CN111487407A

CN111487407A - Detection kit for S100B protein and use method thereof

Info

Publication number: CN111487407A
Application number: CN201910079588.7A
Authority: CN
Inventors: 艾冰花; 赵京超; 王立杰; 常青侠; 焉丽波; 陈蒙蒙
Original assignee: Avioq Biology Technology Co ltd
Current assignee: Avioq Biology Technology Co ltd
Priority date: 2019-01-28
Filing date: 2019-01-28
Publication date: 2020-08-04

Abstract

The invention is applicable to the field of biotechnology, and particularly relates to a detection kit of S100B protein and a use method thereof, wherein the kit comprises an enzyme label plate coated with an anti-S100B monoclonal antibody, an anti-S100 polyclonal antibody solution labeled by horseradish peroxidase for detection, a standard diluent, a sample diluent, a concentrated washing solution, a developing solution A, a developing solution B, a stop solution and a detection antibody diluent.

Description

Detection kit for S100B protein and use method thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a detection kit for S100B protein and a use method thereof.

Background

The S100B protein is a specific protein existing in brain colloid cells, has wide biological activity, and plays a plurality of important physiological functions under physiological concentration, including influencing the growth, reproduction and differentiation of glial cells, maintaining calcium homeostasis, possibly playing a certain role in learning and memory, and the like. When the glial cell membrane is damaged due to brain injury, the S100B protein is released to the extracellular space and enters cerebrospinal fluid and blood through the damaged blood brain barrier, and the content of the protein in the serum is closely related to the disease condition and prognosis of some diseases such as nervous system and the like.

At present, the diagnosis of craniocerebral injuries and cerebrovascular diseases mainly depends on imaging (CT and MRI) examination, but the modern imaging technology is not suitable for some critical patients with unstable vital signs or using a respirator, and the examination cannot be performed in time under some special conditions. Therefore, the detection of the protein level of S100B can provide a new means for the diagnosis and prognosis of craniocerebral injury and cerebrovascular diseases, and the accurate, convenient and quick detection of the level of S100B protein in serum is particularly important.

The current detection methods for the serum S100B comprise Radioimmunoassay (RIA), immunoradiometric assay (IRMA), enzyme-linked immunosorbent assay (E L ISA), electrochemiluminescence (EC L IA) and the like, because RIA and IRMA have radioactivity, the half-life of nuclides and the stable period of a kit are short, and the operation is complicated, the clinical application of RIA and IRMA is limited.

The electrochemical luminescence method (EC L IA) is a product combining electrochemical luminescence (EC L) and immunoassay, and the method comprises the steps of connecting streptavidin-coated magnetic beads and biotinylated antibodies with S100B antibodies into a whole to form a double-antibody sandwich, and finally carrying out redox reaction on the surfaces of electrodes through luminescent agents coupled with the antibodies and an electron donor TPA repeatedly, wherein the light intensity is in linear relation with the concentration of the luminescent agents to measure the content of the antigen to be measured.

The enzyme-linked immunosorbent assay (E L ISA) is a test technology with high sensitivity based on immunological reaction and combining specific reaction of antigen and antibody with high-efficiency catalytic action of enzyme on a substrate, the E L ISA method has the advantages of wide linear range, high sensitivity, good repeatability, convenient operation and reliable experiment, is more suitable for clinical detection of patient diseases and judgment of prognosis, and is one of the mainstream detection methods at present, but the detection method of the double-antibody sandwich E L ISA which is widely used clinically at present has lower detection sensitivity, and is mainly related to the fact that the used standard product is mostly S100B/S100A protein dimer, namely the content of the S100B and S100A proteins which are actually measured by the kit, and meanwhile, the specificity and affinity of the antibody are not ideal and are also part of reasons for low sensitivity.

In conclusion, the existing detection method for the serum S100B has the problems of high detection cost, lower detection sensitivity and poor specificity and stability.

Disclosure of Invention

The embodiment of the invention provides a detection kit for S100B protein, and aims to solve the problems of high detection cost, low detection sensitivity and poor specificity and stability of the existing detection method for serum S100B.

