CN112485445B - Kit for quantitatively detecting GFAP and application thereof - Google Patents
Kit for quantitatively detecting GFAP and application thereof Download PDFInfo
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Abstract
The invention discloses a kit for quantitatively detecting GFAP and application thereof, wherein the kit comprises a GFAP antibody solution marked by magnetic particles, and a buffer solution consists of the following components: na (Na)2HPO4·12H2O 5.6‑5.9g/L、NaH2PO40.55-0.60g/L, NaCl 9.0.0 g/L, bovine serum albumin 1.0-50g/L, sucrose 80.0-140g/L, cellulose salt or cellulose derivative 1.0-5.0g/L, gelatin 5.0-50 g/L; the pH value is 6.2-8.0. The invention improves the fluidity and the sedimentation performance of the magnetic particle antibody conjugate by improving the buffer system of the magnetic particle carrier in the magnetic particle luminescence method, improves the accuracy and the repeatability of the kit, has good stability, low detection limit and good linear relation, and meets the industrial standard.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a kit for quantitatively detecting GFAP and application thereof.
Background
Traumatic Brain Injury (TBI) is brain injury caused by external forces that disrupt normal brain function, resulting in impaired cognitive or physical performance in humans. Among all types of TBI, the most common sequelae are headache (47.9%) and memory abnormalities (42%), with about three patients requiring psychological counseling or neurological treatment. TBI is the most common disease in neurosurgery and is also one of the leading causes of death and disability worldwide, with sequelae that have a permanent impact on patient health. Research shows that the incidence rate of TBI is 790-979 people/10 ten thousand/year in the global scope, and 5400-6000 million people are estimated to suffer from TBI every year in the global scope. Therefore, the number of new TBI patients 1100-1370 ten thousands of people are added every year in China.
The suspected TBI medical care procedure is divided into three steps, first the nerves are evaluated using a 15-minute Glasgow Coma Scale (GCS) to assess the severity of brain injury, then structural neuroimaging examinations are performed, most commonly fractures and intracranial lesions are visualized by head CT scanning, and finally a treatment plan, a stay-in-the-field observation or discharge from a hospital is formulated according to the CT results.
Currently, CT scanning is the only objective, simple and reliable option widely used to help clinicians assess TBI. However, the correctness of the CT result is directly related to the accuracy of the CT device and the level of interpretation of the physician, and is a relatively subjective judgment method compared with other detection methods. CT scans with about 90% mild TBI (sometimes referred to as "concussion") were negative. Less than 1% of these patients require neurosurgical intervention. In view of the very low percentage of CT scan positivity and the unnecessary imaging detection of these patients may increase the risk of radiation-induced carcinogenesis, it is of great clinical and strategic importance to find and develop other brain injury diagnostic methods to accurately determine the extent of craniocerebral injury and to assess prognosis.
Glial Fibrillary Acidic Protein (GFAP) is a type III intermediate filament protein. Human GFAP consists of 432 amino acids, is distributed mainly in astrocytes of the central nervous system, and is involved in cytoskeleton formation and maintenance of its tonicity strength. GFAP is a nervous system specific protein that has a major impact on the recovery of nervous system function in brain injury.
In TBI, GFAP enters the blood through the blood brain barrier within 1 hour, resulting in a significant increase in serum GFAP. Has important significance for early diagnosis, differential diagnosis and prognosis judgment of TBI, and is mainly used for auxiliary diagnosis of brain trauma clinically.
CN109521004A discloses a magnetic particle separation chemiluminescence immunoassay for detecting Glial Fibrillary Acidic Protein (GFAP), the kit composition comprises: the kit comprises a calibrator, a quality control product reagent A, a reagent B, a cleaning solution concentrated solution and a luminescent substrate solution, wherein the calibrator is a Glial Fibrillary Acidic Protein (GFAP) antigen containing a series of concentrations and is used for establishing a standard curve; the quality control product is prepared from a buffer solution containing a certain concentration of Glial Fibrillary Acidic Protein (GFAP) antigen; the reagent A is a colloidal acidic protein (GFAP) antibody solution containing a magnetic particle marker with a certain concentration; the reagent B is a colloidal fiber acidic protein (GFAP) antibody solution containing a certain concentration of alkaline phosphatase label; the concentrated cleaning solution is used for preparing cleaning solution; the luminescent substrate solution is catalyzed by alkaline phosphatase (ALP). The invention improves the signal intensity and sensitivity of immunoreaction, and enables low-content substances to generate strong chemiluminescent signals when carrying out immune combination, thereby providing a more accurate, convenient, rapid and simple method for detecting human Glial Fibrillary Acidic Protein (GFAP).
CN 105300966A discloses a preservation solution, which comprises 0.1-10g/L buffer salt, 100-300g/L antibody stabilizer, 0.1-1g/L antiseptic bacteriostatic agent, 2-12g/L carrageenan and water, wherein the buffer salt, the antibody stabilizer and the antiseptic bacteriostatic agent in the preservation solution act together to ensure the stability of magnetic microparticles or magnetic nanoparticles and antibodies in the whole system, but the system causes the linearity of the detection of the kit to be poor, and the accuracy and stability of the detection result are poor, so that the industrial standard cannot be met.
Disclosure of Invention
In order to solve the technical problems, the invention provides a kit for quantitatively detecting GFAP, which improves the fluidity and the sedimentation performance of a magnetic particle antibody conjugate, improves the accuracy and the repeatability of a detection result of the kit, has good stability, low detection limit and good linear relation and meets the industrial standard by improving a buffer system of a magnetic particle carrier in a magnetic particle luminescence method.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides a GFAP antibody solution marked by magnetic particles, which consists of a GFAP antibody magnetic particle conjugate and a buffer solution 9;
the buffer solution 9 consists of the following components: na (Na)2HPO4·12H2O 5.6-5.9g/L、NaH2PO40.55-0.60g/L, NaCl 9.0.0 g/L, bovine serum albumin 1.0-50g/L, sucrose 80.0-140g/L, cellulose salt or cellulose derivative 1.0-5.0g/L, gelatin 5.0-50 g/L; the pH value is 6.2-8.0.
Preferably, the preparation method of the GFAP antibody magnetic particle conjugate comprises the following steps:
a1, washing the magnetic particles by using a buffer solution 4, and then suspending the magnetic particles to 5mg/mL to obtain a magnetic particle solution; adding a GFAP antibody into the magnetic particle solution according to the mass ratio of the magnetic particles to the antibody of 100:1-10 to obtain a mixed solution 1;
a2, adding a buffer solution 4 into the mixed solution 1 according to the volume mass ratio of the buffer solution 4 to the magnetic particles being 100:1-10, reacting at room temperature for 10min to obtain a mixed solution 2, adding a buffer solution 5 into the mixed solution 2 according to the volume mass ratio of the buffer solution 5 to the magnetic particles being 100:1-10, and reacting at 37 ℃ for 16-24 h to obtain a magnetic particle conjugate;
a3, washing the magnetic particle conjugate with a buffer solution 6, suspending to 5mg/mL, reacting for 16-24 hours at 37 ℃, washing the magnetic particle conjugate with a buffer solution 8, and suspending to 10mg/mL to obtain the GFAP antibody magnetic particle conjugate.
Preferably, the magnetic particles in step a1 are tosyl magnetic beads.
Preferably, the buffer 4 in step a1 consists of: na (Na)2B4O7·10H2O7-10 g/L and pH 9.0-11.0.
Preferably, the buffer 5 in step a2 consists of: k2HPO4470-530 g/L; the pH is 9.0-11.0.
Preferably, the buffer 6 in step a3 consists of: tris 7.5-8.0g/L, NaCl 9.0.0 g/L, bovine serum albumin 3.0-10.0g/L and Tween 205-20 mL; the pH value is 7.3-7.8.
Preferably, the buffer 8 in step a3 consists of: na (Na)2HPO4·12H2O 5.6-5.9g/L、NaH2PO40.55-0.60g/L, NaCl 9.0.0 g/L and bovine serum albumin 1.0-50 g/L; the pH is 7.0-7.6.
The invention also provides a detection reagent strip which comprises the GFAP antibody solution marked by the magnetic particles.
