CN118033151A - Homogeneous chemiluminescent procalcitonin detection reagent constructed by cyclopeptide luminescent coupling molecules - Google Patents
Homogeneous chemiluminescent procalcitonin detection reagent constructed by cyclopeptide luminescent coupling molecules Download PDFInfo
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- CN118033151A CN118033151A CN202410432391.8A CN202410432391A CN118033151A CN 118033151 A CN118033151 A CN 118033151A CN 202410432391 A CN202410432391 A CN 202410432391A CN 118033151 A CN118033151 A CN 118033151A
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- procalcitonin
- cyclopeptide
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- CWCXERYKLSEGEZ-KDKHKZEGSA-N procalcitonin Chemical compound C([C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)NCC(O)=O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCSC)NC(=O)[C@H]1NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)[C@@H](N)CSSC1)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 CWCXERYKLSEGEZ-KDKHKZEGSA-N 0.000 title claims abstract description 134
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Abstract
The invention provides a homogeneous chemiluminescent procalcitonin detection reagent constructed by a cyclopeptide luminescent coupling molecule, which comprises an R1 reagent and an R2 reagent, wherein the R1 reagent comprises a Fe 3O4 nano enzyme-labeled procalcitonin monoclonal antibody; the R2 reagent comprises a procalcitonin monoclonal antibody coupled with a cyclopeptide luminescent coupling molecule, wherein the cyclopeptide luminescent coupling molecule is octacyclopeptide or dodecacyclopeptide coupled acridine salt. In use, the luminous intensity of the procalcitonin detection reagent directly reflects the concentration of procalcitonin PCT in a sample, so that quantitative determination of procalcitonin PCT is realized.
Description
Technical Field
The invention relates to the field of in-vitro diagnosis, in particular to a homogeneous phase chemiluminescent procalcitonin detection reagent constructed by cyclopeptide luminescent coupling molecules.
Background
Microorganisms such as bacteria and viruses have various ways of invading human bodies, such as a way of wound, urinary tract, respiratory tract, digestive tract and the like, and when the organism suffers serious wound and has obviously reduced immunity, serious infection reaction is easily caused. These infections can lead to a series of life threatening complications such as sepsis, septic shock, and multiple organ dysfunction syndrome, and in extreme cases, risk of death.
Procalcitonin (PCT) as a key biomarker plays a vital role in identifying and assessing bacterial infections. Procalcitonin (PCT) shows a very high response sensitivity when the body is subjected to bacterial infections, especially in the face of severe systemic infections such as sepsis, bacterial sepsis, etc. PCT shows high specificity and sensitivity by virtue of the accurate and dynamic change characteristics, and has immeasurable clinical value in the aspects of auxiliary differential diagnosis, disease progress evaluation, prognosis judgment, treatment effect monitoring and the like of sepsis and systemic bacterial infection. Through the continuous monitoring of PCT level, doctor not only can effectively distinguish whether be bacterial infection, can also make more scientific and accurate decision on antibiotic use management.
Currently, procalcitonin (PCT) detection methods have a trend toward a variety of approaches, including, but not limited to, chemiluminescence immunoassay (CLIA), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and colloidal Gold Immunochromatography (GICA). However, in practice, these approaches increasingly expose respective limitations. For example, radioimmunoassay has not only environmental pollution problems due to the use of radioactive substances, but also long detection process, poor reproducibility of results, and short shelf life of the label. Although the ELISA is widely applied, the enzyme is easy to inactivate, and meanwhile, the macromolecular marker can generate steric effect, so that the sensitivity is limited, the linear range is narrow, and the full-automatic operation is difficult to realize. The colloidal gold immunochromatography method has the characteristic of only being capable of carrying out qualitative rather than quantitative detection due to lower detection sensitivity, and limits the accuracy of the colloidal gold immunochromatography method in clinical diagnosis.
In contrast, chemiluminescent immunoassay developed based on enzyme linked immunoassay, exhibited significant advantages: has been widely adopted in recent years, because of its high sensitivity, wide detection linear range, simple operation flow, and high automation. Meanwhile, the magnetic particle immune detection technology is also focused as a novel detection means, and by adopting magnetic solid-phase particles with specific sizes as carriers and fixing immune active components such as antibodies or antigens with specific binding capacity in a physical adsorption or chemical coupling mode and the like, the rapid and efficient separation effect is realized, the repeatability and the simple operation steps are good, and the biological characteristics and functions of detected cells or other biological materials are not influenced.
The PCT chemiluminescent detection kit sold in the market at present mostly comprises a plurality of reagent components such as magnetic beads, biotin-labeled PCT antibodies, acridinium ester-labeled PCT antibodies and the like. However, the process of the kit is relatively complex, the control is difficult, and the cost of the magnetic beads is extremely high. When the detection method is loaded on a chemiluminescent immunoassay analyzer for experiments, the detection method needs to undergo multiple magnetic separation washing steps, reduces precision and has long time consumption period, and constitutes a main technical challenge currently faced by the detection method.
Disclosure of Invention
In order to overcome the key problems of low detection sensitivity, low specificity, reagent composition complexity, difficult accurate control of a preparation process and the like in the prior art, the invention provides a homogeneous chemiluminescent procalcitonin detection reagent constructed by cyclopeptide luminescent coupling molecules, and aims to improve and optimize the measurement of procalcitonin in blood. The solution provided by the invention simplifies the composition of the reagent, so that the detection flow is more controllable and effective, and the improvement of the accurate and efficient detection capability of procalcitonin in blood in clinical and laboratory environments is promoted.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
a homogeneous chemiluminescent procalcitonin detection reagent constructed by a cyclopeptide luminescent coupling molecule, comprising:
r1 reagent: comprises a procalcitonin monoclonal antibody A marked by Fe 3O4 nano enzyme;
R2 reagent: comprises a procalcitonin monoclonal antibody B marked by a cyclopeptide luminescent coupling molecule; the cyclopeptide luminescent coupling molecule is octacyclopeptide or dodecacyclopeptide coupled acridine salt; the amino acid sequence of the octacyclic peptide is Lys-Ser-Gly-Ser-Cys-Ser-Gly-Ser, and the amino acid sequence of the dodecacyclic peptide is Lys-Ser-Gly-Ser-Lys-Ser-Gly-Ser-Cys-Ser-Gly-Ser.
