CN118130780A - Quantitative detection kit for procalcitonin - Google Patents
Quantitative detection kit for procalcitonin Download PDFInfo
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- CN118130780A CN118130780A CN202410259743.4A CN202410259743A CN118130780A CN 118130780 A CN118130780 A CN 118130780A CN 202410259743 A CN202410259743 A CN 202410259743A CN 118130780 A CN118130780 A CN 118130780A
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- procalcitonin
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- magnetic bead
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- 108010048233 Procalcitonin Proteins 0.000 title claims abstract description 111
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Classifications
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- G—PHYSICS
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5302—Apparatus specially adapted for immunological test procedures
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- G—PHYSICS
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention discloses a quantitative detection kit of procalcitonin, which comprises a first reagent and a second reagent, wherein the first reagent comprises a procalcitonin coated antibody marked by biotin and connected with streptavidin magnetic beads, and the second reagent comprises a procalcitonin marked antibody marked by acridinium ester. The invention discloses a kit for quantitatively detecting procalcitonin by a double-antibody sandwich one-step method aiming at magnetic bead chemiluminescence immunoassay, and overcomes an S-shaped reaction curve of procalcitonin antigen by screening proper mass ratio of magnetic beads to antibodies, thereby expanding the linear range. Meanwhile, the diluent of the specific components of the kit, including magnetic bead diluent, antibody diluent, antigen diluent and the like, can provide favorable conditions for the antibody to recognize the antigen, so that the performances of the kit are optimal.
Description
Technical Field
The invention relates to the technical field of in-vitro detection, in particular to a procalcitonin quantitative detection kit.
Background
Procalcitonin (PCT) is a precursor of Calcitonin (CT), a glycoprotein consisting of 116 amino acids, having a molecular weight of about 13KD, comprising an N-terminal of 59 amino acids, an active CT of 32 peptides and a calcitonin of 21 peptides.
Its level in plasma increases when there is severe bacterial, fungal, parasitic infection, sepsis and multiple organ failure. PCT does not rise upon autoimmune, allergic and viral infections. Locally limited bacterial infections, mild infections and chronic inflammation do not lead to an increase. Bacterial endotoxins play a critical role in the induction process. PCT reflects the activity level of the systemic inflammatory response. Factors affecting PCT levels include the size and type of the organ being infected, the type of bacteria, the extent of inflammation, and the condition of the immune response.
At present, PCT level can be used as an evaluation index of therapeutic effect of antibacterial drugs. PCT is continuously raised or does not decline after treatment, which generally suggests poor treatment effects, and antibacterial agents need to be adjusted. In contrast, PCT decline after treatment can be used as a reference indicator of the effectiveness and withdrawal of an antibacterial drug. For patients with non-severe lower respiratory tract infections, it is recommended to deactivate the antibacterial agent when PCT falls below 0.25ng/mL or peak concentration is below 80% and the condition is stable. For patients with severe Intensive Care Unit (ICU) severe infections undergoing antibacterial therapy, withdrawal of the antibacterial agent is recommended when PCT falls below 0.5ng/mL or peak concentration of 80%.
The prior method and kit for detecting the procalcitonin content, such as the application number 202010124910.6, discloses a kit for detecting the procalcitonin content in serum and a use method thereof, wherein the kit comprises the following components: the principle of the system is that the system emits light enzymatically, and the system has unstable defects; the application number 201510070009.4 discloses a method, a reagent and a kit for measuring procalcitonin in human serum, wherein the kit comprises: reagent 1 and reagent 2, the principle of the system is that latex is immune turbidimetry, false positive and other probability occur. In addition, most of the kits on the market at present have the problems that PCT antigen reaction linearity is always S-shaped in the reagent detection process, so that low-end sensitivity is poor, high-end linearity is poor and the linear range is narrow. At the same time, some research practices have shown that PCT is unstable in serum matrices and PCT is destroyed and denatured when a calibrator or quality control containing PCT is stored in a liquid state for a long period of time.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a quantitative detection kit for procalcitonin, which has the advantages of high detection linear range and sensitivity and higher application value.
