CN115290898A - Application of HEPES buffer solution in glial fibrous acidic protein detection kit - Google Patents
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Abstract
The invention provides an application of a HEPES buffer solution in preparation of a glial fibrillary acidic protein detection kit, wherein each 1000mL of the HEPES buffer solution comprises: 0.5-7.2g of 4-hydroxyethyl piperazine ethanesulfonic acid, 9g of sodium chloride, 0.5-5.0g of sodium caseinate, 5-30g of mannitol, 2-4mL of nonionic surfactant and 1mL of preservative. The invention can make the detection sensitivity reach the picogram level (10) ‑12 g/mL), good repeatability, small difference between batches and between bottles, and improved detection linear interval.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of a HEPES buffer solution in a glial fibrous acid protein detection kit.
Background
Glial Fibrillary Acidic Protein (GFAP) is a type iii intermediate filament protein. In Traumatic Brain Injury (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 TBIOf significance [1] 。
Various detection kits or detection methods for GFAP have been disclosed in the prior art, particularly detection methods and kits based on magnetic particle luminescence.
Chinese patent 201811391978.X discloses a chemiluminescence immunoassay method for separating magnetic particles of human glial fibrillary acidic protein, and a kit comprises the following components: 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 colloidal fiber acidic protein antigen containing a series of concentrations and is used for establishing a standard curve; the quality control product is prepared by a buffer solution containing a certain concentration of the glial fibrillary acidic protein antigen; the reagent A is a colloidal fiber acidic protein antibody solution containing magnetic particles with certain concentration for marking; the reagent B is a colloidal fiber acidic protein 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 greatly improves the signal intensity and sensitivity of immunoreaction, enables low-content substances to generate strong chemiluminescent signals when carrying out immune combination, and provides a more accurate, precise, convenient, rapid and simple method for detecting human glial fibrous acidic protein. However, the sensitivity is actually less than 0.05ng/mL, and a larger progress space still exists for the detection of GFAP.
Chinese patent 202011279930.7 discloses a kit for quantitatively detecting GFAP and application thereof, wherein the kit comprises a GFAP antibody solution marked by magnetic particles, and the buffer solution consists of the following components: na (Na) 2 HPO 4 ·12H 2 O 5.6-5.9g/L、NaH 2 PO 4 0.55-0.60g/L, naCl 9.0g/L, bovine serum albumin 1.0-50g/L, sucrose 80.0-140g/L, cellulose salt or cellulose derivative 1.0-5.0g/L, gelatin 5.0-50g/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. But when the kit is used for detectionThe linear interval is smaller, and further optimization space is provided.
Reference documents:
[1] the biological characteristics and clinical research progress of Liuxia, qinxue, glial fibrillary acidic protein [ J ]. J.International inspection medicine 2015,36 (18): 2.
Disclosure of Invention
In order to solve the problems, the invention provides a buffer solution for a glial fibrillary acidic protein detection kit, so as to improve the detection performance of the kit.
In the present invention, "detection performance of the kit" includes, but is not limited to, accuracy, margin, detection limit, linear interval, repeatability, difference between calibrator and quality control vial, intra-batch difference, inter-batch difference, stability, etc.
In the present invention, "HEPES" means hydroxyethylpiperazine ethanethiosulfonic acid.
In the present invention, "HEPES buffer" refers to a buffer containing hydroxyethylpiperazine ethanesulfonic acid, but does not mean that only one component of hydroxyethylpiperazine ethanesulfonic acid is included in the buffer, and other components advantageous for improving the performance of the detection kit may be included.
In one aspect, the invention provides an application of a HEPES buffer solution in preparing a glial fibrillary acidic protein assay kit.
The HEPES buffer solution comprises the following components:
| name of raw materials | Weighing volume |
| 4-hydroxyethyl piperazine ethanesulfonic acid | 0.5-7.2g |
| Sodium chloride | 9g |
| Casein acid sodium salt | 0.5-5.0g |
| Mannitol | 5-30g |
| Nonionic surfactant | 2-4mL |
| Preservative | 1mL |
| pH value | 7.2-8.0 |
| Purified water | The volume is up to 1000mL |
Preferably, the HEPES buffer comprises the following components:
in the HEPES buffer, the nonionic surfactant includes but is not limited to: tween-20.