Further preferably, the R1 reagent further comprises an R1 buffer, the R1 buffer having a pH of 6.0-7.5, comprising 20-100 mM Tris,100 mM NaCl,0.01-0.1 wt.% Tween20,1-5 wt.% BSA,1-5 wt.% trehalose or sucrose, 1-5 wt.% glycerol, and 0.09 wt.% sodium azide or Proclin 300;
The R2 reagent also comprises an R2 buffer, the pH of which is 6.0-8.5, containing 10-50 mM NaH2PO4,10-50 mM Na2HPO4,100 mM NaCl,0.01-0.1wt% Tween20,1-5wt% BSA,1-5wt% trehalose or sucrose, 1-5wt% glycerol, and 0.09wt% sodium azide or Proclin 300.
Further preferably, the R2 reagent contains 0.1-5.0% protein stabilizer, 0.05% -0.2% preservative, 0.01-0.1% surfactant.
Further preferably, the R2 reagent further comprises a background reducing agent.
Further preferably, the background reducing agent is one or more of reducing molecules such as vitamin C and sodium sulfite.
Further preferably, the procalcitonin detection reagent further comprises procalcitonin PCT series calibrator, quality control product and luminescent substrate solution.
The invention also provides a preparation method of the homogeneous chemiluminescent procalcitonin detection reagent, which comprises the following steps:
① Preparing Fe 3O4 nano enzyme marked procalcitonin monoclonal antibody A:
in a buffer solution system, mixing Fe 3O4 nano enzyme in an alkaline environment with an antibody A solution, gently mixing the mixture at 25-37 ℃ for 30-60 min to form stable non-covalent connection between the Fe 3O4 nano enzyme and the antibody A, adding 0.5-3% BSA buffer solution for sealing, gently mixing the mixture at 25-37 ℃ for 30-60 min, then magnetically separating and cleaning the mixture by using PBS buffer solution containing 0.05% Tween 20 to obtain the Fe 3O4 nano enzyme marked procalcitonin monoclonal antibody A, and storing the procalcitonin monoclonal antibody A in an R1 buffer solution.
② Cyclopeptide luminescent coupling molecule marked procalcitonin monoclonal antibody B: concentrating or diluting the antibody B solution to a concentration of 1-3 mg/mL, and then dissolving the cyclopeptide luminescent coupling molecule in DMSO or acetonitrile in advance to obtain the cyclopeptide luminescent coupling molecule: antibody = 1-6: and (3) adding the mixture into an antibody B solution according to the molar ratio of 1, carrying out vortex mixing and light-shielding reaction, and purifying the reaction mixture to obtain the cyclopeptide luminescent coupling molecular labeled procalcitonin monoclonal antibody B.
Further preferably, the method further comprises the steps of preparing procalcitonin calibrator and quality control product:
1) Preparing a calibrator buffer: 0.10-0.27 g/L of monopotassium phosphate, 1.00-1.45 g/L of disodium hydrogen phosphate, 5.0-8.0 g/L of sodium chloride, 0.1-0.2 g/L of potassium chloride, 0.01-5.00 wt% of bovine serum albumin, 0.01-0.1 wt% of preservative sodium azide or Proclin 300, 0.01-0.10 wt% of Tween 20, 1.00-5.00 wt% of sucrose or trehalose, and 3.00-10.00 wt% of glycerol are prepared into a calibrator buffer solution;
2) Preparing a calibrator and a quality control product: the calibrator and the quality control product are prepared by using procalcitonin pure product and calibrator buffer, and the concentrations of the calibrator are respectively as follows: 0.05 ng/mL, 45 ng/mL, 500 ng/mL, 800 ng/mL,1000 ng/mL,10000 ng/mL, and the quality control concentrations are respectively: 50 ng/mL,1200 ng/mL.
A kit for procalcitonin detection, comprising the procalcitonin detection reagent as described above.
The invention has the beneficial effects that: the Fe 3O4 nanoparticles have catalytic activity similar to horseradish peroxidase, and are considered as nanoscale enzymes capable of decomposing hydrogen peroxide to generate oxygen radicals. Unlike natural proteinase molecule, the inorganic matter has high stability, magnetic responsiveness and great advantage in constructing immunological detection system. The technology adopts Fe 3O4 nano enzyme to mark the antibody A. In the actual detection process, procalcitonin PCT in a sample to be detected and procalcitonin monoclonal antibody A marked by Fe 3O4 nano enzyme and procalcitonin monoclonal antibody B marked by cyclopeptide luminescent coupling molecules form a double-antibody sandwich structure. When hydrogen peroxide is added into the system, fe 3O4 nano enzyme in the immune complex system rapidly catalyzes hydrogen peroxide to generate oxygen free radical, and further activates cyclopeptide luminescent coupling molecules to trigger chemiluminescent reaction. The Fe 3O4 nano enzyme which does not participate in immune reaction can not effectively catalyze the light signal emitted by the cyclopeptide luminescence coupling molecular labeled procalcitonin monoclonal antibody B under the non-composite state due to the inhibition effect of the reducing substances in the solution, so that the background noise is lower. In this way, the luminescence intensity directly reflects the concentration of procalcitonin PCT in the sample, thereby realizing quantitative determination of procalcitonin PCT.
Drawings
FIG. 1 is a schematic representation of a thiol-activated octacyclic acridine salt;
FIG. 2 is a schematic representation of thiol-activated dodecapeptide acridine salts.
FIG. 3 is a schematic diagram of procalcitonin detection according to the present invention.
FIG. 4 is a standard dose curve of procalcitonin when the homogeneous chemiluminescent procalcitonin assay reagent of example 1 is used.
Detailed Description
The implementation of the present invention is further explained below.
