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
							multiple holes 1	375825	1375935	374710	1371970	375340	1378860
Multiple holes 2	375620	1373630	372290	1375895	374375	1373295
							Multiple holes 3	374730	1378095	374905	1372460	375390	1370585
Multiple holes 4	367990	1377205	373930	1373390	375345	1372340
							Multiple holes 5	368125	1377525	373540	1376960	371805	1378965
Multiple holes 6	369625	1375510	371110	1372490	375940	1374120
							Multiple holes 7	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

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:

(2) Drawing a standard curve:

(3) detection of sample to be tested