CN112904023B - Procalcitonin chemiluminescence immunoassay kit - Google Patents
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Abstract
The invention provides a procalcitonin chemiluminescence immunoassay kit, which comprises: magnetically separating the reagent, the first reagent, and the second reagent; the magnetic separation reagent is a magnetic particle reagent containing procalcitonin monoclonal antibodies; the first reagent is a procalcitonin monoclonal antibody solution containing a biotin label; the second reagent is alkaline phosphatase solution containing streptavidin marker. The PCT kit provided by the invention has lower PCT detection sensitivity and wider PCT detection range.
Description
Technical Field
The invention belongs to the technical field of in-vitro detection, and relates to a procalcitonin chemiluminescence immunoassay kit.
Background
Bacteria or viruses invade from wound surfaces, respiratory tracts, urinary tracts and digestive tracts, can cause serious infection symptoms of organisms in severe wounds, general resistance reduction, in-vivo normal flora imbalance, bacterial displacement and the like, and are the main reasons for clinical various complications such as sepsis, septic shock, Multiple Organ Dysfunction Syndrome (MODS) and even death. Statistics in the united states of america 2001 show that 75.1 million cases of severe sepsis alone in this year have an average mortality rate of 28.5%, which means that the number of deaths in this year is almost three times that of acquired immunodeficiency syndrome (AIDS). Can diagnose infectious diseases and complications thereof early and treat the infectious diseases and the complications thereof in time, and can effectively reduce the death rate. However, the diagnostic indicators currently used in general (such as leukocyte classification and counting, C-reactive protein, blood routine examination, etc.) are non-specific, and therefore, misdiagnosis or missed diagnosis is likely to occur.
Procalcitonin (PCT) discovered in 1990 has proved that the increase in serum concentration is closely related to the occurrence of infection, and can provide a new laboratory standard for diagnosis, identification, selection of treatment regimen, prognosis, and the like of diseases. In normal and healthy individuals, the serum concentration is very low, only 10-50pg/ml, and is not detected by the general method. The PCT level of serum of patients with systemic bacterial, fungal and parasitic infection, systemic inflammatory response syndrome, septicemia, acute and chronic pneumonia, acute pancreatitis, active hepatitis, wound, etc. is abnormally increased, and the concentration of the PCT level can reach several times or even ten thousand times of the normal level; the serum concentration of patients with virus infection, autoimmune diseases, organ transplantation exclusion reaction and the like is not increased or slightly increased. The high specificity of PCT is determined, so that the PCT can be used for differential diagnosis of diseases, has high clinical value in the aspects of auxiliary differential diagnosis, prognosis judgment, curative effect observation and the like of systemic bacterial infection and sepsis, and can be widely used in ICU wards, hematology departments, surgical departments, internal medicine departments, organ transplantation departments, treatment laboratories and the like.
Procalcitonin (PCT) is derived from a single copy gene located on chromosome 11 (11p15,4) consisting of 280 base pairs, containing 6 exons and 5 introns. After transcription, the protein is translated into procalcitonin precursor (Preprocalcitonin) in the rough endoplasmic reticulum of the thyroid parafollicular cells, and comprises 84 amino acids at the N end, active calcitonin and a calcitonin protein part. Under the action of endogenous polypeptide enzyme, the procalcitonin precursor cuts off a single sequence at the end of the nPro-CT to generate PCT with 116 amino acids, the molecular weight is about 13kD, and the PCT and calcitonin have the same sequence (60-91 sites) with 32 amino acids. PCT is expressed by neuroendocrine cells (C cells including thyroid, lung and pancreatic tissue) and cleaved enzymatically into (immature) calcitonin, carboxy-terminal peptides and amino-terminal peptides.
The methods currently used for detecting PCT are mainly Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), colloidal Gold Immunochromatography (GICA) and chemiluminescence immunoassay (CLIA). RIA has high detection sensitivity, but because the marker has the defects of radioactive hazard, poor stability of the marker, difficult treatment of waste and the like, the RIA gradually exits from the field of clinical examination; the ELISA method adopts horseradish peroxidase (HRP) or alkaline phosphatase to mark the antibody, catalyzes a substrate to generate color change, and has the characteristics of simple operation and long reagent stability, but the detection sensitivity of the ELISA method is low, and the ELISA method is mainly applied to items with low requirements on detection sensitivity, such as infectious disease screening and the like at present; the GICA has the advantages of simple operation, high detection speed and the like, but also has the defects of low sensitivity, unstable reagent, poor repeatability and difficult quantification.
