Gfa protein magnetic microparticle chemiluminescence immunoassay quantitative detection reagent kit

Abstract

The application discloses a quantitative detection reagent kit for glial fibrillary acidic protein (GFAP) by magnetic particle chemiluminescence immunoassay, which comprises a streptomycin affinity substance coated magnetic particle / biotinized GFAP capture antibody compound suspension, alkaline phosphatase labeled GFAP detection antibody solution, recombinant GFAP antigen serial calibration product, whole blood sample diluent, chemiluminescence liquid and cleaning liquid.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, and particularly relates to a glial fibrillary acidic protein magnetic microparticle chemiluminescence immunoassay quantitative detection kit. BACKGROUND

[0002] Exogenous brain injury (TBI) is caused by external force, which can damage the normal function of the brain and cause cognitive impairment or brain function impairment. It is the most common disease in neurosurgery. Its sequelae are mostly headache (47.9%) and memory abnormalities (42%), which have a permanent impact on the health of patients. Currently, Glasgow coma scale is used to evaluate the neurological behavior, and imaging is used for examination. CT scan is the only objective, simple and reliable choice for clinical physician evaluation. Currently, about 90% of CT scans of mild TBI are negative, and only 1% of people need neurosurgical intervention. Given that the percentage of positive CT scans is very low, unnecessary imaging tests may increase the risk of radiation-induced cancer. Therefore, it is of great clinical significance and strategic significance to find and develop brain injury markers. In TBI, GFAP enters the blood within 1h, resulting in a significant increase in GFAP in serum.

[0003] Glial fibrillary acidic protein (GFAP, hereinafter referred to as GFAP) is a class III intermediate filament, which is an acidic protein composed of 432 amino acids, with a relative molecular mass of 50-52KD, and an isoelectric point of 5.7-5.8. GFAP exists in the central nervous system of astrocytes (AS) and the peripheral nervous system, and is an important skeletal protein of AS. As the main skeletal protein in astrocytes, it has multiple biological functions such as maintaining the morphological stability of astrocytes, participating in the formation of the blood-brain barrier, and regulating synaptic function. It plays an important role in neurophysiological activity, neural tissue regeneration, immunity, and the pathogenesis of various neurological diseases.

[0004] Currently, the main methods for GFAP detection are enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), high-performance liquid chromatography, and gas chromatography. However, these methods have certain defects. RIA has high pollution, ELISA has long reaction time, low automation, low sensitivity, and complicated operation, and cannot accurately test whole blood. High-performance liquid chromatography and gas chromatography cannot be widely used in clinical practice. In addition, some patients require immediate diagnosis due to head injury. Therefore, a detection method with low pollution, convenient operation, fast results, good specificity, high sensitivity, and direct whole blood detection is needed for quantitative detection of glial fibrillary acidic protein. SUMMARY

[0005] Therefore, the application provides a GFAP magnetic microparticle chemiluminescence immunoassay quantitative detection kit, which has high sensitivity and specificity and can be directly used for whole blood detection.

[0006] To achieve the above technical purposes, the application adopts the following technical scheme.

[0007] The application provides a GFAP magnetic microparticle chemiluminescence immunoassay quantitative detection kit, which comprises a streptavidin-coated magnetic microparticle / biotinylated GFAP capture antibody complex suspension, an alkaline phosphatase-labeled GFAP detection antibody solution, a recombinant GFAP antigen series calibration product, a whole blood sample diluent, a chemiluminescence solution and a cleaning solution.

[0008] Preferably, the preparation method of the streptavidin-coated magnetic microparticle / biotinylated GFAP capture antibody complex is as follows: after the streptavidin-coated magnetic microparticles and the biotinylated GFAP capture antibody are mixed and coated, magnetic cleaning is performed, and then the mixture is resuspended in a magnetic microparticle coating buffer to obtain the streptavidin-coated magnetic microparticle / biotinylated GFAP capture antibody complex.

[0009] Preferably, the mass ratio of the streptavidin-coated magnetic microparticles to the biotinylated GFAP capture antibody is 1:10-40.

[0010] Preferably, the concentration of the streptavidin-coated magnetic microparticles is 1-10 μg / ml.

[0011] Preferably, the whole blood sample diluent comprises the following components: sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, cysteine, glycerol, PC-300, BSA, Triton X-100 and EDTA·2Na·2H2O.

