CN110794131A - Interleukin-6 detection kit and preparation method thereof - Google Patents
Interleukin-6 detection kit and preparation method thereof Download PDFInfo
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- 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
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Abstract
The invention relates to the technical field of biology, relates to an interleukin-6 detection kit, and particularly relates to a preparation method of the interleukin-6 detection kit, and discloses components and concentrations including a reagent R1 and a reagent R2, wherein the reagent R2 consists of a second buffer solution, a preservation solution and a latex microsphere antibody conjugate, the preservation solution consists of N, N-dihydroxyethyl glycine, a second stabilizer and a second preservative, and the latex microsphere antibody conjugate consists of a latex microsphere, an interleukin-6 antibody and 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride. The invention has the advantages that: the method uses a small amount of samples, has the characteristics of simplicity, accuracy and rapidness compared with other detection methods, and can realize high-throughput detection by batch analysis on a full-automatic biochemical analyzer; the kit has the characteristics of good stability, high sensitivity, wide linear range, high specificity, good precision, short detection time and the like, and is low in price.
Description
Technical Field
The invention relates to the technical field of biology, relates to an interleukin-6 detection kit, and particularly relates to a preparation method of the interleukin-6 detection kit.
Background
Interleukin-6 (IL-6) is a pleiotropic cytokine with a wide range of functions. The interleukin-6 has physiological activity on B cells, T cells, hematopoietic stem cells, liver cells and brain cells, can induce the differentiation of the B cells to generate immunoglobulin, promote the proliferation and growth of the T cells, promote the proliferation of bone marrow hematopoietic stem cells, and enhance the differentiation of blood cells and the anti-tumor effect thereof. The biological function of interleukin-6 plays an important role in resisting infection, tumor, immune diseases, immunoregulation and the like.
Studies have shown that interleukin-6 is rapidly produced during acute inflammatory reactions in the presence of trauma, surgery, stress, infection, brain death, tumor production and other conditions. The continuous monitoring of the interleukin-6 content in the serum or plasma of an intensive care patient can effectively evaluate the severity of Systemic Inflammatory Response Syndrome (SIRS), and the prognosis of sepsis and septic shock. Interleukin-6 can be used as early warning index of sepsis, and simultaneously, interleukin-6 also plays an important role in chronic inflammatory response.
At present, in China, the detection of interleukin-6 mainly takes foreign imported reagents and enzyme-linked immunosorbent assay (ELISA) as main clinical application, the imported reagents are expensive, the economic burden is large for patients, and the primary popularization is not facilitated; the ELISA method has low detection sensitivity, narrow linear range and long detection process, is usually only suitable for manual operation and is not beneficial to detection of a full-automatic detection instrument, thereby limiting the popularization and application of the method. At present, the domestic interleukin-6 immunochemiluminescence kit with independent intellectual property rights is less, so that the interleukin-6 domestic kit with high analysis sensitivity and wide linear range has important practical significance in clinical application.
Disclosure of Invention
The purpose of the invention is: aiming at the defects, the interleukin-6 detection kit and the preparation method thereof are provided, wherein the interleukin-6 detection kit has the advantages of good stability, high sensitivity, wide linear range, high specificity, good precision and short detection time.
In order to achieve the purpose, the invention adopts the technical scheme that:
the interleukin-6 detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components in percentage by weight:
the reagent R2 consists of a second buffer solution, a preservation solution and a latex microsphere antibody conjugate, wherein the preservation solution consists of N, N-dihydroxyethyl glycine, a second stabilizing agent and a second preservative, the latex microsphere antibody conjugate consists of a latex microsphere, an interleukin-6 antibody and 1- (3-dimethylamino propyl) -3-ethyl carbodiimide hydrochloride, and the concentration of each component is as follows:
0.01-0.3g/L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.
The first buffer solution in the reagent R1 comprises one or more of PBS buffer solution, HEPES buffer solution, MES buffer solution and Tris buffer solution.