The invention also provides a kit for quantitatively detecting GFAP, which comprises the detection reagent strip.
A kit for quantitatively detecting GFAP comprises a detection reagent strip, a calibrator, a quality control product and a two-dimensional code, wherein the detection reagent strip is integrated by a series of solutions and accessories and can independently detect a sample; the calibrator is prepared from GFAP antigens with two concentrations and buffer solution and is used for calibrating a standard curve; the quality control product is prepared from GFAP antigen and buffer solution; the standard curve of the current batch is recorded in the two-dimensional code.
Preferably, the detection reagent strip comprises a reagent A, a reagent B, a cleaning solution and a luminescent substrate, wherein the reagent A is a GFAP antibody solution containing a certain concentration of alkaline phosphatase label; the reagent B is GFAP antibody solution containing magnetic particles with certain concentration for marking; the cleaning solution is used for cleaning the reaction process; the luminogenic substrate is an ALP-catalyzed luminogenic substrate.
Preferably, the alkaline phosphatase-labeled GFAP antibody solution is composed of an enzyme-labeled GFAP antibody conjugate and a buffer 8.
Preferably, the preparation method of the enzyme-labeled GFAP antibody conjugate comprises the following steps:
b1, activation of antibody:
b1.1 activation step: weighing 4-8mg of 2-iminothiolane hydrochloride (2-IT), dissolving the hydrochloride into 13.76mg/mL by using a buffer solution 1, adding the 2IT solution into an antibody solution according to the molar ratio of the 2-IT to the antibody of 15-30:1 for activation (namely adding 1mg of the antibody into 10-20 mu L of the 2IT solution), shaking and uniformly mixing, and reacting at room temperature for 30 minutes to obtain an activated antibody pre-product;
b1.2 termination of the activation step: adding buffer solution 2 into the activated antibody pre-product solution according to the proportion of adding 1mg antibody into 5-20 μ L buffer solution 2, reacting at room temperature for 10min, removing excessive 2IT by using a PD10 desalting column, collecting the activated antibody to obtain the activated antibody, adding the activated antibody into an ultrafiltration concentration tube, and performing high-speed low-temperature concentration to make the final concentration of the activated antibody be 1-4mg/ml, thereby obtaining the concentrated activated antibody.
B2 activation of alkaline phosphatase (ALP)
B2.1 activation step: weighing 2-4mg (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), dissolving the SMCC in Dimethylformamide (DMF) to 6.69mg/mL, adding an SMCC solution (namely, adding 1mg ALP into 8.5-34.5 muL of SMCC solution) into an ALP solution according to the molar ratio of SMCC to ALP of 15-60:1, shaking, uniformly mixing, and reacting at room temperature for 30 minutes to obtain an alkaline phosphatase (ALP) activated pre-product;
b2.2 termination of the activation step: adding buffer solution 2 into ALP solution at a ratio of 1mg ALP to 10-50 μ L buffer solution 2, reacting at room temperature for 10min, removing excessive SMCC with PD10 desalting column, collecting activated ALP to obtain activated ALP, and concentrating at high speed and low temperature in ultrafiltration concentration tube to obtain concentrated activated ALP with final concentration of 1-4 mg/ml;
linkage of B3, activating antibody and activating ALP
Mixing the concentrated activated antibody obtained in the step B1.2 with the concentrated activated ALP obtained in the step B2.2 according to the mass ratio of 1:2-1 (namely adding 1.0mg of antibody into 1.0-2.0mg of ALP), shaking and uniformly mixing, and reacting the mixture in an environment at the temperature of 2-8 ℃ for 12-18 hours to obtain an antibody conjugate pre-product;
b4 termination and purification of antibody conjugates
Weighing 1-10mg of maleimide, dissolving the maleimide with DMF to 9.7mg/mL, and diluting with buffer solution 1 according to the proportion of 1/10 to obtain 0.97mg/mL of maleimide solution; adding 10 mu L of 0.97mg/mL maleimide solution into 1mg antibody, and reacting for 15 minutes at room temperature; dissolving 6 μ L ethanolamine with buffer solution 1 to 100mM, adding 994 μ L buffer solution 1 into 6 μ L ethanolamine, adding ethanolamine solution according to the proportion of adding 1mg antibody into 10-50 μ L100 mM ethanolamine solution, shaking and mixing; concentrating the antibody conjugate to be purified to 0.5-2mg/mL by using an ultrafiltration concentration tube; and (3) purifying the antibody by using a purified protein analyzer and a Superdex 200 preparative grade 2.6/60 gel column, wherein the eluent is a buffer solution 2, and purifying to obtain the enzyme-labeled antibody conjugate.
The invention also provides application of the kit in detecting the content of human peripheral blood and/or serum GFAP.
The invention also provides a detection method of the kit, which comprises the following steps:
s1, immune response: sequentially adding a 30uL sample, a 50uL reagent B and a 50uL reagent A into the reaction hole, and reacting for 20min at 37 ℃;
s2, magnetic separation and cleaning: adding 300 mu L of cleaning solution into a cleaning hole position M1, sucking the mixture containing magnetic particles out of the reaction hole position by using magnetic force, and demagnetizing at a cleaning hole position M1; after cleaning for 2min, respectively carrying out 1-time magnetic separation and cleaning on a cleaning hole position M2 and a cleaning hole position M3;
s3, reading: adding 150uL of luminescent substrate into the reading hole, sucking the mixture containing magnetic particles out of the cleaning hole position M3 by magnetic force, demagnetizing the reading hole, and detecting relative luminescence intensity (RLU) after the luminescent substrate catalyzed by alkaline phosphatase emits light;
s4, obtaining a GFAP concentration-luminous value standard curve according to the detected value of the calibrator, wherein the standard curve is fitted by using a four-parameter Logistic equation;
and S5, enabling the detection value of the sample to correspond to the concentration value on the standard curve, and realizing the concentration detection of the sample.
The invention has the beneficial effects that:
(1) the invention uses immunological detection means to carry out blood detection on traumatic brain injury, compared with imaging detection means (mainly CT), the invention can objectively reflect the real situation of a sample, and reduces misjudgment and missed judgment caused by subjective judgment;
(2) the magnetic particle chemiluminescence method used in the invention controls the calibration material, the quality control material and the specific components of the kit A and the kit B, and the specific buffer solution is used for dilution, so that the detection sensitivity can reach the picogram level (10)-12g/mL), even if CT is negative, normal people and mild TBI patients can be effectively distinguished;
(3) the invention uses a full-automatic instrument for detection, and accurate results can be obtained only by adding a serum sample for 30 minutes. The CT detection time is long, and the detection result can be obtained only by waiting for at least 4 hours;
(4) the concentration value is used for judgment, and the obtained result can indicate whether the patient is ill, so that the objectivity is strong, and the missed judgment and the misjudgment are not easy to cause;
(5) the magnetic particles being of a massGranules mainly containing FeO and Fe2O3The diameter of the magnetic particle antibody conjugate is 1-4 microns, the magnetic particle antibody conjugate is insoluble in water, the magnetic particle antibody conjugate has certain hydrophilicity due to the connection of protein, the magnetic particle antibody conjugate can quickly sink in an aqueous medium under the action of gravity, hardening can occur after a certain time, and the difficulty of uniformly mixing the hardened magnetic particle antibody conjugate again is high. The invention improves the fluidity and the sedimentation performance of the magnetic particle antibody coupling substance by improving the buffer system of the magnetic particle carrier in the magnetic particle luminescence method, improves the stability of the detection result, and has good repeatability, lower detection limit and good linear relation.
Drawings
FIG. 1 is a flow chart of an immune reaction.
FIG. 2 is a schematic diagram of a GFAP detection reagent strip.
Wherein: a-reaction hole site, M1-cleaning hole site, M2-cleaning hole site, M3-cleaning hole site and B-reading hole site.
Detailed Description
The following description of the embodiments is only intended to aid in the understanding of the method of the invention and its core ideas. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention. The following description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The water used in the present invention is purified water, and the buffer solution used is filtered through a 0.22 μm filter, unless otherwise specified.