The 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 diluent, a sample diluent, a concentrated washing solution, a developing solution A, a developing solution B, a stop solution and a detection antibody diluent.

The embodiment of the invention also provides a use method of the 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 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;

diluting the concentrated washing liquid with distilled water or deionized water according to a volume ratio of 1:20 to obtain washing liquid;

(2) drawing a standard curve:

respectively adding the standard substance solutions with the concentration gradient change of 50 mu L into corresponding holes of the ELISA plate coated with the anti-S100B monoclonal antibody, and lightly shaking and uniformly mixing;

sealing the plate with a sealing plate film, then incubating for 30 minutes at 37 ℃, fully washing for 5 times with the washing solution, and drying;

adding 100 mu L of anti-S100 polyclonal antibody solution for detection labeled by horseradish peroxidase into each hole, and lightly shaking and uniformly mixing;

adding A, B color development liquid 50 μ L into each well, mixing by gentle shaking, and developing at 37 deg.C in dark for 15 min;

adding 50 mu L of stop solution into each hole, tapping and mixing uniformly, setting the wavelength of an enzyme-labeling instrument to be 450nm, adjusting the zero point by using a blank hole, and then measuring the optical density value of each hole;

performing data processing by using a log-log linear fitting mode, and drawing by taking the logarithm of the optical density value of each calibrator as a vertical coordinate (Y axis) and the logarithm of the concentration of each calibrator as a horizontal coordinate (X axis) to obtain a standard curve;

(3) detection of sample to be tested

Slightly oscillating and uniformly mixing samples to be detected with 50 mu L in corresponding holes of the ELISA plate coated with the anti-S100B monoclonal antibody;

adding 100 mu L of an anti-S100 polyclonal antibody solution for detection marked by horseradish peroxidase into the hole, and lightly shaking and uniformly mixing;

adding 50 mu L of color development liquid A, B into the holes, lightly shaking and uniformly mixing, and developing for 15 minutes at 37 ℃ in a dark place;

adding a stop solution of 50 mu L into the hole, tapping and uniformly mixing, setting the wavelength of an enzyme-labeling instrument to be 450nm, adjusting the zero point by using a blank hole, and then determining the optical density value;

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 kit has the advantages of high sensitivity, low minimum detection limit of 1pg/m L, good uniformity, high stability, less than 10 percent of variation coefficient in batches and less than 5 percent of variation coefficient in batches, good specificity, low cross reaction between the kit and serum total bilirubin TBI L, triglyceride TG and total cholesterol TC which are easy to interfere with the test in serum, no interference on the measurement of S100B, good clinical trial effect and lower single detection cost than the kits of the same type in the market.

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 embodiment of the invention provides a detection kit of S100B protein, which 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 diluent, a sample diluent, a concentrated washing solution, a developing solution A, a developing solution B, a stop solution and a detection antibody diluent.

In the present example, the concentration of the horseradish peroxidase-labeled detection anti-S100 polyclonal antibody in the horseradish peroxidase-labeled detection anti-S100 polyclonal antibody solution is 2 to 3mg/m L, and the solvent of the horseradish peroxidase-labeled detection anti-S100 polyclonal antibody solution is a phosphate buffer solution with a concentration of 0.15 mol/L and a pH of 7.4.

In the embodiment of the invention, the sample diluent comprises 0.2% of gelatin by mass, 0.2% of casein by mass, 150 mmol/L of NaCl by mass, 0.01% of Tween-20 by mass, 5% of cane sugar by mass, 0.5/1 ten thousand of bromopotash phenol purple by mass, 0.1% of preservative by mass and the balance of PBS buffer solution with the concentration of 20 mmol/L and the pH value of 6.0.

In the examples of the present invention, Na was contained in the concentrated washing solution₂HPO₄12H2O at a concentration of 116 g/L₂PO₄·2H₂The concentration of O is 11.84 g/L, the concentration of O is 180 g/L, the concentration of Tween 20 is 5m L/L, the concentration of preservative is 1m L/L, and the solvent is water.

