CN116298311A - Magnetic particle chemiluminescence kit for quantitatively detecting interleukin 18 and application thereof - Google Patents
Magnetic particle chemiluminescence kit for quantitatively detecting interleukin 18 and application thereof Download PDFInfo
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- G01—MEASURING; TESTING
- 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
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- 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
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Abstract
The invention discloses a magnetic particle chemiluminescence kit for quantitatively detecting interleukin 18 and application thereof, and belongs to the technical field of immunodetection. The magnetic particle chemiluminescence kit for quantitatively detecting interleukin 18 comprises streptavidin magnetic particle working solution, a chemiluminescent substance-marked IL-18 monoclonal antibody, a biotin-marked IL-18 monoclonal antibody, an interleukin 18 standard substance and a standard substance diluent. The magnetic particle chemiluminescence kit for quantitatively detecting interleukin 18 is applied to quantitatively detecting interleukin 18. The invention also discloses a quantitative detection method of interleukin 18. The magnetic particle chemiluminescence kit for quantitatively detecting IL-18 has stable performance, good specificity and high sensitivity; the method has low requirements on sample pretreatment, realizes full-automatic detection, can rapidly detect a large number of samples with high flux, is single-tube detection, and can detect samples randomly, and the detection method is convenient and easy to implement, effectively shortens the detection time and improves the detection efficiency.
Description
Technical Field
The invention relates to a magnetic particle chemiluminescence kit for quantitatively detecting interleukin 18 and application thereof, and belongs to the technical field of immunodetection.
Background
Interleukin 18 (Interleukin 18, IL-18, interleukin 18) is a substance of molecular weight 18-19 KD purified in 1995 from mouse liver extract co-treated with heat-inactivated Propionibacterium acnes and lipopolysaccharides, which has structural similarity to IL-1 family proteins, belongs to the IL-1 family, and is an important regulator of innate and acquired immune responses, also known as interferon-gamma inducer (inter feron gamma inducing factor, IGIF).
Interleukin 18, a recently discovered multifunctional cytokine, has been involved in the pathogenesis of a variety of immune-related diseases. IL-18 levels in normal humans are generally in the range of 100-200 pg/mL, and when a patient is ill, the levels in the body are significantly increased, thus playing a very important role in pathological research and clinical diagnosis of many diseases. For example, the IL-18 level is closely related to the occurrence and development of type 2 diabetic nephropathy and can be used as an evaluation index of the disease development degree and prognosis of type 2 diabetic patients; in post-operative treatment of breast cancer, IL-18 contributes to the prognosis of patients undergoing operative treatment for breast cancer in anti-cancer immunity induced by cytotoxic T cells; in the treatment of ischemic stroke, the level of IL-18 is helpful to judge the development and severity of ischemic stroke, and can be used as an important reference for early detection of ischemic stroke; in the pathogenesis of systemic lupus erythematosus, the content of IL-18 in the body is increased, and is also a disease sign of the disease; IL-18 can be used as a detection index for the development of heart failure, and when heart failure is aggravated, the concentration in a patient can be increased, and after treatment is improved, IL-18 can be reduced.
At present, no in vitro diagnosis and detection reagent for the IL-18 in human body exists clinically. The existing IL-18 detection kit is only in a scientific research stage and mainly adopts an ELISA method, so that development of a detection kit capable of rapidly, accurately and highly sensitively detecting the content of IL-18 in a human body is urgently needed for prediction, diagnosis and prognosis monitoring of diseases related to IL-18.
Therefore, how to provide a kit and a detection mode for rapidly and quantitatively detecting IL-18 for clinical staff is a problem to be solved by the technicians in the field, and the kit can be used for predicting, diagnosing and prognostic monitoring of diseases related to IL-18.
Disclosure of Invention
Aiming at the prior art, the invention provides a magnetic particle chemiluminescence kit for quantitatively detecting interleukin 18 and application thereof. The kit has the advantages of strong specificity, high sensitivity and good stability, and can rapidly, accurately and fully automatically detect the content of IL-18 in human bodies in a large batch.
