Magnetic microsphere electrochemiluminescence immunoassay kit for detecting acidic protein in glial fibers and preparation method thereof
阅读说明：本技术 用于胶质纤维酸性蛋白检测的磁微球电化学发光免疫检测试剂盒及其制备方法 (Magnetic microsphere electrochemiluminescence immunoassay kit for detecting acidic protein in glial fibers and preparation method thereof ) 是由 秦军 谢元东 谢良思 于 2020-12-24 设计创作，主要内容包括：本发明涉及电化学检测领域,特别涉及用于胶质纤维酸性蛋白(GFAP)检测的磁微球电化学发光免疫检测试剂盒。本发明采用方法为电化学发光法,采用吡啶钌作为化学发光标记物具有明显优势,主要表现在：稳定性更好,钌是金属离子,分子量小,不影响抗体的空间位阻。生产过程短,重复性好,检测范围宽。电化学发光反应可控,降低信号采集难度。(The invention relates to the field of electrochemical detection, in particular to a magnetic microsphere electrochemical luminescence immunoassay kit for detecting Glial Fibrillary Acidic Protein (GFAP). The method adopted by the invention is an electrochemical luminescence method, and the ruthenium pyridine adopted as the chemiluminescent marker has obvious advantages, which are mainly shown in that: the stability is better, ruthenium is metal ion, the molecular weight is small, and the steric hindrance of the antibody is not influenced. Short production process, good repeatability and wide detection range. The electrochemical luminescence reaction is controllable, and the signal acquisition difficulty is reduced.)
1. The composition for detecting the Glial Fibrillary Acidic Protein (GFAP) is characterized by comprising a GFAP reagent Ra, a GFAP reagent Rb and streptavidin superparamagnetic microspheres;
the GFAP reagent Ra comprises an anti-GFAP monoclonal antibody containing a biotin label;
the GFAP reagent Rb comprises an anti-GFAP monoclonal antibody marked by terpyridyl ruthenium;
the streptavidin superparamagnetic microspheres comprise superparamagnetic microspheres coated with streptavidin on the surfaces, and the particle size of the magnetic microspheres is 1.5-5.0 microns.
2. The composition according to claim 1, wherein the amount of biotin molecular markers per antibody molecule surface is 2 to 5 in the GFAP reagent Ra; in the GFAP reagent Rb, the labeling quantity of ruthenium molecules on the surface of each antibody molecule is 2-10.
3. The composition of claim 1 or 2, wherein the GFAP reagent Ra is prepared by: mixing an anti-GFAP monoclonal antibody with biotin in the presence of a buffer solution to prepare a GFAP reagent Ra; the buffer solution includes phosphate buffer solution with pH 7.4 and 20 mM-200 mM or trihydroxymethyl aminomethane buffer solution with pH 7.4 and 20 mM-200 mM.
4. The composition of any one of claims 1 to 3, wherein the GFAP agent Rb is prepared by: mixing an anti-GFAP monoclonal antibody with ruthenium terpyridyl in the presence of a buffer solution to prepare a GFAP reagent Rb; the buffer solution includes phosphate buffer solution with pH 7.4 and 20 mM-200 mM or trihydroxymethyl aminomethane buffer solution with pH 7.4 and 20 mM-200 mM.
5. The composition of any one of claims 1 to 4, further comprising a sealer and/or a rinse; the cleaning solution comprises tripropylamine with the concentration of 180mmol/L and phosphate buffer solution with the concentration of 300 mmol/L; or
Dibutylethanolamine at a concentration of 90mmol/L and phosphate buffer at a concentration of 300 mmol/L.
6. The composition of any one of claims 1 to 4, wherein the volume ratio of the GFAP reagent Ra, the GFAP reagent Rb to the streptavidin superparamagnetic microspheres is (70-80): (70-80): 40.
7. use of a composition according to any one of claims 1 to 6 for the preparation of a magnetic microsphere electrochemiluminescence immunoassay kit for Glial Fibrillary Acidic Protein (GFAP).
