CN115684432A - Detection method and detection kit for content of calprotectin in blood - Google Patents
Detection method and detection kit for content of calprotectin in blood Download PDFInfo
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Abstract
The invention relates to the technical field of biological detection, in particular to a detection method and a detection kit for the content of calprotectin in blood. The detection method provided by the invention is used for enriching calprotectin in a sample to be detected by using an antigen-antibody combination method and then analyzing by using a liquid chromatography-tandem mass spectrometry method. The detection method has high sensitivity and covers the whole content range from healthy people to virus infected people. The detection method has simple pretreatment operation, shortens the analysis time and improves the analysis speed.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a detection method and a detection kit for the content of calprotectin in blood.
Background
Calprotectin is a calcium and zinc binding protein with the molecular weight of 36 kD and abundant content, belongs to a sub-S100 family, and is heterotrimer formed by connecting calcium ions by two heavy chains (S100A 9) with the molecular weight of 14 kD and a light chain (S100A 8) with the molecular weight of 8 kD through covalent bonds. Calprotectin accounts for 5% of total protein and 45% of neutrophil cytoplasmic protein, has antibacterial and antifungal characteristics, is increased under many inflammatory conditions, such as arthritis, chronic Obstructive Pulmonary Disease (COPD) and Inflammatory Bowel Disease (IBD), is the strongest independent prediction factor for IBD diagnosis, can accurately predict IBD inflammatory burden by combining other blood biomarkers, and has the potential of predicting diseases and outcome thereof, and the calprotectin detection becomes the 'gold standard' for judging the therapeutic effect of IBD treatment at present.
Calprotectin is widely distributed in human tissues, body fluids and cells, the calprotectin content of different parts is different, and the calprotectin content in excrement is highest, so the calprotectin is the most commonly used detection matrix at present, but the defects of poor patient compliance, unstable detection result and the like exist in the excrement calprotectin detection, so researchers at home and abroad begin to try to use blood serum as the matrix for detecting the calprotectin content determination.
For the quantitative detection of macromolecular proteins, the traditional methods such as an enzyme-linked immunosorbent assay, a chemiluminescence method and a latex turbidimetric method are mainly used. The traditional method for realizing quantitative determination based on the principle of antigen-antibody combination is difficult to avoid the problem of non-specific combination with structural similarity protein, so the result accuracy is low and the accurate assignment is difficult. Moreover, the content of calprotectin in the blood plasma is low, for example, the content of calprotectin in the blood plasma of healthy adults is 0.1-0.6 ng/muL, and the content of virus infected persons is 0.1-11.4 ng/muL; the concentration of the bacteria infected patients is 0.6-11.0 ng/. Mu.L. Therefore, the sensitivity requirement for the determination of the calprotectin content in the plasma is high.
In view of this, the invention is particularly proposed.
Disclosure of Invention
In order to solve the technical problems, the invention provides a detection method and a detection kit for the content of calprotectin in blood.
The invention provides a method for detecting the content of calprotectin in blood, which is characterized in that after the calprotectin in a sample to be detected is enriched by an antigen-antibody combination method, the calprotectin is analyzed by a liquid chromatography-tandem mass spectrometry method.
The detection method at least comprises the following steps:
s1, preparing a standard curve equation, which comprises the following steps:
s11, preparing a standard working solution and an internal standard working solution with gradient concentration; the standard working solution contains calprotectin recombinant protein standard, and the internal standard working solution contains at least two characteristic peptide internal standards;
s12, preparing a standard solution: respectively adding blank plasma matrixes into the standard working solution with the gradient concentration to obtain standard solutions with serial concentrations;
s13, enriching calprotectin recombinant proteins in the standard solutions with the series of concentrations by using an antigen-antibody combination method to obtain standard solution enrichment solutions with the series of concentrations;
s14, pretreating the standard solution enrichment solution with the series of concentrations to obtain a standard solution loading solution with the concentration;
s15, detecting the sample loading liquid of the standard solution by using a high performance liquid chromatography-mass spectrometer to obtain a standard curve equation;
s2, processing to obtain a sample loading liquid of the sample to be detected, comprising:
s21, enriching a sample to be detected by using an antigen-antibody binding method to obtain an enriched liquid of the sample to be detected;
s22, preprocessing the enrichment solution of the sample to be detected to obtain a sample loading solution of the sample to be detected;
s3, detecting a sample to be detected, comprising: and detecting at least two characteristic peptide fragments in the sample loading solution of the sample to be detected by using a high performance liquid chromatography-mass spectrometer, and substituting the detection result of the quantitative peptide fragments into a standard curve equation to obtain the concentration of the calprotectin in the sample to be detected.
The invention also provides a kit for quantitatively detecting calprotectin in blood, which comprises the following components:
a protein extraction reagent comprising: an anti-calprotectin monoclonal antibody reagent marked by magnetic beads and an antigen-antibody conjugate dissociating agent;
pretreatment reagents: protein reducing agents, protein alkylating agents, proteolytic enzymes;
standard solution reagents: the volume ratio of the standard solution to the blank plasma matrix is 1:79 of a solvent; the standard working solution is a solution with water as a solvent and a calprotectin recombinant protein standard as a solute;
an internal standard working solution reagent; taking an aqueous solution containing 30-40% by volume of acetonitrile and 0.05-0.2% by volume of formic acid as a solvent, and comparing the mass ratio of a quantitative characteristic peptide internal standard substance to a qualitative characteristic peptide internal standard substance by 1:1, the mixture is a solution of solute; wherein, calprotectin quantitative characteristic peptide fragment internal standard substance: peptide fragment LGHPDTLNQGEFK (isotope labeled peptide of peptide fragment 4): calprotectin qualitative characteristic peptide fragment internal standard adopts peptide fragment LTWASHEK (isotope labeled peptide of peptide fragment 5);
mobile phase reagent: phase A: water containing 0.05-0.2% by volume of formic acid, phase B: acetonitrile containing 0.05-0.2% by volume of formic acid.
Compared with the prior art, the technical scheme provided by the embodiment of the invention has the following advantages:
the detection method has high sensitivity, enables the quantitative range to reach 0.049 ng/muL-12.5 ng/muL, and covers the full range of contents from healthy people to virus infected people.
The detection method disclosed by the invention is simple in pretreatment operation and reduces the requirements on technicians.
The detection method shortens the analysis time and improves the analysis speed, wherein the enrichment and the pretreatment can be completed within 5 hours, the liquid chromatography-tandem mass spectrometry detection can be completed within 9.5 minutes, the whole detection process can be completed within 5.5 hours, the pretreatment process is simplified, the experiment period is shortened, and the requirements on experimenters are reduced. The detection method provided by the invention has the advantages of high sensitivity, good reproducibility and high recovery rate, the quantitative range is 0.049 ng/muL-12.5 ng/muL, the full content range from healthy people to virus infected people is covered, and the detection performance is better than that of the traditional method.
In the preferred technical scheme of the invention, the plasma matrix is added into the standard curve working solution, so that the consistency of the pretreatment method is ensured.