[0012] Preferably, the preparation method of the alkaline phosphatase-labeled GFAP detection antibody solution is as follows: the N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate activated GFAP capture antibody is mixed with Traunt's reagent activated alkaline phosphatase, and then column purification is performed, and the mixture is dispersed in an enzyme-labeled buffer.

[0013] Preferably, the enzyme-labeled buffer comprises the following components: MES, NaCl, MgCl2, PVP, ZnCl2, BSA, Tween-20 and PC-300.

[0014] Preferably, the GFAP detection antibody and the GFAP capture antibody are different mouse-derived monoclonal antibodies.

[0015] Preferably, the preparation method of the recombinant GFAP antigen series calibration sample is that the recombinant GFAP antigen is dissolved in a calibration sample buffer, and the concentration of the recombinant GFAP antigen series calibration sample is 30 pg / ml and 900 pg / ml.

[0016] Preferably, the components of the cleaning solution include Tris, Tween-20, a preservative, and the pH value of the cleaning solution is 8-9.

[0017] The beneficial effects of the present application are as follows: the present scheme combines the double-antibody sandwich method and the magnetic microparticle chemiluminescence technology to prepare a kit for measuring the content of GFAP in whole blood samples, which can achieve rapid detection and has the advantages of multiple use conditions, high sensitivity, good accuracy, good stability, and automatic operation; the detection is rapid, the reagent range is wide, and the linear range is wide. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The standard curve of the kit GFAP of Example 1. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0020] The present application provides a glial fibrillary acidic protein magnetic microparticle chemiluminescence immunoassay quantitative detection kit, which comprises a streptomycin affinity substance coated magnetic microparticle / biotinized GFAP capture antibody complex suspension, an alkaline phosphatase labeled GFAP detection antibody solution, a recombinant GFAP antigen series calibration sample, a whole blood sample diluent, a chemiluminescence solution, and a cleaning solution.

[0021] The principle of the present scheme is: adopting double antibody sandwich method combined with magnetic particle chemiluminescence technology, through the development of reagent process, GFAP in serum sample and whole blood sample can be quantitatively detected; the streptavidin-coated magnetic particles and biotinylated GFAP antibody are coated by affinity and biotin to form a magnetic bead-antibody complex, and a working solution is formed according to a certain concentration; the GFAP detection sample or the recombinant GFAP antigen series calibrator is added, through antigen-antibody reaction, a complex of magnetic particle-affinity-biotin-GFAP capture antibody-GFAP is formed, and then the alkaline phosphatase-labeled GFAP detection antibody is added, through antigen-antibody reaction, the GFAP detection antibody specifically binds to another binding site of GFAP to form a complex structure of magnetic particle-affinity-biotin-GFAP capture antibody-GFAP-GFAP detection antibody-alkaline phosphatase, through magnetic separation and washing process, the complex is adsorbed in the sample addition structure, and the chemiluminescence solution is added, and the instrument detects the luminescence value (RLU) of the solution, and the concentration of GFAP in the test sample is quantitatively calculated according to the standard curve.

[0022] In the process of determining the whole blood sample, the magnetic bead-antibody complex formed by the streptavidin-coated magnetic particle / biotinylated GFAP capture antibody complex of the present application has stronger anti-interference ability, higher accuracy and stronger sensitivity compared with the reaction system formed by synchronously adding streptavidin-coated magnetic particles, biotinylated GFAP capture antibody and other components in the detection process, which avoids the defects of low binding efficiency of streptavidin-coated magnetic particles and biotinylated GFAP capture antibody in the detection process, false negative or false positive caused by interference of whole blood sample, and more accurately utilizes the streptavidin-biotin system for signal amplification to improve sensitivity.

[0023] The preparation method of the streptavidin-coated magnetic particle / biotinylated GFAP capture antibody complex is as follows: after mixing and coating the streptavidin-coated magnetic particles and biotinylated GFAP capture antibody, magnetic cleaning and purification are performed, and then the complex is resuspended in a magnetic particle coating buffer to obtain the streptavidin-coated magnetic particle / biotinylated GFAP capture antibody complex.

[0024] In some embodiments, the working concentration of the magnetic particle coating buffer is 1-10 μg / ml, which contains 0.1 M PBS, and the specific components include Na2HPO4·12H2O, NaH2PO4·2H2O, NaCl, Tween-20, BSA, gelatin, and BND.