8. Kit for the magnetomicrosphere electrochemiluminescence immunoassay of Glial Fibrillary Acidic Protein (GFAP) comprising a composition according to any one of claims 1 to 6 and reagents acceptable for the detection.
9. Kit for the magnetomicrosphere electrochemiluminescence immunoassay of Glial Fibrillary Acidic Protein (GFAP), based on the composition according to any one of claims 1 to 6 or the kit according to claim 8, the method of use of which comprises the following steps:
step 1: taking a sample, sequentially adding a GFAP reagent Ra and a GFAP reagent Rb, incubating for 9min at 37 ℃, finally adding streptavidin superparamagnetic microspheres, and incubating for 9min at 37 ℃ to obtain a reaction solution; wherein the volume ratio of the sample, the GFAP reagent Ra, the GFAP reagent Rb to the streptavidin superparamagnetic microspheres is 15: (70-80): (70-80): 40;
step 2: adsorbing the reaction solution by using a magnet;
and step 3: taking the cleaning solution, cleaning the ruthenium-labeled antibody and the sample which are not bonded to the superparamagnetic microspheres, electrifying, and enabling the terpyridyl ruthenium to emit light under the condition of the presence of the cleaning solution;
and 4, step 4: the luminescence values were optically recorded and the concentration of GAFP in the sample was obtained from the established standard curve.
10. The kit for magnetic microsphere electrochemiluminescence immunoassay according to claim 9, wherein the kit is used in a method in which the incubation is performed at 37 ℃ for 9 min.
Technical Field
The invention relates to the field of electrochemical detection, in particular to a magnetic microsphere electrochemical luminescence immunoassay kit for detecting Glial Fibrillary Acidic Protein (GFAP).
Background
GFAP is an English abbreviation of glial fibrillary acidic protein (glial fibrillary acidic protein) and is a marker of astrocyte activation. Glial fibrillary acidic protein. Glial Fibrillary Acidic Protein (GFAP) is a type iii intermediate filament protein that exists in monomeric form. Among humans, 8 isoforms are found, with relative molecular masses between (40-53). times.10 ^ 3. The human GFAP gene is located in the band 1 of the long arm 2 region of chromosome 17 and consists of 9 exons and 8 introns.
GFAP is distributed mainly in astrocytes of the central nervous system, and is involved in the formation of cytoskeleton and maintains its tonicity strength. It is also expressed in chondrocytes, fibroblasts, myoepithelial cells, lymphocytes, hepatic stellate cells.
Detecting GFAP level in serum of brain injury patient can be used as biochemical marker for central nervous system injury range and prognosis in clinic. But also in gliomas. Can be used for reflecting the malignancy degree of glioma.
Up to now, methods for detecting GFAP in human serum are mainly: enzyme linked immunosorbent assay (ELISA) and enzymatic magnetic particle chemiluminescence. However, the sensitivity is not high, the linear range is narrow, the detection time is too long, and particularly the antibody treatment time is too long, so that the detection efficiency is influenced.
Disclosure of Invention
Detecting GFAP level in serum of brain injury patient can be used as biochemical marker for central nervous system injury range and prognosis in clinic. But also in gliomas. Can be used for reflecting the malignancy degree of glioma. The increased concentration of GFAP in the serum of CNS-infected patients may reflect the prognosis of CNS infection.
In view of the above, the invention provides a GFAP magnetic particle electrochemiluminescence kit and a preparation and detection method thereof, and the kit has the advantages of high production efficiency, short detection time, suitability for full-automatic detection, higher sensitivity, wide linear range and the like.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a composition for detecting Glial Fibrillary Acidic Protein (GFAP), which comprises a GFAP reagent Ra, a GFAP reagent Rb and streptavidin superparamagnetic microspheres;
the GFAP reagent Ra comprises an anti-GFAP monoclonal antibody containing a biotin label;
the GFAP reagent Rb comprises an anti-GFAP monoclonal antibody marked by terpyridyl ruthenium;
the streptavidin superparamagnetic microspheres comprise superparamagnetic microspheres coated with streptavidin on the surfaces, and the particle size of the magnetic microspheres is 1.5-5.0 microns.