Drawings
FIG. 1 is a mass spectrum of 2 calprotectin-characteristic peptide fragments in a plasma sample;
FIG. 2 is a mass spectrum of calprotectin characteristic peptide fragment 4 isotope internal standard in a plasma sample;
fig. 3 is a calprotectin characteristic peptide fragment 5 isotope internal standard mass spectrogram in a plasma sample;
FIG. 4 is a curve of quantification of calprotectin standard solution in a plasma sample;
FIG. 5 is a mass spectrum of a characteristic peptide fragment of a plasma sample at a column temperature of 20 ℃, wherein the right arrow represents a peptide fragment 4, and the left arrow represents a peptide fragment 5;
FIG. 6 is a mass spectrogram of a characteristic peptide fragment of a plasma sample at a column temperature of 40 ℃, wherein a right arrow represents a peptide fragment 4, and a left arrow represents a peptide fragment 5;
FIG. 7 is a mass spectrogram of a characteristic peptide fragment of a plasma sample at a column temperature of 60 ℃, wherein a right arrow represents a peptide fragment 4, and a left arrow represents a peptide fragment 5;
FIG. 8 is a mass spectrum of a characteristic peptide fragment of a plasma sample when a chromatographic column A is adopted;
FIG. 9 is a standard curve using column A;
FIG. 10 is a mass spectrum of a characteristic peptide fragment of a plasma sample using a chromatographic column B;
FIG. 11 is a standard curve using column B;
FIG. 12 is a mass spectrum of a characteristic peptide fragment of a plasma sample using column C;
FIG. 13 is a standard curve using column C;
FIG. 14 is a mass spectrum of a characteristic peptide fragment of a plasma sample using column D;
FIG. 15 is a standard curve using column D;
FIG. 16 is a mass spectrum of a characteristic peptide fragment of a plasma sample using column E;
FIG. 17 is a standard curve using column E;
FIG. 18 is a mass spectrum of a characteristic peptide fragment of a plasma sample under mobile phase condition 1;
FIG. 19 is a standard curve using mobile phase condition 1;
FIG. 20 is a mass spectrum of a characteristic peptide fragment of a plasma sample under mobile phase condition 2;
FIG. 21 is a standard curve using mobile phase condition 2;
FIG. 22 is a mass spectrum of a characteristic peptide fragment of a plasma sample under mobile phase condition 3;
FIG. 23 is a standard curve using mobile phase condition 3;
FIG. 24 is a mass spectrum of a characteristic peptide fragment in a plasma sample when an elution gradient with a detection duration of 9 min is used;
FIG. 25 is a standard curve of elution gradient at a detection time of 9 min;
figure 26 is a standard curve using 0.060 μ g/μ L of anti-calprotectin monoclonal antibody.
Detailed Description
In order that the above objects, features and advantages of the present invention may be more clearly understood, a solution of the present invention will be further described below. It should be noted that the embodiments of the present invention and features of the embodiments may be combined with each other without conflict.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those described herein; it is to be understood that the embodiments described in this specification are only some embodiments of the invention, and not all embodiments.
Because the content of calprotectin is low, the traditional detection method has low detection accuracy and poor sensitivity and is difficult to accurately assign. With the development of mass spectrometric detection technology, it is possible to detect macromolecular proteins in samples by using a liquid mass method. However, the liquid method has high requirements on the sample, so that accurate experimental results are difficult to obtain if the pretreatment treatment is inappropriate, or the requirement of large-flux detection cannot be met due to the complex pretreatment method and long experimental period. In view of this, the embodiment of the invention provides a mass spectrometry-based calprotectin quantitative detection technology, and a novel pretreatment method is initiated, wherein an antigen-antibody binding method is used for enriching calprotectin in a sample to be detected, then the calprotectin in the sample to be detected is enriched, and a liquid chromatography-tandem mass spectrometry method is used for analyzing. The detection method utilizes the specificity of antigen-antibody combination, simplifies the pretreatment process, shortens the experimental period, reduces the requirements on experimenters, can also improve the sensitivity, widens the linear range, and achieves better detection performance than the traditional method.
As an improvement of the embodiment of the invention, the antigen-antibody combination method specifically adopts an anti-calprotectin monoclonal antibody marked by magnetic beads to combine with calprotectin in a serum sample to be detected, obtains an antigen-antibody combination by magnetic sorting, dissociates the antigen-antibody combination after obtaining the combination, and further completes the enrichment of the calprotectin in the serum sample to be detected. The embodiment of the invention utilizes the specificity of antigen-antibody combination, improves the purity of the pre-treated sample, and ensures the reliability, accuracy and sensitivity of detection.
As an improvement of the embodiment of the invention, the method for detecting the content of the calprotectin in the blood by combining the antigen-antibody binding method with the liquid chromatography-tandem mass spectrometry method at least comprises the following steps:
s1, preparing a standard curve equation, which comprises the following steps:
s11, preparing a standard working solution and an internal standard working solution with gradient concentration; the standard working solution contains calprotectin recombinant protein standard, and the internal standard working solution contains internal standards of at least two characteristic peptide sections;
s12, preparing a standard solution: respectively adding blank plasma matrixes into the standard working solution with the gradient concentration to obtain standard solutions with series concentrations;
s13, enriching calprotectin recombinant proteins in the standard solutions with the series of concentrations by using an antigen-antibody combination method to obtain enriched solutions of the standard solutions with the series of concentrations;
s14, preprocessing the standard solution enrichment solutions with the series of concentrations to obtain standard solution loading solutions with the series of concentrations;
s15, detecting the sample loading liquid of the standard solution by using a high performance liquid chromatography-mass spectrometer to obtain a standard curve equation;
s2, processing to obtain a sample loading solution of the sample to be detected, which comprises the following steps:
s21, enriching a sample to be detected by using an antigen-antibody combination method to obtain an enrichment solution of the sample to be detected;
s22, preprocessing the enrichment solution of the sample to be detected to obtain a sample loading solution of the sample to be detected;
s3, detecting a sample to be detected, comprising the following steps: and detecting at least two characteristic peptide fragments in the sample loading solution of the sample to be detected by using a high performance liquid chromatography-mass spectrometer, and substituting the detection result of the quantitative characteristic peptide fragments into a standard curve equation to obtain the concentration of calprotectin in the sample to be detected.
The embodiment of the invention utilizes the serum sample as the detection matrix, thereby ensuring the consistency of the pretreatment method. As an improvement of the embodiment of the invention, the characteristic peptide segment has 8-15 amino acids; preferably, the characteristic peptide fragments comprise at least one qualitative characteristic peptide fragment and at least one quantitative characteristic peptide fragment; the internal standard substance of the characteristic peptide fragment is taken as an isotope label of the characteristic peptide fragment.
Specifically, the quantitative characteristic peptide fragment can be selected from peptide fragment LGHPDTLNQGEFK (peptide fragment 4 for short), and the qualitative characteristic peptide fragment can be selected from peptide fragment LTWASHEK (peptide fragment 5 for short). The quantitative characteristic peptide fragment internal standard substance is an isotope labeled peptide of the quantitative characteristic peptide fragment, and the qualitative characteristic peptide fragment internal standard substance is an isotope labeled peptide of the qualitative characteristic peptide fragment.
In the embodiment of the invention, the plasma matrix is added into the standard yeast working solution, so that the consistency of the pretreatment method is ensured.
As an improvement of the embodiment of the invention, the preparation method of the standard working solution comprises the following steps: the calprotectin recombinant protein standard is prepared into a solution with gradient concentration by adopting water as a solvent. Specifically, 1 mug/muL calprotectin recombinant protein can be used as a standard substance, and is diluted by purified water for 9 times step by step to prepare standard working solution with the concentration of 3.9063, 7.8125, 15.625, 31.25, 62.5, 125, 250, 500 and 1000 ng/muL.