The chemiluminescence immunoassay method is an immunoassay technology developed on the basis of an enzyme-linked immunoassay method, and has the advantages of high sensitivity, wide detection linear range, simple and convenient operation, high automation degree and the like. The chemiluminescence immunoassay technology is widely applied due to the advantages. The magnetic separation chemiluminescence immunoassay method comprehensively adopts two mainstream immunoassay technologies of suspension magnetic particle carrier technology and chemiluminescence detection technology in the world at present, so that the system can fully meet the clinical requirements on detection results.
The biotin-avidin system is applied to the field of chemiluminescence reagents, and the detection sensitivity level of the reagents is obviously improved. The biotin (also called vitamin H) has a relative molecular mass of 244.31, is in a ring structure, and is divided into a ring I and a ring II, wherein the ring I is the main part for binding avidin, and the ring II is the only structure for labeling antibodies and enzymes, and can effectively bind different antigens or proteins such as antibodies. Streptavidin (Streptavidin, abbreviation SA) is a slightly acidic protein, the relative molecular mass of which is 65000, and which is composed of 4 identical peptide chains, each peptide chain can be combined with 1 biotin molecule, namely 1 SA molecule can be combined with 4 biotin molecules, so that biotin is adopted to be combined with various markers to play an effective biological amplification role, and a plurality of 'antenna' polyvalent reagents formed by biotinylation enable the whole reaction system to have multistage amplification reaction, and the Streptavidin has extremely high sensitivity and high reaction speed, and shows excellent performance.
At present, for the detection of PCT, a chemiluminescence product using a biotin avidin system has appeared on the market, which employs SA-labeled magnetic particles, two monoclonal antibodies against PCT, one of which is labeled with biotin and is bound to SA, and the other is labeled with a luminescent substance or an enzyme for luminescence reaction. For example, chinese patent CN103901203B discloses a kit for detecting procalcitonin, which comprises the following reagents: procalcitonin series of calibrators; magnetic separation reagent: a streptavidin-coupled suspension of magnetic particles; a first reagent: a procalcitonin monoclonal antibody solution containing an N-hydroxysuccinimide biotin ester label; a second reagent: contains alkaline phosphatase-labeled procalcitonin monoclonal antibody solution. Although the sensitivity is improved (0.008ng/mL), the kit specificity is poor, and the detection range is general (0-100 ng/mL). Another Chinese patent CN102359958B discloses a PCT chemiluminescence detection kit, which takes a magnetic particle coupled antibody as a fixed separation phase, a biotin-streptavidin multistage amplification system and horseradish peroxidase as a catalytic enzyme to catalyze a luminescent substrate to emit light, but the detection sensitivity is only 0.05 ng/mL.
Disclosure of Invention
Aiming at the defects of PCT detection in the prior art, the invention provides a procalcitonin chemiluminescence immunoassay kit, which improves the detection sensitivity of PCT and widens the detection range of PCT.
One aspect of the present invention provides a procalcitonin chemiluminescent immunoassay kit, comprising: magnetically separating the reagent, the first reagent, and the second reagent;
the magnetic separation reagent is a magnetic particle reagent containing procalcitonin monoclonal antibody (PCT monoclonal antibody);
the first reagent is a procalcitonin monoclonal antibody solution containing a biotin label;
the second reagent is alkaline phosphatase solution containing Streptavidin (SA) label.
The invention adopts the magnetic particles coated with the PCT monoclonal antibody, the biotinylated PCT monoclonal antibody and the 'post-amplification' mode of SA-labeled alkaline phosphatase, when a sample is detected, the magnetic particles firstly form a 'sandwich' compound with the coated antibody, an object to be detected and the biotinylated labeled antibody, meanwhile, biotin on the labeled antibody is specifically combined with SA on the alkaline phosphatase, and finally, the alkaline phosphatase catalyzes a substrate to carry out a luminous reaction. By adopting the biotin-avidin system, the detection sensitivity of PCT can be effectively improved, and the detection range of PCT is widened.
Preferably, the concentration of the magnetic particles coated with the procalcitonin monoclonal antibody in the magnetic separation reagent is 0.05-0.5mg/ml, the concentration of the biotin-labeled procalcitonin monoclonal antibody in the first reagent is 0.05-3.0. mu.g/ml, and the concentration of the streptavidin-labeled alkaline phosphatase solution in the second reagent is 0.5-2. mu.g/ml.