In the HEPES buffer, preservatives include, but are not limited to: proclin300.
The preparation method of the HEPES buffer solution comprises the following steps: 0.5-7.2g of HEPES, 9.0g of NaCl, 0.5-5.0g of sodium caseinate, 5-30g of mannitol, 2-4mL of nonionic surfactant and 1mL of preservative are weighed and added into a certain amount of purified water to be stirred until the sodium caseinate, the mannitol and the nonionic surfactant are completely dissolved, the pH value is adjusted to be 7.2-8.0, and the volume is adjusted to be 1000mL. Filtration was performed with a 0.22 μm filter.
The detection kit for the acidic protein in the glial fibers is realized by a magnetic particle chemiluminescence immunoassay method.
The kit also comprises other reagents required in the magnetic particle chemiluminescence immunoassay method for detecting the glial fibrillary acidic protein.
Such other agents include, but are not limited to: sodium borohydride solution, zinc chloride solution, magnesium chloride hexahydrate solution, acetic acid solution, TMB solution, GFAP antibody solution, quality control product, calibrator, cleaning concentrated solution and sulfuric acid.
In another aspect, the invention provides a kit for detecting collagen fibrin.
The kit comprises a HEPES buffer solution, wherein the HEPES buffer solution comprises the following components:
preferably, the HEPES buffer comprises the following components:
| name of raw materials | Weighing volume |
| 4-hydroxyethyl piperazine ethanesulfonic acid | 3-7g |
| Chlorination ofSodium salt | 9g |
| Sodium caseinate | 2-4g |
| Mannitol | 10-20g |
| Nonionic surfactant | 2-4mL |
| Preservative | 1mL |
| pH value | 7.2-8.0 |
| Purified water | The volume is up to 1000mL |
In the HEPES buffer, the non-ionic surfactant includes but is not limited to: tween-20; such preservatives include, but are not limited to: proclin300.
The preparation method of the HEPES buffer solution comprises the following steps: HEPES, naCl, sodium caseinate, mannitol, a non-ionic surfactant and a preservative are weighed and added into a certain amount of purified water to be stirred until the materials are completely dissolved, the pH value is adjusted, and the volume is fixed to 1000mL.
The glial fibrous acidic protein detection kit is realized by a magnetic particle chemiluminescence immunoassay method.
The kit also comprises other reagents required in a magnetic particle chemiluminescence immunoassay method for detecting the glial fibrillary acidic protein.
Such other agents include, but are not limited to: sodium borohydride solution, zinc chloride solution, magnesium chloride hexahydrate solution, acetic acid solution, TMB solution, GFAP antibody solution, quality control product, calibrator, cleaning concentrated solution and sulfuric acid.
The invention has the beneficial effects that:
the invention can make the detection sensitivity reach the level of picogram (10) -12 g/mL), good repeatability, small difference between batches and between bottles, and improved detection linear interval.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples, unless otherwise specified, and experimental methods not specified in specific conditions in the examples, are generally commercially available according to conventional conditions, and materials, reagents, and the like used in the following examples, unless otherwise specified.
The various buffers used in the examples of this application are specifically as follows:
the formula of the buffer solution 1 is as follows:
| name of raw materials | Weighing volume |
| Disodium hydrogen phosphate dodecahydrate | 2.0-3.8g |
| Sodium dihydrogen phosphate | 0.01-0.8g |
| pH value | 6.5-7.6 |
| Purified water | The volume is up to 1000mL |
The preparation method of the buffer solution 1 comprises the following steps: weighing 2.0-3.8g of Na 2 HPO 4 ·12H 2 O, 0.01-0.8g of NaH 2 PO 4 Adding into a certain amount of purified water, stirring until the mixture is completely dissolved, adjusting the pH value to be between 6.5 and 7.6, and metering to 1000mL. Filtration was performed with a 0.22 μm filter.