The first stabilizer of the reagent R1 and the second stabilizer of the reagent R2 are one or more of casein, mannitol and bovine serum albumin.
The electrolyte in the reagent R1 is one or a mixture of more than two of sodium chloride, potassium chloride, magnesium chloride and magnesium sulfate.
The surfactant in the reagent R1 is one or more of Tween 20, Triton, polyvinylpyrrolidone and octyl phenyl polyoxyethylene ether.
The first preservative in the reagent R1 and the second preservative in the reagent R2 are respectively one of sodium azide, Proclin-950, Proclin-300 and thimerosal.
The second buffer solution in the reagent R2 is one or more of PBS buffer solution, HEPES buffer solution, glycine buffer system, MES buffer solution and Tris buffer solution.
The latex microsphere in the reagent R2 has a selected particle size of 80-250nm, and the surface modification group is one of carboxyl, hydroxyl, aldehyde group and amino group.
A preparation method of an interleukin-6 detection kit comprises the following steps: A) reagent R1 preparation:
adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding a first buffer solution, a first stabilizer, an electrolyte, a surfactant, polyethylene glycol 6000 and a first preservative while stirring, stirring for 20-30 minutes until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 8.30-10.50, finally fixing the volume to the final volume, storing in a finished product tank, and marking;
B) reagent R2 preparation:
preparation of B1 buffer: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding a second buffer solution while stirring, stirring for 20-30 minutes until the materials are completely dissolved, adjusting the pH value to 6.00-7.50, continuously stirring for 5-10 minutes until the solution is clear and transparent, keeping the bottom of the liquid preparation tank free of precipitate, fixing the volume to the final volume, storing the solution for later use, and marking;
preparation of B2 preservative solution: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding N, N-dihydroxyethylglycine, a second stabilizer and a second preservative while stirring, stirring for 20-30 minutes until the materials are completely dissolved, adjusting the pH value to 6.00-7.50, continuously stirring for 5-10 minutes until all the materials are completely dissolved, keeping the solution clear and transparent, keeping the bottom of the liquid preparation tank free of precipitate, fixing the volume to the final volume, storing the solution for later use, and marking;
b3 preparation of latex microsphere antibody conjugate: diluting the latex microspheres to the concentration of 0.1-2.0% by using a buffer solution, diluting the interleukin-6 antibody to the concentration of 0.5-3.0g/L by using the buffer solution, uniformly mixing the two, dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the concentration of 0.01-0.3g/L by using the buffer solution, dripping the mixture into the mixed solution while shaking the mixture, reacting the mixture at room temperature for 120 minutes, adding a second stabilizer for sealing, shaking the mixture for uniformly mixing, reacting the mixture at room temperature for 60 minutes, centrifuging the mixed solution at the rotating speed of 14800rpm for 30 minutes, sucking a supernatant, adding a preservation solution, ultrasonically resuspending the mixture, repeatedly centrifuging and cleaning the mixture for 3 times, centrifuging the supernatant for the last time, adding a resuspension solution, ultrasonically suspending the suspension, filling the prepared R2 into a finished product tank, and marking the finished product tank.
Compared with the prior art, the invention achieves the technical effects that: the invention realizes the detection of the interleukin-6 content on a biochemical analyzer for the first time, and the detection range can reach 1.5-3000 pg/mL; the method uses a small amount of samples, has the characteristics of simplicity, accuracy and rapidness compared with other detection methods, and can realize high-throughput detection by batch analysis on a full-automatic biochemical analyzer; the kit has the characteristics of good stability, high sensitivity, wide linear range, high specificity, good precision, short detection time and the like, is low in price, and can be widely applied to large, medium and small hospitals.
Drawings
FIG. 1 is a calibration curve diagram of indirect coupling of latex microspheres with interleukin-6 antibody in example 4 of the present invention. Wherein the X-axis represents standard concentration and the Y-axis represents absorbance.