The invention is realized in the following way:
A kit for quantitative detection of procalcitonin comprising: a first reagent and a second reagent;
The first reagent comprises a procalcitonin coated antibody marked by biotin and connected with streptavidin magnetic beads, and the procalcitonin coated antibody comprises two procalcitonin antibodies aiming at different epitopes of the same antigen to be detected;
the second reagent comprises an acridinium ester marked procalcitonin marked antibody, and the procalcitonin marked antibody comprises two procalcitonin antibodies aiming at different epitopes of the same antigen to be detected.
In some embodiments, the first reagent comprises a first reagent A and a first reagent B, wherein the mass ratio of the streptavidin magnetic beads to the biotin-labeled procalcitonin coated antibody in the first reagent is 1:0.01-0.06.
In some embodiments, the mass ratio of streptavidin magnetic beads to biotin-labeled procalcitonin coated antibody in the first reagent a is 1:0.01.
In some embodiments, the mass ratio of streptavidin magnetic beads to biotin-labeled procalcitonin coated antibody in first reagent B is 1:0.06.
In some embodiments, the mass ratio of procalcitonin coated antibody to biotin in the first reagent is 1:10-20.
In some embodiments, the first reagent a comprises a streptavidin magnetic bead-linked biotin-labeled procalcitonin monoclonal antibody, wherein the mass ratio of procalcitonin coated antibody to biotin is 1:10.
In some embodiments, the first reagent B comprises a streptavidin magnetic bead-linked biotin-labeled procalcitonin monoclonal antibody, wherein the mass ratio of procalcitonin coated antibody to biotin is 1:20.
In some embodiments, the volume ratio of first reagent a to first reagent B in the first reagent is 1:1.
In some embodiments, the first reagent further comprises a magnetic bead diluent, wherein the magnetic bead diluent has a ph=7.0 to 7.4, and the magnetic bead diluent comprises the following components: tris, BSA, tween 20, proclin-300.
In some embodiments, the method of preparing the first reagent comprises: the first reagent A and the first reagent B are mixed in proportion to obtain the first reagent.
In some embodiments, the method of preparing the first reagent a comprises: and (3) adding a procalcitonin coated antibody A after the magnetic bead is resuspended, performing magnetic separation after suspension reaction, then obtaining an antibody A-magnetic bead cross-linked matter, adding a blocking agent into the magnetic bead cross-linked matter for blocking, washing, adding a magnetic bead diluent, obtaining a streptavidin magnetic bead solution of the procalcitonin coated antibody A after the magnetic bead is resuspended, and then adding the magnetic bead diluent to dilute the streptavidin magnetic bead solution of the procalcitonin coated antibody A to a target concentration.
In some embodiments, the method of preparing the first reagent B comprises: and (3) adding a procalcitonin coated antibody B after the magnetic bead is resuspended, performing magnetic separation after suspension reaction, then obtaining an antibody B-magnetic bead cross-linked matter, adding a blocking agent into the magnetic bead cross-linked matter for blocking, washing, adding a magnetic bead diluent, obtaining a streptavidin magnetic bead solution of the procalcitonin coated antibody B after the resuspension, and diluting the streptavidin magnetic bead solution of the procalcitonin coated antibody B to a target concentration after adding the magnetic bead diluent.
In some embodiments, the molar ratio of procalcitonin coated antibody to acridinium ester in the second reagent is 1:20.
In some embodiments, the second reagent further comprises an antibody diluent, wherein the ph=7.0-7.4 of the antibody diluent comprises PBS, BSA, tween-20, proclin300.
In some embodiments, the method of preparing the second reagent comprises: and mixing the second reagent A and the second reagent B in proportion to obtain a second reagent.
In some embodiments, the method of preparing the second reagent a comprises: adding acridine ester into procalcitonin labeled antibody A in proportion, adding lysine to terminate reaction after suspension reaction, removing acridine ester by ultrafiltration or dialysis, and diluting the procalcitonin labeled antibody A with the acridine ester to the target concentration by using an antibody diluent.