The formula of the buffer solution 2 is as follows:
| name of raw materials | Weighing amount |
| Sodium periodate | 10-28g |
| pH value | 6.5-7.6 |
| Buffer solution 1 | The volume is up to 1000mL |
The preparation method of the buffer solution 2 comprises the following steps: weighing 10-28g of NaIO, adding the NaIO into a certain amount of buffer solution 1, stirring until the NaIO is completely dissolved, adjusting the pH value to be 6.5-7.6, and metering to 1000mL. Filtration was performed with a 0.22 μm filter.
The formula of the buffer solution 3 is as follows:
| name of raw materials | Weighing volume |
| Magnesium chloride hexahydrate | 203.3g |
| Purified water | The volume is up to 1000mL |
The preparation method of the buffer solution 3 comprises the following steps: 203.3g of MgCl were weighed 2 ·6H 2 And adding O into a certain amount of purified water, stirring until the O is completely dissolved, and metering to 1000mL. Filtration was performed with a 0.22 μm filter.
The formula of the buffer solution 4 is as follows:
the preparation method of the buffer solution 4 comprises the following steps: 136.3g of ZnCl were weighed 2 Adding into a certain amount of purified water, stirring until the solution is completely dissolved, and fixing the volume to 1000mL. Filtration was performed with a 0.22 μm filter.
The formula of the buffer solution 5 is as follows:
| name of raw materials | Weighing volume |
| Acetic Acid (AA) | 0.02-0.57g |
| Purified water | The volume is up to 1000mL |
The preparation method of the buffer solution 5 comprises the following steps: 0.02-0.57g of CH COOH is weighed and added into a certain amount of purified water to be stirred until the CH COOH is completely dissolved, and the volume is up to 1000mL. Filtration was performed with a 0.22 μm filter.
The formula of the buffer solution 6 is as follows:
| name of raw materials | Weighing volume |
| Sodium carbonate | 8.5-52g |
| Sodium bicarbonate | 14-86g |
| pH value | 9.0-10.0 |
| Purified water | The volume is up to 1000mL |
The preparation method of the buffer solution 6 comprises the following steps: weighing 8.5-52g of Na 2 CO 3 14-86g of NaHCO 3 Adding into a certain amount of purified water, stirring until completely dissolving, adjusting pH value to 9.0-10.0, and metering to 1000mL. Filtration was performed with a 0.22 μm filter.
The formula of the buffer solution 7 is as follows:
| name of raw materials | Weighing volume |
| Sodium borohydride | 1-5mg |
| Purified water | The volume is up to 1000mL |
The preparation method of the buffer solution 7 comprises the following steps: weighing 1-5mg NaBH 4 Adding into a certain amount of purified water, stirring until the solution is completely dissolved, and fixing the volume to 1000mL. Filtration was performed with a 0.22 μm filter.
The formula of the buffer solution 8 is as follows:
the preparation method of the buffer solution 8 comprises the following steps: weighing 2.0-3.8g of Na 2 HPO 4 ·12H 2 O, 0.01-0.8g of NaH 2 PO 4 6-10g of NaCl and 0.1-0.6g of KCl are added into a certain amount of purified water and stirred until the NaCl and the KCl are completely dissolved, the pH value is adjusted to be 7.0-7.8, and the volume is adjusted to 1000mL. Filtration was performed with a 0.22 μm filter.
The formula of the buffer solution 10 is as follows:
| name of raw materials | Weighing amount |
| Tris (hydroxymethyl) aminomethane | 1.0-1.5g |
| Sodium chloride | 9g |
| Nonionic surfactant | 0.2-5mL |
| pH value | 7.0-7.5 |
| Purified water | The volume is up to 1000mL |
The preparation method of the buffer solution 10 comprises the following steps: weighing 1.0-1.5g of Tris, 9g of NaCl and 0.2-5mL of nonionic surfactant, adding into a certain amount of purified water, stirring until the mixture is completely dissolved, adjusting the pH value to 7.0-7.5, and metering to 1000mL. Filtration was performed with a 0.22 μm filter.