Preferably, in the magnetic separation reagent, the mass ratio of the magnetic particles to the procalcitonin monoclonal antibody is (50-100): 1.
the magnetic particle is colloidal composite material formed by combining magnetic nano particles with organic molecules or inorganic molecules, and the colloidal composite material can be uniformly dispersed in a certain base solution and has high stability, and the surface of the magnetic particle contains one or more active functional groups such as tosyl, amino, carboxyl, hydroxyl, ethylene oxide and the like.
Preferably, the magnetic particles used in the present invention are Fe2O3Or Fe3O4The magnetic particle surface contains tosyl or carboxyl, and the particle diameter of the magnetic particle is 0.1-3 μm.
Preferably, in the first reagent, the molar ratio of the biotin to the procalcitonin monoclonal antibody is (10-20):1, the ratio of the biotin to the antibody to be labeled is proper, and the excessive dosage of the biotin can cause the blocking of the antigen binding site of the antibody to be labeled and the inactivation of the antibody.
Preferably, the biotin is N-hydroxysuccinimide ester activated biotin, i.e., N-hydroxysuccinimide biotin ester.
Preferably, in the second reagent, 1 is (1-2):1 between streptavidin and alkaline phosphatase.
Preferably, the alkaline phosphatase is one or more of shrimp alkaline phosphatase, heat-sensitive alkaline phosphatase, calf intestinal alkaline phosphatase, purity > 95 wt%, and activity > 1000 glycine U/mg protein. Definition of activity units: 1 mmol of p-nitrophenol phosphate hydrolyzed 1 minute at 25 ℃ at pH 9.6 (glycine buffer).
Preferably, the preparation method of the magnetic separation reagent comprises the following steps:
adding procalcitonin monoclonal antibody after the magnetic particles are resuspended, then adding a reaction catalyst, carrying out suspension reaction for 2-12h at 15-40 ℃, carrying out magnetic separation, then sealing with a sealing agent, and then carrying out resuspension to obtain a magnetic separation reagent;
the preparation method of the first reagent comprises the following steps:
mixing biotin and procalcitonin monoclonal antibodies, standing at 15-40 ℃ for 40-60min, adding a terminator to terminate the reaction, and finally adding a buffer solution to dilute to obtain a first reagent;
the preparation method of the second reagent comprises the following steps:
adding streptavidin into the activated alkaline phosphatase, stirring and mixing for 4-6h at 15-20 ℃, then adding glycine solution or bovine serum albumin solution for sealing, continuing mixing and reacting for 0.5-3h, and then adding buffer solution for dilution to obtain a second reagent.
The preparation method of the magnetic separation reagent specifically comprises the following steps:
resuspending the magnetic particles with buffer solution (such as 0.03-0.10mol/L boric acid buffer solution) with pH of 7.5-8.0, wherein the concentration of the magnetic particles after resuspension is 5-10 mg/ml; adding the procalcitonin monoclonal antibody to ensure that the mass ratio of the magnetic particles to the procalcitonin monoclonal antibody is (50-100): 1, uniformly mixing, adding a reaction catalyst which can be an ammonium sulfate solution or a sodium borate solution, and then suspending and reacting at 15-40 ℃ for 2-12 h; performing magnetic separation after the reaction, removing supernatant, adding blocking agent into the precipitate for blocking reaction, and suspending at 15-40 deg.C for 0.5-3 hr, wherein the blocking agent is exemplified by Blockmaster produced by JSR Life Science corporationTMCE510, CE210, DB1130 and PA1080, adding 200-300. mu.L of blocking agent per 10mg of magnetic beads; after the magnetic particles are cleaned, 0.05-0.08 wt% of bovine serum albumin and 0.005-0.015mol/L, pH 7.4.4-7.8 of trihydroxymethyl aminomethane buffer solution are added, so that the concentration of the magnetic particles coated with the procalcitonin monoclonal antibody is 0.05-0.5 mg/ml.