In some embodiments, the method of preparing the second reagent B comprises: adding acridine ester into procalcitonin labeled antibody B in proportion, adding lysine to stop reaction after suspension reaction, removing acridine ester by ultrafiltration or dialysis, and diluting the procalcitonin labeled antibody B marked by acridine ester to target concentration by using an antibody diluent.
In some embodiments, the quantitative detection kit further comprises a calibrator and a quality control product, wherein the calibrator and the quality control product are solutions with different concentrations prepared by diluting procalcitonin antigen with antigen diluent.
The invention has the following beneficial effects:
The invention discloses a kit for quantitatively detecting procalcitonin by a double-antibody sandwich one-step method aiming at magnetic bead chemiluminescence immunoassay, and overcomes an S-shaped reaction curve of procalcitonin antigen by screening proper mass ratio of magnetic beads to antibodies, thereby expanding the linear range. Meanwhile, the diluent of the specific components of the kit, including magnetic bead diluent, antibody diluent, antigen diluent and the like, can provide favorable conditions for the antibody to recognize the antigen, so that the performances of the kit are optimal. In addition, the invention adopts the freeze-dried calibrator state, can obviously stabilize antigen storage and avoid damage and denaturation of PCT.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The invention solves the problem that PCT antigen reaction linearity is always in an S shape in the detection process, so that low-end sensitivity is poor, high-end linearity is poor, and linearity range is narrow.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Coated antibody A was purchased from Baiqiao Rui Jing, cat# AB042-5;
coated antibody B was purchased from Yinqiao Shenzhou, cat# AB042-1;
labeled antibody A was purchased from Baiqiao Rui Jing, cat# AB042-8;
Labeled antibody B was purchased from Tianchen, cat# AB042-10.
Example 1
The embodiment provides a procalcitonin quantitative detection kit, which comprises a first reagent, a second reagent, a calibrator and a quality control product.
The first reagent comprises a streptavidin magnetic bead-connected biotin-labeled procalcitonin coated antibody, the second reagent comprises an acridinium ester-labeled procalcitonin labeled antibody, and the calibrator and the quality control product are solutions with different concentrations prepared by diluting procalcitonin antigen with antigen diluent.
The preparation method of the first reagent in the kit comprises the following steps:
(1) And (5) resuspension: 10mg of streptavidin magnetic beads and a 2mL centrifuge tube are taken, the supernatant is removed, the mixture is washed 3 times with 1mL of cross-linking buffer, the supernatant is removed, and finally the beads are resuspended with 1mL of cross-linking buffer, the cross-linking buffer is 1 XCB, and the pH is 9.2.
(2) Crosslinking: adding PCT monoclonal antibody 1 (coated antibody A) 1:10 into magnetic beads, suspending at 25deg.C for 30min, and removing supernatant to obtain antibody-magnetic bead conjugate.
(3) Adding 1mL of blocking agent into the antibody-magnetic bead cross-linked substance, carrying out suspension reaction for 2h at 25 ℃ for blocking, removing supernatant after blocking, washing for 3 times by using a washing solution, removing supernatant, adding 1mL of magnetic bead diluent for resuspension to obtain streptavidin magnetic bead solution coated with PCT monoclonal antibody, adding a proper amount of magnetic bead diluent for diluting the antibody solution to 0.2mg/mL to obtain a first reagent A, and storing at 2-8 ℃ for standby.
The components of the magnetic bead diluent are 0.05MTris, 1% BSA, 1% Tween 20 and 1% Proclin-300, and the pH of the magnetic bead diluent is 7.0-7.4.
The mass ratio of the magnetic beads to the PCT monoclonal antibody in the first reagent A is 1:0.01.
(4) The first reagent B was prepared as the first reagent a except that PCT monoclonal antibody was changed to PCT monoclonal antibody 2 (coated antibody B) in the crosslinking step, and the mass ratio of PCT monoclonal antibody to biotin was changed to 1:20.
The mass ratio of the magnetic beads to the PCT monoclonal antibody in the first reagent B is 1:0.06.