The formulation of buffer 11 was:
| name of raw materials | Weighing volume |
| Tris (hydroxymethyl) aminomethane | 12.0-15.0g |
| Bovine serum albumin | 5.0-50g |
| Glycine | 1.0-30g |
| pH value | 7.6-8.8 |
| Purified water | The volume is up to 1000mL |
The preparation method of the buffer solution 11 comprises the following steps: weighing 12.0-15.0g of Tris, 5.0-50g of bovine serum albumin and 1.0-30g of glycine, adding into a certain amount of purified water, stirring until the mixture is completely dissolved, adjusting the pH value to 7.6-8.8 and fixing the volume to 1000mL. Filtration was performed with a 0.22 μm filter.
The formulation of buffer 12 was:
| name of raw materials | Weighing volume |
| 4-hydroxyethyl piperazine ethanesulfonic acid | 5.6-5.9g |
| Sodium chloride | 9.0g |
| Bovine IgG | 5.0-30g |
| Buffer solution 3 | 1-10mL |
| Buffer 4 | 1-10mL |
| Enzymatically hydrolyzed gelatin | 1.0-20g |
| Enzyme stabilizer | 30-120mL |
| Polyoxyethylene lauryl ether | 0.2-5g |
| Preservative | 1mL |
| pH value | 7.0-7.6 |
| Purified water | The volume is up to 1000mL |
The preparation method of the buffer solution 12 comprises the following steps:
weighing 2.3-23.8g of HEPES, 9.0g of NaCl, 5.0-30g of bovine IgG, 1-10mL of buffer solution 3, 1-10mL of buffer solution 4, 1-20g of enzyme hydrolyzed gelatin, 30-120mL of enzyme stabilizer, 0.2-5g of polyoxyethylene lauryl ether and 1mL of preservative, adding into a certain amount of purified water, stirring until the mixture is completely dissolved, adjusting the pH value to be between 7.0 and 7.6, and fixing the volume to 1000mL. Filtration was performed with a 0.22 μm filter.
The formulation of buffer 13 was:
| name of raw materials | Weighing volume |
| Tris (hydroxymethyl) aminomethane | 10-15g |
| Sodium chloride | 90g |
| Nonionic surfactant | 2-50mL |
| pH value | 7.0-7.5 |
| Purified water | The volume is up to 1000mL |
The preparation method of the buffer solution 13 comprises the following steps: weighing 10-15g of Tris, 90g of NaCl and 2-50mL of nonionic surfactant, adding into a certain amount of purified water, stirring until the Tris and the NaCl are completely dissolved, adjusting the pH value to 7.0-7.5, and metering to 1000mL. Filtration was performed with a 0.22 μm filter.
Example 1 human glial fibrillary acidic protein GFAP magnetic particle chemiluminescence immunoassay method and kit
1. Kit Components of the present example
The GFAP kit consists of a coated plate, a reagent A, a calibrator, a quality control product, a cleaning concentrated solution (buffer solution 13), a developing solution and a stop solution. Wherein the coating plate is a 96-hole microporous plate. The reagent A is a GFAP antibody solution containing a certain concentration of horseradish peroxidase (HRP) mark; the calibrator is prepared from GFAP antigen with six concentrations and buffer solution and is used for calibrating a standard curve; the quality control product is prepared from GFAP antigen with two concentrations and buffer solution; the cleaning concentrated solution is used for cleaning in the reaction process, and the concentration of the working solution is 10 times of the diluted concentrated solution; the color development liquid is TMB solution (Solibao, PR 1200); the stop solution was 2M sulfuric acid. The following were used:
| the main components of the kit | Loading capacity |
| Coating plate (96 holes) | 1 is provided with |
| Reagent A | 30mL |
| Quality control product | 1mL×1 |
| Calibrator 1-6 | 1mL×1 |
| Cleaning concentrate | 10mL |
| Color developing liquid | 30mL |
| Stopping liquid | 30mL |
2. Principle of detection
The kit adopts a double-antibody sandwich method to determine the content of GFAP. GFAP in the sample is combined with an enzyme-labeled antibody 1 (enzyme-labeled GFAP antibody conjugate) in a reagent A and an antibody 2 in a coating plate to form a sandwich structure. After washing, the reaction is carried out by adding color developing solution. The color developing solution is converted into blue color under the catalysis of peroxidase and is converted into final yellow color under the action of acid. The shade of the color is positively correlated with the concentration of GFAP in the sample. OD values were measured at a wavelength of 450nm and the sample concentration was calculated.