The preparation method of the first reagent specifically comprises the following steps:
passing procalcitonin monoclonal antibody through gel column (such as G-25 gel column) with PBS buffer solution of 0.01-0.03mol/L, pH of 7.0-7.5 to obtain procalcitonin monoclonal antibody solution; dissolving N-hydroxysuccinimide biotin ester in dimethyl sulfoxide to prepare N-hydroxysuccinimide biotin ester solution, wherein the mass concentration of N-hydroxysuccinimide biotin ester is 10-15 mmol/L; adding the N-hydroxysuccinimide biotin ester solution into the procalcitonin monoclonal antibody solution to ensure that the molar ratio of the N-hydroxysuccinimide biotin ester to the procalcitonin monoclonal antibody is (10-20):1, uniformly mixing, and standing at 15-40 ℃ for 40-60 min; then adding a terminator to terminate the reaction, wherein the terminator is 1.0-1.5mol/L, pH 7.0.0-7.5 trihydroxymethyl aminomethane buffer solution; then diluting the solution by using a trihydroxymethyl aminomethane buffer solution containing 2.0-3.5% of bovine serum albumin and 0.01-0.03mol/L, pH of 7.2-7.6 to ensure that the concentration of the procalcitonin monoclonal antibody marked by the N-hydroxysuccinimide biotin ester is 0.05-3.0 mu g/ml, thus obtaining the first reagent.
The preparation method of the activated alkaline phosphatase comprises the following steps: adding alkaline phosphatase into carbodiimide coupling agent, standing at 15-25 deg.C for 30-40min, passing through gel column, and collecting the activated alkaline phosphatase.
The preparation method of the second reagent specifically comprises the following steps:
diluting alkaline phosphatase to 0.1-0.5mg/mL with 10mM MES solution, adding into carbodiimide coupling agent with concentration of 10-15mg/mL, standing at 15-25 deg.C for 30-40min, desalting with gel column (such as G-25 gel column), and collecting activated alkaline phosphatase; 0.5-0.8mg/mL of SA was added to the activated alkaline phosphatase solution so that the molar ratio of streptavidin to alkaline phosphatase was (1-2):1, stirring and mixing for 4-6h at 15-20 ℃; then adding 0.5-2mol/L glycine solution or 2-5% BSA solution for blocking, stirring and mixing for 0.5-3 h; then diluting with HEPES buffer solution containing 0.1-0.5% bovine serum albumin and 0.05-0.10mol/L, pH 6.5.5-7.0 until the concentration of streptavidin-labeled alkaline phosphatase solution is 0.5-2 μ g/ml, thus obtaining the second reagent.
The carbodiimide coupling agent used in the preparation of the second reagent may be exemplified by one or more of DCC (dicyclohexylcarbodiimide), DIC (N, N' -diisopropylcarbodiimide), and EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride).
Preferably, the kit further comprises a procalcitonin series calibrator which is obtained by diluting procalcitonin to a plurality of series concentrations by using a protective agent buffer solution.
Preferably, the protective agent buffer has a pH of 7.0-7.5 and comprises one or more of bovine serum albumin, trehalose, carboxymethyl cellulose and cyclodextrin.
Preferably, the protective agent buffer comprises 1.5-2.0 wt% of bovine serum albumin, or the protective agent buffer comprises 2.5-3.0 wt% of trehalose, or the protective agent buffer comprises 1 wt% of carboxymethyl cellulose, or the protective agent buffer comprises 2.5-3.0 wt% of cyclodextrin.
Preferably, the protective buffer comprises 0.5-1.0 wt% of cyclodextrin and 0.5 wt% of carboxymethylcellulose.
The detection method of the procalcitonin chemiluminescence immunoassay kit provided by the invention comprises the following steps:
s1, adding a sample to be detected into a test tube, then adding a magnetic separation reagent and a first reagent, uniformly mixing, and carrying out water bath at 36-38 ℃ for 8-12min, wherein the volume ratio of a sample stock solution to be detected, the magnetic separation reagent and the first reagent is 1: 0.8-1.2: 0.8-1.2;
s2, adding a second reagent, uniformly mixing, and carrying out water bath at 36-38 ℃ for 4-5min, wherein the volume ratio of the sample stock solution to be detected to the second reagent is 1: 0.8-1.2;
s3, placing the test tube into a magnetic separator, adding a magnetic field, settling in the magnetic field for 2-4min, removing supernatant, and repeatedly cleaning with cleaning solution for several times;
and S4, adding a luminescent substrate which is catalyzed and luminesced by alkaline phosphatase, suspending for 2-5S, and detecting by using a luminescence detector.