In some embodiments of the invention, the amount of the biotin molecular marker on the surface of each antibody molecule in the GFAP reagent Ra is 2 to 5; in the GFAP reagent Rb, the labeling quantity of ruthenium molecules on the surface of each antibody molecule is 2-10.
In some embodiments of the invention, the GFAP reagent Ra is prepared by: mixing an anti-GFAP monoclonal antibody with biotin in the presence of a buffer solution to prepare a GFAP reagent Ra; the buffer solution comprises phosphate buffer solution with pH value of 6.5-7.4 and 20 mM-200 mM or trihydroxymethyl aminomethane buffer solution with pH value of 7.0-8.4 and 20 mM-200 mM.
In some embodiments of the invention, the GFAP reagent Ra is prepared by: after 2.0mg of an antibody for labeling a biotin Glial Fibrillary Acidic Protein (GFAP) was taken out, the buffer was changed to a phosphate buffer (PH 7.8) using a desalting column PD10, the buffer was concentrated using an ultrafiltration tube and adjusted to a concentration of 2.0mg/mL, 30 to 80 μ g of biotin (dissolved in DMF) was added thereto, and the mixture was mixed and reacted for 10 to 60 minutes, and then the unlabeled biotin was removed using a desalting column PD 10. A Glial Fibrillary Acidic Protein (GFAP) antibody labeled with biotin was diluted to 0.2 to 1mg/L with a phosphate buffer solution (PH 6.5 to 7.4) containing 0.1 to 2% bovine serum albumin as a GFAP reagent Ra.
In some embodiments of the invention, the GFAP agent Rb is prepared by: mixing an anti-GFAP monoclonal antibody with ruthenium terpyridyl in the presence of a buffer solution to prepare a GFAP reagent Rb; the buffer solution comprises phosphate buffer solution with pH value of 6.5-7.4 and 20 mM-200 mM or trihydroxymethyl aminomethane buffer solution with pH value of 7.0-8.4 and 20 mM-200 mM.
In some embodiments of the invention, the GFAP agent Rb is prepared by: after 2.0mg of a Glial Fibrillary Acidic Protein (GFAP) antibody for labeling ruthenium terpyridyl was taken, the buffer was changed to phosphate buffer (PH 7.8) using desalting column PD10, the buffer was concentrated using an ultrafiltration tube and adjusted to 2.0mg/mL, 30 to 80 μ g of ruthenium succinamide terpyridyl (dissolved in DMF) was added thereto, and the mixture was mixed and reacted for 30 minutes, and then unlabeled ruthenium was removed using desalting column PD 10. A ruthenium-labeled Glial Fibrillary Acidic Protein (GFAP) antibody was diluted to 1mg/L with a phosphate buffer (PH 6.5-7.4) containing 0.1-2% bovine serum albumin as the GFAP reagent Rb.
In some embodiments of the invention, the compositions provided herein further comprise a taggant and/or a cleaning solution; the cleaning solution comprises tripropylamine with the concentration of 180mmol/L and phosphate buffer solution with the concentration of 100-300 mmol/L; or dibutylethanolamine with the concentration of 90mmol/L and phosphate buffer with the concentration of 100-300 mmol/L.
In some embodiments of the invention, the volume ratio of the GFAP reagent Ra, the GFAP reagent Rb to the streptavidin superparamagnetic microspheres is (50-80): (50-80): 40.
on the basis of the research, the invention also provides application of the composition in preparing a magnetic microsphere electrochemiluminescence immunoassay kit for Glial Fibrillary Acidic Protein (GFAP).
The invention also provides a magnetic microsphere electrochemiluminescence immunoassay kit for Glial Fibrillary Acidic Protein (GFAP), which comprises the composition and a detection acceptable reagent.