FIG. 2 is a graph showing the measurement of the linear range in example 4 of the present invention.
Detailed Description
The invention is further described with reference to the following figures and examples:
the first embodiment is as follows:
the interleukin-6 detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components in percentage by weight: 6.0g/L of tris (hydroxymethyl) aminomethane serving as a first buffer solution, 0.5g/L of bovine serum albumin serving as a first stabilizer, 5.8g/L of sodium chloride serving as an electrolyte, 1ml/L of Tween 20 serving as a surfactant, 40g/L of polyethylene glycol 6000 serving as a first preservative, and 0.8ml/L of Proclin-950 serving as a first preservative;
the reagent R2 is composed of 2- (N-morpholine) ethanesulfonic acid monohydrate serving as a second buffer solution, a preservation solution and a latex microsphere antibody conjugate, wherein the preservation solution is composed of N, N-dihydroxyethylglycine, bovine serum albumin serving as a second stabilizer and Proclin-950 serving as a second preservative, the latex microsphere antibody conjugate is composed of latex microspheres, interleukin-6 antibody and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the concentration of each component is as follows: 2- (N-morpholine) ethanesulfonic acid monohydrate with the concentration of 10.00g/L, N, N-dihydroxyethylglycine with the concentration of 1.26g/L, bovine serum albumin with the concentration of 0.5g/L, Proclin-950 with the concentration of 0.8ml/L, latex microspheres with the concentration of 0.1 percent, interleukin-6 antibody with the concentration of 0.5g/L, and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride with the concentration of 0.01 g/L.
The latex microsphere in the reagent R2 has a selected particle size of 80nm, and the surface modification group is one of carboxyl, hydroxyl, aldehyde group and amino group.
A preparation method of an interleukin-6 detection kit comprises the following steps: A) reagent R1 preparation:
adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding tris (hydroxymethyl) aminomethane, bovine serum albumin, sodium chloride, tween 20, polyethylene glycol 6000 and Proclin-950 while stirring, stirring for 20 minutes until the materials are completely dissolved, the solution is clear and transparent, no sediment is left at the bottom of the liquid preparation tank, adjusting the pH value to 8.30, finally fixing the volume to the final volume, storing in a finished product tank, and identifying;
B) reagent R2 preparation:
preparation of B1 buffer: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding 2- (N-morpholine) ethanesulfonic acid monohydrate while stirring, stirring for 20 minutes until the material is completely dissolved, adjusting the pH value to 6.00, continuously stirring for 5 minutes until the solution is clear and transparent, keeping no precipitate at the bottom of the liquid preparation tank, fixing the volume to the final volume, and storing the solution for later use for marking;
preparation of B2 preservative solution: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding N, N-dihydroxyethylglycine, bovine serum albumin and Proclin-950 while stirring, stirring for 20 minutes until the materials are completely dissolved, adjusting the pH value to 6.00, continuously stirring for 5 minutes until all the materials are completely dissolved, keeping the solution clear and transparent, keeping the bottom of the liquid preparation tank free of precipitate, fixing the volume to the final volume, storing the solution for later use, and marking;
b3 preparation of latex microsphere antibody conjugate: diluting latex microspheres to the concentration of 0.1 by using a buffer solution, diluting an interleukin-6 antibody to the concentration of 0.5g/L by using the buffer solution, uniformly mixing the latex microspheres and the buffer solution, dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the concentration of 0.01g/L by using the buffer solution, shaking uniformly and dripping into the mixed solution while stirring, reacting at room temperature for 120 minutes, adding bovine serum albumin for sealing, shaking uniformly, reacting at room temperature for 60 minutes, centrifuging the mixed solution at the rotating speed of 14800rpm for 30 minutes, sucking off a supernatant, adding a preservative solution, carrying out ultrasonic resuspension, repeating the centrifugal cleaning for 3 times, centrifuging the supernatant for the last time, adding the preservative solution, carrying out ultrasonic resuspension, filling the prepared R2 into a finished product tank, and marking.