Compared with the prior art, the invention has the following beneficial effects:
(1) the kit is prepared by adopting the magnetic particle reagent coated with the procalcitonin monoclonal antibody, the procalcitonin monoclonal antibody solution containing the biotin label and the alkaline phosphatase solution containing the streptavidin label, so that the analysis sensitivity is as low as 5pg/mL, the accuracy is good, the precision is high, the operation is simple and time-saving, the detection range is wide, and the detection range is 0-200 ng/mL;
(2) the PCT calibrator is in a solution form, so that a freeze-drying step is omitted, and time and labor are saved;
(3) the PCT calibrator is stored by adopting one or more protective buffer solutions comprising bovine serum albumin, trehalose, carboxymethyl cellulose and cyclodextrin, so that the stability of the procalcitonin calibrator is greatly improved, and the quality guarantee period and the accuracy of a kit are improved;
(4) the PCT calibration product is stored by adopting a protective agent buffer solution containing 0.5-1.0 wt% of cyclodextrin and 0.5 wt% of carboxymethyl cellulose, so that the PCT calibration product can be stored for 15 months in a liquid state, the step of diluting a freeze-dried product is omitted, and the labor and the time are greatly saved.
Drawings
FIG. 1 is a regression curve of concentration versus RLU value for 0ng/mL calibrator and 0.2ng/mL calibrator in the kits of example 1 of the present invention;
figure 2 is a regression curve of measured concentration versus theoretical concentration for PCT calibrators at different concentrations.
Detailed Description
The technical solution of the present invention is further described below with reference to the following specific examples and the accompanying drawings, but the present invention is not limited thereto. The raw materials used in the examples of the present invention are those commonly used in the art, and the methods used in the examples are those conventional in the art, unless otherwise specified.
Magnetic particles: ThermoFisher Scientific, DynabeadsTM M-280 Tosylactivated；
Procalcitonin monoclonal antibody: shenzhen fenpeng bio-shares, ltd, PCT pairings 1;
a sealing agent: blockmaster manufactured by SR Life Science corporationTM CE210；
G-25 gel column: GE Healthcare, Superdex pre-column (gel filtration pre-column) (Sephadex G-25M);
n-hydroxysuccinimide biotin ester: purchased from Sigma, cat #: h1759
Alkaline phosphatase (alkaline phosphatase): purchased from BBI Solutions, cat #: ALPI 8G;
streptavidin: Sigma-Aldrich, Cat number: s4762;
EDC: thermo fisher Scientific, cat #: 22981;
the pure procalcitonin product comprises the following components: guangzhou, Greyrelin Biotechnology Inc., Cat.No. 7010.
Example 1
The kit of this embodiment includes a magnetic separation reagent, a first reagent, a second reagent, and a procalcitonin series calibrator.
The preparation method of the magnetic separation reagent comprises the following steps:
resuspending the magnetic particles with 0.05mol/L, pH 8.0.0 boric acid buffer solution, wherein the concentration of the resuspended magnetic particles is 6 mg/ml; adding the procalcitonin monoclonal antibody to ensure that the mass ratio of the magnetic particles to the procalcitonin monoclonal antibody is 80: 1, uniformly mixing, adding 1mol/L ammonium sulfate solution, adding 1/2 with the volume being the sum of the volumes of the magnetic particles and the PCT monoclonal antibody, and suspending and reacting for 8 hours at 25 ℃; after the reaction is finished, carrying out magnetic separation, removing supernatant, adding a blocking agent CE210 into the precipitate, carrying out suspension reaction at 25 ℃ for 1h, and adding 250 mu L of the blocking agent CE210 into each 10mg of magnetic beads; then, the magnetic particles were washed 3 times with Tris buffer (pH 7.4) containing 1% Tween20, and finally, Tris buffer containing 0.06 wt% bovine serum albumin and 0.007mol/L, pH 7.4.4 was added so that the concentration of the magnetic particles coated with procalcitonin monoclonal antibody was 0.1 mg/ml.
The preparation method of the first reagent comprises the following steps:
the procalcitonin monoclonal antibody is treated with PBS buffer solution with the concentration of 0.02mol/L, pH being 7.2 by a G-25 gel column to obtain procalcitonin monoclonal antibody solution; dissolving N-hydroxysuccinimide biotin ester in dimethyl sulfoxide to prepare N-hydroxysuccinimide biotin ester solution, wherein the mass concentration of N-hydroxysuccinimide biotin ester is 12 mmol/L; adding the N-hydroxysuccinimide biotin ester solution into the procalcitonin monoclonal antibody solution, so that the molar ratio of the N-hydroxysuccinimide biotin ester to the procalcitonin monoclonal antibody is 15: 1, uniformly mixing, standing at 25 ℃ for 50min, adding 1.5mol/L, pH 7.4.4 of tris buffer solution, and stopping reaction; then, the solution was diluted with 2.2 wt% bovine serum albumin and 0.03mol/L, pH 7.4.4 tris buffer solution so that the concentration of the N-hydroxysuccinimide biotin-labeled procalcitonin monoclonal antibody was 2.8. mu.g/ml.