(5) And mixing the first reagent A and the first reagent B according to a ratio of 1:1 to obtain the first reagent.
The preparation method of the second reagent in the kit comprises the following steps:
(1) 100 mu gPCT of monoclonal antibody (labeled antibody A) was added to the corresponding amount of acridine ester in a molar mass ratio of 1:20, and the reaction was suspended at 25℃for 60min, followed by termination with 1mg of lysine.
(2) The uncrosslinked acridinium ester was removed by ultrafiltration tube or dialysis bag, only acridinium ester-labeled antibody complex was retained as much as possible, and diluted to 0.06. Mu.g/mL by addition of antibody diluent to give second reagent A.
The components of the antibody diluent are 1 XPBS, 1% BSA, 1 permillage Tween-20 and 1 permillage Proclin300, and the pH of the antibody diluent is 7.0-7.4.
(3) The preparation method of the second reagent B is the same as that of the second reagent A, and only the labeled antibody A is replaced by the labeled antibody B, and then the antibody diluent is added to dilute the labeled antibody B to 0.04 mug/mL to obtain the second reagent B.
(4) And mixing the second reagent A and the second reagent B according to a ratio of 1:1 to obtain the second reagent.
The preparation method of the calibrator in the kit comprises the following steps:
Procalcitonin antigen was formulated with antigen dilutions of 0ng/mL, 2.5ng/mL, 10ng/mL, 25ng/mL, 50ng/mL and 100ng/mL.
The antigen diluent comprises 1 XPBS, 10mM EDTA, 2% trehalose, 3% BSA, 1 millTween-20, 1 millproclin-300, and the pH of the antigen diluent is 7.0-7.4.
The preparation method of the quality control product in the kit comprises the following steps:
Antigen dilutions of procalcitonin antigen were formulated as 5ng/mL (low value quality control), 15ng/mL (median quality control) and 35ng/mL (high value quality control).
The antigen diluent comprises 1 XPBS, 10mM EDTA, 2% trehalose, 3% BSA, 1 millTween-20, 1 millproclin-300, and the pH of the antigen diluent is 7.0-7.4.
Example 2
The present example is a method for quantitatively detecting procalcitonin by using the kit of example 1, and specifically comprises the following steps:
(1) Taking 10 mu L of a sample to be detected, 50 mu L of a first reagent and 50 mu L of a second reagent, and mixing the two reagents to form a reaction mixture of capture antibody crosslinked magnetic microsphere-antigen-acridinium ester labeled detection antibody; full-automatic chemiluminescence immunoassay instrument Spring C2200 and Spring C1200 manufactured by Hongte Biotechnology Co., ltd.
(2) After the reaction is finished, cleaning liquid (supplied by Shandong Hongte Biotechnology Co., ltd., record number: lu Jixie is 20220101) is taken and cleaned, then substrate liquid (substrate liquid for a full-automatic immunoassay system: supplied by Shandong Hongte Biotechnology Co., ltd., record number: lu Jixie is 20220102; contains substrate liquid A and substrate liquid B) is added into the reaction mixture, and chemiluminescent reaction is excited;
(3) The concentration of PCT in a sample to be detected is proportional to the relative luminescence value (RLU) detected by the system, and the concentration of PCT is calculated through a batch calibration curve fitted by an instrument by calibrating a corresponding batch of calibrator;
(4) The accuracy of the calibration was evaluated using the quality control in example 1.
Comparative example 1
The difference from example 1 is that the first reagent component comprises only the first reagent a and the second reagent component comprises only the second reagent a.
Comparative example 2
The difference from example 1 is that the first reagent component comprises only the first reagent a.
The kits of example 1 and comparative examples 1 to 2 were tested on a full-automatic chemiluminescence immunoassay analyzer, and indexes such as sensitivity, precision, linear range and the like of the kit prepared by the invention were examined to perform comprehensive evaluation.
Wherein the sensitivity mainly examines the blank limit and the detection limit.