Example two:
the interleukin-6 detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components in percentage by weight: tris (hydroxymethyl) aminomethane as a first buffer solution with a content of 9g/L, mannitol as a first stabilizer with a content of 1.2g/L, potassium chloride as an electrolyte with a content of 12g/L, triton as a surfactant with a content of 2ml/L, polyethylene glycol 6000 with a content of 40-120g/L, Proclin-300 as a first preservative with a content of 1.4 ml/L;
the reagent R2 consists of 2- (N-morpholine) ethanesulfonic acid monohydrate serving as a second buffer solution, a preservation solution and a latex microsphere antibody conjugate, wherein the preservation solution consists of N, N-dihydroxyethylglycine, mannitol serving as a second stabilizing agent and Proclin-300 serving as a second preservative, the latex microsphere antibody conjugate consists of latex microspheres, interleukin-6 antibody and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the concentration of each component is as follows: 2- (N-morpholine) ethanesulfonic acid monohydrate with a concentration of 18g/L, N, N-dihydroxyethylglycine with a concentration of 5g/L, mannitol with a concentration of 1g/L, Proclin-300 with a concentration of 1.4ml/L, latex microspheres with a concentration of 1%, interleukin-6 antibody with a concentration of 2g/L, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride with a concentration of 0.2 g/L.
The latex microsphere in the reagent R2 has a selected particle size of 160nm, and the surface modification group is one of carboxyl, hydroxyl, aldehyde and amino.
A preparation method of an interleukin-6 detection kit comprises the following steps: A) reagent R1 preparation:
adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding tris (hydroxymethyl) aminomethane, mannitol, potassium chloride, triton, polyethylene glycol 6000 and Proclin-300 while stirring, stirring for 25 minutes until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 9.3, finally fixing the volume to the final volume, storing in a finished product tank, and marking;
B) reagent R2 preparation:
preparation of B1 buffer: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding 2- (N-morpholine) ethanesulfonic acid monohydrate while stirring, stirring for 25 minutes until the material is completely dissolved, adjusting the pH value to 6.5, continuously stirring for 7 minutes until the solution is clear and transparent, keeping no precipitate at the bottom of the liquid preparation tank, fixing the volume to the final volume, and storing the solution for later use for marking;
preparation of B2 preservative solution: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding N, N-dihydroxyethyl glycine, mannitol and Proclin-300 while stirring, stirring for 25 minutes until the materials are completely dissolved, adjusting the pH value to 6.5, continuously stirring for 7 minutes until all the materials are completely dissolved, enabling the solution to be clear and transparent, enabling the bottom of the liquid preparation tank to have no precipitate, fixing the volume to a final volume, storing the solution for later use, and marking;
b3 preparation of latex microsphere antibody conjugate: diluting latex microspheres to the concentration of 1% by using a buffer solution, diluting an interleukin-6 antibody to the concentration of 2g/L by using the buffer solution, mixing the two solutions uniformly, dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the concentration of 0.2g/L by using the buffer solution, shaking uniformly and dripping into the mixed solution while shaking uniformly, reacting for 120 minutes at room temperature, adding Proclin-300, sealing, shaking uniformly, reacting for 60 minutes at room temperature, centrifuging the mixed solution at the rotating speed of 14800rpm for 30 minutes, sucking off supernatant, adding a preservative solution, carrying out ultrasonic resuspension, repeating the centrifugal cleaning for 3 times, centrifuging the supernatant for the last time, adding the preservative solution, carrying out ultrasonic resuspension, and filling the prepared R2 into a finished product tank for marking.