3. Production process
3.1 production of calibrator and quality control Material
The GFAP recombinant protein (Abcam, ab 114149) was used as a starting material for a calibrator. The samples were dissolved in buffer 11 and mixed well to prepare 6 calibrators at concentrations of 0pg/mL, 20pg/mL, 40pg/mL, 80pg/mL, 160pg/mL, and 320pg/mL.
The GFAP recombinant protein is used as a raw material of a quality control product. Dissolving with buffer solution 11, and mixing to obtain quality control product. The concentrations were 20pg/mL and 80pg/mL.
3.2 production of reagent A
An enzyme-labeled GFAP antibody conjugate was used as a raw material of the reagent a. The reagent A was prepared by thoroughly mixing the mixture with buffer solution 12.
4. Detection process
Adopts Beijing Meiliantaceae biotechnology limited company MS-Fast
Series and MS-Fast
pro series of instruments, in particular MS-Fast
、MS-Fast
aceso 80A、MS-Fast
pro 80、MS-Fast
pro160、MS-Fast
pro 240. The sample size required by the reaction is 100 mu L, and the automatic detection process comprises the following steps:
immune reaction: and sequentially adding 100uL of sample and 100uL of reagent A into the hole of the microporous plate, and reacting at 37 ℃ for 40min.
Cleaning: the microplate is cleaned by the automatic cleaning process of the instrument.
Reading value: adding 100 mu L of color development liquid into each hole, reacting for 15min at room temperature in a dark place, and adding 50 mu L of stop solution into each hole. After developing the color of the developing solution catalyzed by the horseradish peroxidase, absorbance (OD value) at the wavelength of 450nm is detected by a self-developed instrument within 5min.
And obtaining a GFAP concentration-luminous value standard curve according to the detected value of the calibrator. The curve was fitted using a four parameter Logistic equation.
The detection value of the sample can correspond to the unique concentration value obtained on the curve, so that the concentration detection of the unknown sample is realized.
5. Detecting the index
5.1 accuracy
A Glial Fibrillary Acidic Protein (GFAP) solution (A) with a concentration of about 800pg/mL (tolerance. + -. 10%) was added to a sample B with a concentration ranging from 0pg/mL to 10pg/mL, and the recovery R was calculated according to equation (1) at a volume ratio between the added GFAP antigen and the sample B of 1.
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-average of 3 measurements of serum sample B fluid;
CS-concentration of sample A solution.
5.2 blank limit
Repeating the test for 20 times on the sample without any analyte to obtain concentration values of 20 test results, and calculating the average value
And Standard Deviation (SD). Mean value of
The blank limit is obtained, and the result is less than or equal to 10pg/mL.
5.3 Linear region
Mixing a high value sample close to the upper limit of the linear region and a low value sample close to the lower limit of the linear region or a zero concentration sample to be not less than 5 dilution concentrations, wherein the low value concentration sample is close to the lower limit of the linear region. Repeating the test 3 times for each concentration sample to obtain luminescence value, recording the measurement result of each sample, and calculating the average value (y) of the 3 measurement values of each sample i ). In diluted concentration (x) i ) As independent variable, the mean value (y) of the results is determined i ) Linear regression equations were solved for the dependent variables. Calculating the correlation coefficient (r) of the linear regression according to the formula (2), wherein the correlation coefficient r is more than or equal to 0.990 in a linear interval of 10-320 pg/mL.
In the formula:
r- — -correlation coefficient;
x i -dilution ratio;
y i -mean value of individual sample measurements;
5.4 repeatability
The quality control product is repeatedly tested 10 times by the same batch number kit, and the average value of 10 test results is calculated
And standard deviation SD. The Coefficient of Variation (CV) was calculated according to equation (3) and the result was CV ≦ 10%.