Basic embodiment
As shown in fig. 1 and 2, the present invention provides a kit for quantitatively detecting GFAP, which comprises a detection reagent strip, a calibrator, a quality control material and a two-dimensional code, wherein the detection reagent strip comprises a series of solutions and accessories to form a whole, and can independently detect a sample; the calibrator is prepared from GFAP antigens with two concentrations and buffer solution and is used for calibrating a standard curve; the quality control product is prepared from GFAP antigen and buffer solution; the standard curve of the current batch is recorded in the two-dimensional code.
The detection reagent strip comprises a reagent A, a reagent B, a cleaning solution, a luminescent substrate, a reading hole, an elution sleeve and a suction head, wherein the reagent A is a GFAP antibody solution containing alkaline phosphatase marks with a certain concentration; the reagent B is GFAP antibody solution containing magnetic particles with certain concentration for marking; the cleaning solution is used for cleaning the reaction process; the luminescent substrate is an ALP catalyzed luminescent substrate; the reading hole is used for final detection reading;
the preparation method of the reagent A comprises the following steps: dissolving the enzyme-labeled GFAP antibody conjugate in a buffer solution 8, and uniformly mixing; the buffer solution 8 consists of the following components: na (Na)2HPO4·12H2O5.6-5.9 g/L, NaCl 9.0.0 g/L and bovine serum albumin 1.0-50g/L, and pH is 7.0-7.6.
The preparation method of the enzyme-labeled GFAP antibody conjugate comprises the following steps: the method comprises the following steps:
b1, activation of antibody:
b1.1 activation step: weighing 4-8mg of 2-iminothiolane hydrochloride (2-IT), dissolving the hydrochloride into 13.76mg/mL by using a buffer solution 1, adding the 2IT solution into an antibody solution according to the molar ratio of the 2-IT to the antibody of 15-30:1 for activation (namely adding 1mg of the antibody into 10-20 mu L of the 2IT solution), shaking and uniformly mixing, and reacting at room temperature for 30 minutes to obtain an activated antibody pre-product;
b1.2 termination of the activation step: adding the buffer solution 2 into the activated antibody pre-product solution according to the proportion that 1mg of antibody is added into 5-20 mul of buffer solution 2, reacting for 10min at room temperature, removing excessive 2IT by using a PD10 desalting column, collecting the activated antibody to obtain the activated antibody, adding the activated antibody into an ultrafiltration concentration tube for high-speed low-temperature concentration to ensure that the final concentration is between 1-4mg/mL, and obtaining the concentrated activated antibody;
b2 activation of alkaline phosphatase (ALP)
B2.1 activation step: weighing 2-4mg (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), dissolving the SMCC in Dimethylformamide (DMF) to 6.69mg/mL, adding an SMCC solution (namely, adding 1mg ALP into 8.5-34.5 muL of SMCC solution) into an ALP solution according to the molar ratio of SMCC to ALP of 15-60:1, shaking, uniformly mixing, and reacting at room temperature for 30 minutes to obtain an alkaline phosphatase (ALP) activated pre-product;
b2.2 termination of the activation step: adding buffer solution 2 into ALP solution at a ratio of 1mg ALP to 10-50 μ L buffer solution 2, reacting at room temperature for 10min, removing excessive SMCC with PD10 desalting column, collecting activated ALP to obtain activated ALP, and concentrating at high speed and low temperature in ultrafiltration concentration tube to obtain concentrated activated ALP with final concentration of 1-4 mg/mL;
linkage of B3, activating antibody and activating ALP
Mixing the concentrated activated antibody obtained in the step B1.2 with the concentrated activated ALP obtained in the step B2.2 according to the mass ratio of 1:2-1 (namely adding 1.0mg of antibody into 1.0-2.0mg of ALP), shaking and uniformly mixing, and reacting the mixture in an environment at the temperature of 2-8 ℃ for 12-18 hours to obtain an antibody conjugate pre-product;
b4 termination and purification of antibody conjugates
Weighing 1-10mg of maleimide, dissolving the maleimide with DMF to 9.7mg/mL, and diluting with buffer solution 1 according to the proportion of 1/10 to obtain 0.97mg/mL of maleimide solution; adding 10 mu L of 0.97mg/mL maleimide solution into 1mg antibody, and reacting for 15 minutes at room temperature; dissolving 6 μ L ethanolamine with buffer solution 1 to 100mM, adding 994 μ L buffer solution 1 into 6 μ L ethanolamine, adding ethanolamine solution according to the proportion of adding 1mg antibody into 10-50 μ L100 mM ethanolamine solution, shaking and mixing; concentrating the antibody conjugate to be purified to 0.5-2mg/mL by using an ultrafiltration concentration tube; purifying the antibody by using a purified protein analyzer and a Superdex 200 preparative 2.6/60 gel column, wherein the eluent is a buffer solution 2, and purifying to obtain an enzyme-labeled antibody conjugate;
the buffer solution 1 consists of the following components: ethanolamine 14.8-15.1g/L and NaCl 5.8-6.0g/L, and pH 7.3-7.6;
the buffer solution 2 consists of the following components: 75g/L of glycine;
the preparation method of the reagent B comprises the following steps: dissolving the GFAP antibody magnetic particle conjugate in buffer solution 9, and uniformly mixing;
the buffer solution 9 consists of the following components: na (Na)2HPO4·12H2O 5.6-5.9g/L、NaH2PO40.55-0.60g/L, NaCl 9.0.0 g/L, bovine serum albumin 1.0-50g/L, sucrose 80.0-140g/L, cellulose salt or cellulose derivative 1.0-5.0g/L, gelatin 5.0-50 g/L; the pH value is 6.2-8.0.
The preparation method of the GFAP antibody magnetic particle conjugate comprises the following steps:
a1, washing the magnetic particles by using a buffer solution 4, and then suspending the magnetic particles to 5mg/mL to obtain a magnetic particle solution; adding a GFAP antibody into the magnetic particle solution according to the mass ratio of the magnetic particles to the antibody of 100:1-10 to obtain a mixed solution 1;
a2, adding a buffer solution 4 into the mixed solution 1 according to the volume mass ratio of the buffer solution 4 to the magnetic particles being 100:1-10, reacting at room temperature for 10min to obtain a mixed solution 2, adding a buffer solution 5 into the mixed solution 2 according to the volume mass ratio of the buffer solution 5 to the magnetic particles being 100:1-10, and reacting at 37 ℃ for 16-24 h to obtain a magnetic particle conjugate;
a3, washing the magnetic particle conjugate by using a buffer solution 6, resuspending to 5mg/mL, reacting for 16-24 hours at 37 ℃, washing the magnetic particle conjugate by using a buffer solution 8, and resuspending to 10mg/mL to obtain the GFAP antibody magnetic particle conjugate;
the buffer solution 4 consists of the following components: na (Na)2B4O7·10H2O7.0-10.0 g/L; the pH is 9.0-11.0;
the buffer solution 5 consists of the following componentsConsists of the following components: k2HPO4470-530 g/L; the pH is 9.0-11.0;
the buffer solution 6 consists of the following components: tris 7.5-8.0g/L, NaCl 9.0.0 g/L, bovine serum albumin 3.0-10g/L, Tween 205-20 mL; the pH value is 7.3-7.8.
The calibrator is a GFAP recombinant protein solution with the concentration of 20pg/mL and 160pg/mL, and the preparation method comprises the following steps: dissolving GFAP recombinant protein in buffer solution 7, and mixing uniformly;
the quality control product is a GFAP recombinant protein solution with the concentration of 40pg/mL, and the preparation method comprises the following steps: dissolving GFAP recombinant protein in buffer solution 7, and mixing uniformly;
the buffer solution 7 consists of the following components: tris 12.0-15.0g/L, bovine serum albumin 5.0-50g/L, glycine 1.0-30 g/L; the pH value is 7.6-8.8.
