CN111487409A - Chemiluminescence detection kit for S100B protein and use method thereof - Google Patents
Chemiluminescence detection kit for S100B protein and use method thereof Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/532—Production of labelled immunochemicals
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2871—Cerebrovascular disorders, e.g. stroke, cerebral infarct, cerebral haemorrhage, transient ischemic event
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Abstract
The invention is applicable to the field of biotechnology, and particularly relates to a chemiluminescence method detection kit for S100B protein and a use method thereof, wherein the detection kit for S100B protein comprises an ELISA plate coated with an anti-S100B monoclonal antibody, an anti-S100 polyclonal antibody solution for detection marked by horseradish peroxidase, a standard substance diluent, a sample diluent, a concentrated washing solution, a luminescent solution A, a luminescent solution B and a detection antibody diluent. The reaction time of a to-be-detected product (a standard product or a serum sample) of the kit provided by the embodiment of the invention and a detection anti-S100 polyclonal antibody solution marked by horseradish peroxidase is 2 hours; the clinical common pathological jaundice and lipemia specimens have no obvious interference on the determination of the serum S100B protein; the average variation in batch and between batches is less than 5.0 percent, and the method has the technical effects of simplicity, convenience, rapidness, specificity, sensitivity and suitability for clinical routine application.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a chemiluminescence method detection kit for S100B protein and a using method thereof.
Background
The S100B protein is an acidic calcium binding protein with a molecular weight of 21 KD. Because the molecular weight is small and the molecular weight exists in the central nervous system in a large amount, the compound plays a plurality of biological functions in vivo, and therefore, the compound has close relationship with blood brain barriers and brain injuries. At present, a great deal of research on the S100B protein, particularly on the clinical significance of the dynamic change level of the protein in serum in the aspects of disease diagnosis and prognosis, is carried out at home and abroad.
The detection of S100B is actually the detection of S100B subunit, the detection of S100B protein mainly adopts immunological method, at present, there are Radioimmunoassay (RIA) or immunoradiometric assay (IRMA), Fluorescence Immunoassay (FIA), enzyme-linked immunosorbent assay (E L ISA) and electrochemiluminescence (EC L IA) mainly, which are used for detecting the content of S100B protein in cerebrospinal fluid, blood, urine, amniotic fluid and the like, thereby further clinical diagnosis is carried out.
The basic principle of Radioimmunoassay (RIA) or immunoradiometric assay (IRMA) is that a labeled antigen competes with an unlabeled antigen to be detected for a specific binding site on a limited antibody in a reaction system, and the sensitivity is 0.2 mu g/L.
The basic principle of the Fluorescence Immunoassay (FIA) is that a specific antibody marked by a fluorescent pigment is combined with an antigen to be detected, the intensity of fluorescence is measured to calculate the content of the antigen in a sample, Wiesmann and the like adopt the FIA method, a microporous plate is coated by an anti-S100B protein monoclonal antibody, and the monoclonal antibody is combined with the antigen to be detected and a biotin-marked rabbit anti-S100B antibody to form a double-antibody sandwich compound.
The enzyme-linked immunosorbent assay (E L ISA) adopts S100B expressed by a prokaryotic system as a standard substance and prepares an anti-S100B monoclonal antibody, and the established double-antibody sandwich method has the advantages of high sensitivity of 0.0125 mu g/L and linear range of 0-3.2 mu g/L < 6.9%, compared with RIA and FIA, the E L ISA method has the advantages of high sensitivity, no pollution, simple and convenient operation, quick report result and the like, and has great application value in clinic.
The electrochemical luminescence method (EC L IA) is that streptavidin-coated magnetic beads and biotinylated antibodies are connected with S100B antibodies to form a double-antibody sandwich, and finally, a luminescent agent coupled with the antibodies and an electron donor TPA are carried out on the surface of an electrode repeatedly through oxidation-reduction reaction to generate a plurality of photons, a photomultiplier is used for detecting light intensity, and the light intensity is in a linear relation with the concentration of the luminescent agent, so that the content of the antigen to be detected can be detected.
Therefore, in order to meet the clinical detection requirement and realize the quantitative detection of the S100B protein in the serum more quickly and accurately, an in vitro diagnostic kit for quantitatively determining the concentration level of the S100B protein in the serum is urgently needed, and help is provided for the diagnosis and prognosis judgment of craniocerebral injury and cerebrovascular diseases.