The invention is realized by the following technical scheme:
a magnetic particle chemiluminescence kit for quantitatively detecting interleukin 18 comprises streptavidin magnetic particle working solution, a chemiluminescent substance-labeled IL-18 monoclonal antibody, a biotin-labeled IL-18 monoclonal antibody, an interleukin 18 standard substance and a standard substance diluent.
The working solution of the streptavidin magnetic particles is an R1 reagent prepared by diluting the streptavidin magnetic particles with a phosphate buffer solution containing a protein stabilizer, wherein the concentration of the streptavidin magnetic particles is 0.04-0.1 mg/mL, and the particle size of the streptavidin magnetic particles is 2-3 mu m; the components of the phosphate buffer solution containing the protein stabilizer are as follows: 20mmol/L PB (20 mmol/L for disodium hydrogen phosphate and sodium dihydrogen phosphate), 150mmol/L NaCl,1% BSA (bovine serum albumin), 0.5% casein sodium salt, and water for the rest, with pH 7.2.
The chemiluminescent substance-labeled IL-18 monoclonal antibody is prepared by the following method: adding IL-18 monoclonal antibody into chemiluminescent substance solution, mixing uniformly, and marking at 37 ℃ for 0.5-1 h; after the marking reaction is finished, adding a sealing buffer solution, and sealing for 0.5-1 h at 37 ℃; performing ultrafiltration and centrifugation after sealing to obtain an intermediate 1; diluting the intermediate 1 with a labeling buffer solution to prepare an R2 reagent, wherein the concentration of the IL-18 monoclonal antibody is 0.2-0.4 mug/mL; the chemiluminescent substance is selected from acridinium esters or acridinium ester derivatives; the marking buffer solution comprises the following components: 20mmol/L PB,150mmol/L NaCl,1% BSA,1% trehalose, 0.5% casein sodium salt, 0.1% preservative (such as proclin 300), balance water, pH 6.8; the sealing buffer solution comprises the following components: 20mmol/L PB,100mmol/L NaCl,5% glycine, the balance water.
The biotin-labeled IL-18 monoclonal antibody is prepared by the following method: adding IL-18 monoclonal antibody into biotin solution, mixing uniformly, and marking at 37 ℃ for 0.5-2 h; after the marking reaction is finished, adding a sealing buffer solution, and sealing for 0.5-2 h at 37 ℃; performing ultrafiltration and centrifugation after sealing to obtain an intermediate 2; diluting the intermediate 2 with a labeling buffer solution to prepare an R3 reagent, wherein the concentration of the IL-18 monoclonal antibody is 0.2-0.4 mu g/mL; the labeling buffer and the blocking buffer are the same.
The interleukin 18 standard substance is interleukin 18 solution with the concentration of 1ng/mL.
The components of the standard substance diluent are as follows: 20mmol/L PB,100mmol/L NaCl,1% BSA,1% trehalose, 0.1% proclin300, the balance water.
The magnetic particle chemiluminescence kit for quantitatively detecting interleukin 18 is applied to quantitatively detecting interleukin 18.
A quantitative detection method of interleukin 18 comprises the following steps: adding a serum or plasma sample to be detected and a biotin-labeled IL-18 monoclonal antibody into a streptavidin magnetic particle working solution, mixing the three components in a volume ratio of 1:1:1, vibrating and uniformly mixing, and reacting for 15min at 37 ℃; after the reaction is finished, carrying out magnetic separation, and washing 3 times by using a washing liquid; adding a chemiluminescent substance marked IL-18 monoclonal antibody, shaking and mixing uniformly, and reacting for 10min at 37 ℃; after the reaction is finished, carrying out magnetic separation, and washing 3 times by using a washing liquid; adding a luminescent substrate solution, shaking and mixing uniformly, reacting for 1-5 min at room temperature, and detecting chemiluminescence intensity (RLU); and drawing a standard curve, and calculating the concentration of interleukin 18 in the serum or plasma sample to be detected.
The washing liquid comprises the following components: 0.2g KH 2 PO 4 ，2.9g Na 2 HPO 4 ·12H 2 O,8g NaCl,0.2g KCl,0.5mL Tween-20, purified water was added to 1000mL.