[0025] The mass ratio of streptomycin affinity coated magnetic particles to biotinylated GFAP capture antibody is 1:10-40, preferably 1:20, within this range, the biotinylated GFAP capture antibody is kept in slight excess to enhance the degree of streptomycin affinity-biotin binding, and excessive biotinylated GFAP capture antibody will cause difficulties in purification or waste, etc.

[0026] The concentration of streptomycin affinity coated magnetic particle antibody is 1-10 μg / ml, too high will cause raw material waste, too low will cause insufficient sensitivity of reagent, preferably 2 μg / ml.

[0027] The whole blood sample diluent includes the following components: sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, cysteine, glycerol, PC-300, BSA, Triton X-100, EDTA·2Na·2H2O, the whole blood sample diluent of the present application can protect the integrity of blood cells in the reaction by optimizing the process, reduce the interference of whole blood components on reagent testing, and reduce the whole blood testing CV.

[0028] The preparation method of alkaline phosphatase labeled GFAP detection antibody solution is as follows: mixing N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate activated GFAP capture antibody with Traunt's reagent activated alkaline phosphatase, then column purification, and dispersing in enzyme-labeled buffer, in some embodiments, the concentration of alkaline phosphatase labeled GFAP detection antibody solution is 0.1-4.0 ug / mL, preferably 1.0 ug / mL, the enzyme-labeled buffer includes 0.05M MES and 1% BSA, in some embodiments, the enzyme-labeled buffer includes the following components: MES, NaCl, MgCl2, PVP, ZnCl2, BSA, Tween-20, PC-300, the PVP in the enzyme-labeled buffer of the present application significantly reduces non-specific interference of the reagent under the support of other components, improves the low-end CV, preferably the pH value of the enzyme-labeled buffer is 6.

[0029] The GFAP detection antibody and the GFAP capture antibody are different mouse monoclonal antibodies.

[0030] The preparation method of recombinant GFAP antigen series calibrator is to dissolve the recombinant GFAP antigen in calibrator buffer, the concentration of recombinant GFAP antigen series calibrator is 30 pg / ml, 900 pg / ml, the calibrator buffer contains 0.1M Tris and 2% BSA, and the pH value is 7.4.

[0031] The components of the washing solution include Tris, Tween-20, preservatives, and the pH value of the washing solution is 8-9.

[0032] Chemiluminescent solution is AMPPD substrate solution of 2-amino-2-methyl-1-propanol.

[0033] The preparation method of the biotin-labeled GFAP capture antibody is as follows: the GFAP capture antibody is column-purified, the purified GFAP capture antibody is mixed with biotin-NHS at a molar ratio of 1:1, and incubated at 25 DEG C for 45 min, and then column-purified again after incubation.

[0034] The present application is further described below through specific examples. The reagents and raw materials used in the present application are common and well-known commercial reagents used by those skilled in the art.

[0035] Example 1

[0036] A magnetic microparticle chemiluminescence immunoassay quantitative detection kit for glial fibrillary acidic protein comprises a streptavidin-coated magnetic microparticle / biotinylated GFAP capture antibody complex suspension, an alkaline phosphatase-labeled GFAP detection antibody solution, a recombinant GFAP antigen series calibration product, a whole blood sample diluent, a chemiluminescent solution, and a cleaning solution.

[0037] 1. Streptavidin-coated magnetic microparticle / biotinylated GFAP capture antibody complex suspension

[0038] Preparation of biotinylated GFAP capture antibody: the GFAP capture antibody is purified by a commercial purification column (Thermo; item number: 89882), the purified antibody is collected, the GFAP capture antibody is mixed with commercial biotin-NHS (Thermo; item number: 21336) at a molar ratio of 1:1, and incubated at 25 DEG C for 45 min, and then the biotin-GFAP capture antibody conjugate is purified by a purification column to obtain a GFAP-Biotion mother liquor (biotinylated GFAP capture antibody);

[0039] Preparation of complex suspension: the streptavidin-coated magnetic microparticle mother liquor (purchased from Invitrogen Company) is added to the magnetic microparticle coating buffer to form a streptavidin-coated magnetic microparticle suspension with a concentration of 2 mg / ml, and biotinylated GFAP antibody with a final concentration of 40 ug / ml is added; mixed for 30 min; washed three times, resuspended in the magnetic microparticle coating buffer, and the biotin antibody is adjusted to a working solution concentration of 2 ug / ml. The formula of the magnetic microparticle coating buffer is shown in Table 1, and after preparation, NaOH or HCl is used to adjust the pH value to 7.4±0.1.