The invention also provides a detection method of the kit, which comprises the following steps:
s1, immune response: sequentially adding a 30uL sample, a 50uL reagent B and a 50uL reagent A into the reaction hole, and reacting for 20min at 37 ℃;
s2, magnetic separation and cleaning: adding 300 mu L of cleaning solution into a cleaning hole position M1, sucking the mixture containing magnetic particles out of the reaction hole position by using magnetic force, and demagnetizing at a cleaning hole position M1; after cleaning for 2min, respectively carrying out 1-time magnetic separation and cleaning on a cleaning hole position M2 and a cleaning hole position M3;
s3, reading: adding 150uL of luminescent substrate into the reading hole, sucking the mixture containing magnetic particles out of the cleaning hole position M3 by magnetic force, demagnetizing the reading hole, and detecting relative luminescence intensity (RLU) after the luminescent substrate catalyzed by alkaline phosphatase emits light;
s4, obtaining a GFAP concentration-luminous value standard curve according to the detected value of the calibrator, wherein the standard curve is fitted by using a four-parameter Logistic equation;
and S5, enabling the detection value of the sample to correspond to the concentration value on the standard curve, and realizing the concentration detection of the sample.
In order to facilitate understanding of the present invention by those skilled in the art, the technical solutions and effects of the present invention are further described below with reference to specific embodiments.
Example 1
A kit for quantitatively detecting GFAP comprises a detection reagent strip, a calibrator, a quality control product and a two-dimensional code (shown in table 1), wherein the detection reagent strip is integrated by a series of solutions and accessories and can independently detect a sample; the calibrator is prepared from GFAP antigens with two concentrations and buffer solution and is used for calibrating a standard curve; the quality control product is prepared from GFAP antigen and buffer solution; the standard curve of the current batch is recorded in the two-dimensional code.
TABLE 1 component Table of kit
The detection reagent strip comprises a reagent A, a reagent B, a cleaning solution, a luminescent substrate, a reading hole, an elution sleeve and a suction head, wherein the reagent A is a GFAP antibody solution containing alkaline phosphatase marks with a certain concentration; the reagent B is GFAP antibody solution containing magnetic particles with certain concentration for marking; the cleaning solution is used for cleaning the reaction process; the luminescent substrate is an ALP catalyzed luminescent substrate; the reading hole is used for final detection reading;
the preparation method of the reagent A comprises the following steps: dissolving the enzyme-labeled GFAP antibody conjugate in a buffer solution 8, and uniformly mixing; the buffer solution 8 consists of the following components: na (Na)2HPO4·12H2O5.6 g/L, NaCl 9.0.0 g/L and bovine serum albumin 1.0g/L, and the pH is 7.0-7.6.
The preparation method of the enzyme-labeled GFAP antibody conjugate comprises the following steps: the method comprises the following steps:
b1, activation of antibody:
b1.1 activation step: weighing 4-8mg of 2-iminothiolane hydrochloride (2-IT), dissolving the hydrochloride into 13.76mg/mL by using a buffer solution 1, adding the 2IT solution into an antibody solution according to the molar ratio of the 2-IT to the antibody of 15:1 for activation (namely adding 1mg of the antibody into 10 mu L of the 2IT solution), shaking and uniformly mixing, and reacting at room temperature for 30 minutes to obtain an activated antibody pre-product;
b1.2 termination of the activation step: adding the buffer solution 2 into the activated antibody pre-product solution according to the proportion that 1mg of the antibody is added into 5 mu L of the buffer solution 2, reacting for 10min at room temperature, removing excessive 2IT by using a PD10 desalting column, collecting the activated antibody to obtain the activated antibody, adding the activated antibody into an ultrafiltration concentration tube to perform high-speed low-temperature concentration, and enabling the final concentration to be 1-4mg/mL to obtain the concentrated activated antibody;
b2 activation of alkaline phosphatase (ALP)
B2.1 activation step: weighing 2-4mg (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), dissolving the SMCC in Dimethylformamide (DMF) to 6.69mg/mL, adding an SMCC solution (namely, adding 8.5 mu L of SMCC solution into 1mg ALP) into an ALP solution according to the molar ratio of SMCC to ALP of 15:1, shaking and uniformly mixing, and reacting at room temperature for 30 minutes to obtain an alkaline phosphatase (ALP) activated pre-product;
b2.2 termination of the activation step: adding buffer solution 2 into ALP solution at a ratio of 1mg ALP to 10 μ L buffer solution 2, reacting at room temperature for 10min, removing excessive SMCC with PD10 desalting column, collecting activated ALP to obtain activated ALP, and concentrating the activated ALP at high speed and low temperature in ultrafiltration concentration tube to obtain concentrated activated ALP with final concentration of 1-4 mg/mL;
linkage of B3, activating antibody and activating ALP
Mixing the concentrated activated antibody obtained in the step B1.2 and the concentrated activated ALP obtained in the step B2.2 according to the mass ratio of 1:2 (namely adding 1.0mg of antibody into 2.0mg of ALP), shaking and uniformly mixing, and reacting the mixture at the temperature of 2 ℃ for 18 hours to obtain an antibody conjugate pre-product;
b4 termination and purification of antibody conjugates
Weighing 1-10mg of maleimide, dissolving the maleimide with DMF to 9.7mg/mL, and diluting with buffer solution 1 according to the proportion of 1/10 to obtain 0.97mg/mL of maleimide solution; adding 10 mu L of 0.97mg/mL maleimide solution into 1mg antibody, and reacting for 15 minutes at room temperature; dissolving 6 μ L ethanolamine with buffer solution 1 to 100mM, adding 994 μ L buffer solution 1 into 6 μ L ethanolamine, adding ethanolamine solution according to the proportion of adding 1mg antibody into 10 μ L100 mM ethanolamine solution, shaking and mixing; concentrating the antibody conjugate to be purified to 0.5-2mg/mL by using an ultrafiltration concentration tube; purifying the antibody by using a purified protein analyzer and a Superdex 200 preparative 2.6/60 gel column, wherein the eluent is a buffer solution 2, and purifying to obtain an enzyme-labeled antibody conjugate;
the buffer solution 1 consists of the following components: ethanolamine 14.8g/L and NaCl 5.8g/L, and pH 7.3-7.6;
the buffer solution 2 consists of the following components: 75g/L of glycine;
the preparation method of the reagent B comprises the following steps: dissolving the GFAP antibody magnetic particle conjugate in buffer solution 9, and uniformly mixing;
the buffer solution 9 consists of the following components: na (Na)2HPO4·12H2O 5.6g/L、NaH2PO40.55g/L, NaCl 9.0.0 g/L, bovine serum albumin 1.0g/L, sucrose 80.0g/L, cellulose salt or cellulose derivative 1.0g/L, gelatin 50 g/L; the pH value is 6.2-8.0.
The preparation method of the GFAP antibody magnetic particle conjugate comprises the following steps:
a1, washing the magnetic particles by using a buffer solution 4, and then suspending the magnetic particles to 5mg/mL to obtain a magnetic particle solution; adding a GFAP antibody into the magnetic particle solution according to the mass ratio of the magnetic particles to the antibody of 100:1 to obtain a mixed solution 1;
a2, adding a buffer solution 4 into the mixed solution 1 according to the volume mass ratio of the buffer solution 4 to the magnetic particles being 100:1, reacting at room temperature for 10min to obtain a mixed solution 2, adding a buffer solution 5 into the mixture 2 according to the volume mass ratio of the buffer solution 5 to the magnetic particles being 100:1, and reacting at 37 ℃ for 16 h to obtain a magnetic particle conjugate;
a3, washing the magnetic particle conjugate by using a buffer solution 6, carrying out resuspension to 5mg/mL, reacting for 16 hours at 37 ℃, washing the magnetic particle conjugate by using a buffer solution 8, and carrying out resuspension to 10mg/mL to obtain the GFAP antibody magnetic particle conjugate;
the buffer solution 4 consists of the following components: na (Na)2B4O7·10H2O7.0 g/L; the pH is 9.0-11.0;
the buffer solution 5 consists of the following components: k2HPO4470 g/L; the pH is 9.0-11.0;
the buffer solution 6 consists of the following components: tris 7.5g/L, NaCl 9.0.0 g/L, bovine serum albumin 3.0g/L, Tween 205 mL; the pH value is 7.3-7.8.
The calibrator 1 is a 20pg/mL GFAP recombinant protein solution, the standard 2 is a 160pg/mL GFAP recombinant protein solution, and the preparation method comprises the following steps: the GFAP recombinant protein was dissolved in buffer 7 and mixed well.