The luminescent substrate liquid comprises: the pre-excitation liquid comprises the following components: 50mmol/L fluoboric acid, 10mmol/L carbamide peroxide, 2mmol/L disodium ethylenediamine tetraacetate, proclin300 with the volume ratio of 0.1% and the balance of water. The excitation liquid comprises the following components: 100mmol/L sodium hydroxide, NP-10 at 2% by volume, DMF at 0.1% by volume, proclin300 at 0.1% by volume, the remainder being water.
The standard curve is generated by the following method: the interleukin 18 standard is subjected to multiple ratio dilution by using the standard diluent to obtain the diluent with the following gradient concentration: 1ng/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/mL, 15.625pg/mL, 0pg/mL, wherein 0pg/mL is a standard diluent; and detecting the diluent according to a detection mode of a sample to be detected, reading the chemiluminescence intensity, and drawing a standard curve.
The detection principle of the magnetic particle chemiluminescence kit for quantitatively detecting IL-18 provided by the invention is as follows: the invention adopts a chemiluminescence immunoassay technology based on a double-antibody sandwich method, adds a biotin-marked IL-18 monoclonal antibody and a human serum or plasma sample to be detected into a streptavidin magnetic particle working solution, wherein human IL-18 in the sample to be detected can be fully combined with the biotin-marked IL-18 monoclonal antibody, and the biotin-marked IL-18 monoclonal antibody can be fully combined with the streptavidin magnetic particle to form an antigen-antibody-magnetic particle compound; magnetic separation and cleaning of magnetic particles, and adding chemiluminescent substance-marked IL-18 monoclonal antibody to form chemiluminescent substance-antibody-antigen-antibody-magnetic particle compound; and (3) magnetically separating and cleaning magnetic particles, adding chemiluminescent substrate liquid, and detecting the chemiluminescent intensity. The concentration of IL-18 in the sample is proportional to the RLU, and the concentration of IL-18 in the sample can be calculated by drawing a standard curve and four-parameter fitting software.
The magnetic particle chemiluminescence kit for quantitatively detecting IL-18 has the advantages of stable reagent performance, good specificity, high sensitivity, high detection speed and good repeatability. The kit of the invention uses a biotin-avidin system, which avoids the aggregation of magnetic particles caused by directly coating the magnetic particles with antibodies, solves the problem of stability, and connects the biotin-labeled antibodies to the magnetic particles through the specific interaction between streptavidin and biotin. The standard substance of the invention is dry powder containing IL-18 recombinant antigen; the standard diluent is phosphate buffer solution containing bovine serum albumin, sucrose, casein sodium salt and preservative. The invention has the following beneficial effects:
1. the invention combines the chemiluminescence technology and the immunomagnetic particles to detect interleukin 18, provides a reaction environment close to a liquid phase, has more complete and rapid reaction, has higher sensitivity, specificity and precision, can effectively shorten the detection time and improves the detection efficiency; meanwhile, the full-automatic luminous equipment is used for detection, errors possibly generated by manual operation are avoided, and the repeatability is better.
2. The invention uses biotin-streptavidin system, which greatly improves the reaction specificity of the detection sensitivity box.
3. The invention provides possibility for rapidly and accurately detecting interleukin 18, has low requirements on sample pretreatment, can rapidly and high-flux detect a large number of samples, is single-tube detection, can realize random detection of the samples, is convenient and feasible, and fills the blank of the prior art.
The various terms and phrases used herein have the ordinary meaning known to those skilled in the art.
Drawings
Fig. 1: standard curve for IL-18 standard.
Fig. 2: linear analysis of the kit.
Detailed Description
The invention is further illustrated below with reference to examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof.
The instruments, reagents and materials used in the examples below are conventional instruments, reagents and materials known in the art and are commercially available. The experimental methods and detection methods in the following examples are conventional experimental methods and detection methods in the prior art unless otherwise specified.
In the embodiment of the invention, the streptavidin magnetic particles are commercial products. The washing liquid and the luminescent substrate liquid are commercial products (the commercial products are named as cleaning liquid and substrate liquid for a full-automatic immune inspection system) which are packaged and sold separately by a matched magnetic particle full-automatic chemiluminescence instrument.