The preparation method of the second reagent comprises the following steps:
adding 0.3mg/ml alkaline phosphatase into 10mg/ml EDC coupling agent, standing at 20 deg.C for 30 min, desalting with G-25 gel column, and collecting activated alkaline phosphatase; adding 0.6mg/mL of SA into the activated alkaline phosphatase solution to ensure that the molar ratio of the streptavidin to the alkaline phosphatase is 1:1, and stirring and mixing for 5 hours at 20 ℃; adding 1.0mol/L glycine solution, stirring and mixing for 1 h; then, 0.3 wt% bovine serum albumin, 0.06mol/L, pH 6.8.8 in HEPES buffer was added to dilute the solution to a concentration of 1.5. mu.g/ml of streptavidin-labeled alkaline phosphatase.
The preparation method of the procalcitonin series calibration product comprises the following steps:
diluting the pure procalcitonin into a calibrator concentration by using a protective agent buffer solution: 0ng/ml, 0.2ng/ml, 1ng/ml, 5ng/ml, 25ng/ml, 100ng/ml, 200 ng/ml. Protective agent buffer solution: tris buffer containing 1.5 wt% bovine serum albumin, 0.08mol/L, pH 7.4.
1. Example 1 kit Performance index determination
Manufacturers of luminescent substrates, washing solutions: beckmann coulter
1.1 detection method
Adding 50 μ L of sample to be tested and calibrator into corresponding test tube, adding 50 μ L of magnetic separation reagent and 50 μ L of first reagent, mixing, and water bathing at 37 + -0.5 deg.C for 10 min; then adding 50 mu L of second reagent, mixing uniformly, and bathing for 4min at 37 +/-0.5 ℃; placing the test tube in a magnetic separator, adding a magnetic field, settling in the magnetic field for 2min, removing supernatant, and repeatedly cleaning with cleaning solution for 3 times;
add 100. mu.L of a luminogenic substrate that emits light catalyzed by alkaline phosphatase, suspend for 3s, and then measure with a luminometer. And (4) according to the relative luminous intensity RLU of the sample to be detected, calculating the concentration of the PCT in the sample from the standard curve.
1.2 sensitivity
Taking a 0ng/mL calibrator in the kit as a sample to be measured, repeatedly measuring for 20 times to obtain an RLU value of 20 measurement results, and calculating an average value (M) and a Standard Deviation (SD) of the RLU value to obtain an RLU value corresponding to M +2SD, wherein the result is shown in Table 2; and measuring the RLU value of the 0.2ng/mL calibrator, wherein the result is shown in Table 3, two-point regression fitting is carried out according to the concentration-RLU value result between the 0ng/mL calibrator and the 0.2ng/mL calibrator, a fitting curve is shown in FIG. 1, a linear regression equation y is obtained as 68845x +8221, the RLU value of M +2SD is substituted into the equation, and the corresponding concentration value is obtained, wherein the concentration value is the minimum detection limit. The experiment was repeated 3 times, and the sensitivity of the kit to PCT analysis was finally determined to be 5 pg/mL.
TABLE 1
TABLE 2
| Mean(S0) | 8221 |
| SD | 156 |
| M+2SD | 8533 |
TABLE 3
1.3 precision
1.3.1 the PCT kit is used for testing samples with low, medium and high concentrations, each concentration is tested repeatedly for 10 times, the precision of the kit is calculated, the result is shown in a table 4, and the result shows that all coefficients of variation CV of the kit are less than 5%.
TABLE 4
| Sample concentration (ng/ml) | Intra-lot CV (%) |
| 0.3 | 4.6 |
| 5 | 4.2 |
| 20 | 3.1 |
Note: n-10
1.3.2 five batches of the kit are taken, each batch of the kit is used for measuring samples with three different concentrations, namely low, medium and high, each concentration is repeatedly tested for 10 times, each sample obtains 50 concentration measurement values, the inter-batch variation coefficient of statistical analysis is 3.7-4.8%, and the results are shown in Table 5.