The invention discloses a homogeneous phase chemiluminescent procalcitonin detection reagent constructed by a cyclopeptide luminescence coupling molecule, which comprises the following components:
R1 reagent: fe 3O4 nanometer enzyme marked procalcitonin monoclonal antibody A and R1 buffer (pH 6.0-7.5, containing 20-100 mM Tris,100 mM NaCl,0.01-0.1wt% Tween20,1-5wt% BSA,1-5wt% trehalose or sucrose, 1-5wt% glycerol, and 0.09wt% sodium azide or Proclin 300);
R2 reagent: the cyclopeptide luminescent coupling molecule marked procalcitonin monoclonal antibodies B and R2 buffer (pH is 6.0-8.5, containing 10-50 mM NaH2PO4,10-50 mM Na2HPO4,100 mM NaCl,0.01-0.1wt% Tween20,1-5wt% BSA,1-5wt% trehalose or sucrose, 1-5wt% glycerol, and 0.09wt% sodium azide or procalin 300); the cyclopeptide luminescent coupling molecule is octacyclopeptide or dodecacyclopeptide coupled acridine salt; the R2 reagent may contain 0.1-5.0wt% protein stabilizer, 0.05-0.2 wt% preservative, 0.01-0.1 wt% surfactant; the R2 reagent can contain a background reducing agent, wherein the background reducing agent is any one or more of reducing molecules such as vitamin C, sodium sulfite and the like;
procalcitonin (PCT) series calibrator, quality control and luminescent substrate solution.
The preparation method of the homogeneous chemiluminescent procalcitonin detection reagent comprises the following steps:
① Preparing Fe 3O4 nano enzyme marked procalcitonin monoclonal antibody A:
1) Antibody a desalting: desalting the labeled antibody A by using a PD10 desalting column to remove substances interfering with subsequent reactions;
2) Pretreatment of Fe 3O4 nano enzyme: controlling the pH value to be in the range of 8.6-10.0, carrying out alkaline treatment on Fe 3O4 nano enzyme, and keeping the pH value to be the maximum on the premise of ensuring that nano particles are stable and not aggregated, thereby being used for efficiently marking the antibody A;
3) Labeling reaction: mixing Fe 3O4 nano enzyme in alkaline environment with antibody solution in a proper buffer solution system, gently mixing at 25-37 ℃ for 30-60 min to form stable non-covalent connection between Fe 3O4 nano enzyme and antibody A, adding 0.5-3 wt% BSA buffer solution for blocking, gently mixing at 25-37 ℃ for 30-60 min, then using PBS buffer solution containing 0.05 wt% Tween 20 for magnetic separation, washing for three times, and storing in R1 buffer solution;
4) Potency determination and validation: after the coupling is completed, ELISA identification is carried out on the prepared Fe 3O4 nano enzyme marked procalcitonin monoclonal antibody A, and the original specific binding capacity is reserved and the prepared procalcitonin monoclonal antibody A has good enzyme activity;
5) Split charging and preserving: the obtained Fe 3O4 nano enzyme marked procalcitonin monoclonal antibody A is split-packed in a proper R1 buffer solution and stored at a low temperature (such as-20 ℃ or lower) so as to keep the stability of the monoclonal antibody A for subsequent experiments.
② Cyclopeptide luminescent coupling molecule marked procalcitonin monoclonal antibody B:
1) Pre-activation treatment of antibody B: removing metal ions and impurities containing primary amino groups in an antibody solution by means of dialysis and the like, avoiding influencing subsequent coupling, replacing a coupling buffer solution with a PBS buffer solution with the pH of 7.6-8.6, adding an SM (PEG) 2 activator with the molar quantity of 6 times that of the antibody, modifying the antibody B with maleimide groups, and then desalting and purifying to remove redundant activators;
2) Chemiluminescent coupling molecules: dissolving cyclopeptide luminescent coupling molecules in DMSO or acetonitrile solution with the concentration of 2-4 mg/mL, and simultaneously adding TCEP of 1mM to avoid disulfide bond generation;
3) Labeling reaction: concentrating or diluting the antibody B solution to a concentration of 1-3 mg/mL, and then dissolving the cyclopeptide luminescent coupling molecules in DMSO or acetonitrile in advance according to a molar ratio of 1-6:1, adding the mixture into an antibody B solution (mercapto activated octacyclopeptide acridinium salt: antibody B=1-6:1), carrying out vortex mixing, carrying out light-shielding reaction at 25 ℃ for 2 hours, and purifying the reaction mixture by a molecular sieve to obtain a cyclopeptide luminescent coupling molecular labeled procalcitonin monoclonal antibody B;
4) Labeling efficiency and activity detection: and (3) detecting the luminous efficiency of the obtained cyclopeptide luminous coupling molecular labeled procalcitonin monoclonal antibody B, and simultaneously evaluating whether the original specificity and affinity of the antibody B are influenced in the labeling process or not, wherein the evaluation can be performed through enzyme dynamics and sensitivity.
③ Preparing procalcitonin calibrator and quality control product:
1) Preparing a calibrator buffer: 0.10-0.27 g/L of monopotassium phosphate, 1.00-1.45 g/L of disodium hydrogen phosphate, 5.0-8.0 g/L of sodium chloride, 0.1-0.2 g/L of potassium chloride, 0.01-5.00 wt% of bovine serum albumin, 0.01-0.1 wt% of preservative sodium azide or Proclin 300, 0.01-0.10 wt% of Tween 20, 1.00-5.00 wt% of sucrose or trehalose, and 3.00-10.00 wt% of glycerol are prepared into a calibrator buffer solution;
2) Preparing a calibrator and a quality control product: the calibrator and the quality control product are prepared by using procalcitonin pure product and calibrator buffer, and the concentrations of the calibrator are respectively as follows: 0.05 ng/mL, 45 ng/mL, 500 ng/mL, 800 ng/mL,1000 ng/mL,10000 ng/mL, and the quality control product concentration is: 50 ng/mL, 1200/ng/mL.