Disclosure of Invention
The embodiment of the invention provides a chemiluminescence detection kit for S100B protein, which is used for meeting the clinical detection requirement and realizing quantitative detection of S100B protein in serum more quickly and accurately.
The chemiluminescence detection kit for the S100B protein provided by the embodiment of the invention comprises: the kit comprises an ELISA plate coated with an anti-S100B monoclonal antibody, an anti-S100 polyclonal antibody solution for detecting horseradish peroxidase labeling, a standard product diluent, a sample diluent, a concentrated washing solution, a luminescent solution A, a luminescent solution B and a detection antibody diluent.
The embodiment of the invention also provides a use method of the chemiluminescence detection kit for the S100B protein, which comprises the following steps:
(1) the following solutions were prepared:
diluting a detection anti-S100 polyclonal antibody solution marked by horseradish peroxidase by using a detection antibody diluent, wherein the ratio of the detection anti-S100 polyclonal antibody solution marked by horseradish peroxidase to the detection antibody diluent is 1: 10000;
diluting a detection anti-S100 polyclonal antibody solution marked by horseradish peroxidase by using a detection antibody diluent, wherein the volume ratio of the detection anti-S100 polyclonal antibody marked by the horseradish peroxidase to the detection antibody diluent is 1:5000, so as to obtain an enzyme conjugate;
dissolving a standard substance by using a standard substance diluent to obtain a plurality of standard substance solutions with gradient changes in concentration;
and diluting the concentrated washing solution with distilled water or deionized water according to the volume ratio of 1:20 to obtain the washing solution.
(2) Drawing a standard curve:
respectively adding 50 mu L of calibrator solution into corresponding holes of an ELISA plate coated with the anti-S100B monoclonal antibody, then respectively adding 100 mu L of horseradish peroxidase-labeled anti-S100 polyclonal antibody solution for detection into each hole, and lightly shaking and uniformly mixing;
sealing the plate with a sealing plate film, then incubating for 2 hours at 37 ℃, fully washing for 5 times with the washing solution, and drying;
adding 25 mu L of luminous liquid A, B into each hole, and lightly shaking and uniformly mixing;
adjusting the zero point by using a blank hole, and measuring the optical density value of each hole by using a luminometer;
performing data processing by using a log-log linear fitting mode, and drawing by using the log of the optical density value of each calibrator as a vertical coordinate and the log of the concentration of each calibrator as a horizontal coordinate to obtain a standard curve;
(3) detection of sample to be tested
Respectively adding a sample to be detected to 50 mu L into corresponding holes of an ELISA plate coated with an anti-S100B monoclonal antibody, then adding a horseradish peroxidase-labeled detection anti-S100 polyclonal antibody solution to 100 mu L, and lightly oscillating and uniformly mixing;
sealing the plate with a sealing plate film, then incubating for 2 hours at 37 ℃, fully washing for 5 times with the washing solution, and drying;
adding 25 mu L of luminous liquid A, B liquid respectively, and lightly shaking and uniformly mixing;
adjusting the zero point by using a blank hole, and measuring the optical density value by using a luminometer;
and substituting the optical density value of the sample solution to be detected into the standard curve to obtain the content of S100B in the sample solution to be detected.
According to the kit provided by the embodiment of the invention, the reaction time of the anti-S100 polyclonal antibody solution for detecting the horseradish peroxidase label of the to-be-detected substance (standard substance or serum sample) is 2 hours; the clinical common pathological jaundice and lipemia specimens have no obvious interference on the determination of the serum S100B protein; the average variation in batch and between batches is less than 5.0 percent, and the method has the technical effects of simplicity, convenience, rapidness, specificity, sensitivity and suitability for clinical routine application.
Drawings
FIG. 1 is a schematic diagram of a calibration curve provided by an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The chemiluminescence detection kit for the S100B protein provided by the embodiment of the invention comprises: the kit comprises an ELISA plate coated with an anti-S100B monoclonal antibody, an anti-S100 polyclonal antibody solution for detecting horseradish peroxidase labeling, a standard product diluent, a sample diluent, a concentrated washing solution, a luminescent solution A, a luminescent solution B and a detection antibody diluent.