The calprotectin recombinant protein standard can be selected from the following specific examples: anti-Calprotectin monoclonal antibody Anti-S100A9 + Calprotectin (S100A 8/A9 complex) antibody (MAC 387).
As an improvement of the embodiment of the invention, the internal standard working solution can adopt an aqueous solution containing 30-40% by volume of acetonitrile and 0.05-0.2% by volume of formic acid as a diluent, preferably an aqueous solution containing 35% by volume of acetonitrile and 0.1% by volume of formic acid as a diluent, and the internal standard substances of two characteristic peptide segments are mixed according to the weight ratio of 1:1 as internal standard, diluting to 10 ng/. Mu.L as internal standard, and storing the internal standard working solution at-20 ℃.
Specifically, the internal standard substance of two characteristic peptide fragments can be selected from: calprotectin quantitative characteristic peptide fragment internal standard: peptide stretch LGHPDTLNQGEFK (isotopically labeled peptide of peptide stretch 4): calprotectin qualitative characteristic peptide fragment internal standard substance: peptide segment ltwashiek (isotopically labeled peptide of peptide segment 5).
As an improvement of the embodiment of the present invention, in S12, the volume ratio of the standard working solution to the blank plasma matrix is 1:79. specifically, 2.5. Mu.L of each of the 9 standard solutions with different concentrations can be added to 197.5. Mu.L of blank plasma matrix to prepare 9 standard solutions with final concentrations of 0.049, 0.098, 0.195, 0.391, 0.781, 1.563, 3.125, 6.25 and 12.5 ng/. Mu.L.
As a refinement of the embodiment of the present invention, the enrichment in S13 comprises the following steps: combining the anti-calprotectin monoclonal antibody marked by the magnetic beads with calprotectin recombinant protein in the standard solution, obtaining an antigen-antibody conjugate through magnetic sorting, and dissociating the antigen-antibody conjugate after washing to obtain standard solution enrichment liquid.
As an improvement of the embodiments of the present invention, the enrichment in S21 comprises the steps of: combining the anti-calprotectin monoclonal antibody marked by the magnetic beads with a serum sample to be detected, obtaining an antigen-antibody conjugate through magnetic sorting, and dissociating the antigen-antibody conjugate after washing to obtain the enrichment solution of the sample to be detected.
In the enrichment process, the enrichment can be completed in about 45 min after a single sample is processed.
Wherein, the calprotectin-resistant monoclonal antibody marked by the magnetic beads can be prepared by adopting the prior art. As an improvement of the embodiment of the present invention, the preparation method of the magnetic bead labeled calprotectin-resistant monoclonal antibody can adopt the following steps: activating magnetic beads, combining the anti-calprotectin monoclonal antibody with the activated magnetic beads, and cleaning to remove the unbound antibody; preferably, the calprotectin-resistant monoclonal antibody is diluted to 0.075-0.5 mu g/mu L by adopting a binding buffer solution and then incubated with the activated magnetic beads; preferably, the mass-to-volume ratio of the anti-calprotectin monoclonal antibody to the magnetic bead is 1-10 μ g: 10.μ L, preferably 1.5 μ g: 10. mu L; the binding buffer contained: HEPES 20 mM, potassium acetate 110 mM, sodium acetate 5mM, magnesium acetate 2 mM, EDTA 1 mM, potassium hydroxide to adjust pH to 7.3; preferably, the combination condition is incubation for 10-30 minutes at room temperature; the washing conditions were resuspension in combination with buffer, gentle pipetting, placing the tubes on a magnetic rack, and discarding the supernatant.
The specific technical scheme can be as follows:
(1) Dynabeads activation: taking out Dynabeads Protein G10 muL, incubating for 5min by rotation, placing the test tube on a magnetic frame, and discarding the supernatant;
(2) Preparing an antibody: mu.L (1. Mu.g/. Mu.L) of anti-calprotectin monoclonal antibody is diluted with 20. Mu.L of binding buffer;
(3) Antibody binding: adding the diluted anti-calprotectin monoclonal antibody into activated Dynabeads for affinity reaction, incubating for 10 min at room temperature, placing the test tube on a magnetic frame, and discarding the supernatant;
(4) Removal of unbound antibody and residue: resuspend with 20. Mu.L of binding buffer, gently pipette, place the tube on a magnetic rack, and discard the supernatant.
As an improvement of the embodiment of the invention, the mass-to-volume ratio of the anti-calprotectin monoclonal antibody to the serum sample to be tested is 1.5 mug: 200. μ L. Specific conditions can be as follows: incubate at room temperature for 30 min with rotation.
As an improvement of the embodiment of the present invention, dissociation employs non-denaturing elution, specifically: adding an antigen-antibody conjugate dissociating agent to the antigen-antibody conjugate to separate the calprotectin from the anti-calprotectin monoclonal antibody, thereby collecting the calprotectin. Specifically, the following can be adopted: the complex was dissociated by rotary incubation at room temperature for 2 min.
The antigen-antibody conjugate dissociation agent can be acetic acid with volume percentage of 5%.
The specific technical scheme can be as follows:
(5) And (3) immunoprecipitation: adding 200 mu L of serum sample into the Dynabeads-Ab conjugate, performing rotary incubation at room temperature for 30 min, placing the test tube on a magnetic frame, and removing the supernatant;
(6) Removal of interference in serum that does not bind specifically to antibodies: add 50. Mu.L of washing buffer (binding buffer + 0.5% Triton X100) to gently resuspend, place the tube on a magnet, discard the supernatant, repeat three times;
(7) Non-denaturing elution: after removing the washing buffer, 10. Mu.L of 5% acetic acid was added to the Dynabeads-Ab-Ag conjugate, and placed on a magnetic frame, and the supernatant was retained.
As an improvement of the embodiment of the present invention, the preprocessing in S14 includes: adding internal standard working solution into the standard solution enrichment solution, drying, adding a protein reducing agent and a protein alkylating reagent for reductive alkylation, and then adding proteolytic enzyme for enzymolysis; stopping the reaction after the enzymolysis reaction is finished, and diluting the desalted sample solution by using a sample loading buffer solution to obtain a sample loading solution of the standard solution;
as an improvement of the embodiment of the present invention, the preprocessing in S21 includes: adding internal standard working solution into the enrichment solution of the sample to be tested, drying, adding a protein reducing agent and a protein alkylating reagent for reductive alkylation, and then adding proteolytic enzyme for enzymolysis; and stopping the reaction after the enzymolysis reaction is finished, and diluting the sample by using a sample loading buffer solution after desalting to obtain the sample loading solution of the sample to be detected.
Preferably, the ratio of the added solution after enriching every 200 μ L of the serum sample to be tested is as follows: adding 10 mu L of protein reduction reagent, 2 mu L of protein alkylation reagent and 0.05 mu g of trypsin into 1 mu L of internal standard working solution;
more preferably, the conditions for adding the protein reducing agent are as follows: heating in water bath at 37 deg.C for 30 min, adding protein alkylating reagent, and reacting at room temperature in dark place for 30 min; the conditions for adding trypsin are as follows: incubate at 45 ℃ for 3 hours, pH 7.4.