In the formula: s-standard deviation of sample test values;
5.5 run-to-run Difference
The quality control materials are tested repeatedly for 10 times by using the kits with 3 batch numbers respectively, and the average value of the test results of 30 times is calculated
And standard deviation SD, obtaining Coefficient of Variation (CV) according to formula (3), and obtaining the result CV of less than or equal to 15%.
5.6 difference between calibrator and quality control bottle
Detecting 10 bottles of calibrator (or quality control material) of the same batch for 1 time respectively, calculating according to formula (5), and determining the mean value of the results
And standard deviation (S1). 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 (S2), calculating the repeatability CV% between bottles according to the formulas (6) and (7), and measuring the result to be less than 10%.
(Note: when S1< S2, CV bottles are made to have a spacing = 0.)
In the formula:
s-standard deviation.
6. Method for treating antibody 1 and antibody 2
6.1 enzyme-labeled antibody 1 and purification
6.1.1 detection of Horseradish peroxidase (HRP)
The ultra-micro ultraviolet spectrophotometer was zeroed using purified water, and the OD values of HRP at 275nm and 403nm were measured. RZ value is calculated, RZ = OD403nm/OD275nm, and HRP is available when RZ is more than or equal to 3.0.
6.1.2 pretreatment of concentrated centrifuge tubes by ultrafiltration
Two ultrafiltration concentration centrifuge tubes are taken, and the molecular cut-off is more than or equal to 10KD. Soaking the inner tube and the outer tube in purified water for more than 5min. To the inner tube was added 500. Mu.L of purified water. The centrifuge parameters were set as: centrifuging at 12000rpm for 5min at 2-8 deg.C. After the supernatant of the outer tube was discarded, the above steps of adding liquid, centrifuging, and discarding the supernatant were repeated 5 times in total. And (4) emptying liquid from the two treated ultrafiltration concentration centrifugal tubes, and covering a cover for later use.
6.1.3 activation of Horseradish peroxidase (HRP)
5mg of HRP is weighed, dissolved in 1.2mL of purified water, added with 0.3mL of freshly prepared buffer 2 and mixed for 20min at room temperature. The HRP solution was transferred to the inner tube of a treated ultrafiltered concentration centrifuge tube, and 500. Mu.L of buffer 5 was added to the inner tube. The centrifuge parameters were set as: centrifuging at 12000rpm for 5min at 2-8 deg.C. After the supernatant of the outer tube is discarded, the steps of adding liquid, centrifuging and discarding the supernatant are repeated for 5 times in total. Collecting the concentrated solution in the inner tube, and storing at 2-8 deg.C in dark.
6.1.4 activation of antibody 1
The buffer 6 is added into the antibody 1 solution for activation according to the volume ratio of the antibody 1 (Abcam, EPR 19996) to the buffer 6 being 1 to 5-1, and after shaking and mixing, the pH value of the mixed solution is immediately tested, wherein the pH value is between 9 and 10.
6.1.5 conjugation of antibody 1 to HRP
HRP solution was added to the antibody 1 solution at a ratio of antibody 1 to HRP mass ratio of 1. After shaking and mixing, immediately testing the pH value of the mixed solution, wherein the pH value is 9-10, and if the pH value is not satisfactory, adjusting by using a buffer solution 6. And (3) uniformly mixing the mixture at the temperature of between 20 and 40 ℃ in a dark place, and reacting for 1 to 3.5 hours.
6.1.6 termination and purification of antibody 1 conjugates
The solution was added in a ratio of 1mg of antibody 1 to 100. Mu.L of buffer 7, and the mixture was reacted at 2 to 8 ℃ for 2 hours. Transferring the reaction solution into another treated inner tube of the ultrafiltration concentration centrifuge tube. Add 500. Mu.L of buffer 8 to the inner tube. The centrifuge parameters were set as: centrifuging at 12000rpm for 5min at 2-8 deg.C. After the supernatant of the outer tube is discarded, the steps of adding liquid, centrifuging and discarding the supernatant are repeated for 5 times in total. The concentrate in the inner tube was collected. The concentration of the concentrate, i.e., antibody 1 conjugate, should be between 1-4mg/mL, if not in range, it should be re-concentrated or diluted with buffer 8.