In the embodiment of the invention, the anti-S100 polyclonal antibody solution for detecting the horseradish peroxidase marker is prepared by purifying and quantifying the antibody, dialyzing the antibody in a carbonate buffer solution with the pH of 9.0-9.5 and the concentration of 0.01 mol/L to 5mg/m L, preparing a horseradish peroxidase aqueous solution with the concentration of 5mg/m L, and adding NaIO (sodium iodide oxide) with the concentration of 37.5mg/m L into the horseradish peroxidase aqueous solution with the concentration of 0.4m L4Stirring at room temperature in dark for 20min to obtain oxidized enzyme solution, dialyzing the oxidized enzyme solution in acetate buffer solution of 1 mmol/L, pH 4.4 and 4 deg.C overnight, changing solution for 3 times, collecting the solution in dialysis bag, adding 0.2 mol/L Na2CO3Adjusting the pH of the buffer solution to 9.0-9.5, quickly mixing the buffer solution with purified and quantified antibody 1:1, slightly stirring the mixture for 2 hours at room temperature in a dark place, adding 100ul of L NaBH4 solution at 6mg/m, uniformly mixing, reacting for 2 hours at 4 ℃, dialyzing the reduced reaction system in PBS (0.15 mol/L and pH 7.4) at 4 ℃ overnight, changing the solution for 3 times, adding BSA (bovine serum albumin) until the final concentration is 2-3 mg/m L, uniformly mixing, × 3 minutes at 10000rpm, collecting supernatant, subpackaging and freezing.
In the present example, the standard is the S100B antigen.
In the present example, the solvent of the sample diluent is a phosphate buffered saline solution with 20 mmol/L and pH 6.0, and the solute and its mass concentration in the sample diluent are 0.2% gelatin, 0.2% casein, 150 mmol/L NaCl, 0.01% Tween-20, 5% sucrose, 0.5/ten thousand bromopotassium phenol violet, and 0.1% preservative.
In the embodiment of the invention, the solvent of the concentrated washing solution is water, and the solute and Na with the concentration of 116 g/L in the concentrated washing solution are2HPO4·12H2O、11.84g/L NaH2PO42H2O, 180 g/L NaCl, 5m L/L Tween 20 and 1m L/L preservative.
In the embodiment of the invention, the solvent of the luminescent liquid A is Tris-HCl buffer solution with pH of 8.0 and 0.2 mol/L, the solute and the concentration of the solute in the luminescent liquid A are 3mg/m L of luminol, 0.25mg/m L of p-iodophenol and 0.5mg/m L of sodium tetraphenylborate;
in the embodiment of the invention, the solvent of the luminescent liquid B is Tris-HCl buffer solution with the pH value of 8.0 and 0.2 mol/L, and the solute and carbamide peroxide with the concentration of 0.5mg/m L in the luminescent liquid B are used as solutes.
In the embodiment of the invention, the solvent of the standard dilution is a phosphate buffered saline solution with 20 mmol/L and pH7.4, the solute and the concentration thereof in the standard dilution are casein with the mass percentage of 1%, trehalose with the mass percentage of 8%, mannitol with the mass percentage of 3%, EDTA with the mass percentage of 1 mmol/L, glycine with the mass percentage of 0.5%, NaCl with the mass percentage of 150 mmol/L and preservative with the mass percentage of 0.1%.
In the embodiment of the present invention, the solvent of the test antibody diluent is a phosphate buffered saline solution with a pH of 7.4 of 20 mmol/L, and the solute and the concentration thereof in the test antibody diluent are bovine serum albumin with a mass percentage of 2%, casein with a mass percentage of 0.1%, NaCl with a mass percentage of 150 mmol/L, tween 20 with a mass percentage of 0.01%, aminopyrine with a mass percentage of 5/1 ten thousand, dye with a mass percentage of 1/20 ten thousand, and preservative with a mass percentage of 0.1%.
As follows, the embodiment of the present invention provides a method for drawing a standard curve, in which an Optical Density (OD) value of a sample solution to be measured is substituted into the standard curve, so as to obtain a content of S100B in the sample solution to be measured.
(1) The following solutions were prepared:
diluting a detection anti-S100 polyclonal antibody solution marked by horseradish peroxidase by using a detection antibody diluent, wherein the ratio of the detection anti-S100 polyclonal antibody solution marked by horseradish peroxidase to the detection antibody diluent is 1: 10000;
diluting a detection anti-S100 polyclonal antibody solution marked by horseradish peroxidase by using a detection antibody diluent, wherein the volume ratio of the detection anti-S100 polyclonal antibody marked by the horseradish peroxidase to the detection antibody diluent is 1:5000, so as to obtain an enzyme conjugate;
dissolving the standard substance by using the standard substance diluent to obtain standard substance solutions with the concentrations of 0pg/m L, 10pg/m L, 100pg/m L, 200pg/m L, 500pg/m L and 1000pg/m L respectively;
and diluting the concentrated washing solution with distilled water or deionized water according to the volume ratio of 1:20 to obtain the washing solution.