Example three:
the interleukin-6 detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components in percentage by weight: 12.2g/L of tris (hydroxymethyl) aminomethane as a first buffer, 2g/L of casein as a first stabilizer, 20.0g/L of magnesium chloride as an electrolyte, 3ml/L of polyvinylpyrrolidone as a surfactant, 120g/L of polyethylene glycol 6000 as a first preservative, and 2.0ml/L of sodium azide as a first preservative;
the reagent R2 is composed of 2- (N-morpholine) ethanesulfonic acid monohydrate serving as a second buffer solution, a preservation solution and a latex microsphere antibody conjugate, wherein the preservation solution is composed of N, N-dihydroxyethylglycine, casein serving as a second stabilizer and sodium azide serving as a second preservative, the latex microsphere antibody conjugate is composed of latex microspheres, interleukin-6 antibody and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the concentration of each component is as follows: 2- (N-morpholine) ethanesulfonic acid monohydrate with a concentration of 25.30g/L, N, N-dihydroxyethylglycine with a concentration of 9.83 g/L, casein with a concentration of 1.5g/L, sodium azide with a concentration of 2.0ml/L, latex microspheres with a concentration of 2.0%, interleukin-6 antibody with a concentration of 3.0g/L, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride with a concentration of 0.3 g/L.
The latex microsphere in the reagent R2 has a selected particle size of 250nm, and the surface modification group is one of carboxyl, hydroxyl, aldehyde and amino.
A preparation method of an interleukin-6 detection kit comprises the following steps: A) reagent R1 preparation:
adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding tris (hydroxymethyl) aminomethane, casein, magnesium chloride, polyvinylpyrrolidone, polyethylene glycol 6000 and sodium azide while stirring, stirring for 30 minutes until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 10.50, finally fixing the volume to the final volume, storing in a finished product tank, and identifying;
B) reagent R2 preparation:
preparation of B1 buffer: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding 2- (N-morpholine) ethanesulfonic acid monohydrate while stirring, stirring for 30 minutes until the material is completely dissolved, adjusting the pH value to 7.50, continuously stirring for 10 minutes until the solution is clear and transparent, keeping no precipitate at the bottom of the liquid preparation tank, fixing the volume to the final volume, and storing the solution for later use for marking;
preparation of B2 preservative solution: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding N, N-dihydroxyethyl glycine, casein and sodium azide while stirring, stirring for 30 minutes until the materials are completely dissolved, adjusting the pH value to 7.50, continuing stirring for 10 minutes until all the materials are completely dissolved, keeping the solution clear and transparent, keeping the bottom of the liquid preparation tank free of precipitate, fixing the volume to the final volume, storing the solution for later use, and marking;
b3 preparation of latex microsphere antibody conjugate: diluting latex microspheres to the concentration of 2.0% by using a buffer solution, diluting an interleukin-6 antibody to the concentration of 3.0g/L by using the buffer solution, uniformly mixing the two, dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the concentration of 0.3g/L by using the buffer solution, shaking uniformly and dripping into the mixed solution while shaking, reacting at room temperature for 120 minutes, adding casein for sealing, shaking uniformly, reacting at room temperature for 60 minutes, centrifuging the mixed solution at the rotating speed of 14800rpm for 30 minutes, sucking off supernatant, adding a preservation solution, ultrasonically resuspending, repeatedly centrifuging and cleaning for 3 times, centrifuging the supernatant for the last time, adding a preservation solution, ultrasonically resuspending, filling prepared R2 into a finished product tank, and marking.
Example four:
the latex microspheres were indirectly coupled to interleukin-6 antibody and calibrated using standards, the results are shown in table 1 and fig. 1.
Table 1: the result of the calibration of the latex microsphere indirect coupling interleukin-6 antibody is as follows:
selecting a sample to be detected to dilute the sample by using physiological saline, and generating a sample close to the low-limit concentration level of the linear range of the method, wherein the concentration levels are generally 4, and the concentration levels are respectively as follows: 1.0pg/mL, 1.2pg/mL, 1.5pg/mL and 2.0pg/mL, each concentration level sample is repeatedly measured for 10 times, CV is less than or equal to 8% as an acceptable threshold, and the lowest concentration level with the CV value equal to or less than the acceptable threshold is selected from the data as the lower limit of the detection range, and the results are shown in Table 2.