In the embodiment of the invention, the solvent of the color developing liquid A is water, and the solute and the concentration thereof in the color developing liquid A are 7.28 g/L of citric acid, 23.75 g/L of disodium hydrogen phosphate and 0.5 g/L of hydrogen peroxide urine;

the solvent of the color developing solution B is water, and the solute and the concentration thereof in the color developing solution B are as follows, methanol is 100m L/L, polyvinyl alcohol is 1 g/L, Na₂S₂O₃1.5 g/L, hydrochloric acid 1m L/L, 3,3',5,5' -tetramethyl benzidine 0.03% by weight, dimethyl sulfoxide 1% by weight,

in the embodiment of the invention, the solvent of the stop solution is water, and the solute and the concentration thereof in the stop solution are 200m L/1.8L of sulfuric acid and 1.35 g/1.8L of ethylene diamine tetraacetic acid.

In the embodiment of the invention, the solvent of the standard dilution is PBS buffer solution with 20 mmol/L and pH7.4, and the solute and the concentration thereof in the standard dilution are casein with the mass percent of 1%, trehalose with the mass percent of 8%, mannitol with the mass percent of 3%, 1 mmol/L EDTA, glycine with the mass percent of 0.5%, 150 mmol/L NaCl and a preservative Proclin-300 with the mass percent of 0.1%.

In the embodiment of the invention, the solvent of the detection antibody diluent is PBS buffer solution with the concentration of 20 mmol/L and the pH value of 7.4, and the solute and the concentration thereof in the detection antibody diluent are as follows, bovine serum albumin with the mass percentage of 2%, casein with the mass percentage of 0.1%, NaCl with the mass percentage of 150 mmol/L, Tween 20 with the mass percentage of 0.01%, amino oxazoline with the mass percentage of 5/1 ten thousand, dye with the mass percentage of 1/20 ten thousand, and preservative Proclin-300 with the mass percentage of 0.1%

In the present example, the standard is S100B protein.

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:

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 in sequence;

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:

sealing the plate with a sealing plate membrane, then incubating for 30 minutes at 37 ℃, fully washing for 5 times with the washing solution after fully reacting the antibody and the antigen, and drying;

sealing the plate with a sealing plate membrane, then incubating for 30 minutes at 37 ℃, fully reacting the antibody and the antigen, fully washing for 5 times with the washing solution, and drying;

the data was processed using a log-log linear fit, plotted with the log of the optical density value of each calibrator as the ordinate (Y-axis) and the log of the concentration of each calibrator as the abscissa (X-axis), to give a standard curve, which, as shown in fig. 1, had the equation Y-0.5716X-1.2989 and a correlation coefficient of 0.9996.

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.

in the embodiment of the invention, the sample to be detected is fresh serum or a fresh plasma sample, the separation is carried out within 24 hours after the venous blood collection, the storage time of the serum plasma sample at 4 ℃ is not more than 1 week, if the measurement can not be carried out within 1 week after the blood collection, the serum sample is sealed and then placed below-20 ℃, and repeated freeze thawing is avoided. Severely hemolyzed, lipemic samples were not available for detection.

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, slightly oscillating and uniformly mixing, placing the plate film at the rear part of 37 ℃ for incubation for 30 minutes, fully washing the plate film for 5 times and drying the plate film, adding 100 mu L of an anti-S100 polyclonal antibody solution for detection marked by horseradish peroxidase into each hole, slightly oscillating and uniformly mixing, placing the plate film at the rear part of 37 ℃ for incubation for 30 minutes, fully washing the plate film for 5 times and drying the plate film, adding 50 mu L color development liquid A and 50 mu L color development liquid B into each hole, keeping the plate film dark and developing the plate film for 15 minutes at 37 ℃, adding 50 mu L of stop solution into each hole, and detecting the plate film on an ELISA reader by using 450/630nm double-wavelength detection.

Wherein, each kit to be tested, the blank standard substance solution is provided with 20 multiple holes, other standard substances are provided with double holes, and the light absorption value of each hole is shown in table 1.

Table 1:

calculating the Mean (Mean) and Standard Deviation (SD) of the absorbance values corresponding to the 50 mu L S0 solution of 20 wells, and calculating the concentration 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.