1mL of glycerol (i.e., volume to 1: 1) was added to 1mL of antibody 1 conjugate, and 5-20. Mu.g of bovine serum albumin (i.e., mass to volume ratio 200. Mixing, and storing at-20 deg.C.
6.2 antibody 2 coating and blocking
6.2.1 pretreatment of concentrated centrifuge tubes by ultrafiltration
Taking an ultrafiltration concentration centrifugal tube, wherein the molecular cut-off is more than or equal to 10KD. Soaking the inner tube and the outer tube in purified water for more than 5min. To the inner tube was added 500. Mu.L of purified water. The centrifuge parameters were set as: centrifuging at 12000rpm for 5min at 2-8 deg.C. After the supernatant of the outer tube was discarded, the above steps of adding liquid, centrifuging, and discarding the supernatant were repeated 5 times in total. And (4) emptying the liquid from the treated ultrafiltration concentration centrifugal tube, and covering a cover for standby.
6.2.2 pretreatment of antibody 2
1mg of antibody 2 (Abcam, EPR 19996-132) solution was measured and transferred to the inner tube of a treated ultrafiltered concentration centrifuge tube, to which 500. Mu.L of buffer 8 was added. The centrifuge parameters were set as: centrifuging at 12000rpm for 5min at 2-8 deg.C. After the supernatant of the outer tube was discarded, the above steps of adding liquid, centrifuging, and discarding the supernatant were repeated 5 times in total. The concentration of the concentrate in the inner tube is collected and should be between 0.5-2mg/mL, if out of range, it should be re-concentrated or diluted with buffer 8. Storing at 2-8 deg.C.
6.2.3 coating, washing and blocking of antibody 2
The treated antibody 2 was diluted with buffer 8 at a concentration of 0.1-4. Mu.g/mL. Adding the diluted antibody 2 solution into a 96-well microplate, wherein the addition amount of each well is 50-150 mu L, and reacting for 12-16 hours at the temperature of 2-8 ℃ in the dark or reacting for 0.5-2 hours at the temperature of 35-42 ℃ in the dark. The reaction solution was then discarded from each well and buffer 10 was added to each well in an amount of 200. Mu.L per well. The microplate was shaken horizontally and evenly for 2min. The reaction solution was then discarded and the process repeated for a total of 3-5 times. Finally, the reaction solution in each well is discarded and the solution is drained. Then add intoBuffer 9 (HEPES buffer)The amount added per well was 200. Mu.L. Reacting for 0.5-2 hours at 35-42 ℃ in the dark.
6.2.4 washing and preservation of antibody 2-coated plates
After the reaction, the reaction solution in each well was discarded. And buffer 10 was added to each well in an amount of 200. Mu.L per well. The microplate was shaken horizontally and evenly for 2min. Then the reaction solution is discarded and the process is repeated for a total of 3-5 times. Finally, the reaction liquid in each hole is discarded and the liquid is drained, and the reaction is carried out for 0.5 to 2 hours in a dark place at the temperature of between 35 and 42 ℃. And vacuumizing and sealing the treated coated plate by using a light-resistant aluminum film bag, and storing at the temperature of 2-8 ℃.
Examples 2-7 HEPES buffer formulations
In all the following formulations, the preservative was Proclin300 (Solarbio, P6840) in an amount of 1mL; the using amount of sodium chloride is 9g; the nonionic surfactant is Tween-20 (Samerfei, 85113).
The preparation method comprises the following steps: adding HEPES, naCl, sodium caseinate, mannitol, a non-ionic surfactant and a preservative into a certain amount of purified water, stirring until the materials are completely dissolved, adjusting the pH value, and fixing the volume to 1000mL. Filtration was performed with a 0.22 μm filter.