The calibrants a were tested for 20 times simultaneously with the test of example 1 and comparative example 1-2, RLU values of 20 measurement results were obtained, the average value and Standard Deviation (SD) thereof were calculated, and the corresponding RLU values were substituted into the primary equation obtained by two-point regression fitting of the concentration RLU values of the calibrant a and the adjacent low concentration calibrant B, and the corresponding concentration values were obtained as a blank Limit (LOB), and the specific results are shown in table 1.
TABLE 1 blank data for example 1, comparative examples 1-2
As can be seen from the data in table 1, the blank results of example 1 are lower than those of comparative examples 1-2, demonstrating that the kits of the present invention have higher sensitivity.
And 5 samples with low values, the concentration of which is approximately the detection limit, are detected by using the embodiment 1 and the comparative examples 1-2, each sample is detected 5 times, the detection results are ordered according to the sizes, and when the number of the detection results which are lower than the blank limit value is less than or equal to 3, the detection limit can be considered to meet the requirements. The blank limit of the kit was 0.02ng/mL. The specific results are shown in Table 2.
TABLE 2 detection limit data for example 1, comparative examples 1-2
As can be seen from the data in Table 2, the detection limit luminescence value of example 1 is higher and the CV value of concentration is smaller than that of comparative examples 1-2, demonstrating that the kit of the present invention has higher sensitivity.
And (3) precision detection: three concentration levels of quality control and 0.02-100ng/mL serum samples were tested simultaneously during two time periods of the day using example 1, comparative examples 1-2, and the precision total CV of the test concentration results was calculated. The results are shown in Table 3.
TABLE 3 precision data for example 1, comparative examples 1-2
As can be seen from the data in Table 3, example 1 has lower CV values than comparative examples 1-2, with better precision, whether it is a low value quality control, a medium value quality control, or a high value quality control, or the detection of different serum samples.
Linear range: the high-value serum samples were simultaneously tested for samples diluted with zero concentration serum gradient using example 1 and comparative examples 1-2, and the linear correlation coefficients were calculated, and the results are shown in table 4.
TABLE 4 Linear Range data for example 1, comparative examples 1-2
As can be seen from the data in table 4, the linearity of each example was satisfactory, but the overall light emission value of example 1 was higher and the correlation was better.
Specific data of the anti-jamming capability are shown in table 5:
TABLE 5 anti-tamper capability test results
As can be seen from the data in Table 5, the anti-jamming capability of example 1 was satisfactory.
Experimental example 1
The experimental example is to compare the detection effect of the kit prepared by the connection ratio of different antibodies to biotin and the coupling ratio of different antibodies to acridinium ester, other reagents are the same as in example 1, the operation method is the same as in example 2, and the comparison effect is shown in table 6:
TABLE 6 comparison of detection effects of coated and labeled antibodies from different sources
From Table 6, MP: ab is 1:10 and AE: when Ab is 1:20, the luminous value can meet the experimental requirement, and the PN ratio is good.
Experimental example 2
The experimental example is to compare the detection effect of the kit prepared by magnetic beads and antibodies in different proportions, other reagents are the same as those in the example 1, the operation method is the same as that in the example 2, and the comparison effect is shown in table 7:
TABLE 7 comparison of detection effects of magnetic beads and antibodies at different ratios
From the results in Table 7, MP: ab is 1:6 and AE: when Ab is 1:20, the luminous value can meet the requirement, and the PN ratio is good.
Experimental example 3
This experimental example is to verify the difference between the linear range of the test of the mixed antibody, example 1, and the comparative monoclonal antibody, comparative examples 1-2, as compared to the monoclonal antibody set under the same conditions, and the test results are shown in table 8:
TABLE 8 detection Effect of Mixed antibodies compared to monoclonal antibodies
From the results in Table 8, it can be seen that example 1 is significantly broader than the linear range of comparative examples 1-2, demonstrating that the mixed antibodies are more linear than the single antibody combination test procalcitonin.
Experimental example 4
The test example is a comparison of the detection effect of the clinical samples detected by the kits of example 1 and comparative examples 1-2, respectively, wherein the clinical samples are fresh clinical samples taken from Shandao two-national institute and are over 18 years old. To ensure the test requirements, the sample size is required to be not less than 500. Mu.L. Severely hemolyzed, lipidemic and severely contaminated samples are excluded.