The invention also provides a magnetic microsphere electrochemiluminescence immunoassay method for Glial Fibrillary Acidic Protein (GFAP), which is based on the composition or the kit and comprises the following steps:
step 1: taking a sample, sequentially adding a GFAP reagent Ra and a GFAP reagent Rb, incubating for 9min at 37 ℃, finally adding streptavidin superparamagnetic microspheres, and incubating for 9min at 37 ℃ to obtain a reaction solution; wherein the volume ratio of the sample, the GFAP reagent Ra, the GFAP reagent Rb to the streptavidin superparamagnetic microspheres is 15: (50-80): (50-80): 40;
step 2: adsorbing the reaction solution by using a magnet;
and step 3: taking the cleaning solution, cleaning the ruthenium-labeled antibody and the sample which are not bonded to the superparamagnetic microspheres, electrifying, and enabling the terpyridyl ruthenium to emit light under the condition of the presence of the cleaning solution;
and 4, step 4: the luminescence values were optically recorded and the concentration of GAFP in the sample was obtained from the established standard curve.
In some embodiments of the invention, the incubation is at 37 ℃ for 9 min.
In some embodiments of the present invention, the detection method specifically comprises:
step 1: adding 15ul of sample into a reaction tube, sequentially adding a GFAP reagent Ra40-70ul and a GFAP reagent Rb40-70ul, incubating at 37 ℃ for 9min, finally adding 40ul of streptavidin superparamagnetic microspheres, and incubating at 37 ℃ for 9 min;
step 2: sucking the reaction tube after the incubation reaction into an electrochemical flow cell through a liquid absorption steel needle, and adsorbing the reaction tube by a magnet of the flow cell;
and step 3: and (3) sucking a cleaning solution (tripropylamine or DBAE) by a liquid suction steel needle, cleaning the ruthenium-labeled antibody which is not bound to the superparamagnetic microspheres and the sample, electrifying the flow cell, and emitting light by the terpyridyl ruthenium under the condition that the tripropylamine or DBAE exists.
And 4, step 4: the photomultiplier tube records the luminescence value and calculates the concentration of GAFP in the sample according to the established standard curve.
The magnetic particles can be used as carriers of biological macromolecules, the antibody-coated magnetic particles are called immune magnetic particles, and the immune magnetic particles have the characteristics of antigen combination and magnetism, so that the immune magnetic particles have more advantages in the aspects of separating, purifying and concentrating target microorganisms, cells, biological macromolecules and the like from complex samples, and comprise rapidness, strong specificity, simple and convenient operation, wide application range and the like. The nanometer material is a new material which is rapidly developed after 90 years in the 20 th century, and the nanometer magnetic particles (the particle size is less than 10 nm-100 nm) are greatly different from the common magnetic particles in the aspects of magnetic structure and magnetism: the nano magnetic particles have more particles per unit volume and larger specific surface area; the magnetic material has superparamagnetism, and the magnetic interaction is weak; it can move directionally under the action of external magnetic field to separate, concentrate or purify some special components. The magnetic particle chemiluminescence method established by the invention has the advantages of high sensitivity, strong specificity, accuracy, rapidness, short detection time and higher accuracy and repeatability of a detection result.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows the results of a straight line fit of diluted concentration to measured concentration in the reaction method of example 3;
FIG. 2 shows the results of a straight line fit of the diluted concentration to the measured concentration in the reaction method of example 4.
Detailed Description
The invention discloses a magnetic microsphere electrochemiluminescence immunoassay kit for detecting Glial Fibrillary Acidic Protein (GFAP), which can be realized by appropriately improving process parameters by taking the contents as reference by the technical personnel in the field. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention provides the following technical scheme: a GFAP magnetic microsphere electrochemiluminescence kit comprises: GFAP reagent Ra, GFAP reagent Rb, streptavidin superparamagnetic microspheres, a calibration product, tripropylamine cleaning solution and dibutylethanolamine cleaning solution.