In order to make the technical means, creation characteristics, achievement purposes and effects of the present invention easy to understand, the following are combined:
the invention is further illustrated by means of the following examples:
Example 1
A homogeneous chemiluminescent procalcitonin detection reagent constructed by a cyclopeptide luminescent coupling molecule, comprising:
procalcitonin (PCT) series calibrator, quality control and luminescent substrate solution;
R1 reagent: fe 3O4 nanometer enzyme marked procalcitonin monoclonal antibody A and R1 buffer solution;
r2 reagent: cyclopeptide luminescent coupling molecule marked procalcitonin monoclonal antibody B and R2 buffer solution.
Luminescent substrate solution: contains 0.1M Tris-HCl, 0.1-0.3 wt% H 2O2, 1-5 mM p-hydroxy cinnamic acid, and 10-30 mM tetraiodophenol.
The preparation method of the homogeneous chemiluminescent procalcitonin detection reagent comprises the following steps:
① Preparing Fe 3O4 nano enzyme marked procalcitonin monoclonal antibody A:
1) Antibody a desalting: desalting the labeled antibody A by using a PD10 desalting column to remove substances interfering with subsequent reactions;
2) Pretreatment of Fe 3O4 nano enzyme: controlling the pH value to be in the range of 8.6-10.0, carrying out alkaline treatment on Fe 3O4 nano enzyme, and keeping the pH value to be the maximum on the premise of ensuring that nano particles are stable and not aggregated, thereby being used for efficiently marking the antibody A;
3) Labeling reaction: mixing Fe 3O4 nano enzyme in alkaline environment with antibody solution in a proper buffer solution system, gently mixing at 30 ℃ for 45min to form stable non-covalent connection between Fe 3O4 nano enzyme and antibody A, adding 2 wt% BSA buffer solution for sealing, gently mixing at 30 ℃ for 45min, then magnetically separating by using PBS buffer solution containing 0.05 wt% Tween 20, washing for three times, and storing in R1 buffer solution;
4) Potency determination and validation: after the coupling is completed, ELISA identification is carried out on the prepared Fe 3O4 nano enzyme marked procalcitonin monoclonal antibody A, and the original specific binding capacity is reserved and the prepared procalcitonin monoclonal antibody A has good enzyme activity;
5) Split charging and preserving: the obtained Fe 3O4 nano enzyme marked procalcitonin monoclonal antibody A is split-packed in a proper R1 buffer solution and stored at a low temperature (such as-20 ℃ or lower) so as to keep the stability of the monoclonal antibody A for subsequent experiments.
② Cyclopeptide luminescent coupling molecule marked procalcitonin monoclonal antibody B:
1) Pre-activation treatment of antibody B: removing metal ions and impurities containing primary amino groups in an antibody solution by means of dialysis and the like, avoiding influencing subsequent coupling, replacing a coupling buffer solution with a PBS buffer solution with the pH of 7.6-8.6, adding an SM (PEG) 2 activator with the molar quantity of 6 times that of the antibody, modifying the antibody B with maleimide groups, and then desalting and purifying to remove redundant activators;
2) Chemiluminescent coupling molecules: dissolving sulfhydryl activated octacyclic peptide acridinium salt in DMSO or acetonitrile solution with concentration of 2-4 mg/mL, and simultaneously adding TCEP of 1mM to avoid disulfide bond formation; the amino acid sequence of the octacyclic peptide in the sulfhydryl activated octacyclic peptide acridinium salt is Lys-Ser-Gly-Ser-Cys-Ser-Gly-Ser, which is abbreviated as KSGSCSGS, and is shown as SEQ ID NO.1 of an amino acid sequence table;
3) Labeling reaction: the antibody B solution was concentrated or diluted to a concentration of 2mg/mL, and then the thiol-activated octacyclic peptide acridine salt, pre-dissolved in DMSO or acetonitrile, was added in a molar ratio of 3:1, adding the mixture into an antibody B solution (mercapto activated octacyclopeptide acridinium salt: antibody B=3:1), carrying out vortex mixing, carrying out light-shielding reaction at 25 ℃ for 2 hours, and purifying the reaction mixture by a molecular sieve to obtain a cyclopeptide luminescent coupling molecular labeled procalcitonin monoclonal antibody B (see figure 1);
4) Labeling efficiency and activity detection: and (3) detecting the luminous efficiency of the obtained cyclopeptide luminous coupling molecular labeled procalcitonin monoclonal antibody B, and simultaneously evaluating whether the original specificity and affinity of the antibody B are influenced in the labeling process or not, wherein the evaluation can be performed through enzyme dynamics and sensitivity.
③ Preparing procalcitonin calibrator and quality control product:
1) Preparing a calibrator buffer: 0.19 The calibrator buffer was prepared from g/L of potassium dihydrogen phosphate, 1.23 g/L of disodium hydrogen phosphate, 6.5 g/L of sodium chloride, 0.15 g/L of potassium chloride, 2.5 wt% of bovine serum albumin, 0.05 wt% of preservative sodium azide, 0.05 wt% of Tween 20, 3.00 wt% of sucrose or trehalose, and 6.50 wt% of glycerol;
2) Preparing a calibrator and a quality control product: the calibrator and the quality control product are prepared by using procalcitonin pure product and calibrator buffer, and the concentrations of the calibrator are respectively as follows: 0.05 ng/mL, 45 ng/mL, 500 ng/mL, 800 ng/mL,1000 ng/mL,10000 ng/mL, and the quality control product concentration is: 50 ng/mL, 1200/ng/mL.
Wherein, the preparation of the sulfhydryl activated octacyclic peptide acridine salt comprises the following steps:
S11, synthesizing octapeptide by utilizing a polypeptide solid-phase synthesis method, and connecting amino groups and carboxyl groups at two ends of the octapeptide to obtain octacyclic peptide; the amino acid sequence of the octacyclic peptide is Lys-Ser-Gly-Ser-Cys-Ser-Gly-Ser, which is abbreviated as KSGSCSGS, and is shown as SEQ ID NO.1 of an amino acid sequence table; the octacyclic peptide comprises the following components in a molar ratio of 1:1 with a cysteine residue; lys represents lysine, ser represents serine, thr represents threonine, gly represents glycine, ala represents alanine, cys represents cysteine.