Wherein the protein reducing reagent is selected from a solution containing 6-8M of urea and 5-10 mM of dithiothreitol; the protein alkylating reagent is selected from iodoacetamide solution, and the concentration is preferably 10-20 mM; the protease is selected from trypsin with concentration of 0.05-0.2 μ g/μ L, preferably 0.1 μ g/μ L. The reagent for terminating the enzymolysis reaction is acid, preferably 10% formic acid by volume.
The specific technical scheme can be as follows: adding 1 μ L internal standard working solution into the enriched calprotectin, blowing to dry with nitrogen, adding 10 μ L protein reducing reagent (8M urea, 5mM dithiothreitol), vortex, mixing, dissolving, and heating in water bath at 37 deg.C for 30 min; continuously adding 10 mM iodoacetamide for alkylation for 2 mu L, and reacting for 30 min at room temperature in a dark place; after the reductive alkylation of the protein was completed, 50 mM ammonium bicarbonate was added in a volume of 3 times to adjust pH to 7.4, 0.5. Mu.L of trypsin was added at a concentration of 0.1. Mu.g/. Mu.L, the mixture was incubated at 45 ℃ for 3 hours, the experiment was terminated with 10. Mu.L of 10% formic acid, and the mixture was evaporated to dryness in vacuo.
As an improvement of the embodiment of the invention, the desalting step is further included, and preferably, the desalting treatment is performed by using a micro-scale chromatography column, such as Millipore ZipTip @ micro-scale chromatography column (Millipore, ZTC18S 096), and the desalting step can be performed by referring to the specification.
As an improvement of the embodiment of the present invention, the chromatographic conditions in the liquid chromatography-tandem mass spectrometry include:
mobile phase: phase A: water containing 0.1% formic acid, phase B: acetonitrile containing 0.1% formic acid;
the elution mode is gradient elution;
0.01 to 1.00 minutes: a:97%, B:3 percent;
1.01 to 4.00 minutes: a:55%, B:45 percent;
4.01 to 4.10 minutes: a:15%, B:85 percent;
4.11 to 5.00 minutes: a:15%, B:85 percent;
5.01 to 5.10 minutes: a:97%, B:3 percent;
5.11 to 9.50 minutes: a:97%, B:3 percent;
the flow rate is 0.2-0.4 mL/min, preferably 0.3 mL/min; the column temperature is 40-60 ℃, and preferably 40 ℃; the sample volume is 1 to 5 muL, and preferably 4 muL; analysis time 9.5 min.
As an improvement of the embodiment of the invention, the conditions in the liquid chromatography-tandem mass spectrometry adopt an electrospray ion source, a positive ion mode, a Full scan (Full MS) + a secondary scan (dd-MS 2) + multiple reaction monitoring (PRM);
ion pairing:
calprotectin quantitative characteristic peptide fragment: peptide stretch LGHPDTLNQGEFK (peptide stretch 4): parent ion 485.9124-daughter ion 480.2453; calprotectin qualitative characteristic peptide segment: peptide LTWASHEK (peptide 5): parent ion 324.5030-daughter ion 413.2143;
calprotectin quantitative characteristic peptide fragment internal standard substance: peptide stretch LGHPDTLNQGEFK (isotopically labeled peptide of peptide stretch 4): parent ion 488.5838-daughter ion 488.2595;
calprotectin qualitative characteristic peptide fragment internal standard substance: peptide fragment ltwashk (isotope-labeled peptide of peptide fragment 5): parent ion 327.1744-daughter ion 421.2285.
The embodiment of the invention also comprises a kit for quantitatively detecting calprotectin in blood, which comprises the following components:
a protein extraction reagent comprising: an anti-calprotectin monoclonal antibody reagent marked by magnetic beads and an antigen-antibody conjugate dissociating agent;
pretreatment reagents: protein reducing agents, protein alkylating agents, proteolytic enzymes;
preparation of standard solution reagent: the volume ratio is 1:79 standard working fluid and blank plasma matrix; the standard working solution is a solution with water as a solvent and a calprotectin recombinant protein standard as a solute;
an internal standard working solution reagent; taking an aqueous solution containing 30-40% by volume of acetonitrile and 0.05-0.2% by volume of formic acid as a solvent, and taking the mass ratio of a quantitative characteristic peptide internal standard substance to a qualitative characteristic peptide internal standard substance as 1:1 the mixture is a solution of solute; the solvent is preferably an aqueous solution containing 35% by volume acetonitrile and 0.1% by volume formic acid;
mobile phase reagent: phase A: water containing 0.05-0.2% by volume of formic acid, phase B: acetonitrile containing 0.05-0.2% formic acid by volume. Preferably, the mobile phase reagents: phase A: water containing formic acid at a concentration of 0.1% by volume, phase B: acetonitrile containing 0.1% formic acid by volume. The reagent consumables used in the experiment are as follows:
table 1: main reagent consumable list
| Name of article | Brand | Goods number |
| Dithiothreitol (DTT) | aladdin | D104861 |
| Iodoacetamide (IAM) | aladdin | I131590 |
| Ammonium bicarbonate | aladdin | A110537 |
| Urea (Urea) | AMERSCO | 0379-5KG |
| Recombinant human S100A9 protein | abcam | ab95909 |
| Anti-S100A9 + Calprotectin ( |
abcam | ab22506 |
| Dynabeads™ Protein G | Thermo Scientific™ | 10003D |
| DynaMag-2 magnetic frame | Thermo Scientific™ | 12321D |
| Sequencing Grade Modified Trypsin | Promega | V5117 |
| Pierce Formate, LC-MS grade | Thermo Scientific™ | 85178 |
| Isotopically labeled peptide fragment LGHPDTLNQGEFK standard | Anhuoguoping pharmaceutical industry | / |
| Isotopically labeled peptide fragment LTWASHEK standard | Anhuoguoping pharmaceutical industry | / |
| Millipore ZipTip @ micro-chromatography column | Millipore | ZTC18S096 |
| Protein LoBind @, 0.5 mL, PCR clean grade, colorless | Eppendorf | 0030108094 |
| N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid (HEPES) | aladdin | H109408 |
| Potassium acetate | aladdin | P108325 |
| Sodium acetate buffer solution | aladdin | S378199 |
| Magnesium acetate tetrahydrate | aladdin | M108884 |
| EDTA buffer solution | aladdin | E196386 |
| Potassium hydroxide | aladdin | P301749 |
Example 1
The embodiment provides a liquid chromatography-tandem mass spectrometry method for detecting the content of calprotectin in blood, which comprises the following steps:
calibration of a standard solution
1.1 preparation of standard working solution: the calprotectin recombinant protein with the concentration of 1 mug/muL is taken as a standard substance, purified water is used for diluting for 9 times step by step, calprotectin working solution with the concentration of 3.9063, 7.8125, 15.625, 31.25, 62.5, 125, 250, 500 and 1000 ng/muL is prepared, and the calprotectin working solution is prepared just in use.