Selecting a sample to be detected to dilute the sample by using physiological saline, and generating a sample close to the high-limit concentration level of the linear range of the method, wherein the concentration levels are generally 4, and the concentration levels are respectively as follows: the samples at each concentration level were tested 10 times repeatedly at 2500pg/mL, 3000pg/mL, 3500pg/mL and 4000pg/mL, with CV ≦ 8% as an acceptable threshold, and the highest concentration level with a CV value equal to or less than the acceptable threshold was selected from the data as the upper limit of the detection range, with the results shown in Table 3.
Table 2:
CV > 8% when the sample concentration is less than 1.2 pg/mL; CV < 8% when the sample concentration is above 1.5 pg/mL; therefore, the linear range is limited to 1.5 pg/mL.
Table 3:
CV > 8% when the sample concentration is higher than 3500 pg/mL; CV < 8% when the sample concentration is below 3000 pg/mL; therefore, the linear range is limited to 3000 pg/mL.
In conclusion, the linear range of the invention can be made to be 1.5-3000 pg/mL.
Example five:
and (3) precision CV detection:
the latex microspheres are indirectly coupled with the interleukin-6 antibody, the detection is repeated for 10 times on five samples, and the detection results are shown in table 4:
table 4:
| 1 | 1.7 | 84.4 | 393.4 | 1497.2 | 2879.9 |
| 2 | 1.8 | 83.3 | 370.5 | 1403.0 | 2689.6 |
| 3 | 1.7 | 76.4 | 368.0 | 1405.0 | 2909.0 |
| 4 | 1.6 | 80.0 | 380.3 | 1442.2 | 2719.9 |
| 5 | 1.9 | 76.8 | 386.8 | 1469.1 | 2854.7 |
| 6 | 1.9 | 79.6 | 388.2 | 1470.8 | 2979.3 |
| 7 | 1.7 | 79.6 | 359.6 | 1416.3 | 2726.9 |
| 8 | 1.9 | 75.1 | 400.0 | 1421.1 | 2703.7 |
| 9 | 1.9 | 79.4 | 391.3 | 1438.2 | 2779.2 |
| 10 | 1.7 | 77.4 | 396.6 | 1465.0 | 2971.6 |
| mean value | 1.8 | 79.2 | 383.5 | 1442.8 | 2821.4 |
| SD | 0.11 | 2.95 | 13.44 | 31.86 | 111.52 |
| CV | 6.38％ | 3.73％ | 3.51％ | 2.21％ | 3.95％ |
Precision CV was small, within 8%.
The existing interleukin-6 reagent is tested for relevance to the present invention:
the contrast reagent and the kit of the invention simultaneously detect 20 cases of clinical serum according to respective parameters, carry out correlation analysis on the determination result, the result is shown in the figure, R2 is more than 0.95, which is in line with clinical practice
In summary, the following steps: compared with the prior art, the invention achieves the technical effects that: the method realizes the detection of the content of the interleukin-6 on a biochemical analyzer for the first time, and the detection range can reach 1.5-3000 pg/mL; the method uses a small amount of samples, has the characteristics of simplicity, accuracy and rapidness compared with other detection methods, and can realize high-throughput detection by batch analysis on a full-automatic biochemical analyzer; the kit has the characteristics of good stability, high sensitivity, wide linear range, high specificity, good precision, short detection time and the like, is low in price, and can be widely applied to large, medium and small hospitals.
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (9)
1. An interleukin-6 detection kit, which is characterized in that: the reagent comprises a reagent R1 and a reagent R2, wherein each component and concentration of the reagent R1 comprises:
the reagent R2 is composed of a second buffer solution, a preservation solution and a latex microsphere antibody conjugate, wherein the preservation solution is composed of N, N-dihydroxyethyl glycine, a second stabilizing agent and a second preservative, the latex microsphere antibody conjugate is composed of latex microspheres, interleukin-6 antibody and 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride, and the concentration of each component is as follows:
2. the interleukin-6 detection kit according to claim 1, wherein the kit comprises: the first buffer solution in the reagent R1 comprises one or more of PBS buffer solution, HEPES buffer solution, MES buffer solution and Tris buffer solution.