(2) Drawing a standard curve:
respectively adding 50 mu L of calibrator solution into corresponding holes of an ELISA plate coated with the anti-S100B monoclonal antibody, then respectively adding 100 mu L of horseradish peroxidase-labeled anti-S100 polyclonal antibody solution for detection into each hole, and lightly shaking and uniformly mixing;
sealing the plate with a sealing plate film, then incubating for 2 hours at 37 ℃, fully washing for 5 times with the washing solution, and drying;
adding 25 mu L of luminous liquid A, B into each hole, and lightly shaking and uniformly mixing;
adjusting the zero point by using a blank hole, and measuring the optical density value of each hole by using a luminometer;
performing data processing by using a log-log linear fitting mode, and drawing by using the log of the optical density value of each calibrator as a vertical coordinate and the log of the concentration of each calibrator as a horizontal coordinate to obtain a standard curve; as shown in fig. 1, the equation of the standard curve is 0.8818x +3.6761, and the correlation coefficient is 0.9995.
As follows, the embodiment of the present invention further provides a method for detecting the content of S100B in a sample solution to be detected.
Respectively adding a sample to be detected to 50 mu L into corresponding holes of an ELISA plate coated with an anti-S100B monoclonal antibody, then adding a horseradish peroxidase-labeled detection anti-S100 polyclonal antibody solution to 100 mu L, and lightly oscillating and uniformly mixing;
sealing the plate with a sealing plate film, then incubating for 2 hours at 37 ℃, fully washing for 5 times with the washing solution, and drying;
adding 25 mu L of luminous liquid A, B liquid respectively, and lightly shaking and uniformly mixing;
adjusting the zero point by using a blank hole, and measuring the optical density value by using a luminometer;
and substituting the optical density value of the sample solution to be detected into the standard curve to obtain the content of S100B in the sample solution to be detected.
The kits provided in the examples of the present invention were subjected to performance tests by the following methods. And respectively selecting three batches of kits, namely a batch one, a batch two and a batch three, and carrying out performance test.
Firstly, the method comprises the following steps: minimum limit of detection
Adding 50 mu L standard substance into each hole of an ELISA plate coated with an anti-S100B monoclonal antibody, then adding 100 mu L of an anti-S100 polyclonal antibody solution for detection labeled by horseradish peroxidase into each hole, incubating at 37 ℃ for 2 hours, fully washing for 5 times by using a washing solution, draining, adding 25 mu L luminescent solution A and 25 mu L luminescent solution B into each hole, and measuring the luminescent value of each hole by using a luminometer.
Wherein, each kit to be tested, the blank standard substance solution is provided with 20 compound holes, other standard substances are provided with double holes, the light absorption value of each hole is shown in table 1, and the light emitting value of each hole is shown in table 1.
Calculating the Mean value (Mean) and Standard Deviation (SD) of the corresponding luminescence values of the 50 mu L S0 solution of the 20 holes, and calculating the concentration value of the luminescence value of Mean +2 × SD as the lowest detection limit by a fitting equation.
The lowest detection limit of the three batches of kits is below 1pg/m L.
Table 1:
II, secondly: repeatability and lot-to-lot difference
The repeatability is the Coefficient of Variation (CV) obtained by measuring a sample by using the same batch of kit, each standard sample needs to be subjected to 10-hole precision measurement randomly in one plate, the average concentration (Mean) and the Standard Deviation (SD) of the measurement result are calculated, and the Coefficient of Variation (CV) in the batch is SD/Mean × 100%
The batch difference is the repeatability among different batches of kits, three batches of kits are randomly drawn, the standard substance is measured for 3 times by using the kits, the average concentration (Mean) and the Standard Deviation (SD) of the measurement result are calculated, the batch variation Coefficient (CV) is SD/Mean × 100% 100, and the standard substance is S100B.