1.2 preparation of internal standard working solution: using an aqueous solution of 35% acn and 0.1% formic acid as a diluent, and adding the peptide fragment 4 isotope-labeled peptide and the peptide fragment 5 isotope-labeled peptide in a weight ratio of 1:1 as an internal standard, diluting to 10 ng/mu L as an internal standard, and storing the internal standard working solution at-20 ℃;
1.3 standard solution treatment (plasma matrix): transferring 2.5 μ L of 9 standard working solutions with different concentrations by a pipette, adding into 197.5 μ L of blank plasma matrix to obtain 9 standard solutions with final concentrations of 0.049, 0.098, 0.195, 0.391, 0.781, 1.563, 3.125, 6.25, and 12.5 ng/μ L,
1.4 the calprotectin in the standard solution is enriched by utilizing the antigen-antibody combination principle, and the specific steps are as follows (the whole process of single sample enrichment is 45 min):
1.41 Dynabeads activation: taking out Dynabeads Protein G10. Mu.L, rotary incubating for 5min, placing the test tube on a magnetic frame, and discarding the supernatant;
1.42 Preparing an antibody: anti-calprotectin monoclonal antibody (1. Mu.g/. Mu.L) 1.5. Mu.L was diluted with binding buffer 20. Mu.L (20 mM HEPES, 110 mM potassium acetate, 5mM sodium acetate, 2 mM magnesium acetate, 1 mM EDTA, potassium hydroxide adjusted to pH 7.3);
1.43 Antibody binding: adding the diluted anti-calprotectin monoclonal antibody into activated Dynabeads for affinity reaction, incubating for 10 min at room temperature, placing the test tube on a magnetic frame, and removing the supernatant;
1.44 Removal of unbound antibody and residue: resuspending with 20 μ L of binding buffer, gently blowing and sucking, placing the test tube on a magnetic frame, and discarding the supernatant;
1.45 And (3) immunoprecipitation: adding 200 mu L of serum sample into the Dynabeads-Ab conjugate, performing rotary incubation for 30 min at room temperature, placing the test tube on a magnetic frame, and discarding the supernatant;
1.46 Removal of interference in serum that does not bind specifically to antibodies: add washing buffer 50 uL (binding buffer + 0.5% Triton X100) gently resuspended, put the tube on the magnet, discard the supernatant, repeat three times;
1.47 Non-denaturing elution: after removing the washing buffer, 10. Mu.L of 5% acetic acid was added to the Dynabeads-Ab-Ag conjugate, and the complex was dissociated by rotary incubation at room temperature for 2 min, placed on a magnetic frame, and the supernatant was retained.
Adding 1 μ L of internal standard working solution into the enriched calprotectin, blowing to dry with nitrogen, adding 10 μ L of protein reducing reagent (8M urea, 5mM dithiothreitol), vortex, mixing, dissolving completely, and heating in water bath at 37 deg.C for 30 min; continuously adding 2 mu L of 100 mM iodoacetamide for alkylation, and reacting for 30 min at room temperature in a dark place; after the reductive alkylation of the protein was completed, 50 mM ammonium bicarbonate was added in a volume of 3 times to adjust the pH to 7.4, 0.5. Mu.L of trypsin was added at a concentration of 0.1. Mu.g/. Mu.L, the mixture was incubated at 45 ℃ for 3 hours, the experiment was terminated with 10. Mu.L of 10% formic acid, and the mixture was evaporated to dryness in vacuo. The desalting treatment was carried out using Millipore ZipTip @ micro-chromatography column (Millipore, ZTC18S 096), the desalting step being described in the specification:
re-dissolving the desalted dried sample in 10 mu L of sample buffer solution (1% ACN +1% FA aqueous solution), uniformly mixing by vortex, detecting the solution by using a high performance liquid chromatography-mass spectrometer, obtaining nine calprotectin quantitative characteristic peptide segments (peptide segment 4, LGHPDTLNQGEFK) with different concentrations and standard solution chromatograms of internal standards, respectively obtaining peak areas of the calprotectin characteristic peptide segments and the internal standards from the standard solution chromatograms of the calprotectin characteristic peptide segments and the internal standards, and respectively taking the ratios of the peak areas of the characteristic peptide segments and the internal standards of the nine calprotectin standard solutions with different concentrations as the longitudinal coordinate y of a standard curve equation 1 The concentration of the calprotectin standard working solution is used as the abscissa x of a standard curve equation 1 Performing linear regression on the nine data with different concentrations obtained by detection, and fitting to obtain a standard curve equation of y 1 = a*x 1 + b, and linear equation coefficients a and b are obtained; the standard working solution is calprotectin solution added with plasma matrix, the internal standard working solution is isotope labeling characteristic peptide section solution of calprotectin, and the low-protein adsorption tube is used in the whole experiment.
(II) treatment of the sample to be tested
Transferring 200 mu L of a serum or plasma sample to be detected by using a liquid transfer gun, wherein the enrichment step, the sample pretreatment step and the enzyme digestion step of the target protein are consistent with the standard solution treatment step (I), and finally re-dissolving in 10 mu L of loading buffer solution to obtain the sample to be detected;
(III) detection of sample to be detected
Detecting the sample to be detected in the step (II) by using a high performance liquid chromatography-mass spectrometer to obtain a chromatogram of calprotectin quantitative characteristic peptide segment (peptide segment 4, LGHPDTLNQGEFK) and an internal standard of the sample to be detected, obtaining the peak areas of the quantitative characteristic peptide segment and the internal standard from the chromatogram of the quantitative characteristic peptide segment and the internal standard, and determining the ratio y of the peak areas of the characteristic peptide segment and the internal standard 1 Substituting into the standard curve equation y in the step (one) 1 =a*x 1 + b, calculating to obtain the calprotectin concentration x in the sample to be detected 1 。
The chromatographic column used for the chromatographic analysis was Agilent ZORBAX Eclipse Plus-C18 (95A, 1.8 μm, 2.1X 50 mm); the mobile phase is water (0.1% formic acid) and acetonitrile (0.1% formic acid), gradient elution is carried out (table 2), the flow rate is 0.3 mL/min, the column temperature is 40 ℃, and the sample injection amount is 4 mu L; analysis time 9.5 min.
Table 2: gradient elution conditions
The mass spectrometer is a Thermo QE plus detector, and adopts electrospray ionization (ESI) and positive ion mode, the ion source parameters are shown in Table 3, full scan (Full MS) + secondary scan (dd-MS) 2 ) + multiple reaction monitoring (PRM), process parameters as shown in table 4:
table 3: parameters of ion source
Table 4: parameters of mass spectrometry method
Ion pair parameters: calprotectin quantitation of characteristic peptide fragment (LGHPDTLNQGEFK, peptide fragment 4): parent ion 485.9124-daughter ion 480.2453; calprotectin qualitative characteristic peptide (ltwashiek, peptide 5): parent ion 327.1744-daughter ion 421.2285; calprotectin quantitation feature peptide internal standard (LGHPDTLNQGEFK, internal standard peptide 4): parent ion 488.5838-daughter ion 488.2595; calprotectin qualitative characteristic peptide internal standard (ltwasrek, internal standard peptide 5): parent ion 327.1744-daughter ion 421.2285.
Example 2
1. Example 1 method Linearity
Transferring 200 mu L of a serum or plasma sample to be detected by using a liquid transfer gun, carrying out enrichment and pretreatment according to the method in the embodiment 1 to obtain a loading liquid, and redissolving the loading liquid in 10 mu L of loading buffer solution to obtain the sample to be detected; mu.L of each concentration of calprotectin standard solution obtained above was injected into 4. Mu.L sample for LC-MS/MS analysis. The concentration of calprotectin is in the range of 0.049-12.500 ng/mu L, according to the determination conditions of the embodiment, the determination is carried out according to the concentration from low to high, the ratio of the chromatographic peak area of calprotectin characteristic peptide segment to the chromatographic peak area of the internal standard substance-the concentration of calprotectin is used for drawing to obtain a standard curve; the results show that the plasma calprotectin concentration is in the range of 0.049 ng/mu L to 12.500 ng/mu L, the linearity is good, and the correlation coefficient R 2 ﹥0.99。
2. Recovery and precision of the spiked Process of example 1
The method comprises the steps of respectively taking 3 concentration points of the calprotectin standard solution, namely low, medium and high concentration points, carrying out standard addition recovery rate experiments and precision experiments, carrying out determination according to the method of the embodiment, repeatedly analyzing and determining for 5 batches, wherein the calprotectin recovery rate and the precision are respectively shown in table 5, the average recovery rate of calprotectin in the low, medium and high 3 concentration ranges is 98.07% -118.22%, the relative standard deviation is 12.80% -2.53%, and various technical indexes such as standard addition recovery rate and precision meet requirements.