Instrument: full-automatic chemiluminescence immunoassay instrument Shine i2910 produced by Shenzhen Yingkai biotechnology limited company; reagent: procalcitonin (PCT) assay kit (magnetic particle chemiluminescence method).
The results of the measurements are shown in tables 9-10:
TABLE 9 detection results of clinical samples
TABLE 10 detection results of clinical samples
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From the above table, it can be seen that example 1 has a good correlation coefficient with the control reagent.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A kit for quantitative detection of procalcitonin, comprising: a first reagent and a second reagent;
The first reagent comprises a biotin-marked procalcitonin coated antibody connected with streptavidin magnetic beads, and the procalcitonin coated antibody comprises two procalcitonin antibodies aiming at different epitopes of the same antigen to be detected;
The second reagent comprises an acridinium ester marked procalcitonin marked antibody, and the procalcitonin marked antibody comprises two procalcitonin antibodies aiming at different epitopes of the same antigen to be detected.
2. The quantitative detection kit according to claim 1, wherein the first reagent comprises a first reagent a and a first reagent B; the mass ratio of the streptavidin magnetic beads to the biotin-labeled procalcitonin coated antibody in the first reagent is 1:0.01-0.06;
Preferably, the mass ratio of the streptavidin magnetic beads to the biotin-labeled procalcitonin coated antibody in the first reagent A is 1:0.01;
preferably, the mass ratio of the streptavidin magnetic beads to the biotin-labeled procalcitonin coated antibody in the first reagent B is 1:0.06.
3. The quantitative detection kit according to claim 2, wherein the molar ratio of procalcitonin coated antibody to biotin in the first reagent is 1:10-20;
preferably, the first reagent A contains a biotin-labeled procalcitonin monoclonal antibody connected with streptavidin magnetic beads, and the molar ratio of the procalcitonin coated antibody to the biotin is 1:10;
preferably, the first reagent B contains a biotin-labeled procalcitonin monoclonal antibody connected with streptavidin magnetic beads, and the molar ratio of the procalcitonin coated antibody to the biotin is 1:20;
preferably, the volume ratio of the first reagent A to the first reagent B in the first reagent is 1:1.
4. A quantitative test kit according to claim 3, wherein the first reagent further comprises a magnetic bead dilution solution, and the magnetic bead dilution solution has a ph=7.0 to 7.4, and comprises the following components: 0.05MTris, 1% BSA, 1% Tween 20, 1% Proclin-300.
5. The quantitative detection kit according to claim 4, wherein the preparation method of the first reagent comprises: mixing the first reagent A and the first reagent B in proportion to obtain the first reagent;
Preferably, the preparation method of the first reagent A comprises the following steps: adding a procalcitonin coated antibody A after magnetic bead resuspension, performing magnetic separation after suspension reaction, then obtaining an antibody A-magnetic bead cross-linked matter, adding a blocking agent into the magnetic bead cross-linked matter for blocking, washing, adding a magnetic bead diluent, obtaining a streptavidin magnetic bead solution of the procalcitonin coated antibody A after resuspension, and then adding the magnetic bead diluent to dilute the streptavidin magnetic bead solution of the procalcitonin coated antibody A to a target concentration;
Preferably, the preparation method of the first reagent B comprises the following steps: and (3) adding a procalcitonin coated antibody B after the magnetic bead is resuspended, performing magnetic separation after suspension reaction, then obtaining an antibody B-magnetic bead cross-linked matter, adding a blocking agent into the magnetic bead cross-linked matter for blocking, washing, adding a magnetic bead diluent, obtaining a streptavidin magnetic bead solution of the procalcitonin coated antibody B after the resuspension, and diluting the streptavidin magnetic bead solution of the procalcitonin coated antibody B to a target concentration after the magnetic bead diluent is added.
6. The kit of claim 5, wherein the molar ratio of procalcitonin coated antibody to acridinium ester in the second reagent is 1:20.