The quality control product is a GFAP recombinant protein solution with the concentration of 40pg/mL, and the preparation method comprises the following steps: the GFAP recombinant protein was dissolved in buffer 7 and mixed well.
The buffer solution 7 consists of the following components: tris 12.0g/L, bovine serum albumin 5.0g/L, glycine 1.0 g/L; the pH value is 7.6-8.8.
Example 2
A kit for quantitatively detecting GFAP comprises a detection reagent strip, a calibrator, a quality control product and a two-dimensional code (shown in table 2), wherein the detection reagent strip is integrated by a series of solutions and accessories and can independently detect a sample; the calibrator is prepared from GFAP antigens with two concentrations and buffer solution and is used for calibrating a standard curve; the quality control product is prepared from GFAP antigen and buffer solution; the standard curve of the current batch is recorded in the two-dimensional code.
TABLE 2 kit Components Table
| The main components of the kit | Loading capacity |
| Detection reagent strip | 10 strips |
| Quality control product | 200μL×1 |
| Calibration article 1 | 200μL×1 |
| Calibration article 2 | 200μL×1 |
| Box label two-dimensional code | 1 is provided with |
The detection reagent strip comprises a reagent A, a reagent B, a cleaning solution, a luminescent substrate, a reading hole, an elution sleeve and a suction head, wherein the reagent A is a GFAP antibody solution containing alkaline phosphatase marks with a certain concentration; the reagent B is GFAP antibody solution containing magnetic particles with certain concentration for marking; the cleaning solution is used for cleaning the reaction process; the luminescent substrate is an ALP catalyzed luminescent substrate; the reading hole is used for final detection reading;
the preparation method of the reagent A comprises the following steps: dissolving the enzyme-labeled GFAP antibody conjugate in a buffer solution 8, and uniformly mixing; the buffer solution 8 consists of the following components: na (Na)2HPO4·12H2O5.9 g/L, NaCl 9.0.0 g/L and bovine serum albumin 50g/L, and the pH is 7.0-7.6.
The preparation method of the enzyme-labeled GFAP antibody conjugate comprises the following steps: the method comprises the following steps:
b1, activation of antibody:
b1.1 activation step: weighing 4-8mg of 2-iminothiolane hydrochloride (2-IT), dissolving the hydrochloride into 13.76mg/mL by using a buffer solution 1, adding the 2IT solution into an antibody solution according to the molar ratio of the 2-IT to the antibody of 30:1 for activation (namely adding 1mg of the antibody into 20 mu L of the 2IT solution), shaking and uniformly mixing, and reacting at room temperature for 30 minutes to obtain an activated antibody pre-product;
b1.2 termination of the activation step: adding the buffer solution 2 into the activated antibody pre-product solution according to the proportion that 1mg of the antibody is added into 20 mu L of the buffer solution 2, reacting for 10min at room temperature, removing excessive 2IT by using a PD10 desalting column, collecting the activated antibody to obtain the activated antibody, adding the activated antibody into an ultrafiltration concentration tube to perform high-speed low-temperature concentration, and enabling the final concentration to be 1-4mg/mL to obtain the concentrated activated antibody;
b2 activation of alkaline phosphatase (ALP)
B2.1 activation step: weighing 2-4mg (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), dissolving the SMCC in Dimethylformamide (DMF) to 6.69mg/mL, adding an SMCC solution (namely, adding 34.5 mu L of SMCC solution into 1mg ALP) into an ALP solution according to the molar ratio of SMCC to ALP of 60:1, shaking and uniformly mixing, and reacting at room temperature for 30 minutes to obtain an alkaline phosphatase (ALP) activated pre-product;
b2.2 termination of the activation step: adding buffer solution 2 into ALP solution at a ratio of 1mg ALP to 50 μ L buffer solution 2, reacting at room temperature for 10min, removing excessive SMCC with PD10 desalting column, collecting activated ALP to obtain activated ALP, and concentrating the activated ALP at high speed and low temperature in ultrafiltration concentration tube to obtain concentrated activated ALP with final concentration of 1-4 mg/mL;
linkage of B3, activating antibody and activating ALP
Mixing the concentrated activated antibody obtained in the step B1.2 and the concentrated activated ALP obtained in the step B2.2 according to the mass ratio of 1:1 (namely adding 1.0mg of antibody into 1.0mg of ALP), shaking and uniformly mixing, and reacting the mixture in an environment at 8 ℃ for 12 hours to obtain an antibody conjugate pre-product;
b4 termination and purification of antibody conjugates
Weighing 1-10mg of maleimide, dissolving the maleimide with DMF to 9.7mg/mL, and diluting with buffer solution 1 according to the proportion of 1/10 to obtain 0.97mg/mL of maleimide solution; adding 10 mu L of 0.97mg/mL maleimide solution into 1mg antibody, and reacting for 15 minutes at room temperature; dissolving 6 μ L ethanolamine with buffer solution 1 to 100mM, adding 994 μ L buffer solution 1 into 6 μ L ethanolamine, adding ethanolamine solution according to the proportion of adding 1mg antibody into 50 μ L100 mM ethanolamine solution, shaking and mixing; concentrating the antibody conjugate to be purified to 0.5-2mg/mL by using an ultrafiltration concentration tube; purifying the antibody by using a purified protein analyzer and a Superdex 200 preparative 2.6/60 gel column, wherein the eluent is a buffer solution 2, and purifying to obtain an enzyme-labeled antibody conjugate;
the buffer solution 1 consists of the following components: ethanolamine 15.1g/L and NaCl 6.0g/L, and pH 7.3-7.6;
the buffer solution 2 consists of the following components: 75g/L of glycine;
the preparation method of the reagent B comprises the following steps: dissolving the GFAP antibody magnetic particle conjugate in buffer solution 9, and uniformly mixing;
the buffer solution 9 consists of the following components: na (Na)2HPO4·12H2O 5.9g/L、NaH2PO40.60g/L, NaCl 9.0.0 g/L, bovine serum albumin 50g/L, sucrose 140g/L, cellulose salt or cellulose derivative 5.0g/L, gelatin 5.0 g/L; the pH value is 6.2-8.0.
The preparation method of the GFAP antibody magnetic particle conjugate comprises the following steps:
a1, washing the magnetic particles by using a buffer solution 4, and then suspending the magnetic particles to 5mg/mL to obtain a magnetic particle solution; adding a GFAP antibody into the magnetic particle solution according to the mass ratio of the magnetic particles to the antibody of 100:10 to obtain a mixed solution 1;
a2, adding a buffer solution 4 into the mixed solution 1 according to the volume mass ratio of the buffer solution 4 to the magnetic particles being 100:10, reacting at room temperature for 10min to obtain a mixed solution 2, adding a buffer solution 5 into the mixture 2 according to the volume mass ratio of the buffer solution 5 to the magnetic particles being 100:10, and reacting at 37 ℃ for 24 h to obtain a magnetic particle conjugate;
a3, washing the magnetic particle conjugate by using a buffer solution 6, carrying out resuspension to 5mg/mL, reacting for 24 hours at 37 ℃, washing the magnetic particle conjugate by using a buffer solution 8, and carrying out resuspension to 10mg/mL to obtain the GFAP antibody magnetic particle conjugate;
the buffer solution 4 consists of the following components: na (Na)2B4O7·10H2O10.0 g/L; the pH is 9.0-11.0;
the buffer solution 5 consists of the following components: k2HPO4530 g/L; the pH is 9.0-11.0;
the buffer solution 6 consists of the following components: tris 8.0g/L, NaCl 9.0.0 g/L, bovine serum albumin 10g/L, Tween 2020 mL; the pH value is 7.3-7.8.
The calibrator 1 is a 20pg/mL GFAP recombinant protein solution, the standard 2 is a 160pg/mL GFAP recombinant protein solution, and the preparation method comprises the following steps: the GFAP recombinant protein was dissolved in buffer 7 and mixed well.
The quality control product is a GFAP recombinant protein solution with the concentration of 40pg/mL, and the preparation method comprises the following steps: the GFAP recombinant protein was dissolved in buffer 7 and mixed well.
The buffer solution 7 consists of the following components: 15.0g/L of Tris, 50g/L of bovine serum albumin and 30g/L of glycine; the pH value is 7.6-8.8.