The GFAP magnetic microsphere electrochemiluminescence kit comprises a GFAP reagent Ra, a buffer solution and a magnetic microsphere, wherein the GFAP reagent Ra is an anti-GFAP monoclonal antibody containing biotin labels, the labeling quantity of biotin molecules on the surface of each antibody molecule is 2-5, the buffer solution is 20 mM-200 mM phosphate buffer solution, the pH value is 6.5-7.4 or 20 mM-200 mM tris (hydroxymethyl) aminomethane buffer solution, and the pH value is 7.0-8.5. The GFAP reagent Rb is an anti-GFAP monoclonal antibody containing a terpyridyl ruthenium label, the labeling quantity of ruthenium molecules on the surface of each antibody molecule is 2-10, and the buffer solution is 20 mM-200 mM phosphate buffer solution, pH is 6.5-7.4 or 20 mM-200 mM tris (hydroxymethyl) aminomethane buffer solution, and pH is 7.0-8.5.
The GFAP magnetic microsphere electrochemiluminescence kit comprises a magnetic microsphere coated with streptavidin, wherein the streptavidin superparamagnetic microsphere is coated with the streptavidin, the particle size of the magnetic microsphere is 1.5-5.0 micrometers, the magnetic particle coating buffer solution is 20 mM-200 mM phosphate buffer solution, the pH value is 6.5-7.4 or 20 mM-200 mM tris (hydroxymethyl) aminomethane buffer solution, and the pH value is 7.0-8.5.
The GFAP magnetic microsphere electrochemiluminescence kit comprises a kit body, a kit body and a kit body, wherein the GFAP antibody marked by biotin is a monoclonal antibody;
the detection method of the GFAP magnetic microsphere electrochemiluminescence kit comprises the following steps that the cleaning solution is tripropylamine with the concentration of 180mmol/L, and the phosphate buffer solution with the concentration of 100-300mmol/L is contained. Or 90mmol/L dibutylethanolamine containing 100-300mmol/L phosphate buffer
A detection method of a GFAP magnetic microsphere electrochemiluminescence kit comprises the following steps:
1) adding 15ul of sample into a reaction tube, sequentially adding a GFAP reagent Ra40-70ul and a GFAP reagent Rb40-70ul, incubating at 37 ℃ for 9min, finally adding 40ul of streptavidin superparamagnetic microspheres, and incubating at 37 ℃ for 9 min;
2) sucking the reaction tube after the incubation reaction into an electrochemical flow cell through a liquid absorption steel needle, and adsorbing the reaction tube by a magnet of the flow cell;
3) and (3) sucking a cleaning solution (tripropylamine or DBAE) by a liquid suction steel needle, cleaning the ruthenium-labeled antibody which is not bound to the superparamagnetic microspheres and the sample, electrifying the flow cell, and emitting light by the terpyridyl ruthenium under the condition that the tripropylamine or DBAE exists.
4) The photomultiplier tube records the luminescence value and calculates the concentration of GAFP in the sample according to the established standard curve.
The streptavidin and the biotin have high-specificity binding capacity, and the streptavidin and the biotin-labeled high-purity antibody are specifically bound through non-covalent bonds, so that the streptavidin-labeled high-purity antibody has the effect of cascade amplification, and the reaction is highly specific. Therefore, the sensitivity is improved, non-specific interference is not increased, and the binding property is not affected by the high dilution of the reaction reagent, so that the non-specific action of the reaction reagent can be reduced to the maximum extent in practical application.
The invention combines the high specificity of antibody-antigen reaction with the high sensitivity of ruthenium terpyridyl luminescence, utilizes the photons generated by ruthenium terpyridyl under tripropylamine or DBAE to detect the product concentration, and has the characteristics of higher sensitivity, short reaction time, simple operation and high anti-interference performance.
The magnetic microsphere electrochemiluminescence immunoassay kit for detecting the Glial Fibrillary Acidic Protein (GFAP) provided by the invention is commercially available in raw materials and reagents.