[0040] Table 1 Formula of magnetic microparticle coating buffer

[0041] Material name Concentration Na2HPO4-12H2O 0.01M NaH2PO4.2H2O 0.01M NaCl 0.15M Tween-20 0.05％ BSA 1％ Gelatin 1％ BND 1％

[0042] 2. Preparation of alkaline phosphatase-labeled GFAP detection antibody solution

[0043] Commercially available N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (Thermo; item number 22322) was used to activate commercially available GFAP detection antibody at room temperature, and alkaline phosphatase activated with Traunt's reagent (Thermo; item number: 21336) at room temperature was mixed at a ratio of 1:1.2, and GFAP-AP (alkaline phosphatase-labeled GFAP detection antibody) mother liquor was obtained by separation and purification with a purification column. The coupled GFAP-AP mother liquor was diluted with enzyme marker buffer to a working solution with a concentration of 1.0 ug / mL for use, wherein the enzyme marker buffer was optimized, and the addition of PVP significantly reduced non-specific interference of reagents and improved low-end CV; after the enzyme marker buffer was prepared according to the formula in Table 2, NaOH or HC1 was used to adjust the pH value to 6.0±0.1.

[0044] Table 2 Formula of enzyme marker buffer

[0045] Material name Concentration MES 50 mM NaCl 0.9％ MgCl2 5 mM PVP 0.2％ ZnCl2 0.1 mM BSA 1％ Tween-20 0.1％ PC-300 0.05％

[0046] 3. Recombinant GFAP antigen series calibrator

[0047] The recombinant GFAP antigen series calibrator was prepared into working solutions with concentrations of about 10 ng / mL and 45 ng / mL, respectively, using calibrator buffer. After the calibrator buffer was prepared according to the formula in Table 4, NaOH or HC1 was used to adjust the pH value to 7.4±0.1.

[0048] Table 4 Formula of calibrator buffer

[0049] Material name Concentration Tris 0.1M BSA 2％ Trehalose 2％ PC-300 0.3％

[0050] 4. Whole blood sample diluent

[0051] By optimizing the sample diluent, the integrity of blood cells in the reaction was protected, the interference of whole blood components on reagent testing was reduced, and the whole blood testing CV was reduced. The sample diluent was prepared according to Table 3, and then NaOH or HC1 was used to adjust the pH value to 7.6.

[0052] Table 3 Formula of sample diluent

[0053]

[0054]

[0055] 5. Chemiluminescence solution

[0056] The chemiluminescent solution was prepared according to Table 5, and then the pH value was adjusted to 10.0 ± 0.1 using NaOH or HC1.

[0057] Table 5 Formulation of chemiluminescent solution

[0058] Material name Concentration 2-Amino-2-methyl-1-propanol 0.5M AMPPD 0.1％ Rhodamine 6G 0.01％ MgSO4 0.01M ZnCl2 0.01M PC-300 0.1％

[0059] 6. Washing solution

[0060] The washing solution was prepared according to Table 6, and then the pH value was adjusted to 8.7 ± 0.1 using NaOH or HC1.

[0061] Table 6 Formulation of washing solution

[0062] Material name Concentration NaCl 0.9％ Tris 0.01M Tween-20 0.1％ Triton X-100 0.05％ BND 0.1％

[0063] The streptavidin-coated magnetic microparticle suspension, alkaline phosphatase-labeled GFAP detection antibody solution, sample diluent, recombinant GFAP antigen series calibrators, chemiluminescent solution, and washing solution were assembled into a kit and stored at 2-8 °C.

[0064] Comparative Example 1

[0065] A glial fibrillary acidic protein magnetic microparticle chemiluminescent immunoassay quantitative detection kit includes streptavidin-coated magnetic microparticle suspension, biotinylated GFAP capture antibody complex suspension, alkaline phosphatase-labeled GFAP detection antibody solution, recombinant GFAP antigen series calibrators, whole blood sample diluent, chemiluminescent solution, and washing solution. The preparation and preparation methods of other components are the same as those of Example 1, except that the preparation methods of streptavidin-coated magnetic microparticle suspension and biotinylated GFAP capture antibody complex suspension are different.

