CN117310182A - Sample processing kit for detecting Cys C by MALDI-TOF MS and application thereof - Google Patents

Sample processing kit for detecting Cys C by MALDI-TOF MS and application thereof Download PDF

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CN117310182A
CN117310182A CN202311207060.6A CN202311207060A CN117310182A CN 117310182 A CN117310182 A CN 117310182A CN 202311207060 A CN202311207060 A CN 202311207060A CN 117310182 A CN117310182 A CN 117310182A
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cys
solution
buffer
acid
kit
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马雪婷
门丽影
周晓光
李运涛
奚欣格
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Beijing Rongzhi Youpu Biotechnology Co.,Ltd.
Rongzhi Biotechnology Qingdao Co ltd
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G01N27/626—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using heat to ionise a gas
    • G01N27/628—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using heat to ionise a gas and a beam of energy, e.g. laser enhanced ionisation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
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    • G01—MEASURING; TESTING
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N2001/2893—Preparing calibration standards
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • G01N2333/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin

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Abstract

The invention discloses a sample processing kit and a detection kit for detecting Cys C by using MALDI-TOF MS, wherein the sample processing kit comprises the following reagents: an internal standard Cys C solution; a first buffer; anti-Cys C antibodies-magnetic beads; eluting the solution. The kit can simultaneously realize qualitative and quantitative detection of the wild Cys C and the variants thereof, has good repeatability and consistency of detection results, stable methodology, accurate detection results, high sensitivity, large detection range, high flux, simple and convenient operation, low cost and high application value.