The specific detection process is as follows:
adding 50 mu L specific solution into each well of an ELISA plate coated with an anti-S100B monoclonal antibody, then adding 100 mu L enzyme conjugate into each well, incubating for 2 hours at 37 ℃, fully washing for 5 times by using washing liquid, drying, adding 25 mu L luminescent liquid A and 25 mu L luminescent liquid B into each well, and measuring the luminescent value of each well by using a luminometer.
The specific solution is a specific solution A or a specific solution B.
The specific solution A is an S0 solution (the concentration of a standard is 0pg/m L), an S1 solution (the concentration of the standard is 10pg/m L), an S2 solution (the concentration of the standard is 100pg/m L), an S3 solution (the concentration of the standard is 200pg/m L), an S4 solution (the concentration of the standard is 500pg/m L) and an S5 solution (the concentration of the standard is 1000pg/m L).
The specific solution B is a standard solution with different concentration from the specific solution A.
The standard solution is prepared by dissolving the standard in 20mM PBS buffer (pH7.4) to obtain 100 pg/L low-concentration standard solution (QC1) and 600 pg/L high-concentration standard solution (QC2), and each specific solution B is provided with 10 multiple wells for each test kit.
The luminescence values of the wells with the particular solution A are shown in Table 2.
Table 2:
the luminescence values of the wells with the particular solution B are shown in Table 3.
Table 3:
| QC1 | QC2 | QC1 | QC2 | QC1 | QC2 | |
| |
375825 | 1375935 | 374710 | 1371970 | 375340 | 1378860 |
| |
375620 | 1373630 | 372290 | 1375895 | 374375 | 1373295 |
| |
374730 | 1378095 | 374905 | 1372460 | 375390 | 1370585 |
| Multiple holes 4 | 367990 | 1377205 | 373930 | 1373390 | 375345 | 1372340 |
| |
368125 | 1377525 | 373540 | 1376960 | 371805 | 1378965 |
| |
369625 | 1375510 | 371110 | 1372490 | 375940 | 1374120 |
| |
376525 | 1372685 | 372700 | 1376720 | 373930 | 1378440 |
| Multiple holes 8 | 369915 | 1378400 | 375335 | 1370655 | 376070 | 1376705 |
| Multiple holes 9 | 374190 | 1372405 | 373235 | 1373610 | 372190 | 1373470 |
| Multiple holes 10 | 370595 | 1374980 | 375795 | 1372515 | 375755 | 1370730 |
The data was processed using a log-log linear fit to calculate the concentration of S100B in a particular solution b from the standard curve, and the results are shown in tables 4 and 5. The three batches of the kit are used for measuring two quality controls of high concentration and low concentration, and the variation coefficient in the batch is less than 5 percent, which shows that the kit has good uniformity and repeatability of results.
Table 4:
table 5:
| QC1 | QC2 | |
| first batch | 135.42 | 618.40 |
| Second batch | 138.81 | 617.96 |
| Third batch | 140.30 | 617.99 |
| Mean value of | 138.18 | 618.12 |
| SD | 2.501 | 0.246 |
| CV | 1.81％ | 0.04％ |
Three batches of kits are used for measuring two standard products with high concentration and low concentration, and the batch-to-batch variation coefficient is less than 5 percent, which shows that the kits in different batches have small variation and the measurement result has repeatability.
In summary, the main performance indexes of the kit provided by the invention have the following standards:
the lowest detection limit is not higher than 1pg/m L;
repeatability: the intra-batch variation coefficient is not higher than 5%;
inter-batch difference: the inter-batch coefficient of variation is not higher than 5%.
Third, specificity
Adding 50 mu L specific solution into each hole of an ELISA plate coated with an anti-S100B monoclonal antibody, then adding 100 mu L anti-S100 polyclonal antibody solution for detection labeled by horseradish peroxidase into each hole, incubating at 37 ℃ for 30 minutes, fully washing for 5 times by using a washing solution, drying, adding 25 mu L luminescent solution A and 25 mu L luminescent solution B into each hole, and measuring the luminescent value of each hole by using a luminometer.
The specific solutions were S0 solution (concentration of standard was 0pg/m L), S1 solution (concentration of standard was 10pg/m L), S2 solution (concentration of standard was 100pg/m L), S3 solution (concentration of standard was 200pg/m L), S4 solution (concentration of standard was 500pg/m L), S5 solution (concentration of standard was 1000pg/m L), sample solution with serum total bilirubin TBI L of 40. mu. mol/L, and sample solution with triglyceride TG of 4 mmol/L and total cholesterol TC of 9 mmol/L.