Table 5: calprotectin standard solution standard adding recovery rate and precision
The mass spectrogram of 2 characteristic peptide fragments of calprotectin in the plasma sample is shown in figure 1, and the mass spectrogram of the isotope internal standard of calprotectin characteristic peptide fragment 4 in the plasma sample is shown in figure 2; the isotope internal standard mass spectrogram of the calprotectin characteristic peptide segment 5 in the plasma sample is shown in figure 3, the calprotectin quantitative standard curve is shown in figure 4, the retention time of the calprotectin characteristic peptide segment 4 and the internal standard thereof is 3.79 min, and the retention time of the calprotectin characteristic peptide segment 5 and the internal standard thereof is 3.59 min.
3. Minimum detection limit of the method of example 1: the lowest detection was 0.0244 ng/. Mu.L.
Example 3
This example serves to illustrate the effect of the choice of column temperature on the detection effect:
the assay was carried out as in example 1, with the difference that: the column temperature gradient of 20 ℃, 40 ℃ and 60 ℃ is set.
The experimental results are shown in FIGS. 5 to 7. Wherein, the chromatogram map of the column temperature of 20 ℃ is shown in figure 5, and the retention time of the peptide fragment 5 and the peptide fragment 4 is respectively 3.74 min and 3.90 min, and the difference value is 0.16 min. The chromatogram at 40 ℃ is shown in FIG. 6, and it can be seen from FIG. 6 that the retention times of peptide fragment 5 and peptide fragment 4 are 3.59 min and 3.79 min, respectively, and the difference is 0.20 min. The chromatogram at 60 ℃ is shown in FIG. 7, and it can be seen that the retention times of peptide fragment 5 and peptide fragment 4 are 3.49 min and 3.71 min, respectively, and the difference is 0.22 min.
According to the experimental results, as the column temperature rises, the retention time of the peptide fragments is advanced, the separation degree of the two peptide fragments is higher, wherein the separation of the two peptide fragments is incomplete under the condition that the column temperature is 20 ℃, so that the column temperature is selected to be 40-60 ℃, and based on the service life and protection of an instrument, the column temperature can be further preferably 40 ℃.
Example 4
This example serves to illustrate the effect of the choice of chromatography column on the detection effect:
the assay was carried out as in example 1, with the difference that: different chromatographic columns are selected.
Column A was an Agilent Poroshell 300SB-C18 (300A, 2.1X 75 mm,5 μm);
column B was Agilent extended-C18 (2.1X 50 mm,1.8 μm);
the chromatographic column C is Agilent ZORBAX SB-C18 Rapid Resolution HT (2.1X 50 mm, 1.8-Micron);
column D is a Waters ACQUITY UPLC BEH C18 IVD Column (2.1X 50 mm,1.7 μm);
column E was an Agilent ZORBAX Eclipse Plus-C18 (95A, 1.8 μm, 2.1X 50 mm).
The chromatogram of column A is shown in FIG. 8 and FIG. 9, the retention time of peptide 5 and peptide 4 is 3.02 min and 3.52 min, respectively, and the standard R is 2 0.9923, wherein the signal intensity of the peptide segment 5 is too low, so that the peptide segment is not selected;
the chromatogram of column B is shown in FIG. 10 and FIG. 11, the retention time of peptide 5 and peptide 4 is 3.42 min and 3.69 min, and the label R is 2 Is 0.9085, wherein the peptide segment 4 has a peak shape difference under the condition of low concentration, and the linearity is less than 0.99, so that the peptide segment is not selected;
chromatogram of chromatographic column C is shown in FIG. 12 and FIG. 13, with peptide 5 and peptide 4 retention times of 3.60 min and 3.82 min, respectively, and standard R 2 0.8069, linearity less than 0.99, so it is not selected;
chromatogram of chromatographic column D is shown in FIG. 14 and FIG. 15, retention time of peptide 5 and peptide 4 is 3.72 min and 3.91 min, respectively, and standard R is labeled 2 0.8372 and linearity less than 0.99, so the method is not selected;
the chromatogram of column E is shown in FIGS. 16 and 17, with peptide 5 and 4 having retention times of 3.59 min and 3.79 min, respectively, and labeled R 2 The number of the chromatographic column is 0.9934, and the chromatographic column is the optimal chromatographic column in 5 candidate chromatographic columns, so the chromatographic column is selected as a chromatographic column for subsequent experiments.
Example 5
This example serves to illustrate the effect of the choice of mobile phase on the detection effect:
for the selection of the mobile phase, methanol or acetonitrile is usually selected as the organic phase, and whether acidic substances are added to adjust the ionic strength in the solution is selected according to the circumstances, and for the experiment, the following attempts are respectively made:
mobile phase condition 1: selecting methanol and water as mobile phase, adding 0.1% of FA as ion enhancer, respectively;
mobile phase condition 2: acetonitrile and water are selected as mobile phases, and no acid reagent is added;
mobile phase condition 3: acetonitrile and water were selected as mobile phases, and 0.1% by weight of FA was added as an ion enhancer, respectively.
The chromatogram under mobile phase condition 1 is shown in FIG. 18 and FIG. 19, the retention time of peptide fragment 5 and peptide fragment 4 is 4.38 min and 4.91 min, respectively, and R is labeled 2 The elution time is 0.9738, and the gradient of an 85% organic phase is obtained within 4-5 min, which indicates that the solubility of the peptide fragment in methanol is poor, the elution effect of the methanol on the peptide fragment is poor, and the peptide fragment is easy to flow out together with a large amount of impurities and interference in actual sample detection, so the peptide fragment is not selected;
the chromatogram under mobile phase condition 2 is shown in FIG. 20 and FIG. 21, the retention time of peptide fragment 5 and peptide fragment 4 is 3.55 min and 3.78 min, respectively, and R is labeled 2 Is 0.6409, and as can be seen from the figure, the peak baseline is too high, the linearity is less than 0.99, which indicates that the mass spectrum behavior difference of peptide fragments with different concentrations is too large and is not selected,
chromatogram for mobile phase condition 3 is shown in FIG. 22 and FIG. 23, the retention time of peptide fragment 5 and peptide fragment 4 is 3.59 min and 3.79 min, respectively, and the standard R is 2 0.9934 is the optimum mobile phase choice, so case 3 was chosen as the final mobile phase condition.
Example 6
This embodiment is used to explain the influence of the detection duration on the detection effect:
the assay was carried out as in example 1, with the difference that: the detection duration is different.