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 hole of an ELISA plate coated with an anti-S100B monoclonal antibody, slightly shaking and uniformly mixing, placing the plate at 37 ℃ for incubation for 30 minutes, fully washing the plate for incubation for 5 times by using washing liquid, adding 100 mu L of horseradish peroxidase-labeled anti-S100 polyclonal antibody for detection into each hole, placing the plate for incubation for 30 minutes at 37 ℃, fully washing the plate for 5 times by using the washing liquid, draining the plate, adding 50 mu L developing liquid A and 50 mu L developing liquid B into each hole, developing the plate for 15 minutes in a dark place at 37 ℃, adding 50 mu L of stop solution into each hole, and detecting the plate on an ELISA reader by using 450/630nm double-wavelength.

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 500 pg/L high-concentration standard solution (QC2), and each test kit is provided with 10 multiple wells for each specific solution B.

The absorbance values for each well using the particular solution A are shown in tables 2 and 3.

Table 2:

TABLE 3

And performing data processing by using a log-log linear fitting mode, calculating the concentration of S100B in the specific solution B according to a standard curve, wherein the results are shown in tables 4 and 5, the three batches of the kit are used for measuring high-concentration quality control and low-concentration quality control, and the variation coefficient in batches is less than 10%, so that the kit is good in uniformity and has repeatability.

Table 4:

	QC1	QC2	QC1	QC2	QC1	QC2
							multiple holes 1	104.78	561.35	112.32	557.05	112.36	518.78
Multiple holes 2	101.23	535.41	95.06	519.50	109.76	591.03
							Multiple holes 3	97.50	556.65	112.57	544.06	105.14	531.57
Multiple holes 4	97.25	557.17	106.42	582.90	115.51	543.97
							Multiple holes 5	100.48	541.07	109.74	526.11	109.50	572.32
Multiple holes 6	97.99	567.13	95.78	545.10	104.38	544.48
							Multiple holes 7	100.48	594.79	96.75	508.41	103.88	528.49
Multiple holes 8	114.12	556.12	98.22	578.11	95.45	546.56
							Multiple holes 9	105.29	577.17	98.46	572.81	105.65	540.34
Multiple holes 10	109.92	516.05	97.48	562.28	110.02	589.42
							Mean value of	102.90	556.29	102.28	549.63	107.17	550.70
SD	5.618	21.983	7.138	25.565	5.569	25.121
							CV	5.46％	3.95％	6.98％	4.65％	5.20％	4.56％

Table 5:

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 10%;

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, slightly shaking and uniformly mixing, incubating at 37 ℃ for 30 minutes, fully washing for 5 times by using washing liquid, drying, adding 100 mu L anti-S100 polyclonal antibody solution for detection marked by horseradish peroxidase into each hole, incubating at 37 ℃ for 30 minutes, fully washing for 5 times by using the washing liquid, drying, adding 50 mu L developing solution A and 50 mu L developing solution B into each hole, developing for 15 minutes in a dark place at 37 ℃, adding 50 mu L stopping solution into each hole, and detecting on an ELISA reader by using 450/630nm double-wavelength.

Wherein, the specific solutions are S0 solution (the concentration of the standard is 0pg/m L), S1 solution (the concentration of the standard is 10pg/m L), S2 solution (the concentration of the standard is 100pg/m L), S3 solution (the concentration of the standard is 200pg/m L), S4 solution (the concentration of the standard is 500pg/m L), S5 solution (the concentration of the standard is 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. each kit is provided with two compound holes.

L og-L og fit was made based on the OD values of each calibration point and the corresponding concentrations, the S100B measurement of each cross-reactive material was calculated from the standard curve, which is the intersection of the reagent with TBI L and the intersection of the reagent with TG and TC.

The absorbance and back-extrapolated concentration for each well are shown in Table 6.

Table 6: analysis of the results of the specificity experiment

The specificity is to detect the interference of other substances in blood on the measurement of the kit, substances which easily interfere the measurement of S100B in human blood are serum total bilirubin TBI L, triglyceride TG and total cholesterol TC, and experiments are carried out by using high concentrations of the substances, as shown in tables 6 and 7, and the results show that the kit has low cross reaction with the substances and does not interfere the measurement of S100B.