TABLE 5
| Sample concentration (ng/ml) | Batch CV (%) |
| 0.52 | 4.8 |
| 8 | 4.6 |
| 18 | 3.7 |
Note: n-10
1.4 accuracy
In 2 human samples, different amounts of PCT calibrator were added to form samples at 3 concentration levels, the volume of the additive was less than 1/10 of the total volume, the sample concentration was measured, and the recovery rate was calculated according to the following formula, and the results are shown in Table 6, and it can be seen that the recovery rate of the method is between 90% and 110%.
R: recovering rate;
v: volume of calibrant added;
v0: a volume of a human sample;
c: detecting the concentration of the human source sample after the human source sample is added into the calibrator;
c0: detecting the concentration of the human sample;
cs: the concentration of the calibrator.
TABLE 6
1.5 Linear Range
Diluting 200ng/mL of the high-value calibrator to 50, 12.5, 3.12, 0.78, 0.19, 0.048 and 0.012ng/mL, determining the concentration of the PCT diluted calibrator, and making a regression curve of the measured concentration and a theoretical value, as shown in figure 2, to obtain the linear range of the kit, namely 0-200 ng/mL.
1.6 specificity
Selecting procalcitonin (Humankatalcalcin), calcitonin (Humancalcotonin), calcitonin gene-related peptide b (Humanalpha-CGRPb) and calcitonin gene-related peptide (Humanbeta-CGRP) which have similar structures with PCT to prepare a sample with a concentration higher than physiological concentration, measuring by using the method, and calculating the cross reaction rate. The results are shown in Table 6, and the cross-reactivity rates of the method with Humankatalcalcalcalin, Humancalceton, Humanalpha-CGRPb and Humanbeta-CGRP are all less than 0.03%.
TABLE 7
1.8 correlation
Using the PCT kit of example 1 of the invention and Soeling
BRAHMS
II GEN was performed simultaneously on 210 human serum samples. The serum PCT concentration measured by the invention is used as a horizontal coordinate, the result measured by the Soling kit is used as a vertical coordinate to perform regression analysis, and the related equation is as follows: y is 1.078x-0.5736, and the correlation coefficient is: 0.9949. the results of statistical treatment show that the invention is implementedThe PCT kit of example 1 correlates well with the clinical sample measurements of the solilodine kit.
2. PCT calibrator protectant buffer screening
2.1 screening of bovine serum albumin concentration in protectant buffer
0.5 percent of 1 percent of trihydroxymethyl aminomethane buffer solution with the concentration of 0.08mol/L and the pH value of 7.4 are respectively added,
The results of the test of the signal to noise ratios of different BSA concentration buffers with 1.5%, 2% and 3% bovine serum albumin are shown in Table 8, and the results show that the BSA concentration has a better signal to noise ratio between 1.5 and 2%.
TABLE 8
2.2 screening of trehalose, carboxymethyl cellulose and Cyclodextrin concentrations in protectant buffers
1%, 2%, 2.5% and 3% trehalose, carboxymethyl cellulose and cyclodextrin are respectively added into 0.08mol/L trihydroxymethyl aminomethane buffer solution with pH of 7.4, the results of testing the signal to noise ratio of the buffer solutions with different concentrations are shown in tables 9-11, and the results show that the trehalose concentration is 2.5-3.0%, the carboxymethyl cellulose concentration is 1.0% and the cyclodextrin concentration is 2.5-3.0% and have better signal to noise ratio.
TABLE 9
Watch 10
TABLE 11
2.3 screening of carboxymethyl cellulose and Cyclodextrin combinations in protectant buffers
The signal to noise ratios of the buffers containing different concentrations of carboxymethyl cellulose and cyclodextrin were tested by adding different concentrations of carboxymethyl cellulose and cyclodextrin to 0.08mol/L tris buffer at pH7.4, and the results are shown in Table 12, which indicates that the buffers containing 0.5% cyclodextrin and 0.5% carboxymethyl cellulose, and the buffers containing 1.0% cyclodextrin and 0.5% carboxymethyl cellulose have superior signal to noise ratios.