Example 3
A kit for quantitatively detecting GFAP comprises a detection reagent strip, a calibrator, a quality control product and a two-dimensional code (shown in Table 3), wherein the detection reagent strip is integrated by a series of solutions and accessories and can independently detect a sample; the calibrator is prepared from GFAP antigens with two concentrations and buffer solution and is used for calibrating a standard curve; the quality control product is prepared from GFAP antigen and buffer solution; the standard curve of the current batch is recorded in the two-dimensional code.
TABLE 3 kit Components Table
The detection reagent strip comprises a reagent A, a reagent B, a cleaning solution, a luminescent substrate, a reading hole, an elution sleeve and a suction head, wherein the reagent A is a GFAP antibody solution containing alkaline phosphatase marks with a certain concentration; the reagent B is GFAP antibody solution containing magnetic particles with certain concentration for marking; the cleaning solution is used for cleaning the reaction process; the luminescent substrate is an ALP catalyzed luminescent substrate; the reading hole is used for final detection reading;
the preparation method of the reagent A comprises the following steps: dissolving the enzyme-labeled GFAP antibody conjugate in a buffer solution 8, and uniformly mixing; the buffer solution 8 consists of the following components: na (Na)2HPO4·12H2O5.7 g/L, NaCl 9.0.0 g/L and bovine serum albumin 25.5g/L, and the pH is 7.0-7.6.
The preparation method of the enzyme-labeled GFAP antibody conjugate comprises the following steps: the method comprises the following steps:
b1, activation of antibody:
b1.1 activation step: weighing 4-8mg of 2-iminothiolane hydrochloride (2-IT), dissolving the hydrochloride into 13.76mg/mL by using a buffer solution 1, adding the 2IT solution into an antibody solution according to the molar ratio of the 2-IT to the antibody of 22.5:1 for activation (namely adding 1mg of the antibody into 15 mu L of the 2IT solution), shaking and uniformly mixing, and reacting at room temperature for 30 minutes to obtain an activated antibody pre-product;
b1.2 termination of the activation step: adding buffer solution 2 into the activated antibody pre-product solution according to the proportion that 1mg antibody is added into 12.5 mu L buffer solution 2, reacting for 10min at room temperature, removing excessive 2IT by using a PD10 desalting column, collecting the activated antibody to obtain the activated antibody, adding the activated antibody into an ultrafiltration concentration tube for high-speed low-temperature concentration to ensure that the final concentration is between 1 and 4mg/mL, and obtaining the concentrated activated antibody;
b2 activation of alkaline phosphatase (ALP)
B2.1 activation step: weighing 2-4mg (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), dissolving the SMCC in Dimethylformamide (DMF) to 6.69mg/mL, adding an SMCC solution (namely, adding 21.5 mu L of SMCC solution into 1mg ALP) into an ALP solution according to the molar ratio of SMCC to ALP of 37.5:1, shaking and uniformly mixing, and reacting at room temperature for 30 minutes to obtain an alkaline phosphatase (ALP) activated pre-product;
b2.2 termination of the activation step: adding buffer solution 2 into ALP solution at a ratio of 1mg ALP to 30 μ l buffer solution 2, reacting at room temperature for 10min, removing excessive SMCC with PD10 desalting column, collecting activated ALP to obtain activated ALP, and concentrating the activated ALP at high speed and low temperature in ultrafiltration concentration tube to obtain concentrated activated ALP with final concentration of 1-4 mg/mL;
linkage of B3, activating antibody and activating ALP
Mixing the concentrated activated antibody obtained in the step B1.2 and the concentrated activated ALP obtained in the step B2.2 according to the mass ratio of 1:1.5 (namely adding 1.50mg of ALP into 1.0mg of antibody), shaking and uniformly mixing, and reacting the mixture for 15 hours in an environment at 6 ℃ to obtain an antibody conjugate pre-product;
b4 termination and purification of antibody conjugates
Weighing 1-10mg of maleimide, dissolving the maleimide with DMF to 9.7mg/mL, and diluting with buffer solution 1 according to the proportion of 1/10 to obtain 0.97mg/mL of maleimide solution; adding 10 mu L of 0.97mg/mL maleimide solution into 1mg antibody, and reacting for 15 minutes at room temperature; dissolving 6 mu L of ethanolamine into 100mM buffer solution 1, namely adding 994 mu L of buffer solution 1 into 6 mu L of ethanolamine, adding ethanolamine solution according to the proportion of adding 1mg of antibody into 30 mu L of 100mM ethanolamine solution, and shaking and uniformly mixing; concentrating the antibody conjugate to be purified to 0.5-2mg/mL by using an ultrafiltration concentration tube; purifying the antibody by using a purified protein analyzer and a Superdex 200 preparative 2.6/60 gel column, wherein the eluent is a buffer solution 2, and purifying to obtain an enzyme-labeled antibody conjugate;
the buffer solution 1 consists of the following components: ethanolamine 14.9g/L and NaCl5.9 g/L, and the pH value is 7.3-7.6;
the buffer solution 2 consists of the following components: 75g/L of glycine;
the preparation method of the reagent B comprises the following steps: dissolving the GFAP antibody magnetic particle conjugate in buffer solution 9, and uniformly mixing;
the buffer solution 9 consists of the following components: na (Na)2HPO4·12H2O 5.8g/L、NaH2PO40.58g/L, NaCl 9.0.0 g/L, bovine serum albumin 34.5g/L, sucrose 110g/L, cellulose salt or cellulose derivative 3.0g/L, gelatin 27.5 g/L; the pH value is 6.2-8.0.
The preparation method of the GFAP antibody magnetic particle conjugate comprises the following steps:
a1, washing the magnetic particles by using a buffer solution 4, and then suspending the magnetic particles to 5mg/mL to obtain a magnetic particle solution; adding a GFAP antibody into the magnetic particle solution according to the mass ratio of the magnetic particles to the antibody of 100:5 to obtain a mixed solution 1;
a2, adding a buffer solution 4 into the mixed solution 1 according to the volume mass ratio of the buffer solution 4 to the magnetic particles being 100:5, reacting at room temperature for 10min to obtain a mixed solution 2, adding a buffer solution 5 into the mixture 2 according to the volume mass ratio of the buffer solution 5 to the magnetic particles being 100:5, and reacting at 37 ℃ for 20 h to obtain a magnetic particle conjugate;
a3, washing the magnetic particle conjugate by using a buffer solution 6, carrying out resuspension to 5mg/mL, reacting for 20 hours at 37 ℃, washing the magnetic particle conjugate by using a buffer solution 8, and carrying out resuspension to 10mg/mL to obtain the GFAP antibody magnetic particle conjugate;
the buffer solution 4 consists of the following components: na (Na)2B4O7·10H2O8.5 g/L; the pH is 9.0-11.0;
the buffer solution 5 consists of the following components: k2HPO4500 g/L; the pH is 9.0-11.0;
the buffer solution 6 consists of the following components: tris 7.8g/L, NaCl 9.0.0 g/L, bovine serum albumin 6.5g/L, Tween 2012.5 mL; the pH value is 7.3-7.8.
The calibrator 1 is a 20pg/mL GFAP recombinant protein solution, the standard 2 is a 160pg/mL GFAP recombinant protein solution, and the preparation method comprises the following steps: the GFAP recombinant protein was dissolved in buffer 7 and mixed well.
The quality control product is a GFAP recombinant protein solution with the concentration of 40pg/mL, and the preparation method comprises the following steps: the GFAP recombinant protein was dissolved in buffer 7 and mixed well.
The buffer solution 7 consists of the following components: 13.5g/L Tris, 27.5g/L bovine serum albumin and 15.5g/L glycine; the pH value is 7.6-8.8.
Comparative example 1
This comparative example differs from example 3 in that the buffer 9 used in the preparation of reagent B consists of the following components: na (Na)2HPO4·12H2O 4.5g/L、NaH2PO40.5g/L, NaCl 4.0.0 g/L, bovine serum albumin 100g/L, sucrose 70g/L, cellulose salt or cellulose derivative 8.0g/L, gelatin 22g/L and carrageenan 8 g/L; the pH is 6.2-8.0。
Comparative example 2
This comparative example differs from example 3 in that the buffer 9 used in the preparation of reagent B consists of the following components: na (Na)2HPO4·12H2O 6.5g/L、NaH2PO40.64g/L, NaCl 7.0.0 g/L, bovine serum albumin 60g/L, sucrose 150g/L, cellulose salt or cellulose derivative 1g/L, gelatin 4 g/L; the pH value is 6.2-8.0.