[0066] Biotinylated GFAP capture antibody complex suspension: The GFAP capture antibody was purified by a commercial purification column (Thermo; item number: 89882), and the purified antibody was collected. The GFAP capture antibody was mixed with commercial biotin-NHS (Thermo; item number: 21336) at a molar ratio of 1:1, and incubated at 25 °C for 45 min. After incubation, the biotin-GFAP capture antibody conjugate was purified by a purification column to obtain GFAP-Biotion (biotinylated GFAP capture antibody) mother liquor.

[0067] Streptavidin-coated magnetic microparticle suspension: The streptavidin-coated magnetic microparticle mother liquor (purchased from Invitrogen Company) was added to the magnetic microparticle coating buffer to form a streptavidin-coated magnetic microparticle suspension with a concentration of 2 mg / ml. The components of the magnetic microparticle coating buffer are shown in Table 1.

[0068] Comparative Example 2

[0069] A GFAP magnetic microparticle chemiluminescence immunoassay quantitative detection kit, other contents are the same as Example 1, the difference is that the composition of the whole blood sample diluent does not include cysteine.

[0070] Comparative Example 3

[0071] A GFAP magnetic microparticle chemiluminescence immunoassay quantitative detection kit, other contents are the same as Example 1, the difference is that the enzyme label buffer does not include PVP.

[0072] Evaluation test

[0073] The GFAP magnetic microparticle chemiluminescence immunoassay quantitative detection kits of Example 1 and Comparative Examples 1, 2 and 3 were subjected to method evaluation according to the following method:

[0074] Sample addition and incubation process: 50ul of GFAP calibrator (working calibrator or product calibrator), or fresh sample was added to the reaction strip, followed by 50ul of sample diluent, 50ul of GFAP coated magnetic microparticle suspension, 50ul of alkaline phosphatase labeled GFAP detection antibody solution, and then 37℃ incubation reaction for 5min.

[0075] Magnetic separation and washing process: magnetic separation and washing for 3 times.

[0076] Light emission process: add chemiluminescence solution, measure the number of photons after incubation at 37℃, the content of GFAP in the sample is proportional to the number of photons, draw the standard working curve, and calculate the content of GFAP in the sample according to the standard working curve.

[0077] The detection standards and results of the standard curve, detection limit, specificity, precision, linear range, accuracy, hook effect, and uniformity of the calibrator in Example 1 according to the above operation steps are as follows.

[0078] The standard working curve of Example 1 is shown in Figure 1 The standard curve equation of the kit for detecting GFAP in this scheme is: R 2 = 0.999.

[0079] Detection limit: 5 samples with concentrations close to the lower value of the detection limit were detected, each sample was detected 5 times, and the detection results were sorted according to the size, the results of Example 1 are shown in Table 7, the number of detection results below the blank limit (0.50 pg / mL) should be less than or equal to 3, that is, the detection limit test requirement is met, to determine whether the detection limit of Example 1 can reach 2 pg / ml.

[0080] Table 7 Detection limit test results

[0081]

[0082] The results show that the detection limit of Example 1 can reach 2 pg / ml. The number of detection results lower than the blank limit 0.5 pg / ml of Comparative Example 1 is 4, which does not meet the detection limit requirement

[0083] Specificity: The vimentin with a concentration not less than 354000.00 pg / mL and the desmin with a concentration not less than 127000.00 pg / mL were respectively added to the zero concentration sample, and the detection was repeated for 3 times to take the average value M. The average value Co of the detection results before the addition of the cross-reactants was taken as the average value of the first 3 detection results in the 20 times of measurement of the blank limit of the zero concentration sample. The cross-reactivity was calculated according to formula (1) to judge the specificity. The results of Example 1 are shown in Table 8.

[0084] Rcr = (M-C0) / C x 100% (1), wherein Rcr is the cross-reactivity, M is the average value of the detection results after the addition of the cross-reactants, Co is the average value of the detection results before the addition of the cross-reactants, and C is the labeled value of the cross-reactants.