Example 1 magnetic particle chemiluminescent kit for quantitative detection of IL-18
The magnetic particle chemiluminescence kit for quantitatively detecting IL-18 comprises streptavidin magnetic particle working solution, a chemiluminescent substance-labeled IL-18 monoclonal antibody, a biotin-labeled IL-18 monoclonal antibody, a standard substance and a standard substance diluent.
Preparation of streptavidin magnetic particle working solution: the R1 reagent is prepared by diluting streptavidin magnetic particles with a phosphate buffer solution containing a protein stabilizer, wherein the concentration of the streptavidin magnetic particles is 0.05mg/mL, and the particle size of the streptavidin magnetic particles is 2-3 mu m; the components of the phosphate buffer solution containing the protein stabilizer are as follows: 20mmol/L PB (disodium hydrogen phosphate, sodium dihydrogen phosphate are 20 mmol/L), 150mmol/L NaCl,1% BSA,0.5% casein sodium salt, the balance water, pH 7.2.
Preparation of chemiluminescent substance-labeled IL-18 monoclonal antibody: adding IL-18 monoclonal antibody into chemiluminescent substance solution, mixing well, and labeling at 37 ℃ for 1h; after the marking reaction is finished, adding a sealing buffer solution, and sealing for 1h at 37 ℃; performing ultrafiltration and centrifugation after sealing to obtain an intermediate 1; diluting the intermediate 1 with a labeling buffer solution to prepare an R2 reagent, wherein the concentration of the IL-18 monoclonal antibody is 0.2 mug/mL; the chemiluminescent substance is acridinium ester; the marking buffer solution comprises the following components: 20mmol/L PB,150mmol/L NaCl,1% BSA,1% trehalose, 0.5% casein sodium salt, 0.1% proclin300, balance water, pH 6.8; the sealing buffer solution comprises the following components: 20mmol/L PB,100mmol/L NaCl,5% glycine, the balance water.
Preparation of biotin-labeled IL-18 monoclonal antibody: adding IL-18 monoclonal antibody into biotin solution, mixing well, and labeling at 37 ℃ for 2h; after the marking reaction is finished, adding a sealing buffer solution, and sealing for 2 hours at 37 ℃; performing ultrafiltration and centrifugation after sealing to obtain an intermediate 2; diluting the intermediate 2 with a labeling buffer solution to prepare an R3 reagent, wherein the concentration of the IL-18 monoclonal antibody is 0.2 mug/mL; the labeling buffer and the blocking buffer are the same.
The interleukin 18 standard substance is interleukin 18 solution with the concentration of 1ng/mL.
The components of the standard substance diluent are as follows: 20mmol/L PB,100mmol/L NaCl,1% BSA,1% trehalose, 0.1% proclin300, the balance water.
Example 2 quantitative detection of Interleukin 18 Using the kit
The method comprises the following steps:
(1) Sample addition: adding 50 mu L of human serum or plasma sample to be detected and 50 mu L of biotin-labeled IL-18 monoclonal antibody into 50 mu L of streptavidin magnetic particle working solution, shaking and mixing uniformly, and reacting for 15min at 37 ℃;
(2) Washing: after the reaction, magnetic separation is carried out, and 150 mu L of washing liquid is used for washing 3 times;
(3) Adding 50 mu L of chemiluminescent substance-labeled IL-18 monoclonal antibody, shaking and mixing uniformly, and reacting for 10min at 37 ℃;
(4) Washing: after the reaction, magnetic separation is carried out, and 150 mu L of washing liquid is used for washing 3 times;
(5) Reading: adding 50 mu L of each of the luminescent substrate solutions A and B, shaking and mixing uniformly, reacting for 3min at room temperature, and immediately detecting the chemiluminescence intensity.
(6) And drawing a standard curve, and calculating to obtain the concentration of interleukin 18 in the sample to be detected.