Other information in the formulation is as follows:
the detection results of the detection indexes are as follows:
comparative examples 1 to 5
Comparative examples 1 to 5 were set up with reference to the test methods of examples 1 to 7, and the formulations thereof were as follows:
the detection results of the detection indexes are as follows:
Claims (15)
- the application of the HEPES buffer solution in preparing the glial fibrillary acidic protein detection kit is characterized in that the HEPES buffer solution comprises the following components:
name of raw materials Weighing volume 4-hydroxyethyl piperazine ethanesulfonic acid 0.5-7.2g Sodium chloride 9g Sodium caseinate 0.5-5.0g Mannitol 5-30g Nonionic surfactant 2-4mL Preservative agent 1mL pH value 7.2-8.0 Purified water The volume is up to 1000mL 。 - 2. The use according to claim 1, wherein the HEPES buffer comprises the following components:
name of raw materials Weighing volume 4-hydroxyethyl piperazine ethanesulfonic acid 3-7g Sodium chloride 9g Sodium caseinate 2-4g Mannitol 10-20g Nonionic surfactant 2-4mL Preservative 1mL pH value 7.2-8.0 Purified water The volume is up to 1000mL 。 - 3. The use of claim 1, wherein the non-ionic surfactant is Tween-20.
- 4. The use according to claim 1, wherein the preservative is Proclin300.
- 5. The use according to claim 1, wherein the HEPES buffer is formulated by: 0.5-7.2g of HEPES, 9.0g of NaCl, 0.5-5.0g of sodium caseinate, 5-30g of mannitol, 2-4mL of nonionic surfactant and 1mL of preservative are weighed and added into a certain amount of purified water to be stirred until the mixture is completely dissolved, the pH value is adjusted to be 7.2-8.0, and the volume is fixed to 1000mL.
- 6. Use according to claim 5, characterized in that the volume is brought to constant volume and then filtered through a 0.22 μm filter.
- 7. The use of claim 1, wherein the glial fibrillary acidic protein assay kit is an enzyme-linked immunosorbent assay kit.
- 8. The use of claim 7, wherein the kit further comprises one or more of sodium borohydride solution, zinc chloride solution, magnesium chloride hexahydrate solution, acetic acid solution, TMB solution, GFAP antibody solution, quality control, calibrator, washing concentrate, and sulfuric acid.
- 9. The kit for detecting the colloidal fibrin acidic protein is characterized by comprising a HEPES buffer solution, wherein the HEPES buffer solution comprises the following components:
starting materialsName (R) Weighing volume 4-hydroxyethyl piperazine ethanesulfonic acid 0.5-7.2g Sodium chloride 9g Sodium caseinate 0.5-5.0g Mannitol 5-30g Nonionic surfactant 2-4mL Preservative 1mL pH value 7.2-8.0 Purified water The volume is up to 1000mL 。 - 10. The kit according to claim 9, wherein the HEPES buffer comprises the following components:
- 11. the kit according to claim 9, wherein the HEPES buffer is prepared by the following method: HEPES, naCl, sodium caseinate, mannitol, a non-ionic surfactant and a preservative are weighed and added into a certain amount of purified water to be stirred until the materials are completely dissolved, the pH value is adjusted, and the volume is fixed to 1000mL.
- 12. The kit according to claim 9, wherein the non-ionic surfactant is Tween-20.
- 13. The kit of claim 9, wherein the preservative is Proclin300.
- 14. The kit of claim 9, wherein the glial fibrillary acidic protein assay kit is an enzyme linked immunosorbent assay kit.
- 15. The kit according to claim 9, further comprising: one or more of sodium borohydride solution, zinc chloride solution, magnesium chloride hexahydrate solution, acetic acid solution, TMB solution, GFAP antibody solution, quality control product, calibrator, cleaning concentrate and sulfuric acid.
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| CN112611875A (en) * | 2020-12-29 | 2021-04-06 | 珠海碳云智能科技有限公司 | Method for screening polypeptide for detecting target antibody and application of screened polypeptide |
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| CN109696552A (en) * | 2017-10-20 | 2019-04-30 | 成都蓝瑙生物技术有限公司 | Luminous ELISA external diagnosis reagent case and vitro detection equipment for headstroke |
| CN112611875A (en) * | 2020-12-29 | 2021-04-06 | 珠海碳云智能科技有限公司 | Method for screening polypeptide for detecting target antibody and application of screened polypeptide |
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