[0085] Table 8 Specificity detection results

[0086]

[0087] The specificity of the kit in Example 1 meets the requirements, and the specificity of the kit of the present scheme is good.

[0088] Precision: The high and low concentration levels of the sample were respectively repeated for 10 times, and the average value and the standard deviation of the 10 times of test results were calculated. The coefficient of variation CV was calculated according to formula (2). The batch precision variation coefficient was not more than 8.0%. The specific results of Example 1 are shown in Table 9.

[0089] CV = SD / M x 100% (2), wherein CV is the coefficient of variation, SD is the standard deviation of the 10 times of measurement results, and M is the average value of the 10 times of measurement results.

[0090] Table 9 Precision test results of Example 1

[0091]

[0092]

[0093] The precision of Example 1 meets the requirements, while the batch variation coefficient (CV) of Comparative Example 1 is 10%-12%, indicating that the precision of the kit of the present scheme is high.

[0094] Linear range

[0095] The high value sample near the upper limit of the linear range and the low value sample were mixed in a certain proportion to prepare 6 concentration levels of samples, wherein the concentration of the low value sample should be near the lower limit of the linear range, and each concentration level sample was in the linear interval. Each concentration sample was detected 3 times (conc.-1, conc.-2, conc.-3), and the average value was calculated. The detection data of Example 1 is shown in Table 10. The theoretical concentration was taken as the X axis, the average value of the detection result was taken as the Y axis, the least square method was used for linear fitting, and the linear correlation coefficient (r) was calculated. The results in the range of 2.00-4000.00 pg / mL, the linear correlation coefficient (r) is greater than or equal to 0.990.

[0096] Table 10 Linear range detection results

[0097]

[0098]

[0099] Accuracy

[0100] The high concentration of GFAP liquid (A) was added to the low concentration of serum B, and the volume ratio between the added GFAP (A) and the low concentration of serum B was 1:9. Each was detected 3 times, and the average value was taken. According to formula (3), the recovery rate was calculated, and the results are shown in Table 11.

[0101] Wherein, R is the recovery rate, V is the volume of A liquid, V0 is the volume of serum sample B; c is the detection concentration of serum sample after adding A liquid; c0 is the detection concentration of serum sample B.

[0102] Table 11 Recovery rate detection results

[0103]

[0104] The accuracy of Example 1 is 99%, and the recovery rate of Comparative Example 2 is 85%, although both meet the requirements, but the accuracy of the kit of the present scheme is high.

[0105] HOOK effect

[0106] The luminescence values of the high concentration sample 40000 pg / ml and the diluted sample are greater than the luminescence value of the upper limit of the detection range, and the results are shown in Table 12.

[0107] Table 12 HOOK effect detection results

[0108]

[0109]

[0110] The hook effect of Example 1 meets the requirements, indicating that the hook effect of the present scheme meets the requirements.

[0111] Uniformity of calibrators

[0112] Coefficient of variation CV≤8.0%, inter-bottle uniformity: coefficient of variation CV≤8.0%. Randomly take one kit from a box of kits, take the calibrators in the kit, and measure 10 times for each concentration level. The average value of the test results is calculated according to formulas (4)-(6) Standard deviation (SD) and coefficient of variation (CV), wherein n is the number of tests, and Xi is the i-th test value.

[0113]

[0114] Take 10 kits of the same batch of reagents, take the calibrators in the kit, measure each calibrator in each kit once, and measure a total of 10 determination results for each concentration level. The detection data of Example 1 is shown in Table 13. The average value of the test results is calculated according to formulas (4) and (5) and standard deviation (SD1); take the calibrators in a kit of the same batch of reagents and test according to the method of 3.10.1, and calculate the average value of the test results according to formulas (4) and (5) and standard deviation (SD2); calculate the standard deviation (SD) and coefficient of variation (CV) of the inter-bottle uniformity according to formulas (7) and (8).

[0115]

[0116] When SD1<SD2, CV=0.