Example 3 Generation of a Standard Curve
The interleukin 18 standard is subjected to multiple ratio dilution by using the standard diluent, and the diluent of the standard with the following gradient concentration is obtained: 1ng/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/mL, 15.625pg/mL, 0pg/mL, wherein 0pg/mL is a standard diluent; the diluent is detected according to the detection mode of the sample to be detected, the chemiluminescent intensity is read, and the instrument background is automatically fitted with a standard curve for measuring the concentration of the sample. The results are shown in Table 1.
TABLE 1 Standard test results
Example 4
According to the in vitro diagnostic reagent analysis performance evaluation technical examination guiding principle, the quantitative detection human IL-18 magnetic particle chemiluminescence kit in the embodiments 1, 2 and 3 of the invention has the following performance index analysis, operation method and result:
(1) Blank limit
Using the standard diluent as a blank sample, detecting 1 time a day, continuously measuring for 20 days to obtain a chemiluminescence intensity value (RLU), calculating the mean value X and standard deviation SD of 20 results, and the blank rlu=x+2sd. The blank concentration was calculated to be 5.75pg/mL based on the working curve. The specific results are shown in Table 2.
TABLE 2 blank results
(2) Detection limit
The IL-18 recombinant antigen standard was diluted with standard diluent to obtain 5 low concentration samples: 20pg/mL, 10pg/mL, 5pg/mL, 2.5pg/mL, 0pg/mL, wherein 0pg/mL is the standard diluent. The measurement was performed 2 times a day and 10 times a day, and 20 chemiluminescent intensity values (RLU) were obtained for each sample, and the mean' X and standard deviation SD of the 20 results were calculated. The luminescence average value of 0pg/mL was 7279, the standard deviation was sd=1310, and rlu=x+3sd=7279+1310×3=11209, calculated with 99% confidence. The lowest concentration point with an RLU value higher than 11209 is the lowest detection limit of the reagent, namely 6.25pg/mL. The specific results are shown in Table 3.
TABLE 3 limit of detection results
(3) Linearity of
The standard was diluted in a gradient using a standard diluent to give 10 concentrations: 640pg/mL, 320pg/mL, 160pg/mL, 80pg/mL, 40pg/mL,20pg/mL, 10pg/mL, 5pg/mL, 2.5pg/mL, 0pg/mL, wherein 0pg/mL is a standard diluent, each sample is repeated 3 times, the measured RLU value is subjected to linear analysis, the linear correlation coefficient is calculated, r=0.9962, the results are shown in Table 4, and the linear analysis is shown in FIG. 2.
Table 4 results of the Linear analysis of the kit
(4) Accuracy (recovery test)
Recovery = (average of concentration of sample to be recovered-average of concentration of sample to be recovered)/added concentration x 100%, average recovery = (recovery 1+recovery 2+ … … +recovery n)/n x 100%, wherein added concentration = standard solution concentration x [ standard solution added volume)/(sample volume+standard solution volume). The difference between the recovery per sample and the average recovery should be no more than + -10%.
The volume ratio of the base sample to the added sample in the invention is 10:1. Each sample was tested 3 times in parallel and the average was calculated. The results are shown in Table 5.
Table 5 results of the recovery experiments with the kit
Average recovery = (103.2+98.9)/2×100% = 101.1%
The difference between each sample recovery and the average recovery was calculated:
analysis sample 1:103.2% -101.1% = 2.1%;
analysis sample 2:98.9-101.1% = -2.2%;
the difference between the recovery and the average recovery of both samples was less than + -10%.
Calculating the proportional system error= |100% -101% |=1%, and the recovery experiment (accuracy) of the invention is acceptable.
(5) Precision of
Preparation of precision samples: the standard is diluted by the standard diluent to obtain three samples with different levels of low, medium and high of 100pg/mL, 400pg/mL and 800pg/mL, and the samples are detected as required.
a: the precision in batch is less than or equal to 10 percent.
The operation method is as follows:
taking any batch of the kit, repeatedly detecting 10 holes of each sample, calculating the average value (X) and Standard Deviation (SD) of 10 detection concentrations of each sample, and calculating the variation coefficient CV% = SD/X multiplied by 100% (n=10). The results are shown in Table 6.