Table 7: cross reaction value of kit and high concentration reaction substance

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 serum from a craniocerebral injury patient 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), and the results are shown in table 8.

Table 8:

the reference value range (Mean + -2 SD) of the S100B protein in the serum of a healthy person is 140-380 pg/m L.

In conclusion, the kit has high sensitivity, the minimum detection limit of the kit is as low as 1pg/m L, the kit has good uniformity and high stability, the intra-batch variation coefficient is less than 10 percent, and the inter-batch variation coefficients are less than 5 percent.

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

1. A detection kit of 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 diluent, a sample diluent, a concentrated washing solution, a developing solution A, a developing solution B, a stop solution and a detection antibody diluent.

2. The detection kit for S100B protein according to claim 1, wherein the concentration of the horseradish peroxidase-labeled detection anti-S100 polyclonal antibody in the horseradish peroxidase-labeled detection anti-S100 polyclonal antibody solution is 2-3mg/m L, and the solvent of the horseradish peroxidase-labeled detection anti-S100 polyclonal antibody solution is phosphate buffered saline solution with the concentration of 0.15 mol/L and the pH of 7.4.

3. The S100B protein detection kit, according to claim 1, wherein the sample diluent comprises gelatin 0.2% by mass, casein 0.2% by mass, NaCl 150 mmol/L% by mass, Tween-20 0.01% by mass, sucrose 5% by mass, bromopotash phenol purple 0.5/1 ten thousand by mass, preservative 0.1% by mass, and PBS buffer at a pH of 6.0 at a concentration of 20 mmol/L as the balance.

4. The S100B protein assay kit of claim 1, wherein the concentrated wash solution contains Na₂HPO₄·12H₂The concentration of O is 116 g/L₂PO₄·2H₂The concentration of O is 11.84 g/L, the concentration of O is 180 g/L, the concentration of Tween 20 is 5m L/L, the concentration of preservative is 1m L/L, and the solvent is water.

5. The detection kit for S100B protein according to claim 1,

the solvent of the color developing solution A is water, and the solute and the concentration thereof in the color developing solution A are 7.28 g/L of citric acid, 23.75 g/L of disodium hydrogen phosphate and 0.5 g/L of hydrogen peroxide urine;

the solvent of the color developing solution B is water, and the solute and the concentration thereof in the color developing solution B are as follows, methanol is 100m L/L, polyvinyl alcohol is 1 g/L, Na₂S₂O₃1.5 g/L, hydrochloric acid 1m L/L, 3,3',5,5' -tetramethyl benzidine 0.03% by weight, dimethyl sulfoxide 1% by weight.

6. The kit for detecting the protein S100B, according to claim 1, wherein the solvent of the stop solution is water, and the solutes and the concentrations thereof in the stop solution are 200m L/1.8L for ethylenediaminetetraacetic acid (EDTA) 1.35 g/1.8L.

7. The detection kit of S100B protein according to claim 1, wherein the solvent of the standard dilution is 20mM PBS buffer solution with pH7.4, and the solutes and their concentrations in the standard dilution are 1% by mass of casein, 8% by mass of trehalose, 3% by mass of mannitol, 1 mmol/L EDTA, 0.5% by mass of glycine, 150 mmol/L NaCl, and 0.1% by mass of Proclin-300 as preservative.

8. The kit for detecting S100B protein according to claim 1, wherein the solvent of the diluted detection antibody is 20mM PBS buffer solution with pH7.4, and the solutes and their concentrations in the diluted detection antibody are 2% by mass of bovine serum albumin, 0.1% by mass of casein, 150 mmol/L of NaCl, 0.01% by mass of Tween 20, 5/1 ten thousand of aminopyrine, 1/20 ten thousand of dye, and 0.1% by mass of Proclin-300 as preservative.

9. The detection kit for the S100B protein, according to claim 1, wherein the standard substance is the S100B protein.

10. A method of using the S100B protein detection kit according to any one of claims 1-9, comprising the steps of:

(1) the following solutions were prepared:

(2) drawing a standard curve:

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