All components of the test kit of the present invention can be commercially obtained from biological or chemical reagents companies. The used equipment is a full-automatic chemiluminescence immunoassay analyzer (model UD90DT) produced by Beijing Ongzhong Take technology Limited
The invention is further illustrated by the following examples:
example 1: preparation of biotin-labeled Glial Fibrillary Acidic Protein (GFAP) antibody and reagent Ra
The Glial Fibrillary Acidic Protein (GFAP) antibody for labeling biotin was purchased from North Jing Yuan Tian Ji technology Co., Ltd, with the product number YT-GFAP-002 and the clone number 6E 9.
After 2.0mg of an antibody for labeling a biotin Glial Fibrillary Acidic Protein (GFAP) was taken out, the buffer was changed to a phosphate buffer (PH 7.8) using a desalting column PD10, the resulting mixture was concentrated using an ultrafiltration tube and adjusted to a concentration of 2.0mg/mL, 80 μ g of biotin (dissolved in DMF) was added thereto, the mixture was mixed and reacted for 30 minutes, and unlabeled biotin was removed using a desalting column PD 10. A biotin-labeled Glial Fibrillary Acidic Protein (GFAP) antibody was diluted to 1mg/L with a phosphate buffer solution (PH 7.4) containing 1% bovine serum albumin as a GFAP reagent Ra.
Example 2: preparation of ruthenium-labeled Glial Fibrillary Acidic Protein (GFAP) antibody and reagent Rb
The Glial Fibrillary Acidic Protein (GFAP) antibody for labeling biotin was purchased from North Beijing Tianxin Jane science and technology Ltd, with the product number YT-GFAP-003 and the clone number 5H 4.
After 2.0mg of a Glial Fibrillary Acidic Protein (GFAP) antibody labeled with ruthenium terpyridyl was taken, the buffer was changed to phosphate buffer (PH 7.8) using desalting column PD10, the buffer was concentrated using an ultrafiltration tube and adjusted to 2.0mg/mL, 80 μ g of succinamid ruthenium terpyridyl (dissolved in DMF) was added thereto, and the mixture was mixed and reacted for 30 minutes, and then unlabeled ruthenium was removed using desalting column PD 10. A ruthenium-labeled Glial Fibrillary Acidic Protein (GFAP) antibody was diluted to 1mg/L with a phosphate buffer (PH 7.4) containing 1% bovine serum albumin as the GFAP reagent Rb.
Example 3: preparation of the calibration articles
The antigen for preparing the calibration sample is purchased from Ji Tech Co Ltd of Beijing edge Tian Xin, and has the product number YT-GFAP-001. For recombinant expression of the protein.
The antigen was diluted to 5.0 and 150pg/mL using 1% bovine serum albumin in phosphate buffer (PH 7.4) at the indicated concentrations. Used as a calibrator for establishing a standard curve.
Example 4 preparation of tripropylamine cleaning solution and dibutylethanolamine cleaning solution
300mmol/L phosphate buffer solution is prepared, tripropylamine is added to 180mmol/L, and the mixture is mixed and dissolved. As a tripropylamine cleaning solution.
Preparing 300mmol/L phosphate buffer solution, adding dibutyl ethanolamine to 90mmol/L, and mixing and dissolving. As a cleaning solution of dibutylethanolamine.
Example 5:
the determination of the Glial Fibrillary Acidic Protein (GFAP) adopts a sandwich method, and the detection principle is as follows:
1) adding 15ul of sample into a reaction tube, sequentially adding the GFAP reagent Ra80ul prepared in example 1 and the GFAP reagent Rb80ul prepared in example 2, incubating at 37 ℃ for 9min, finally adding 40ul of streptavidin magnetic microspheres, and incubating at 37 ℃ for 9 min;
2) sucking the reaction tube after the incubation reaction into an electrochemical flow cell through a liquid absorption steel needle, and adsorbing the reaction tube by a magnet of the flow cell;
3) and (3) sucking a cleaning solution (tripropylamine) by a liquid suction steel needle, cleaning the ruthenium-labeled antibody which is not bound to the superparamagnetic microspheres and the sample, electrifying the flow cell, and emitting light by the terpyridyl ruthenium in the presence of the tripropylamine.