S12, dissolving 10 mg octacyclic peptide in phosphate buffer with pH=7.4, adding 100 mu L of NSP-SA-NHS (acridine ester) with the concentration of 5 mg/mL, and mixing by vortex;
S13, after vortex mixing uniformly, carrying out reaction at 37 ℃ and 1h, separating and purifying by High Performance Liquid Chromatography (HPLC), wherein a chromatographic column is C18 (CG 12A-8 um,4 x 50 mm), a mobile phase is acetonitrile, and separating and purifying to obtain the mercapto activated octacyclopeptide acridine salt.
Wherein the solid-phase synthesis method of the polypeptide in the step S11 comprises the following steps:
D1, weighing 0.5g Fmoc-Gly-Wang resin (substitution degree is 1.0 mmol/g), swelling with dichloromethane of about 3 times of resin volume for 30 min, and washing with 2 times of N, N-Dimethylformamide (DMF) for 5 times;
After the washing is finished, adding 3 times of volume of 20% 4-methylpiperidine/DMF, oscillating at 25 ℃ for 30min times for deprotection, and washing with DMF for 5 times;
D3, after washing, a small amount of resin is taken, the resin is washed twice by absolute ethyl alcohol, 500 mu L of ninhydrin color development liquid (1.5 g ninhydrin and 5g phenol are added into 50 mL n-butyl alcohol) is added, the reaction is carried out at 110 ℃ for 5min, the resin turns blue to be deprotected, and the next step is carried out; repeating the deprotection reaction of the step D2 if the resin does not turn blue;
d4, adding 1.5 mmol Fmoc-Ser (tBu) -OH to the deprotected resin, adding 1.8 mol of N, N-diisopropylethylamine and 1.67mol of HBTU peptide coupling reagent respectively, dissolving with DMF, reacting 30min, washing with 2 times of volume of DMF for 5 times;
D5, after washing, taking a small amount of resin, washing twice by absolute ethyl alcohol, adding 500 mu L of ninhydrin color-developing solution, reacting at 110 ℃ for 5 min, condensing until the resin is not changed into blue, and carrying out the next step, otherwise repeating the condensation reaction of the step D4;
D6, repeating the step D2, adding the 3 rd protected amino acid, and condensing Fmoc-Cys (OtBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Gly-OH, fmoc-Ser (tBu) -OH and Fmoc-Lys (Trt) -OH according to the step D4-step D5 respectively; after the condensation and deprotection of the linear octapeptide are completed, adding triethylamine for neutralization, and oscillating at room temperature for 12 h to obtain octacyclic peptide;
D7, adding a cutting fluid (the cutting fluid comprises trifluoroacetic acid, water, triisopropylsilane=95% (2.5% (2.5%) and the volume percentage) into the octacyclic peptide, and reacting 2h; after the reaction is finished, adding the mixture into glacial ethyl ether, performing centrifugal precipitation, washing to obtain an octacyclic peptide crude product, purifying by liquid chromatography, collecting target liquid, and performing freeze-drying to obtain octacyclic peptide freeze-dried powder.
Example 2
A method for preparing a homogeneous chemiluminescent procalcitonin detection reagent, which comprises the following steps:
①Fe3O4 Nano-enzyme labeled procalcitonin monoclonal antibody a:
1) Antibody a desalting: desalting the labeled antibody A by using a PD10 desalting column to remove substances interfering with subsequent reactions;
2) Pretreatment of Fe 3O4 nano enzyme: controlling pH to be in the range of 8.6-10.0, carrying out alkaline treatment on Fe 3O4 nano enzyme, and keeping the pH to be maximum on the premise of ensuring that nano particles are stable and not aggregated, thereby being used for efficiently labeling the antibody.
3) Labeling reaction: in a proper buffer system, fe 3O4 nano enzyme in alkaline environment is mixed with antibody A solution, mild mixing is carried out for 45 min at 30 ℃, stable non-covalent connection is formed between Fe 3O4 nano enzyme and antibody A, 2 wt% BSA buffer solution is added for sealing, mild mixing is carried out for 45 min at 30 ℃, PBS buffer solution containing 0.05 wt% Tween 20 is used for magnetic separation, washing is carried out for three times, and the mixture is stored in R1 buffer solution.
4) Potency determination and validation: after the coupling is completed, ELISA identification is carried out on the prepared Fe 3O4 nano enzyme marked procalcitonin monoclonal antibody A, and the original specific binding capacity is reserved and the prepared procalcitonin monoclonal antibody A has good enzyme activity.
5) Split charging and preserving: the obtained Fe 3O4 nano enzyme marked procalcitonin monoclonal antibody A is split-packed in a proper R1 buffer solution and stored at a low temperature (such as-20 ℃ or lower) so as to keep the stability of the monoclonal antibody A for subsequent experiments.
② Cyclopeptide luminescent coupling molecule marked procalcitonin monoclonal antibody B:
1) Antibody pre-activation treatment: removing metal ions and impurities containing primary amino groups in the antibody solution by means of dialysis and the like, avoiding influencing subsequent coupling, replacing the coupling buffer solution with PBS buffer solution with pH 8.0, adding SM (PEG) 2 activator with 6 times of the molar quantity of the antibody to enable the antibody to modify maleimide groups, and then desalting and purifying to remove redundant activator.
2) Chemiluminescent coupling molecules: preparing thiol-activated dodecacyclic acridine salt, wherein the amino acid sequence of the dodecacyclic peptide in the thiol-activated dodecacyclic acridine salt is Lys-Ser-Gly-Ser-Lys-Ser-Gly-Ser-Cys-Ser-Gly-Ser, which is abbreviated as KSGSKSGSCSGS, as shown in an amino acid sequence table SEQ ID NO. 2; thiol-activated dodecacyclic acridine salt was dissolved in DMSO or acetonitrile at a concentration of 3mg/mL, with the addition of 1mM of ACEP to avoid disulfide formation.