TABLE 12
2.4 PCT calibrator stability assay
PCT standards with different concentrations were subjected to a conventional storage stability test, and protective buffer solutions of the PCT standards respectively contained 2.5% trehalose, 1.0% carboxymethyl cellulose, 3.0% cyclodextrin, 0.5% carboxymethyl cellulose, 1.0% cyclodextrin, and 0.5% carboxymethyl cellulose, and were left at 4 ℃ and tested at 0, 1, 4, 7, 9, 12, and 15 months, respectively, with stability results shown in table 13.
Watch 13
The PCT calibrator containing 3.0% of cyclodextrin, 0.5% of cyclodextrin and 0.5% of carboxymethyl cellulose is stored in a dark environment at 4 ℃ for an effective period of 12 months, the signal value is reduced by less than 8%, and the PCT calibrator containing 1.0% of cyclodextrin and 0.5% of carboxymethyl cellulose has an effective period of 15 months.
The procalcitonin chemiluminescence immunoassay kit prepared by the invention has the advantages of lower detection limit, low analysis sensitivity of 5pg/mL, excellent specificity, high precision and accuracy, and linear range of 0-200 ng/mL. The procalcitonin series calibrators are stored by adopting a protective agent buffer solution containing cyclodextrin and carboxymethyl cellulose, so that the stability of the procalcitonin calibrators is greatly improved, and the quality guarantee period of the kit is prolonged.
The specific embodiments described herein are merely illustrative of the spirit of the invention and do not limit the scope of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
Claims (5)
1. A procalcitonin chemiluminescent immunoassay kit, comprising: magnetically separating the reagent, the first reagent, and the second reagent;
the magnetic separation reagent is a magnetic particle reagent containing procalcitonin monoclonal antibodies;
the first reagent is a procalcitonin monoclonal antibody solution containing a biotin label;
the second reagent is alkaline phosphatase solution containing streptavidin marker;
in the magnetic separation reagent, the mass ratio of the magnetic particles to the procalcitonin monoclonal antibody is (50-100): 1;
the preparation method of the magnetic separation reagent comprises the following steps:
adding procalcitonin monoclonal antibody after the magnetic particles are resuspended, then adding a reaction catalyst, carrying out suspension reaction for 2-12h at 15-40 ℃, carrying out magnetic separation, then sealing with a sealing agent, and then carrying out resuspension to obtain a magnetic separation reagent;
the magnetic particles are magnetic particles with the surfaces containing tosyl groups; the reaction catalyst is ammonium sulfate solution; the blocking agent is CE 210;
the preparation method of the first reagent comprises the following steps:
mixing N-hydroxysuccinimide biotin ester and procalcitonin monoclonal antibody, standing at 15-40 ℃ for 40-60min, adding a terminator to terminate the reaction, and finally adding a buffer solution to dilute to obtain a first reagent;
the preparation method of the second reagent comprises the following steps:
adding streptavidin into activated alkaline phosphatase, stirring and mixing for 4-6h at 15-20 ℃, then adding glycine solution or bovine serum albumin solution for sealing, continuing mixing and reacting for 0.5-3h, and then adding buffer solution for dilution to obtain a second reagent;
the kit also comprises a procalcitonin series calibrator which is obtained by diluting procalcitonin to a plurality of series concentrations by using a protective agent buffer solution;
the pH value of the protective agent buffer solution is 7.0-7.5, and the protective agent buffer solution comprises 0.5-1.0 wt% of cyclodextrin and 0.5 wt% of carboxymethyl cellulose.
2. The procalcitonin chemiluminescent immunoassay kit of claim 1, wherein the concentration of the magnetic particles coated with the procalcitonin monoclonal antibody in the magnetic separation reagent is 0.05 to 0.5mg/ml, the concentration of the biotin-labeled procalcitonin monoclonal antibody in the first reagent is 0.05 to 3.0 μ g/ml, and the concentration of the streptavidin-labeled alkaline phosphatase solution in the second reagent is 0.5 to 2 μ g/ml.
3. The procalcitonin chemiluminescent immunoassay kit of claim 1, wherein the molar ratio of biotin to procalcitonin monoclonal antibody in the first reagent is (10-20): 1.
4. The procalcitonin chemiluminescent immunoassay kit of claim 1, wherein the molar ratio of streptavidin to alkaline phosphatase in the second reagent is (1-2): 1.
5. the procalcitonin chemiluminescent immunoassay kit of claim 1, wherein the activated alkaline phosphatase is prepared by a method comprising: adding alkaline phosphatase into carbodiimide coupling agent, standing at 15-25 deg.C for 30-40min, passing through gel column, and collecting the activated alkaline phosphatase.
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