4) And recording the luminous value by using the photomultiplier, and calculating the concentration of the GAFP in the sample according to a standard curve established after the luminous value of the calibration object is corrected by using the curve provided by the enterprise.
Example 6:
the determination of the Glial Fibrillary Acidic Protein (GFAP) adopts a sandwich method, and the detection principle is as follows:
1)1) sampling 15ul of the sample, adding the sample into a reaction tube, sequentially adding the GFAP reagent Ra80ul prepared in example 1 and the GFAP reagent Rb80ul prepared in example 2, incubating at 37 ℃ for 9min, finally adding 40ul of streptavidin superparamagnetic microspheres, and incubating at 37 ℃ for 9 min;
2) adding a streptavidin-coated superparamagnetic microsphere for incubation, and allowing the formed immune complex to be bound to the superparamagnetic microsphere through the interaction between biotin and streptavidin;
3) after incubation, absorbing the reaction mixture into a measuring cell, adsorbing the superparamagnetic microspheres onto an electrode through a magnet, absorbing the cleaning solution (dibutylethanolamine) by a liquid absorbing steel needle, and absorbing a mark Ru (bpy) which is not combined with the superparamagnetic microspheres3 2+After the antibody and the sample were washed, the flow cell was charged, and Ru (bpy) was performed in the presence of dibutylethanolamine3 2+And (4) emitting light.
4) The photomultiplier records the luminous value, and the concentration of GFAP in the sample is calculated according to a standard curve established after the luminous value of the calibration object is corrected by using the curve provided by the enterprise.
5)
Example 7: margin test
(1) The reaction method in example 3 was used, a zero-concentration diluent was used as a sample to obtain RLU values (relative luminescence values) of 20 measurements, the average (M) and Standard Deviation (SD) thereof were calculated to obtain M +2SD, samples of adjacent concentrations were repeatedly tested 2 times, two-point regression fitting was performed according to the concentration-RLU between the zero-concentration diluent and the adjacent low-concentration samples to obtain a linear equation, and the RLU values of M +2SD were substituted into the above equation to obtain the corresponding concentration values as blank limits.
TABLE 1
(2) The reaction method in example 4 was used, and a zero-concentration diluent was used as a sample to obtain RLU values (relative luminescence values) of 20 measurements, and the average (M) and Standard Deviation (SD) thereof were calculated to obtain M +2SD, and samples of adjacent concentrations were repeatedly tested 2 times, and two-point regression fitting was performed according to the concentration-RLU between the zero-concentration diluent and the adjacent low-concentration samples to obtain a linear equation, and the RLU values of M +2SD were substituted into the above equation to obtain the corresponding concentration value, which was the blank limit.
TABLE 2
Example 8: verification of linear range
(1) Using the reaction method of example 3, high-value samples near the upper limit of the linear range (300ng/ml) were diluted to at least 5 concentrations in a proportion in which the low-value samples had to be near 0.30 ng/ml. And operating according to the kit specification, repeatedly detecting the sample with each concentration for 2 times, calculating the average value of the sample to obtain the measured concentration, performing linear fitting on the diluted concentration and the measured concentration by using a least square method, and calculating a linear correlation coefficient r, wherein the r is not less than 0.99.
TABLE 3
Item
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Concentration of dilution
0.30
60.00
120.00
240.00
300.00
Measured concentration 1
0.30
62.53
119.22
244.34
296.92
Measured concentration 2
0.31
61.38
122.11
237.65
298.26
Measured concentration
0.30
61.96
120.66
241.00
297.59
X axis
0.3
60
120
240
300
y axis
0.303
61.956
120.665
241
297.588
R＝0.99994
(2) The reaction method of example 4, wherein the high value sample near the upper limit of the linear range (300ng/ml) is diluted to at least 5 concentrations in a certain proportion, and the low value sample is near 0.30 ng/ml. And operating according to the kit specification, repeatedly detecting the sample with each concentration for 2 times, calculating the average value of the sample to obtain the measured concentration, performing linear fitting on the diluted concentration and the measured concentration by using a least square method, and calculating a linear correlation coefficient r, wherein the r is not less than 0.99.