B: the precision between batches is less than or equal to 10 percent.
The operation method is as follows:
three batches of the kit were taken, 10 wells per batch of each sample were repeated, the mean value (X) and Standard Deviation (SD) of the measured concentration values were calculated 30 times per sample, and the coefficient of variation was calculated: CV% = SD/X100% (n=10). The results are shown in Table 6.
Table 6 results of the precision test of the kit
According to the result analysis, the precision of the kit in and among batches meets the requirement, and the kit has good repeatability.
(6) Specificity (specificity)
And (3) taking serum of the mixed healthy person, and respectively adding endogenous substance pure products such as hemoglobin, bilirubin and triglyceride to prepare an interference sample, wherein the final concentration of the hemoglobin (Hb) in the interference sample is 300mg/dL, the final concentration of the bilirubin in the interference sample is 20mg/dL, and the final concentration of the triglyceride in the interference sample is 3000mg/dL. The serum with and without the addition of the interfering substances was measured for deviations of less than.+ -. 10%, respectively, and the results are shown in Table 7.
TABLE 7
According to the analysis of the results, the interference substances of hemoglobin (Hb) less than or equal to 300mg/dL, bilirubin less than or equal to 20mg/dL and triglyceride less than or equal to 3000mg/dL have no influence on the detection result.
The foregoing examples are provided to fully disclose and describe how to make and use the claimed embodiments by those skilled in the art, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to a person skilled in the art will be within the scope of the appended claims.
Claims (10)
1. A magnetic particle chemiluminescence kit for quantitatively detecting interleukin 18 is characterized in that: comprises streptavidin magnetic particle working solution, chemiluminescent substance marked IL-18 monoclonal antibody, biotin marked IL-18 monoclonal antibody, interleukin 18 standard and standard diluent;
the concentration of the streptavidin magnetic particles in the streptavidin magnetic particle working solution is 0.04-0.1 mg/mL, and the particle size of the streptavidin magnetic particles is 2-3 mu m;
the concentration of the IL-18 monoclonal antibody in the IL-18 monoclonal antibody marked by the chemiluminescent substance is 0.2-0.4 mug/mL; the chemiluminescent substance is selected from acridinium esters or acridinium ester derivatives;
the concentration of the IL-18 monoclonal antibody in the biotin-marked IL-18 monoclonal antibody is 0.2-0.4 mug/mL.
2. The magnetic particle chemiluminescent kit of claim 1 wherein the magnetic particle chemiluminescent kit for the quantitative detection of interleukin 18 is characterized by: the streptavidin magnetic particle working solution is an R1 reagent prepared by diluting a streptavidin magnetic particle solution with a phosphate buffer solution containing a protein stabilizer; the components of the phosphate buffer solution containing the protein stabilizer are as follows: 20mmol/L PB,150mmol/L NaCl,1% BSA,0.5% casein sodium salt, balance water, pH 7.2.
3. The magnetic particle chemiluminescent kit of claim 1 wherein the chemiluminescent substance-labeled IL-18 monoclonal antibody is prepared by the following method: adding IL-18 monoclonal antibody into chemiluminescent substance solution, mixing uniformly, and marking at 37 ℃ for 0.5-1 h; after the marking reaction is finished, adding a sealing buffer solution, and sealing for 0.5-1 h at 37 ℃; performing ultrafiltration and centrifugation after sealing to obtain an intermediate 1; intermediate 1 was diluted with a labeling buffer to prepare an R2 reagent.
4. The magnetic particle chemiluminescent kit of claim 1 wherein the biotin-labeled IL-18 monoclonal antibody is prepared by the following method: adding IL-18 monoclonal antibody into biotin solution, mixing uniformly, and marking at 37 ℃ for 0.5-2 h; after the marking reaction is finished, adding a sealing buffer solution, and sealing for 0.5-2 h at 37 ℃; performing ultrafiltration and centrifugation after sealing to obtain an intermediate 2; diluting the intermediate product 2 by using a marking buffer solution to prepare an R3 reagent; the labeling buffer and the blocking buffer are the same.