3) Labeling reaction: the antibody solution was concentrated or diluted to a concentration of 1-3 mg/mL, and then thiol-activated dodecapeptide acridine salt, pre-dissolved in DMSO or acetonitrile, was added in a molar ratio of 4:1 is put into an antibody solution, vortex mixing is carried out, and light-shielding reaction is carried out for 2 hours at 25 ℃. The reaction mixture is purified by molecular sieve to obtain the cyclopeptide luminescent coupling molecular labeled procalcitonin monoclonal antibody B, as shown in figure 2.
4) Labeling efficiency and activity detection: and (3) detecting the luminous efficiency of the obtained cyclopeptide luminous coupling molecular labeled procalcitonin monoclonal antibody B, and simultaneously evaluating whether the labeling process influences the specificity and affinity of the procalcitonin or not, wherein the evaluation can be carried out through enzyme dynamics and sensitivity.
The procedure for the preparation of procalcitonin calibrator and quality control was the same as in example 1.
The preparation of thiol-activated dodecacyclopeptide acridinium salt is different from that of the thiol-activated octacyclopeptide acridinium salt in the embodiment 1 in that the cyclopeptide synthesized in the step S11 is dodecacyclopeptide, and the amino acid sequence is Lys-Ser-Gly-Ser-Lys-Ser-Gly-Ser-Cys-Ser-Gly-Ser, and is abbreviated as KSGSKSGSCSGS, as shown in the SEQ ID NO.2 of the amino acid sequence table.
Example 3
A method for preparing a homogeneous chemiluminescent procalcitonin detection reagent, which comprises the following steps:
① Preparing Fe 3O4 nano enzyme marked procalcitonin monoclonal antibody A:
1) Antibody a desalting: desalting the labeled antibody A by using a PD10 desalting column to remove substances interfering with subsequent reactions;
2) Pretreatment of Fe 3O4 nano enzyme: controlling the pH value to be in the range of 8.6-10.0, carrying out alkaline treatment on Fe 3O4 nano enzyme, and keeping the pH value to be the maximum on the premise of ensuring that nano particles are stable and not aggregated, thereby being used for efficiently marking the antibody A;
3) Labeling reaction: mixing Fe 3O4 nano enzyme in alkaline environment with antibody solution in a proper buffer solution system, gently mixing at 25 ℃ for 60 min to form stable non-covalent connection between Fe 3O4 nano enzyme and antibody A, adding 0.5 wt% BSA buffer solution for sealing, gently mixing at 25 ℃ for 60 min, then magnetically separating by using PBS buffer solution containing 0.05 wt% Tween 20, washing for three times, and storing in R1 buffer solution;
4) Potency determination and validation: after the coupling is completed, ELISA identification is carried out on the prepared Fe 3O4 nano enzyme marked procalcitonin monoclonal antibody A, and the original specific binding capacity is reserved and the prepared procalcitonin monoclonal antibody A has good enzyme activity;
5) Split charging and preserving: the obtained Fe 3O4 nano enzyme marked procalcitonin monoclonal antibody A is split-packed in a proper R1 buffer solution and stored at a low temperature (such as-20 ℃ or lower) so as to keep the stability of the monoclonal antibody A for subsequent experiments.
② Cyclopeptide luminescent coupling molecule marked procalcitonin monoclonal antibody B:
1) Pre-activation treatment of antibody B: removing metal ions and impurities containing primary amino groups in an antibody solution by means of dialysis and the like, avoiding influencing subsequent coupling, replacing a coupling buffer solution with a PBS buffer solution with pH of 7.6, adding an SM (PEG) 2 activator with the amount which is 6 times that of the antibody, modifying the antibody B with a maleimide group, and then desalting and purifying to remove the redundant activator;
2) Chemiluminescent coupling molecules: dissolving sulfhydryl activated octacyclic peptide acridinium salt in DMSO or acetonitrile solution with concentration of 2-4 mg/mL, and simultaneously adding TCEP of 1mM to avoid disulfide bond formation;
3) Labeling reaction: the antibody B solution was concentrated or diluted to a concentration of 1mg/mL, and then the thiol-activated octacyclic peptide acridine salt, pre-dissolved in DMSO or acetonitrile, was added in a molar ratio of 1:1, adding the mixture into an antibody B solution (mercapto activated octacyclopeptide acridinium salt: antibody B=1:1), carrying out vortex mixing, carrying out light-shielding reaction at 25 ℃ for 2 hours, and purifying the reaction mixture by a molecular sieve to obtain a cyclopeptide luminescent coupling molecular labeled procalcitonin monoclonal antibody B (see figure 1);
4) Labeling efficiency and activity detection: and (3) detecting the luminous efficiency of the obtained cyclopeptide luminous coupling molecular labeled procalcitonin monoclonal antibody B, and simultaneously evaluating whether the original specificity and affinity of the antibody B are influenced in the labeling process or not, wherein the evaluation can be performed through enzyme dynamics and sensitivity.
③ Preparing procalcitonin calibrator and quality control product:
1) Preparing a calibrator buffer: 0.10 g/L of potassium dihydrogen phosphate, 1.00 g/L of disodium hydrogen phosphate, 5.0 g/L of sodium chloride, 0.1g/L of potassium chloride, 0.01 wt% of bovine serum albumin, 0.01 wt% of Proclin 300, 0.01 wt% of Tween 20, 1.00 wt% of trehalose and 3.00 wt% of glycerol are prepared into a calibrator buffer.
2) Preparing a calibrator and a quality control product: the calibrator and the quality control product are prepared by using procalcitonin pure product and calibrator buffer, and the concentrations of the calibrator are respectively as follows: 0.05 ng/mL, 45 ng/mL, 500 ng/mL, 800 ng/mL, 1000 ng/mL, 10000 ng/mL, and the quality control product concentration is: 50 ng/mL, 1200/ng/mL.