[0117] Table 13 Uniformity detection results of calibrators

[0118]

[0119] Clinical effect verification

[0120] The serum and whole blood samples from the clinical samples diagnosed as brain injury from Tongji Hospital of Huazhong University of Science and Technology (Wuhan Tongji Hospital) were tested, and the results were compared according to the classification results according to their CT diagnosis and clinical manifestations. The experimental results are shown in Table 14. The whole blood samples and serum samples are basically the same, and the reagent realizes consistent detection of whole blood and serum; comparing the brain CT diagnosis results of the hospital with the Mingde GFAP kit test results, it can be seen that in patients with moderate and severe brain injury and positive brain CT diagnosis results, the clinical sensitivity of GFAP can reach 90%, but for patients with mild brain injury with negative CT results, the clinical specificity of GFAP can reach 100%. In view of the current clinical situation that "the number of mild brain injury (TBI) can account for more than 90% of all TBI cases. More than 90% of patients with mild TBI are sent to the emergency department, but the CT results are negative, and at most only 1% of these patients need neurosurgery intervention", the GFAP kit invented by Mingde can distinguish mild TBI patients from moderate and severe TBI patients from the test results, thereby reducing unnecessary brain CT detection of these patients.

[0121] Table 14 Test results of clinical samples

[0122]

[0123] In Example 1, the serum and whole blood concentration comparison is good, while in Comparative Example 3, the whole blood and serum concentration comparison is poor, and the interference of the whole blood sample on the reagent has not been eliminated. The kit of the present solution can effectively shield the interference of the whole blood sample, and accurately test the clinical samples.

[0124] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A quantitative detection reagent kit for a macromolecular protein by a magnetic microparticle chemiluminescent immunoassay, characterized by, The kit comprises streptavidin-coated magnetic particles / biotinylated GFAP capture antibody complex suspension, alkaline phosphatase-labeled GFAP detection antibody solution, recombinant GFAP antigen series calibration, whole blood sample diluent, chemiluminescence solution, cleaning solution; the preparation method of the streptavidin-coated magnetic particles / biotinylated GFAP capture antibody complex is as follows: after mixing and coating streptavidin-coated magnetic particles and biotinylated GFAP capture antibody, magnetic cleaning is carried out, and then the complex is resuspended in a magnetic particle coating buffer to obtain the streptavidin-coated magnetic particles / biotinylated GFAP capture antibody complex; the whole blood sample diluent comprises the following components: sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, cysteine, glycerol, PC-300, BSA, Triton X-100, EDTA·2Na·2H2O.

2. The GFAP-MPLIA quantitative test kit according to claim 1, wherein, The mass ratio of the streptavidin-coated magnetic particles to the biotinylated GFAP capture antibody is 1:10-40.

3. The GFAP-MPLIA quantitative test kit according to claim 1, wherein the GFAP-MPLIA quantitative test kit is a GFAP-MPLIA quantitative test kit for measuring the concentration of GFAP in a sample of a patient with a brain disease. The concentration of the streptavidin-coated magnetic particles is 1-10 μg / ml.

4. The GFAP-MPLIA quantitative test kit according to claim 1, wherein the GFAP-MPLIA quantitative test kit is a GFAP-MPLIA quantitative test kit for measuring the concentration of GFAP in a sample of a patient with a brain disease. The preparation method of the alkaline phosphatase-labeled GFAP detection antibody solution is as follows: mixing N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate activated GFAP capture antibody with Traunt's reagent activated alkaline phosphatase, then performing column purification, and then dispersing in an enzyme-labeled buffer.

5. The GFAP M-PLEX immunoassay quantitative test kit according to claim 4, characterized in that, The enzyme-labeled buffer comprises the following components: MES, NaCl, MgCl2, PVP, ZnCl2, BSA, Tween-20, PC-300.

6. The GFAP-MPLIA quantitative test kit according to claim 1, wherein the GFAP-MPLIA quantitative test kit is a GFAP-MPLIA quantitative test kit for measuring the concentration of GFAP in a sample of a patient with a brain disease. The GFAP detection antibody and the GFAP capture antibody are different mouse monoclonal antibodies.

7. The GFAP M-PLEX immunoassay quantitative test kit according to claim 1, characterized in that, The preparation method of the recombinant GFAP antigen series calibration is as follows: dissolving recombinant GFAP antigen in a calibration buffer, and the concentration of the recombinant GFAP antigen series calibration is 30 pg / ml, 900 pg / ml.

8. The GFAP M-PLEX immunoassay quantitative test kit according to claim 1, characterized in that, The components of the cleaning solution comprise Tris, Tween-20, preservatives, and the pH value of the cleaning solution is 8-9.

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