation buffer comprises an inorganic base and an enhancer, and the pH of the buffer is > 13;
the reinforcing agent is a quaternary ammonium salt cationic surfactant and is selected from one or more of octaalkyltrimethyl ammonium chloride, dodecyltrimethyl ammonium iodide, tetradecyltrimethyl ammonium chloride, hexadecyltrimethyl ammonium bromide, hexadecyltrimethyl ammonium iodide and docosyltrimethyl ammonium chloride; alternatively cetyltrimethylammonium bromide.
2. The magnetic microparticle acridinium ester chemiluminescence detection kit of claim 1, wherein in the acridinium ester labeled first sFlt-1 antibody, the molar ratio of the first sFlt-1 antibody to acridinium ester is 1: 10;
and/or, the acridinium ester-labeled first sFlt-1 antibody, further comprising a labeling buffer comprising the first sFlt-1 antibody for isotyping the acridinium ester label; optionally the concentration of the acridinium ester-labeled first sFlt-1 antibody is 0.5ug/mL, optionally the labeling buffer is a phosphate buffer or a carbonate buffer, optionally a 0.1mol/L carbonate buffer, ph 8.2;
and/or, in the acridinium ester-labeled first PIGF antibody, the molar ratio of the first PIGF antibody to the acridinium ester is 1: 10;
and/or the acridinium ester labeled first PIGF antibody also comprises a labeling buffer solution for fixing the volume of the acridinium ester labeled first PIGF antibody; the concentration of the first PIGF antibody which is optionally marked by the acridinium ester is 0.25ug/mL, the marking buffer solution is phosphate buffer solution or carbonate buffer solution, the carbonate buffer solution is 0.1mol/L, and the pH value is 8.2;
and/or, in the magnetic particles coated with the second sFlt-1 antibody, the mass ratio of the magnetic particles to the second sFlt-1 antibody is 100: (4-8); optionally, MES buffer solution for fixing the magnetic particles coated with the second sFlt-1 antibody is also included, and optionally, the concentration of the magnetic particles of the second sFlt-1 antibody is 0.5 mg/mL; optionally, the concentration of MES buffer is 0.02mol/L, and the pH is 4.5-5.5;
and/or in the magnetic particles coated with the second PIGF antibody, the mass ratio of the magnetic particles to the second PIGF antibody is 100: (4-8); optionally, the kit further comprises an MES buffer solution for fixing the magnetic particles coated with the second PIGF antibody, and optionally, the concentration of the magnetic particles of the second PIGF antibody is 0.5 mg/mL; alternatively, the MES buffer has a concentration of 0.02mol/L and a pH of 4.5 to 5.5.
3. The magnetic particle acridinium ester chemiluminescence detection kit according to claim 1, wherein in the pre-excitation liquid, the acid is nitric acid or hydrochloric acid or sulfuric acid or boric acid; the oxidant is hydrogen peroxide or carbamide peroxide; optionally, in the pre-excitation liquid, the concentration of the acid is 0.01-0.18M, and the mass concentration of the oxidant is 0.1-2.0%; further optionally, the pre-excitation liquid comprises 0.07M nitric acid and 0.6 wt% hydrogen peroxide, and the pH is 1.1;
and/or, in the excitation buffer, the alkali is sodium hydroxide or potassium hydroxide; optionally, in the buffer solution, the concentration of the alkali is 0.2-0.8M, and the mass concentration of the enhancer is 0.2-2.0%; further optionally, the buffer comprises 0.5M potassium hydroxide and 0.478 wt% cetyltrimethylammonium bromide, pH 13.5.
4. A method for preparing a magnetic microparticle acridinium ester chemiluminescence detection kit according to any one of claims 1 to 3, comprising the steps of:
a method for making an acridinium ester-labeled first sFlt-1 antibody comprises: adding a first sFlt-1 antibody and an acridinium ester solution into a labeling buffer solution, uniformly mixing, carrying out a light-shielding oscillation reaction at room temperature, dialyzing, and adding the labeling buffer solution for quantification to obtain an acridinium ester labeled first sFlt-1 antibody reagent;
a method for preparing magnetic microparticles coated with a second sFlt-1 antibody comprises: adding EDC aqueous solution into the magnetic particle suspension for 30-60min, magnetically separating and removing supernatant, re-suspending, adding a second sFlt-1 antibody for coating, and suspending for 16-20 h; magnetically separating the supernatant, and quantifying to obtain a magnetic particle reagent coated with a second sFlt-1 antibody;
the preparation method of the acridinium ester labeled first PIGF antibody comprises the following steps: adding a first PIGF antibody and an acridinium ester solution into a labeling buffer solution, uniformly mixing, carrying out a photophobic oscillation reaction at room temperature, dialyzing, and adding the labeling buffer solution for quantification to obtain an acridinium ester labeled first PIGF antibody reagent; preferably, the labeling buffer is 0.1mol/L carbonate buffer, pH 8.2;
the preparation method of the magnetic particle coated with the second PIGF antibody comprises the following steps: adding EDC aqueous solution into the magnetic particle suspension for 30-60min, magnetically separating and removing supernatant, re-suspending, adding a second PIGF antibody for coating, and suspending for 16-20 h; and (4) magnetically separating the supernatant, and quantifying to obtain the magnetic particle reagent coated with the second PIGF antibody.