TABLE 4
Item
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Concentration of dilution
0.30
60.00
120.00
240.00
300.00
Measured concentration 1
0.29
62.75
116.03
242.85
294.62
Measured concentration 2
0.30
60.08
120.17
238.61
293.79
Measured concentration
0.29
61.41
118.10
240.73
294.21
X axis
0.3
60
120
240
300
y axis
0.294
61.413
118.095
240.729
294.207
R＝0.9998
Summary of the comparative kit conditions:
TABLE 5
The electrochemical luminescence immunoassay technology has the advantages of high sensitivity, rapidness, accuracy, good repeatability, safety, no toxicity, no pollution and the like. Luminol, isoluminol and its derivatives are the first type of chemiluminescent species used, but their application to chemiluminescent immunoassays requires the use of catalysts and enhancers, which leads to an increase in background luminescence, thereby limiting the sensitivity of this technology and its application and development. The acridinium ester luminescent system is simple, does not need a catalyst and is placed in H2O2The acridinium ester can emit light in the solution without a catalytic process or an enhancer, so background light emission is reduced, sensitivity is improved, interference effect is small, but the acridinium ester is easy to hydrolyze, and the light emission of the acridinium ester is released rapidly. The peak value of luminescence is 0.4s, so in-situ sample injection is needed, and the requirement on equipment is high. The ruthenium terpyridyl is easy for protein connection, has small molecular weight, has small influence on the conformation of an antibody after connection, is a metal ion as a marker, has good stability, and can emit light under the condition of electrification, so the light emission is controllable. Therefore, the application of the electrochemical method in the detection of GFAP can improve the sensitivity of the product, shorten the process marking time, improve the linear range and shorten the test time, thereby providing a basis for clinically dealing with the treatment of brain trauma in time.
The property of the electrochemiluminescence marker ruthenium pyridine is very stable, and the luminous efficiency of the electrochemiluminescence marker ruthenium pyridine is not influenced by factors such as temperature, pH and ionic strength. The signal value of the electrochemiluminescence reagent is reduced within 3 percent compared with that of a fresh reagent. The bottle opening period is three months, and the bottle can be stabilized at 2-8 ℃ for more than 15 months.
TABLE 6
Light-emitting system
Horseradish enzyme-luminol
Alkaline phosphatase
Electrochemiluminescence
Time stamping
Greater than 24 hours
Greater than 24 hours
60 minutes
Test time
60 minutes
30 minutes
18 minutes
Expiration date of reagent
12 months old
12 months old
More than 15 months
The electrochemical labeling reaction is rapid, and the whole reaction only needs half an hour. The marking efficiency reaches 70%. The proportion of the mark can be controlled by the feeding ratio, the productivity is improved by more than 50 percent, the molecular weight of ruthenium is small (800D), the steric hindrance is small, and the antibody activity is good. An absorption peak at 455nm, the feed ratio can be controlled to control the batch-to-batch difference.
The steps show that the reaction mode of the sandwich method adopted by the invention utilizes the principle of magnetic microsphere electrochemistry to quantitatively detect the content of Glial Fibrillary Acidic Protein (GFAP) in a human serum or plasma sample, thereby ensuring the detection sensitivity. And is suitable for use in fully automatic equipment. The detection speed and the detection flux are increased, the detection efficiency is improved, and errors caused by manual operation are avoided.
Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to those examples; within the idea of the invention, also technical features in the above embodiments or in different embodiments may be combined and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity. Therefore, any omissions, modifications, substitutions, improvements and the like that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.