L og-L og fitting is carried out according to the luminescence value of each calibration point and the corresponding concentration, and the S100B measured value of each cross-reaction substance is calculated according to a standard curve, wherein the concentration is the intersection value of the reagent and TBI L and the intersection value of the reagent and TG and TC.
The luminescence and the back-concentration of each well are shown in Table 6.
Table 6:
the specificity is to detect the interference of other substances in blood to the measurement of the kit, and the substances which easily interfere with the measurement of S100B in human blood are serum total bilirubin TBI L and triglyceride TG and total cholesterol TC., and the test is carried out with these substances in high concentrations, and referring to tables 6 and 7, the cross-reaction of the kit with these substances is low, and the measurement of S100B is not interfered.
Table 7:
fourthly, the method comprises the following steps: for diagnosis of craniocerebral injury
Clinical samples were collected from hospitals, 100 parts of healthy human serum and 48 parts of craniocerebral injury patient serum 12 hours after injury were collected, and the content of S100B in each serum sample was measured by an S100B quantitative determination kit (enzyme-linked immunosorbent assay), as shown in table 8.
Table 8:
the reference value range of the Mean plus 2 standard deviations Mean + -2 SD is 140-380 pg/m L.
In summary, in the kit provided by the embodiment of the present invention, the reaction time between the sample to be detected (the standard sample or the serum sample) and the horseradish peroxidase-labeled detection anti-S100 polyclonal antibody solution is 2 hours; the clinical common pathological jaundice and lipemia specimens have no obvious interference on the determination of the serum S100B protein; the average variation in batch and between batches is less than 5.0 percent, and the method has the technical effects of simplicity, convenience, rapidness, specificity, sensitivity and suitability for clinical routine application.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A chemiluminescence detection kit for S100B protein, which is characterized by comprising: the kit comprises an ELISA plate coated with an anti-S100B monoclonal antibody, an anti-S100 polyclonal antibody solution for detecting horseradish peroxidase labeling, a standard product diluent, a sample diluent, a concentrated washing solution, a luminescent solution A, a luminescent solution B and a detection antibody diluent.
2. The detection kit of S100B protein according to claim 1, wherein the horseradish peroxidase-labeled detection anti-S100 polyclonal antibody solution is prepared by the following method:
purifying and quantifying the antibody, and dialyzing the antibody in a carbonate buffer solution with the pH of 9.0-9.5 at 0.01 mol/L until the concentration is 5mg/m L;
preparing 5mg/m L horseradish peroxidase aqueous solution;
adding NaIO with concentration of 37.5mg/m L and 0.4m L into the horseradish peroxidase aqueous solution4Stirring at room temperature in dark for 20min to obtain oxidized enzyme solution;
dialyzing the oxidized enzyme solution in acetate buffer solution with the concentration of 1 mmol/L and the pH value of 4.4 at 4 ℃ overnight, and changing the solution for 3 times;
the solution in the dialysis bag was taken out and 0.2 mol/L Na was added2CO3Adjusting the pH of the buffer solution to 9.0-9.5, quickly mixing the buffer solution with the purified and quantified antibody 1:1, and slightly stirring the mixture at room temperature for 2 hours in a dark place;
6mg/m L NaBH was added4Mixing 100ul of the solution uniformly, and reacting for 2h at 4 ℃;
the reaction system after reduction was dialyzed overnight at 4 ℃ in 0.15 mol/L in PBS at pH7.4, and the solution was changed 3 times;
adding BSA to enable the final concentration to be 2-3 mg/m L, uniformly mixing, carrying out 10000rpm × 3min, collecting supernatant, subpackaging and freezing.
3. The detection kit for the S100B protein according to claim 1, wherein the standard substance is the S100B antigen.
4. The S100B protein assay kit according to claim 1, wherein the solvent of the sample diluent is 20 mmol/L phosphate buffered saline solution with pH 6.0, and the solutes and their concentrations in the sample diluent are 0.2% gelatin, 0.2% casein, 150 mmol/L NaCl, 0.01% tween-20, 5% sucrose, 0.5/ten thousand bromopotash phenol violet, and 0.1% preservative.
5. The kit for detecting S100B protein according to claim 1, wherein the solvent of the concentrated washing solution is water, and the solute and its Na concentration in the concentrated washing solution are 116 g/L2HPO4·12H2O、11.84g/LNaH2PO4·2H2O, 180 g/L NaCl, 5m L/L Tween 20 and 1m L/L preservative.