Example 4
A method for preparing a homogeneous chemiluminescent procalcitonin detection reagent, which comprises the following steps:
① Preparing Fe 3O4 nano enzyme marked procalcitonin monoclonal antibody A:
1) Antibody a desalting: desalting the labeled antibody A by using a PD10 desalting column to remove substances interfering with subsequent reactions;
2) Pretreatment of Fe 3O4 nano enzyme: controlling the pH value to be in the range of 8.6-10.0, carrying out alkaline treatment on Fe 3O4 nano enzyme, and keeping the pH value to be the maximum on the premise of ensuring that nano particles are stable and not aggregated, thereby being used for efficiently marking the antibody A;
3) Labeling reaction: mixing Fe 3O4 nano enzyme in alkaline environment with antibody solution in a proper buffer solution system, gently mixing at 37 ℃ for 30 min to form stable non-covalent connection between Fe 3O4 nano enzyme and antibody A, adding 3 wt% BSA buffer solution for sealing, gently mixing at 37 ℃ for 30 min, then magnetically separating by using PBS buffer solution containing 0.05 wt% Tween 20, washing for three times, and storing in R1 buffer solution;
4) Potency determination and validation: after the coupling is completed, ELISA identification is carried out on the prepared Fe 3O4 nano enzyme marked procalcitonin monoclonal antibody A, and the original specific binding capacity is reserved and the prepared procalcitonin monoclonal antibody A has good enzyme activity;
5) Split charging and preserving: the obtained Fe 3O4 nano enzyme marked procalcitonin monoclonal antibody A is split-packed in a proper R1 buffer solution and stored at a low temperature (such as-20 ℃ or lower) so as to keep the stability of the monoclonal antibody A for subsequent experiments.
② Cyclopeptide luminescent coupling molecule marked procalcitonin monoclonal antibody B:
1) Pre-activation treatment of antibody B: removing metal ions and impurities containing primary amino groups in an antibody solution by means of dialysis and the like, avoiding influencing subsequent coupling, replacing a coupling buffer solution with a PBS buffer solution with pH of 8.6, adding an SM (PEG) 2 activator with the amount which is 6 times that of the antibody, modifying the antibody B with a maleimide group, and then desalting and purifying to remove the redundant activator;
2) Chemiluminescent coupling molecules: dissolving sulfhydryl activated octacyclic peptide acridinium salt in DMSO or acetonitrile solution with concentration of 2-4 mg/mL, and simultaneously adding TCEP of 1mM to avoid disulfide bond formation;
3) Labeling reaction: the antibody B solution was concentrated or diluted to a concentration of 3mg/mL, and then the thiol-activated octacyclic peptide acridine salt, pre-dissolved in DMSO or acetonitrile, was added in a molar ratio of 6:1, adding the mixture into an antibody B solution (mercapto activated octacyclopeptide acridinium salt: antibody B=6:1), carrying out vortex mixing, carrying out light-shielding reaction at 25 ℃ for 2 hours, and purifying the reaction mixture by a molecular sieve to obtain a cyclopeptide luminescent coupling molecular labeled procalcitonin monoclonal antibody B (see figure 1);
4) Labeling efficiency and activity detection: and (3) detecting the luminous efficiency of the obtained cyclopeptide luminous coupling molecular labeled procalcitonin monoclonal antibody B, and simultaneously evaluating whether the original specificity and affinity of the antibody B are influenced in the labeling process or not, wherein the evaluation can be performed through enzyme dynamics and sensitivity.
③ Preparing procalcitonin calibrator and quality control product:
1) Preparing a calibrator buffer: 0.27 g/L of potassium dihydrogen phosphate, 1.45 g/L of disodium hydrogen phosphate, 8.0 g/L of sodium chloride, 0.2 g/L of potassium chloride, 5.00 wt% of bovine serum albumin, 0.1 wt% of Proclin 300, 0.10 wt% of Tween 20, 5.00 wt% of sucrose and 10.00 wt% of glycerol are prepared into a calibrator buffer;
2) Preparing a calibrator and a quality control product: the calibrator and the quality control product are prepared by using procalcitonin pure product and calibrator buffer, and the concentrations of the calibrator are respectively as follows: 0.05 ng/mL, 45 ng/mL, 500 ng/mL, 800 ng/mL,1000 ng/mL, 10000 ng/mL, and the quality control product concentration is: 50 ng/mL, 1200/ng/mL.
Example 5
A procalcitonin detection method has a reaction mechanism shown in figure 3, and procalcitonin PCT in a sample to be detected can form a double-antibody sandwich structure with a procalcitonin monoclonal antibody A marked by Fe 3O4 nanometer enzyme and a procalcitonin monoclonal antibody B marked by cyclopeptide luminescent coupling molecules. When hydrogen peroxide is added into the system, fe 3O4 nano enzyme in the immune complex system rapidly catalyzes hydrogen peroxide to generate oxygen free radical, and further activates cyclopeptide luminescent coupling molecules to trigger chemiluminescent reaction. The Fe 3O4 nano enzyme which does not participate in immune reaction can not effectively catalyze the light signal emitted by the cyclopeptide luminescence coupling molecular labeled procalcitonin monoclonal antibody B under the non-composite state due to the inhibition effect of the reducing substances in the solution, so that the background noise is lower. In this way, the luminescence intensity directly reflects the concentration of procalcitonin PCT in the sample, thereby realizing quantitative determination of procalcitonin PCT. Based on the homogeneous chemiluminescent procalcitonin detection reagent obtained in example 1, the procedure was as follows:
Respectively taking 20 mu L of calibrator with different concentrations, uniformly mixing with 40 mu L R of reagent and 40 mu L R of reagent, placing the mixture at 37 ℃ for reaction 15min, adding a luminescent substrate, and using PMT to read 2 s photons to obtain a signal value;
performing four-parameter fitting curve on the concentration value of the calibrator and the photon value obtained by reading (see table 1), wherein SD represents standard deviation, CV represents variation coefficient, and a procalcitonin standard dose curve (shown in figure 4) is obtained;
TABLE 1 photon values read with the homogeneous chemiluminescent procalcitonin detection reagent obtained in example 1
Taking 20 mu L of a sample to be detected, uniformly mixing the sample with a 40 mu L R reagent and a 40 mu L R reagent, placing the mixture at 37 ℃ to react with 15min, adding a luminescent substrate, and using a PMT to read 2 s photons to obtain a signal value; procalcitonin concentrations were calculated from procalcitonin standard dose curves.