5. The method for preparing a magnetic microparticle acridinium ester chemiluminescence detection kit of claim 4, wherein the method for preparing the first acridinium ester-labeled sFlt-1 antibody and the method for preparing the first acridinium ester-labeled PIGF antibody independently comprise: the reaction time is 10-120 minutes under the condition of keeping out of the sun at room temperature and shaking;
and/or the labeling buffer is phosphate buffer or carbonate buffer, optionally 0.1mol/L carbonate buffer, pH 8.2;
and/or, the dialysis method comprises: filling the reaction mixture into a dialysis bag, dialyzing in the dark at the temperature of 2-8 ℃, and replacing the dialysate for 2 h/time, wherein the dialysate is phosphate buffer solution, and the concentration of the dialysate is 0.02 mol/L.
6. The method for preparing a magnetic microparticle acridinium ester chemiluminescence detection kit according to claim 4 or 5, wherein the method for preparing magnetic microparticles coated with the second sFlt-1 antibody and the method for preparing magnetic microparticles coated with the second PIGF antibody independently comprise: adding 0.5-1mL of EDC aqueous solution with the concentration of 10mg/mL into each 100g of magnetic particles; optionally, the magnetic particle suspension is obtained by resuspending the magnetic particles with MES buffer; optionally, using MES buffer for resuspension or quantification; optionally, the concentration of MES buffer is 0.02mol/L, and the pH is 4.5-5.5; alternatively, the quantitative concentration is 0.5 mg/mL; optionally, in the magnetic particle suspension or the resuspension solution, 10mL of MES buffer solution is used for every 100g of magnetic particles; alternatively, the suspension is room temperature suspension.
7. The method for preparing a magnetic microparticle acridinium ester chemiluminescence detection kit according to claim 4, wherein the method for preparing the pre-excitation liquid comprises: mixing purified water, concentrated nitric acid and 30% hydrogen peroxide, fixing the volume, uniformly stirring, and filtering to obtain pre-excitation liquid; the pH of the pre-excitation liquid is 1.10, wherein the concentration of each component is as follows: 0.07M nitric acid and 0.6 wt% hydrogen peroxide.
8. The method for preparing a magnetic microparticle acridinium ester chemiluminescence detection kit according to claim 4, wherein the excitation buffer is prepared by a method comprising: mixing purified water and an enhancer, dissolving, adding potassium hydroxide, dissolving, fixing the volume, and filtering to obtain an excitation buffer solution; optionally, the pH of the excitation buffer is 13.5, optionally, the concentration of potassium hydroxide in the excitation buffer is 0.5M; optionally, the enhancer is cetyltrimethylammonium bromide, optionally with a mass fraction of 0.478 wt%.
9. A magnetic particle acridinium ester chemiluminescence detection method is characterized by comprising the following steps:
s1, adding 20 parts by volume of calibrator, quality control material or sample to be tested into the detection tube;
s2, adding 50 parts by volume of magnetic particles coated with the antibody into a detection tube;
s3, adding 50 parts by volume of acridinium ester labeled antibody into a detection tube;
s4, mixing evenly, and incubating for 15 minutes at 37 +/-0.5 ℃;
s5, adding 300 parts by volume of cleaning solution into a detection tube, and uniformly mixing;
s6, magnetically separating out the supernatant;
s7, repeating the steps S5 and S6 for at least two times;
s8, adding pre-excitation liquid and excitation buffer solution into the detection tube, wherein the addition amount of the pre-excitation liquid and the excitation buffer solution is 200 parts by volume;
detecting the luminous intensity after S9 and 1S;
wherein, when quantitatively detecting soluble fms-like tyrosine kinase-1 in the sample, the antibody in step S2 is a second sFlt-1 antibody, and the antibody in step S3 is a first sFlt-1 antibody; when the placental growth factor in the sample is quantitatively assayed, the antibody in step S2 is a second PIGF antibody, and the antibody in step S3 is a first PIGF antibody;
the volume ratio of the pre-excitation liquid to the excitation buffer liquid is 2: 5-5: 2; alternatively 1: 1;
optionally, the method further comprises: and calculating the ratio of the soluble fms-like tyrosine kinase-1 to the placenta growth factor to be used as a basis for judging early epilepsy.
10. The application of the magnetic particle acridinium ester chemiluminescence detection kit in the preparation of drugs for detecting early epilepsy or preeclampsia is characterized in that the magnetic particle acridinium ester chemiluminescence detection kit according to any one of claims 1 to 3 or the magnetic particle acridinium ester chemiluminescence detection kit prepared by the preparation method according to any one of claims 5 to 8 is adopted.
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