Comparative example 3
This comparative example differs from example 3 in that the buffer 9 used in the preparation of reagent B consists of the following components: na (Na)2HPO4·12H2O 5.0g/L、NaH2PO40.5g/L, NaCl 8.0.0 g/L, bovine serum albumin 1g/L, sucrose 40g/L, cellulose salt or cellulose derivative 6g/L, gelatin 60 g/L; the pH value is 6.2-8.0.
Comparative example 4
The comparative example is different from example 3 in that the preparation method of the GFAP antibody magnetic particle conjugate used as the raw material of the reagent B comprises the following steps:
a1, washing the magnetic particles by using a buffer solution 4, and then suspending the magnetic particles to 5mg/mL to obtain a magnetic particle solution; adding a GFAP antibody into the magnetic particle solution according to the mass ratio of the magnetic particles to the antibody of 100:5 to obtain a mixed solution 1;
a2, adding a buffer solution 4 into the mixed solution 1 according to the volume mass ratio of the buffer solution 4 to the magnetic particles being 100:5, reacting at room temperature for 10min to obtain a mixed solution 2, adding a buffer solution 5 into the mixture 2 according to the volume mass ratio of the buffer solution 5 to the magnetic particles being 100:5, and reacting at 37 ℃ for 20 h to obtain a magnetic particle conjugate;
a3, washing the magnetic particle conjugate by using a buffer solution 6, carrying out resuspension to 5mg/mL, reacting for 20 hours at 37 ℃, washing the magnetic particle conjugate by using a buffer solution 8, and carrying out resuspension to 10mg/mL to obtain the GFAP antibody magnetic particle conjugate;
the buffer solution 4 consists of the following components: na (Na)2B4O7·10H2O12 g/L; the pH is 9.0-11.0;
the buffer solution 5 consists of the following componentsConsists of the following components: k2HPO4450 g/L; the pH is 9.0-11.0;
the buffer solution 6 consists of the following components: tris 6g/L, NaCl 6.0.0 g/L, bovine serum albumin 20g/L, Tween 2015 mL; the pH value is 7.3-7.8.
Example 4
A detection method of a kit for quantitatively detecting GFAP adopts a full-automatic chemiluminescence immunoassay analyzer of Beijing America Thailaceae biotechnology limited company for detection, and comprises the following steps:
s1, immune response: sequentially adding a 30uL sample, a 50uL reagent B and a 50uL reagent A into the reaction hole, and reacting for 20min at 37 ℃;
s2, magnetic separation and cleaning: adding 300 mu L of cleaning solution into a cleaning hole position M1, sucking the mixture containing magnetic particles out of the reaction hole position by using magnetic force, and demagnetizing at a cleaning hole position M1; after cleaning for 2min, respectively carrying out 1-time magnetic separation and cleaning on a cleaning hole position M2 and a cleaning hole position M3;
s3, reading: adding 150uL of luminescent substrate into the reading hole, sucking the mixture containing magnetic particles out of the cleaning hole position M3 by magnetic force, demagnetizing the reading hole, and detecting relative luminescence intensity (RLU) after the luminescent substrate catalyzed by alkaline phosphatase emits light;
s4, obtaining a GFAP concentration-luminous value standard curve according to the detected value of the calibrator, wherein the standard curve is fitted by using a four-parameter Logistic equation;
and S5, enabling the detection value of the sample to correspond to the concentration value on the standard curve, and realizing the concentration detection of the sample.
Example 5 kit Performance analysis
The kits for quantitatively detecting GFAP prepared in examples 1 to 3 and comparative examples 1 to 4 were tested according to the test method of example 4, and the accuracy, detection limit, linearity, reproducibility, and difference between the calibrator and the quality control bottle of the kit were analyzed.
(1) Accuracy of
The experimental requirements are as follows: adding a known concentration of Glial Fibrillary Acidic Protein (GFAP) to the low value samples, wherein the recovery rate is in the range of [85,115 ];
the experimental method comprises the following steps: a Glial Fibrillary Acidic Protein (GFAP) solution (A) with a concentration of 800pg/mL (tolerance. + -. 10%) was added to a sample B with a concentration of 0pg/mL to 20pg/mL, the volume ratio of the added glial fibrillary acidic protein antigen to the sample B was 1:9, and the recovery rate R was calculated according to the formula (1), and the results of the measurements are shown in Table 4.
In the formula:
r-recovery rate;
v is the volume of the sample A liquid;
V0-volume of serum sample B fluid;
c is the average value of 3 measurements after the serum sample B liquid is added into the liquid A;
C0-mean of 3 measurements of serum sample B fluid;
CS-concentration of sample a liquid.
TABLE 4
| Reagent kit | The recovery rate is high |
| Example 1 | 89.22 |
| Example 2 | 110.08 |
| Example 3 | 102.45 |
As can be seen from the above table, the recovery rate of the kits prepared in examples 1 to 3 of the present invention is in the range of [85,115 ]% and meets the requirement.
(2) Margin limit
The experimental requirements are as follows: less than or equal to 10 pg/mL.
The experimental method comprises the following steps: the zero concentration calibrator was used as a sample, and the test was repeated 20 times to obtain the RLU value (relative luminescence value) of the 20 test results, and the average value thereof was calculated
And Standard Deviation (SD). According to the calibration curve equation of the calibrator used in the kit, the average value is calculated
The corresponding RLU value is substituted into the above equation to obtain the corresponding concentration value, which is the blank limit, and the result is shown in table 5.
TABLE 5
As can be seen from the above table, the blank limit of the kit prepared in examples 1-3 of the present invention is not higher than 10pg/mL, which meets the requirement.
(3) Detection limit
The experimental requirements are as follows: the detection limit is not higher than 15 pg/mL.
The experimental method comprises the following steps: samples of 200pg/mL were diluted 8, 10, 12, 14, 16 fold, respectively. And (3) detecting 5 low-value samples with approximate LOD concentration, detecting 5 times for each sample, sequencing the detection results according to the sizes, and if the number of the detection results lower than the blank limit value provided by a production enterprise is less than or equal to 3, determining that the blank limit (10pg/mL) and the detection limit are basically reasonable in setting, wherein the detection results are shown in Table 6.
TABLE 6
| Examples of the invention | Number below blank limit (10pg/mL) |
| Example 1 | 1 |
| Example 2 | 1 |
| Example 3 | 0 |
As can be seen from the above table, the number of the detection results of the kit in embodiments 1 to 3 of the present invention, which is lower than the blank limit value provided by the manufacturing enterprise, is 0 or 1, and the detection limit is set reasonably to meet the requirements.
(4) Linear interval
The experimental requirements are as follows: within the linear range of [0.015,25] ng/mL, the correlation coefficient r should be greater than or equal to 0.990.
The experimental method comprises the following steps: high value samples (25ng/mL) near the upper end of the linear range were mixed with zero concentration samples to 6 dilution concentrations, theoretical concentrations 25ng/mL, 5ng/mL, 1ng/mL, 0.2ng/mL, 0.02ng/mL, 0ng/mL, respectively. Repeating the test 3 times for each sample to obtain concentration value, recording the measurement result of each sample, and calculating the average value (y) of the 3 measurement values of each samplei). In diluted concentration (x)i) As independent variable, the mean value (y) of the results is determinedi) Linear regression equations were solved for the dependent variables. The correlation coefficient (r) of the linear regression was calculated according to the formula (2), and the results are shown in Table 7.