5. The magnetic particle chemiluminescent kit for the quantitative detection of interleukin 18 according to claim 3 or 4, wherein the kit comprises: the marking buffer solution comprises the following components: 20mmol/L PB,150mmol/L NaCl,1% BSA,1% trehalose, 0.5% casein sodium salt, 0.1% proclin300, balance water, pH 6.8; the sealing buffer solution comprises the following components: 20mmol/LPB,100mmol/L NaCl,5% glycine, the balance water.
6. The magnetic particle chemiluminescent kit of claim 1 wherein the magnetic particle chemiluminescent kit for the quantitative detection of interleukin 18 is characterized by: the components of the standard substance diluent are as follows: 20mmol/L PB,100mmol/L NaCl,1% BSA,1% trehalose, 0.1% proclin300, the balance water.
7. The magnetic particle chemiluminescent kit for the quantitative detection of interleukin 18 according to claim 5 or 6, wherein the kit comprises: the preservative is selected from Proclin 300.
8. Use of the magnetic particle chemiluminescent kit for quantitatively detecting interleukin 18 according to any one of claims 1-7 for quantitatively detecting interleukin 18.
9. A method for detecting interleukin 18 using the magnetic particle chemiluminescence kit for quantitatively detecting interleukin 18 according to any one of claims 1-7, wherein: adding a serum or plasma sample to be detected and a biotin-labeled IL-18 monoclonal antibody into a streptavidin magnetic particle working solution, shaking and mixing uniformly, and reacting for 15min at 37 ℃; after the reaction is finished, magnetically separating, and washing by using a washing liquid; adding a chemiluminescent substance marked IL-18 monoclonal antibody, shaking and mixing uniformly, and reacting for 10min at 37 ℃; after the reaction is finished, magnetically separating, and washing by using a washing liquid; adding a luminescent substrate solution, shaking and mixing uniformly, reacting for 1-5 min at room temperature, and detecting the chemiluminescent intensity; and drawing a standard curve, and calculating the concentration of interleukin 18 in the serum or plasma sample to be detected.
10. The method of claim 9, wherein the standard curve is generated by: the interleukin 18 standard is subjected to multiple ratio dilution by using the standard diluent to obtain the diluent with the following gradient concentration: 1ng/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/mL, 15.625pg/mL, 0pg/mL, wherein 0pg/mL is a standard diluent; and detecting the diluent according to a detection mode of a sample to be detected, reading the chemiluminescence intensity, and drawing a standard curve.
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| WO2005012352A1 (en) * | 2003-07-30 | 2005-02-10 | Tomoaki Hoshino | SOLUBLE HUMAN INTERLEUKIN 18 RECEPTOR-α, METHOD OF ASSAYING THE SAME, ASSAY KIT AND MEDICINAL COMPOSITION |
| WO2022121151A1 (en) * | 2020-12-10 | 2022-06-16 | 丹娜(天津)生物科技股份有限公司 | Magnetic particle chemiluminescence-based novel coronavirus antibody detection kit |
| CN114910641A (en) * | 2022-05-09 | 2022-08-16 | 山东中鸿特检生物科技有限公司 | Detection kit for interleukin, detection method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2005012352A1 (en) * | 2003-07-30 | 2005-02-10 | Tomoaki Hoshino | SOLUBLE HUMAN INTERLEUKIN 18 RECEPTOR-α, METHOD OF ASSAYING THE SAME, ASSAY KIT AND MEDICINAL COMPOSITION |
| WO2022121151A1 (en) * | 2020-12-10 | 2022-06-16 | 丹娜(天津)生物科技股份有限公司 | Magnetic particle chemiluminescence-based novel coronavirus antibody detection kit |
| CN114910641A (en) * | 2022-05-09 | 2022-08-16 | 山东中鸿特检生物科技有限公司 | Detection kit for interleukin, detection method and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116908433A (en) * | 2023-06-26 | 2023-10-20 | 广州市妇女儿童医疗中心 | A NEXN chemiluminescence detection kit and its application |
| CN116908433B (en) * | 2023-06-26 | 2024-05-07 | 广州市妇女儿童医疗中心 | A NEXN chemiluminescence detection kit and its application |
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