Gradient elution is finally selected through conditions optimization such as isocratic elution, gradient elution, elution time, organic phase ratio and the like, detection duration is optimized for shortening detection time and optimizing detection efficiency, the final duration is 9.5min and is an optimal result, elution gradient can be seen in a table 2, a chromatogram and a standard curve when the duration is 9 min are only shown below, and a method of 9.5min of elution gradient ratio is used for advancing the gradient elution time by 0.5 min, and is specifically shown in a table 6:
table 6: elution gradient with detection duration of 9 min
The chromatogram under the elution gradient is shown in FIGS. 24 and 25, the retention time of peptide 5 and 4 is 3.47 min and 3.64 min, respectively, and the labeled R 2 0.9817, linearity less than 0.99, not meeting the standard, so under the elution conditions of the column, 9.5min is the optimal detection time.
Example 7
This example serves to illustrate the effect of the amount of anti-calprotectin monoclonal antibody used on the assay effect:
the assay was carried out as in example 1, with the difference that: the amount of anti-calprotectin monoclonal antibody used varies.
For the detection of calprotectin in the method, the enrichment of calprotectin is an important link, the dosage of the antibody in the enrichment process is very important for the accuracy and benefit of the result, the cost of the antibody is meaningless wasted due to excessive dosage, the high-concentration antigen cannot be completely enriched due to too low dosage, and the detection result of the high-concentration sample is lower, the selected anti-calprotectin monoclonal antibody (1 mug/muL) in the embodiment is 1.2 muL, other experimental conditions are consistent, the result is shown in figure 26, the standard curve linearity is less than 0.99, the signal intensity is lower in different degrees at high concentration, which indicates that the 1.2 muL antibody dosage cannot completely enrich the sample to be detected, and the high-concentration detection result is lower, therefore, the dosage of the 1.5 muL antibody is selected as the optimal dosage after optimization.
Comparative example 1
Enriching calprotectin by using a gel electrophoresis method: taking 3 mg of serum protein of a healthy person, carrying out 18% SDS-PAGE gel electrophoresis, staining with Coomassie brilliant blue, cutting, decoloring, dehydrating, reducing and alkylating the adhesive tape within the range of 10-16 kDa, digesting with trypsin for 18 hours, and terminating the enzyme digestion reaction with 1% formic acid. Gradually extracting the polypeptide from the gel by using 60% of acetonitrile and 90% of acetonitrile until the polypeptide is completely extracted, carrying out vacuum drying on the extracted polypeptide, compounding the dried polypeptide by using 40 muL of diluent, adding 10 muL of internal standard (peptide section III with the concentration of 12.5 mug/mL), uniformly mixing, and then taking 10 muL to carry out LC-MS/MS analysis.
The liquid condition is as follows:
(1) Chromatographic conditions are as follows:
mobile phase A liquid: 10 mM ammonium acetate in water (0.1% FA);
mobile phase B liquid: acetonitrile (0.1% fa);
the type of the chromatographic column: agilent SB-C18,1.8 μm,100 mm × 2.1 mm;
flow rate: 0.2 mL/min, and injecting 10 mu L of sample;
the elution conditions used were: 0-3 minutes: 0.5% of B,3 to 12 minutes: 0.5 Percent to 50% B,12 to 12.1 minutes: 50% -85% of B, 12.1-15 minutes: 85% B, 15-15 min: 85% -0.5% by weight, B,15.1 to 20 minutes: 0.5% by weight of B.
(2) Mass spectrum conditions:
an ion source: electrospray ion source (ESI), positive ion mode; ESI acquisition parameters: the spraying voltage is 3500V, the evaporator temperature is 100 ℃, the sheath gas flow rate is 20 Arb, the auxiliary gas flow rate is 10 Arb, the peak width is 0.70 FWHM, the scanning time is 0.05s, and the sample injector is set for 0-2 min: waste, 2-16 min: load, 16-20 min: waste; ion pair parameters of the peptide segment LGHPDTLNQGEFK to be detected: parent ion 728.36-daughter ion 1148.7; parameters of LTWASHEK ion pair of the peptide fragment to be detected: parent ion 486.25-daughter ion 757.38; internal standard LGHPDTLNQGEFK ion pair parameters: parent ion 732.5-daughter ion 1156.5; internal standard ltwashiek ion pair parameters: parent ion 490.27-daughter ion 765.26;
the blood sample of the healthy person is detected by using the conditions, and the detection result is as follows: not detected.
The foregoing are merely exemplary embodiments of the present invention, which enable those skilled in the art to understand or practice the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A method for detecting the content of calprotectin in blood is characterized in that after an antigen-antibody combination method is used for enriching calprotectin in a sample to be detected, a liquid chromatography-tandem mass spectrometry method is used for analyzing.
2. The detection method according to claim 1, characterized in that it comprises at least the following steps:
s1, preparing a standard curve equation, which comprises the following steps:
s11, preparing a standard working solution and an internal standard working solution with gradient concentration; the standard working solution contains calprotectin recombinant protein standard, and the internal standard working solution contains at least two characteristic peptide internal standards;
s12, preparing a standard solution: respectively adding blank plasma matrixes into the standard working solution with the gradient concentration to obtain standard solutions with series concentrations;
s13, enriching calprotectin recombinant proteins in the standard solutions with the series of concentrations by using an antigen-antibody combination method to obtain standard solution enrichment solutions with the series of concentrations;
s14, preprocessing the standard solution enrichment solutions with the series of concentrations to obtain standard solution loading solutions with the series of concentrations;
s15, detecting the standard solution loading liquid by using a high performance liquid chromatography-mass spectrometer to obtain a standard curve equation;
s2, processing to obtain a sample loading liquid of the sample to be detected, comprising:
s21, enriching the sample to be detected by using an antigen-antibody binding method to obtain an enrichment solution of the sample to be detected;
s22, pretreating the enrichment solution of the sample to be detected to obtain a sample loading solution of the sample to be detected;
s3, detecting the sample to be detected, comprising the following steps: and detecting at least two characteristic peptide fragments in the sample loading liquid of the sample to be detected by using a high performance liquid chromatography-mass spectrometry combined instrument, and substituting a quantitative peptide fragment detection result into the standard curve equation to obtain the concentration of the calprotectin in the sample to be detected.
3. The detection method according to claim 2,
s11, preparing a calprotectin recombinant protein standard into a solution with gradient concentration by using water as a solvent in the standard working solution;
the internal standard working solution adopts 30-40% by volume of acetonitrile and 0.05-0.2% by volume of formic acid as solvents, and two characteristic peptide internal standards are mixed according to the mass ratio of 1:1 mixing the raw materials to be used as an internal standard substance for preparation;
in S12, the volume ratio of the standard working solution to the blank plasma matrix is 1:79.
4. the detection method according to claim 2,
enrichment in S13 includes: combining an anti-calprotectin monoclonal antibody marked by magnetic beads with calprotectin recombinant protein in a standard solution, obtaining an antigen-antibody conjugate through magnetic sorting, and dissociating the antigen-antibody conjugate after washing to obtain standard solution enrichment liquid;
in S21, combining the anti-calprotectin monoclonal antibody marked by the magnetic beads with a to-be-detected serum sample, obtaining an antigen-antibody conjugate through magnetic sorting, washing, and dissociating the antigen-antibody conjugate to obtain an enrichment solution of the to-be-detected sample.