In the formula:
r-correlation coefficient;
xidilution ratio;
yi-mean of individual sample measurements;
TABLE 7
| Reagent kit | Coefficient of correlation r |
| Example 1 | 0.9993 |
| Example 2 | 0.9995 |
| Example 3 | 0.9998 |
| Comparative example 1 | 0.7134 |
| Comparative example 2 | 0.8553 |
| Comparative example 3 | 0.8216 |
| Comparative example 4 | 0.8886 |
As can be seen from the above table, the results of the linear correlation coefficient (r) experiment of the kits of examples 1-3 are not less than 0.9900, and thus the linear range of the kits of examples 1-3 can be defined as [0.015,25] ng/mL; the linear correlation coefficient (r) of the kits of comparative examples 1 to 4 was less than 0.9900, and the experimental results of the linear correlation coefficient (r) were less than 0.9900, indicating that when the composition and amount of the buffer solution 9 of the GFAP antibody solution labeled with the reagent B magnetic particles and the preparation method of the raw material GFAP antibody magnetic particle conjugate were changed, the linear relationship of the kits was affected, resulting in deterioration of the linear relationship.
(5) Repeatability of
The experimental requirements are as follows: the high-concentration sample and the low-concentration sample are respectively tested for 10 times, and the CV is less than or equal to 10 percent.
The experimental method comprises the following steps: repeatedly testing high concentration samples (4-6ng/mL) and low concentration samples (0.18-0.22ng/mL) 10 times by the same batch number kit, and calculating the average value of 10 testing results
And standard deviation SD. The Coefficient of Variation (CV) was calculated according to equation (3), and the results are shown in table 8.
In the formula:
SD — standard deviation of sample test values;
TABLE 8
As can be seen from the above table, the reproducibility of the kits of examples 1 to 3 of the present invention was not higher than 10%, whereas the reproducibility of the kits of comparative examples 1 to 4 was higher than 10%, indicating that the composition and amount of buffer solution 9 when the reagent B magnetic particle-labeled GFAP antibody solution was changed and the preparation method of the raw material GFAP antibody magnetic particle conjugate resulted in deterioration of the reproducibility and stability of the kits.
(6) Difference between calibrator and quality control bottle
The experimental requirements are as follows: the difference CV between the calibrator and the quality control product bottle is less than 10 percent.
The experimental method comprises the following steps: detecting 10 bottles of calibrator (or quality control material) of the same batch for 1 time respectively, calculating according to formula (4), and determining the mean value of the results
And Standard Deviation (SD)1). Continuously measuring 1 bottle of the above 10 bottles of calibrator (or quality control) for 5 times, and calculating the mean value of the results
And Standard Deviation (SD)2) The inter-vial reproducibility CV% was calculated according to the equations (5) and (7), and the measurement results are shown in Table 9.
Description of the drawings: when SD1<SD2Let CV beBottle room＝0
In the formula: SD-standard deviation.
TABLE 9
As can be seen from the above table, the reagent kits of examples 1-3 both meet the requirements for detecting the difference CV between the calibrator and the quality control material bottles of less than 10%, while the reagent kits of comparative examples 1-4 both meet the requirements for detecting the difference CV between the calibrator and the quality control material bottles of more than or equal to 10%.
The magnetic particles are particles with a certain mass and mainly comprise Fe0 and Fe2O3The diameter of the magnetic particle antibody conjugate is 1-4 microns, the magnetic particle antibody conjugate is insoluble in water, the magnetic particle antibody conjugate has certain hydrophilicity due to the fact that the protein is connected, the magnetic particle antibody conjugate can sink rapidly in an aqueous medium under the action of gravity, even can be hardened after a certain time, and the difficulty of uniformly mixing the hardened magnetic particle antibody conjugate again is high, so that the detection result of the kit is influenced.
The invention improves the buffer system (buffer solution 9) of the magnetic particle carrier in the magnetic particle luminescence method, so that the invention has the following advantages:
(1) the magnetic particle antibody conjugate can not sink visible to naked eyes within 7 days under a normal temperature environment;
(2) the reagent B keeps good fluidity in a normal-temperature environment, the absorption amount cannot be influenced when an instrument performs absorption operation, and the solution cannot be hung on the wall and remains when being uniformly mixed;
(3) the reagent B can be changed into gel under the environment of 2-8 ℃, and the suspension property of the magnetic particle antibody conjugate can be kept within 6 months;
(4) the experiment was performed at month 13, and although the magnetic particle antibody conjugate had settled completely, it was very well mixed.
As shown in Table 10, the kits of examples 1-3 of the present invention showed a CV of less than 8% in the reproducibility results at month 14, with an absolute deviation of no more than 10% from the accuracy results at month 0. Whereas the results of comparative examples 1-4 at month 14 had a repeatability CV of greater than 30% with an absolute deviation from the accuracy results at month 0 of more than 50%,
wherein, the calculation formula of the deviation from 0 month is as follows: (monthly 14 recovery rate value-monthly 0 recovery rate value) × 100/monthly 0 recovery rate value.
Watch 10
In conclusion, the kit for quantitatively detecting GFAP improves the fluidity and the sedimentation performance of the magnetic particle antibody conjugate, improves the accuracy and the repeatability of detection, has good stability, low detection limit and good linear relation, and meets the industrial standard.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (3)
1. A magnetic particle labeled GFAP antibody solution is characterized by consisting of a GFAP antibody magnetic particle conjugate and a buffer solution 9;
the buffer solution 9 consists of the following components: na (Na)2HPO4·12H2O 5.8g/L、NaH2PO40.58g/L, NaCl 9.0.0 g/L, bovine serum albumin 34.5g/L, sucrose 110g/L, cellulose salt or cellulose derivative 3.0g/L, gelatin 27.5 g/L; the pH value is 6.2-8.0;
the preparation method of the GFAP antibody magnetic particle conjugate comprises the following steps:
(1) washing the magnetic particles by using a buffer solution 4, and then suspending the magnetic particles to 5mg/mL to obtain a magnetic particle solution; adding a GFAP antibody into the magnetic particle solution according to the mass ratio of the magnetic particles to the antibody of 100:5 to obtain a mixed solution 1;
(2) adding a buffer solution 4 into the mixed solution 1 according to the volume mass ratio of the buffer solution 4 to the magnetic particles of 100:5, reacting at room temperature for 10min to obtain a mixed solution 2, adding a buffer solution 5 into the mixed solution 2 according to the volume mass ratio of the buffer solution 5 to the magnetic particles of 100:5, and reacting at 37 ℃ for 20 hours to obtain a magnetic particle conjugate;
(3) washing the magnetic particle conjugate with a buffer solution 6, resuspending to 5mg/mL, reacting for 20 hours at 37 ℃, washing the magnetic particle conjugate with a buffer solution 8, and resuspending to 10mg/mL to obtain the GFAP antibody magnetic particle conjugate;
the magnetic particles in the step (1) are tosyl magnetic beads;
the buffer solution 4 in the step (1) consists of the following components: na (Na)2B4O7·10H2O8.5 g/L, pH 9.0-11.0;
the buffer solution 5 in the step (2) consists of the following components: k2HPO4 500 g/L; the pH is 9.0-11.0;
the buffer solution 6 in the step (3) consists of the following components: tris 7.8g/L, NaCl 9.0.0 g/L, bovine serum albumin 6.5g/L and Tween 2012.5 mL; the pH value is 7.3-7.8;
the buffer solution 8 in the step (3) consists of the following components: na (Na)2HPO4·12H2O5.7 g/L, NaCl 9.0.0 g/L and bovine serum albumin 25.5 g/L; the pH is 7.0-7.6.
2. A test reagent strip comprising the magnetic particle-labeled GFAP antibody solution according to claim 1.
3. A kit for quantitatively detecting GFAP, comprising the detection reagent strip of claim 2, a calibrator, a quality controller, and a two-dimensional code, wherein the detection reagent strip is integrated with a series of solutions and accessories for independent detection of a sample; the calibrator is prepared from GFAP antigens with two concentrations and buffer solution and is used for calibrating a standard curve; the quality control product is prepared from GFAP antigen and buffer solution; the standard curve of the current batch is recorded in the two-dimensional code;
the detection reagent strip comprises a reagent A, a reagent B, a cleaning solution and a luminescent substrate, wherein the reagent A is a GFAP antibody solution marked by alkaline phosphatase; the reagent B is a GFAP antibody solution marked by magnetic particles; the cleaning solution is used for cleaning the reaction process; the luminogenic substrate is an ALP-catalyzed luminogenic substrate.
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