3. The interleukin-6 detection kit according to claim 1, wherein the kit comprises: the first stabilizer of the reagent R1 and the second stabilizer of the reagent R2 are one or more of casein, mannitol and bovine serum albumin.
4. The interleukin-6 detection kit according to claim 1, wherein the kit comprises: the electrolyte in the reagent R1 is one or a mixture of more than two of sodium chloride, potassium chloride, magnesium chloride and magnesium sulfate.
5. The interleukin-6 detection kit according to claim 1, wherein the kit comprises: the surfactant in the reagent R1 is one or a mixture of more than one of Tween 20, Triton, polyvinylpyrrolidone and octyl phenyl polyoxyethylene ether.
6. The interleukin-6 detection kit according to claim 1, wherein the kit comprises: the first preservative in the reagent R1 and the second preservative in the reagent R2 are respectively one of sodium azide, Proclin-950, Proclin-300 and thimerosal.
7. The interleukin-6 detection kit according to claim 1, wherein the kit comprises: the second buffer solution in the reagent R2 is one or more of PBS buffer solution, HEPES buffer solution, glycine buffer system, MES buffer solution and Tris buffer solution.
8. The interleukin-6 detection kit according to claim 1, wherein the kit comprises: the latex microsphere in the reagent R2 has a selected particle size of 80-250nm, and the surface modification group is one of carboxyl, hydroxyl, aldehyde group and amino group.
9. The method for preparing the interleukin-6 detection kit according to any one of claims 1 to 8, wherein the method comprises the following steps: the method comprises the following steps: A) reagent R1 preparation:
adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding a first buffer solution, a first stabilizer, an electrolyte, a surfactant, polyethylene glycol 6000 and a first preservative while stirring, stirring for 20-30 minutes until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 8.30-10.50, finally fixing the volume to the final volume, storing in a finished product tank, and marking;
B) reagent R2 preparation:
preparation of B1 buffer: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding a second buffer solution while stirring, stirring for 20-30 minutes until the materials are completely dissolved, adjusting the pH value to 6.00-7.50, continuously stirring for 5-10 minutes until the solution is clear and transparent, keeping the bottom of the liquid preparation tank free of precipitate, fixing the volume to the final volume, storing the solution for later use, and marking;
preparation of B2 preservative solution: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding N, N-dihydroxyethylglycine, a second stabilizer and a second preservative while stirring, stirring for 20-30 minutes until the materials are completely dissolved, adjusting the pH value to 6.00-7.50, continuously stirring for 5-10 minutes until all the materials are completely dissolved, keeping the solution clear and transparent, keeping the bottom of the liquid preparation tank free of precipitate, fixing the volume to the final volume, storing the solution for later use, and marking;
b3 preparation of latex microsphere antibody conjugate: diluting latex microspheres to the concentration of 0.1-2.0% by using a buffer solution, diluting an interleukin-6 antibody to the concentration of 0.5-3.0g/L by using the buffer solution, uniformly mixing the two, dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the concentration of 0.01-0.3g/L by using the buffer solution, shaking and dripping the mixture into the mixed solution while reacting for 120 minutes at room temperature, adding a second stabilizer for sealing, shaking and uniformly mixing, reacting for 60 minutes at room temperature, centrifuging the mixed solution at the rotating speed of 14800rpm for 30 minutes, sucking a supernatant, adding a preservation solution, ultrasonically resuspending, repeatedly centrifuging and cleaning for 3 times, centrifuging the supernatant after the last time, adding the preservation solution, ultrasonically suspending, filling the prepared R2 into a finished product tank, and marking.
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