6. The kit for detecting the protein S100B, according to claim 1, wherein the solvent of the luminescent solution a is Tris-HCl buffer solution with pH 8.0 and 0.2 mol/L, and the solute and the concentration thereof in the luminescent solution a are 3mg/m L luminol, 0.25mg/m L paraiodophenol and 0.5mg/m L sodium tetrabenate.
7. The kit for detecting S100B protein according to claim 1, wherein the solvent of the luminescent solution B is Tris-HCl buffer solution with pH 8.0 and 0.2 mol/L, and the solute and carbamide peroxide with the concentration of 0.5mg/m L in the luminescent solution B.
8. The S100B protein assay kit according to claim 1, wherein the solvent of the standard dilution is 20 mmol/L phosphate buffered saline solution with pH7.4, the solute and its concentration in the standard dilution are 1% by mass casein, 8% by mass trehalose, 3% by mass mannitol, 1 mmol/L EDTA, 0.5% by mass glycine, 150 mmol/L NaCl, and 0.1% by mass preservative.
9. The S100B protein assay kit according to claim 1, wherein the solvent of the test antibody diluent is a phosphate buffered saline solution with a pH of 7.4 at 20 mmol/L, and the solute and its concentration in the test antibody diluent are bovine serum albumin with a mass percentage of 2%, casein with a mass percentage of 0.1%, NaCl with a mass percentage of 150 mmol/L, tween 20 with a mass percentage of 0.01%, aminopyrine with a mass percentage of 5/1 ten thousand, dye with a mass percentage of 1/20 ten thousand, and preservative with a mass percentage of 0.1%.
10. A method of using the chemiluminescent detection kit of S100B protein according to any one of claims 1-9, comprising the steps of:
(1) the following solutions were prepared:
diluting a detection anti-S100 polyclonal antibody solution marked by horseradish peroxidase by using a detection antibody diluent, wherein the ratio of the detection anti-S100 polyclonal antibody solution marked by horseradish peroxidase to the detection antibody diluent is 1: 10000;
diluting a detection anti-S100 polyclonal antibody solution marked by horseradish peroxidase by using a detection antibody diluent, wherein the volume ratio of the detection anti-S100 polyclonal antibody marked by the horseradish peroxidase to the detection antibody diluent is 1:5000, so as to obtain an enzyme conjugate;
dissolving a standard substance by using a standard substance diluent to obtain a plurality of standard substance solutions with gradient changes in concentration;
and diluting the concentrated washing solution with distilled water or deionized water according to the volume ratio of 1:20 to obtain the washing solution.
(2) Drawing a standard curve:
respectively adding 50 mu L of calibrator solution into corresponding holes of an ELISA plate coated with the anti-S100B monoclonal antibody, then respectively adding 100 mu L of horseradish peroxidase-labeled anti-S100 polyclonal antibody solution for detection into each hole, and lightly shaking and uniformly mixing;
sealing the plate with a sealing plate film, then incubating for 2 hours at 37 ℃, fully washing for 5 times with the washing solution, and drying;
adding 25 mu L of luminous liquid A, B into each hole, and lightly shaking and uniformly mixing;
adjusting the zero point by using a blank hole, and measuring the optical density value of each hole by using a luminometer;
performing data processing by using a log-log linear fitting mode, and drawing by using the log of the optical density value of each calibrator as a vertical coordinate and the log of the concentration of each calibrator as a horizontal coordinate to obtain a standard curve;
(3) detection of sample to be tested
Respectively adding a sample to be detected to 50 mu L into corresponding holes of an ELISA plate coated with an anti-S100B monoclonal antibody, then adding a horseradish peroxidase-labeled detection anti-S100 polyclonal antibody solution to 100 mu L, and lightly oscillating and uniformly mixing;
sealing the plate with a sealing plate film, then incubating for 2 hours at 37 ℃, fully washing for 5 times with the washing solution, and drying;
adding 25 mu L of luminous liquid A, B liquid respectively, and lightly shaking and uniformly mixing;
adjusting the zero point by using a blank hole, and measuring the optical density value by using a luminometer;
and substituting the optical density value of the sample solution to be detected into the standard curve to obtain the content of S100B in the sample solution to be detected.
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Application publication date: 20200804 |