5. A method of detection as claimed in claim 4 wherein the magnetic bead labelled anti-calprotectin monoclonal antibody is prepared by a method comprising:
activating the magnetic beads, combining the anti-calprotectin monoclonal antibody with the activated magnetic beads, and cleaning to remove the unbound antibody;
and/or diluting the calprotectin-resistant monoclonal antibody to 0.075-0.5 mu g/mu L by using a binding buffer solution, and then incubating the diluted calprotectin-resistant monoclonal antibody with the activated magnetic beads;
and/or the mass-volume ratio of the anti-calprotectin monoclonal antibody to the magnetic bead is 1-10 mu g: 50. mu.L;
and/or incubating for 10-30 minutes at room temperature; the washing conditions are that the combined buffer solution is used for resuspension, the test tube is lightly blown and sucked, the test tube is placed on a magnetic frame, and the supernatant is discarded.
6. The assay method according to claim 5, wherein the mass to volume ratio of the anti-calprotectin monoclonal antibody to the serum sample to be assayed is 1 to 10 μ g: 200. mu.L;
the dissociation method adopting non-denaturing elution comprises the following steps: an acetic acid solution was added to the antigen-antibody conjugate, and the incubation was performed at room temperature with rotation.
7. The detection method according to claim 2,
the pretreatment in S14 includes:
adding mixed internal standard working solution into the standard solution enrichment solution, drying, adding a protein reducing agent and a protein alkylating reagent for reductive alkylation, and then adding proteolytic enzyme for enzymolysis; stopping the reaction after the enzymolysis reaction is finished, and diluting the desalted sample solution by using a sample loading buffer solution to obtain a sample loading solution of the standard solution;
the pretreatment in S21 includes:
adding a mixed internal standard working solution into the enrichment solution of the sample to be detected, drying, adding a protein reducing agent and a protein alkylating reagent for reductive alkylation, and then adding proteolytic enzyme for enzymolysis; stopping the reaction after the enzymolysis reaction is finished, and diluting the sample by using a sample loading buffer solution after desalting to obtain a sample loading solution of the sample to be detected;
the protein reducing reagent is selected from a solution containing 6-8 mol/L urea and 5-10 mmol/L dithiothreitol;
the protein alkylation reagent is selected from 10-20 mmol/L iodoacetamide solution;
the proteolytic enzyme is selected from 0.05-0.2 mu g/mu L trypsin.
8. The detection method according to claim 1, wherein the chromatographic conditions in the liquid chromatography-tandem mass spectrometry comprise:
mobile phase: phase A: water containing 0.05-0.2% by volume of formic acid, phase B: acetonitrile containing 0.05-0.2% by volume of formic acid;
the elution mode is gradient elution;
0.01 to 1.00 minutes: a:97%, B:3 percent;
1.01 to 4.00 minutes: a:55%, B:45 percent;
4.01 to 4.10 minutes: a:15%, B:85 percent;
4.11 to 5.00 minutes: a:15%, B:85 percent;
5.01 to 5.10 minutes: a:97%, B:3 percent;
5.11 to 9.50 minutes: a:97%, B:3 percent;
the flow rate is 0.2-0.4 mL/min;
the column temperature is 40-60 ℃;
the sample injection amount is 1-5 muL;
analysis time 9.5 min.
9. The detection method according to claim 1, wherein the conditions in the liquid chromatography-tandem mass spectrometry are electrospray ion source, positive ion mode, full scan + secondary scan + multiple reaction monitoring;
the ion pair of calprotectin quantitative characteristic peptide fragments is as follows: parent ion 485.9124-daughter ion 480.2453;
the ion pair of calprotectin qualitative characteristic peptide segments is as follows: parent ion 324.5030-daughter ion 413.2143;
the ion pair of calprotectin quantitative characteristic peptide fragment internal standard substance is as follows: parent ion 488.5838-daughter ion 488.2595;
the ion pair of calprotectin qualitative characteristic peptide fragment internal standard substance is as follows: parent ion 327.1744-daughter ion 421.2285.
10. A kit for quantitatively detecting calprotectin in blood is characterized by comprising the following components:
a protein extraction reagent comprising: an anti-calprotectin monoclonal antibody reagent marked by magnetic beads and an antigen-antibody conjugate dissociating agent;
pretreatment reagents: protein reducing agents, protein alkylating agents, proteolytic enzymes;
standard solution reagents: the volume ratio of the standard solution to the blank plasma matrix is 1:79 of a solvent; the standard working solution is a solution with water as a solvent and a calprotectin recombinant protein standard as a solute;
an internal standard working solution reagent; taking an aqueous solution containing 30-40% by volume of acetonitrile and 0.05-0.2% by volume of formic acid as a solvent, and comparing the mass ratio of a quantitative characteristic peptide internal standard substance to a qualitative characteristic peptide internal standard substance by 1:1 the mixture is a solution of solute; wherein, calprotectin quantitative characteristic peptide fragment internal standard substance: peptide LGHPDTLNQGEFK internal standard was used: the calprotectin qualitative characteristic peptide internal standard adopts a peptide LTWASHEK internal standard;
mobile phase reagent: phase A: water containing 0.05-0.2% by volume of formic acid, phase B: acetonitrile containing 0.05-0.2% by volume of formic acid.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2249154A1 (en) * | 2009-05-07 | 2010-11-10 | IRCCS San Raffaele Pisana- San Raffaele | Method for high recovery of serum low-abundance, low molecular-weight proteins and their analysis by matrix assisted laser desorption ionization mass spectrometry |
| CN110133275A (en) * | 2019-04-01 | 2019-08-16 | 芜湖森爱驰生物科技有限公司 | A kind of calprotectin detection kit and preparation method thereof rapidly and efficiently |
| CN110850099A (en) * | 2019-11-25 | 2020-02-28 | 中国计量科学研究院 | Method for valuing C-reactive protein in serum |
| WO2021229016A1 (en) * | 2020-05-13 | 2021-11-18 | Nordic Bioscience A/S | Calprotectin assay |
| CN114166966A (en) * | 2021-11-26 | 2022-03-11 | 杭州广科安德生物科技有限公司 | Method for determining value of pro-SFTPB in serum |
-
2023
- 2023-01-04 CN CN202310006487.3A patent/CN115684432B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2249154A1 (en) * | 2009-05-07 | 2010-11-10 | IRCCS San Raffaele Pisana- San Raffaele | Method for high recovery of serum low-abundance, low molecular-weight proteins and their analysis by matrix assisted laser desorption ionization mass spectrometry |
| CN110133275A (en) * | 2019-04-01 | 2019-08-16 | 芜湖森爱驰生物科技有限公司 | A kind of calprotectin detection kit and preparation method thereof rapidly and efficiently |
| CN110850099A (en) * | 2019-11-25 | 2020-02-28 | 中国计量科学研究院 | Method for valuing C-reactive protein in serum |
| WO2021229016A1 (en) * | 2020-05-13 | 2021-11-18 | Nordic Bioscience A/S | Calprotectin assay |
| CN114166966A (en) * | 2021-11-26 | 2022-03-11 | 杭州广科安德生物科技有限公司 | Method for determining value of pro-SFTPB in serum |
Non-Patent Citations (3)
| Title |
|---|
| ADA AITA 等: "Salivary proteomic analysis in asymptomatic and symptomatic SARS-CoV-2 infection: Innate immunity, taste perception and FABP5 proteins make the difference" * |
| 张方彦 等: "人血清中C反应蛋白的磁珠富集与质谱检测" * |
| 肖忠华 等: "血浆蛋白质组学研究中样品前处理技术的研究进展" * |
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