7. The quantitative detection kit according to claim 6, wherein the second reagent further comprises an antibody diluent, and the ph=7.0-7.4 of the antibody diluent comprises 1×pbs, 1% bsa, 1%tween-20, 1%proclin 300.
8. The quantitative detection kit according to claim 7, wherein the preparation method of the second reagent comprises: mixing a second reagent A and a second reagent B in proportion to obtain the second reagent;
preferably, the preparation method of the second reagent A comprises the following steps: adding acridine ester into the procalcitonin marked antibody A according to a certain proportion, adding lysine to terminate the reaction after suspension reaction, removing the acridine ester by ultrafiltration or dialysis, and diluting the procalcitonin marked antibody A marked by the acridine ester to the target concentration by using an antibody diluent;
Preferably, the preparation method of the second reagent B comprises the following steps: adding acridine ester into procalcitonin labeled antibody B in proportion, adding lysine to terminate reaction after suspension reaction, removing acridine ester by ultrafiltration or dialysis, and diluting the procalcitonin labeled antibody B with the acridine ester to the target concentration by using an antibody diluent.
9. The quantitative detection kit according to claim 8, wherein the procalcitonin coated antibody a, procalcitonin coated antibody B, procalcitonin single labeled antibody a and procalcitonin single labeled antibody B are directed against different epitopes of the same antigen to be detected;
Preferably, said procalcitonin coated antibody a is purchased from baiqiao ruijing, cat No. AB042-5; procalcitonin coated antibody B was purchased from Yinqiaoshenzhou, cat# AB042-1; procalcitonin single labeled antibody A was purchased from Baiqiaoruijing, cat# AB042-8; procalcitonin single labeled antibody B was purchased from Tianchen, cat# AB042-10.
10. The quantitative detection kit according to claim 9, further comprising a calibrator and a quality control material, wherein the calibrator and the quality control material are solutions with different concentrations prepared by diluting procalcitonin antigen with an antigen diluent.
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| CN102359958A (en) * | 2011-07-19 | 2012-02-22 | 深圳市国赛生物技术有限公司 | Kit and method for detecting procalcitonin |
| CN107367620A (en) * | 2017-09-21 | 2017-11-21 | 苏州新波生物技术有限公司 | The kit of Procalcitonin and the detection method of Procalcitonin in a kind of detection blood |
| CN107807240A (en) * | 2017-11-03 | 2018-03-16 | 太原瑞盛生物科技有限公司 | A kind of chemiluminescence detection kit of Procalcitonin and preparation method thereof |
| CN109709323A (en) * | 2019-02-15 | 2019-05-03 | 深圳上泰生物工程有限公司 | High quick cardiac muscle troponin I magnetic microparticle chemiluminescence immunity detection reagent, Preparation method and use |
| CN114487442A (en) * | 2022-02-14 | 2022-05-13 | 四川沃文特生物技术有限公司 | Mouse monoclonal antibody coated magnetic bead, preparation method and kit for determining high-sensitivity cardiac troponin I |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102359958A (en) * | 2011-07-19 | 2012-02-22 | 深圳市国赛生物技术有限公司 | Kit and method for detecting procalcitonin |
| CN107367620A (en) * | 2017-09-21 | 2017-11-21 | 苏州新波生物技术有限公司 | The kit of Procalcitonin and the detection method of Procalcitonin in a kind of detection blood |
| CN107807240A (en) * | 2017-11-03 | 2018-03-16 | 太原瑞盛生物科技有限公司 | A kind of chemiluminescence detection kit of Procalcitonin and preparation method thereof |
| CN109709323A (en) * | 2019-02-15 | 2019-05-03 | 深圳上泰生物工程有限公司 | High quick cardiac muscle troponin I magnetic microparticle chemiluminescence immunity detection reagent, Preparation method and use |
| CN114487442A (en) * | 2022-02-14 | 2022-05-13 | 四川沃文特生物技术有限公司 | Mouse monoclonal antibody coated magnetic bead, preparation method and kit for determining high-sensitivity cardiac troponin I |
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