Example 6
The embodiment provides a procalcitonin detection kit, which comprises the procalcitonin detection reagent in embodiment 1.
While embodiments of the present invention have been described in detail hereinabove, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. It is to be understood that such modifications and variations are within the scope and spirit of the present invention as set forth in the following claims. Moreover, the invention described herein is capable of other embodiments and of being practiced or of being carried out in various ways.
Claims (9)
1. A homogeneous chemiluminescent procalcitonin detection reagent constructed by a cyclopeptide luminescent coupling molecule, which is characterized by comprising the following components:
r1 reagent: comprises a procalcitonin monoclonal antibody A marked by Fe 3O4 nano enzyme;
r2 reagent: comprises a procalcitonin monoclonal antibody B marked by a cyclopeptide luminescent coupling molecule;
The cyclic peptide luminous coupling molecule is octacyclic peptide or dodecacyclic peptide coupled acridine salt, the amino acid sequence of the octacyclic peptide is Lys-Ser-Gly-Ser-Cys-Ser-Gly-Ser, and the amino acid sequence of the dodecacyclic peptide is Lys-Ser-Gly-Ser-Lys-Ser-Gly-Ser-Cys-Ser-Gly-Ser.
2. The homogeneous chemiluminescent procalcitonin detection reagent of claim 1 wherein the R1 reagent further comprises an R1 buffer having a pH of 6.0-7.5 comprising 20-100 mM Tris,100 mM NaCl,0.01-0.1 wt.% Tween20,1-5 wt.% BSA,1-5 wt.% trehalose or sucrose, 1-5 wt.% glycerol, and 0.09 wt.% sodium azide or Proclin 300;
The R2 reagent also comprises an R2 buffer, the pH of which is 6.0-8.5, containing 10-50 mM NaH2PO4,10-50 mM Na2HPO4,100 mM NaCl,0.01-0.1wt% Tween20,1-5wt% BSA,1-5wt% trehalose or sucrose, 1-5wt% glycerol, and 0.09wt% sodium azide or Proclin 300.
3. The homogeneous chemiluminescent procalcitonin detection reagent of claim 1 wherein the R2 reagent comprises 0.1-5.0 wt% protein stabilizer, 0.05-0.2% preservative, 0.01-0.1 wt% surfactant.
4. The homogeneous chemiluminescent procalcitonin detection reagent of claim 1 wherein the R2 reagent further comprises a background reducing agent.
5. The homogeneous chemiluminescent procalcitonin detection reagent of claim 4 wherein the background reducing agent is one or both of vitamin C and sodium sulfite.
6. The homogeneous chemiluminescent procalcitonin detection reagent constructed by the cyclopeptide luminescent coupling molecule according to claim 1, wherein the procalcitonin detection reagent further comprises procalcitonin PCT series calibrator, quality control product and luminescent substrate solution.
7. A method of preparing a homogeneous chemiluminescent procalcitonin detection reagent according to any one of claims 1 to 6 comprising:
① Preparing Fe 3O4 nano enzyme marked procalcitonin monoclonal antibody A:
Mixing Fe 3O4 nano enzyme in an alkaline environment with an antibody A solution in a buffer solution system, gently mixing the mixture at 25-37 ℃ for 30-60 min to form stable non-covalent connection between the Fe 3O4 nano enzyme and the antibody A, adding 0.5-3% BSA buffer solution for sealing, gently mixing the mixture at 25-37 ℃ for 30-60 min, then magnetically separating and cleaning the mixture by using PBS buffer solution containing 0.05% Tween 20 to obtain a Fe 3O4 nano enzyme marked procalcitonin monoclonal antibody A, and storing the procalcitonin monoclonal antibody A in an R1 buffer solution;
② Cyclopeptide luminescent coupling molecule marked procalcitonin monoclonal antibody B: concentrating or diluting the antibody B solution to a concentration of 1-3 mg/mL, and then dissolving the cyclopeptide luminescent coupling molecule in DMSO or acetonitrile in advance to obtain the cyclopeptide luminescent coupling molecule: antibody = 1-6: and (3) adding the mixture into an antibody B solution according to the molar ratio of 1, carrying out vortex mixing and light-shielding reaction, and purifying the reaction mixture to obtain the cyclopeptide luminescent coupling molecular labeled procalcitonin monoclonal antibody B.
8. The method of claim 7, further comprising preparing procalcitonin calibrants and quality control:
Preparing a calibrator buffer: 0.10-0.27 g/L of monopotassium phosphate, 1.00-1.45 g/L of disodium hydrogen phosphate, 5.0-8.0 g/L of sodium chloride, 0.1-0.2 g/L of potassium chloride, 0.01-5.00 wt% of bovine serum albumin, 0.01-0.1 wt% of preservative sodium azide or Proclin 300, 0.01-0.10 wt% of Tween 20, 1.00-5.00 wt% of sucrose or trehalose, and 3.00-10.00 wt% of glycerol are prepared into a calibrator buffer solution;
Preparing a calibrator and a quality control product: the calibrator and the quality control product are prepared by using procalcitonin pure product and calibrator buffer, and the concentrations of the calibrator are respectively as follows: 0.05 ng/mL, 45 ng/mL, 500 ng/mL, 800 ng/mL, 1000 ng/mL, 10000 ng/mL, and the quality control concentrations are respectively: 50 ng/mL,1200 ng/mL.
9. A homogeneous chemiluminescent procalcitonin detection kit constructed by a cyclopeptide luminescent coupling molecule, wherein the kit comprises the homogeneous chemiluminescent procalcitonin detection reagent of any one of claims 1-6.
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