Description

Sample processing kit for detecting Cys C by MALDI-TOF MS and application thereof
Technical Field
The invention relates to the technical field of in-vitro detection, in particular to a sample processing kit for detecting Cys C by using MALDI-TOF MS and application thereof.
Background
Serum Cystatin C (Cys C for short) is also called cysteine proteinase inhibitor C, is a low molecular weight alkaline non-glycosylated protein which consists of 120 amino acids and has the molecular weight of 13.3kDa, can be produced by all nucleated cells of an organism, and has the Cys C content of 0.51-1.09 mg/L in normal human serum. Cys C is widely present in nucleated cells and body fluids of various tissues, has constant rate of production and release into blood, does not form a complex with other proteins, can pass through glomerular filtration freely and is totally reabsorbed in proximal tubules and rapidly metabolically decomposed. Therefore, the concentration of Cys C in blood is determined by glomerular filtration rate (glomerular filtration rate, GFR), is not affected by factors such as inflammation, infection, tumor and liver function, is irrelevant to gender, age, diet, body surface area and muscle mass, and is an ideal endogenous marker for reflecting the change of glomerular filtration rate.
Cys C is an index capable of sensitively and accurately reflecting early damage of renal functions, has important clinical application value, and is based on physiological characteristics and clinical application value of Cys C, and the concentration of Cys C in body fluids such as serum, urine and the like is low, so that the clinical sample Cys C content measurement method has high requirements on sensitivity and specificity. The existing Cys C content determination method mainly comprises an enzyme-linked immunoassay, a radioimmunoassay, a fluorogenic enzyme immunoassay, an immunonephelometry and other immunoassays. These methods are all interfered by various factors, and the quantitative results have large fluctuation. Thus, there is a need for a Cys C quantification technique that is accurate, consistent, methodologically stable, high in throughput and low in cost.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent. Therefore, the invention provides a sample processing kit and a detection kit for detecting Cys C by using MALDI-TOF MS, and the kit can simultaneously realize qualitative and quantitative detection of wild Cys C and variants thereof, has good reproducibility and consistency of detection results, stable methodology, accurate detection results, high sensitivity, large detection range, high flux, simple and convenient operation, low cost and high application value.
In a first aspect of the invention, the invention proposes a sample processing kit for detecting Cys C using MALDI-TOF MS. The kit comprises the following reagents:
an internal standard Cys C solution;
a first buffer;
anti-Cys C antibodies-magnetic beads;
eluting the solution.
According to the embodiment of the invention, the sample to be detected, the internal standard Cys C solution and the first buffer solution are mixed by using the sample processing kit to obtain a uniform mixed solution containing the Cys C to be detected and the internal standard Cys C; the mixed solution is contacted with an anti-Cys C antibody-magnetic bead to obtain an antigen antibody-magnetic bead compound enriched with Cys C to be detected and internal standard Cys C, so that the purposes of effectively enriching and removing impurities of a target object are achieved, and the interference of impurities on subsequent detection is avoided. Then, the Cys C to be detected and the internal standard Cys C are dissociated from the antigen-antibody-magnetic bead complex through elution treatment, and the separated solution to be detected containing the Cys C to be detected and the internal standard Cys C can be used for subsequent detection. And the MALDI-TOF MS is used for detecting the Cys C content of the solution to be detected, so that the qualitative and quantitative detection of the wild Cys C and the variant thereof can be realized simultaneously.
Therefore, the kit can simultaneously realize qualitative and quantitative detection of the wild Cys C and the variants thereof, has good reproducibility and consistency of detection results, stable methodology, accurate detection results, high sensitivity, large detection range, high flux, simple and convenient operation, low cost and high application value. The sample treatment kit is used for treating clinical samples, has less reagent consumption and low cost, and is favorable for popularization.
According to an embodiment of the invention, the first buffer is selected from one of MES buffer, PBS buffer, tris-HCl buffer, HEPES buffer, preferably Tris-HCl buffer. Thus, by using the sample processing kit of the invention, a uniform mixed solution containing Cys C to be detected and internal standard Cys C can be obtained through processing.
According to an embodiment of the invention, the pH of the first buffer is between 10.1 and 11.1, preferably between 10.4 and 10.8.
According to an embodiment of the invention, the concentration of the first buffer is between 0.05M and 0.15M, preferably between 0.08M and 0.12M.
The inventor obtains the above-mentioned better pH value and concentration through a large number of experiments, thus Cys C molecules in the obtained mixed solution can fully contact and react with anti-Cys C antibody-magnetic beads, and high-efficiency enrichment of Cys C to be detected and internal standard Cys C is realized, so that stable and consistent antigen-antibody-magnetic bead complexes are obtained.
According to an embodiment of the invention, the internal standard Cys C solution contains BSA. The inventor finds that adding BSA into the internal standard Cys C solution can avoid nonspecific adsorption of experimental consumables (such as a sampling gun head, a reaction EP tube, magnetic beads and the like) on target Cys C, improve the enrichment rate and reduce the sample use amount. Therefore, the accuracy and consistency of the detection result are further improved.
According to an embodiment of the invention, the concentration of BSA is 0.5-5%, preferably 0.5-1.5%, more preferably 1% by mass volume in mg/mL. The inventor obtains the above-mentioned preferred BSA concentration through a large number of experiments, from this, can further improve Cys C enrichment rate, reduce the sample use amount, improve accuracy and uniformity of the detection result.
According to an embodiment of the invention, the internal standard Cys C solution contains 1.0-3.0 μg/mL of internal standard Cys C. The inventor obtains the concentration of the Cys of the preferable internal standard through a large number of experiments, so that the accuracy of a detection result can be further improved by using the kit disclosed by the invention, and the purpose of accurate detection is achieved.
According to an embodiment of the invention, the internal standard Cys C is selected from a Cys C recombinant protein having a tag sequence or an isotopically labeled Cys C protein.
According to an embodiment of the invention, the tag sequence comprises at least one selected from the group consisting of His tag, flag tag, GST tag, MBP tag, C-MYC tag, HA tag, FC tag, AVI tag, MBP tag, DDDDK tag. Thereby facilitating subsequent purification treatments.
According to an embodiment of the invention, the tag sequence is preferably a His tag.
In some embodiments of the invention, the internal standard is a Cys C recombinant protein containing 6 His-Tag tags.
According to an embodiment of the invention, the isotope is 13 C or 15 N。
In some embodiments of the invention, the internal standard is 15 N-labeled Cys C intact protein.
In some embodiments of the invention, a specific volume of internal standard solution is added to a sample to be tested according to the following mass ratio: the mass ratio of the Cys C to be measured to the internal standard Cys C is (0.1-10) to 1.
The inventor obtains the mass ratio of the Cys C to be detected and the internal standard Cys C through research and analysis, so that the kit can further improve the accuracy of a detection result and achieve the purpose of accurate detection.
According to an embodiment of the invention, the anti-Cys C antibody is a monoclonal antibody.
Compared with polyclonal antibody, the monoclonal antibody of the anti-Cys C antibody has high consistency of anti-Cys C antibody-magnetic beads, small batch-to-batch difference and comparable enrichment effect. Therefore, the stability and consistency of the detection method and the comparability of the detection result can be further improved by using the kit.
According to an embodiment of the invention, the kit further comprises a second buffer comprising a non-ionic surfactant, and/or deionized water. The sample containing Cys C is treated by the sample treatment kit, and before the antigen-antibody-magnetic bead complex is eluted, the antigen-antibody-magnetic bead complex is subjected to first washing and/or second washing, wherein the first washing adopts a second buffer solution containing a nonionic surfactant, and the second washing adopts deionized water. Through the nonionic surfactant, the nonspecifically adsorbed impurity proteins on the antigen-antibody-magnetic bead complex and other impurities in the sample can be effectively removed. Impurities can be further removed through deionized water, and the subsequent detection is prevented from being interfered by the impurities. The antigen-antibody-magnetic bead complex subjected to the first washing and the second washing can be directly subjected to elution treatment. Therefore, the accuracy and consistency of the detection result can be further improved by using the kit.
According to an embodiment of the invention, the nonionic surfactant comprises at least one member selected from the group consisting of tween 20, tween 60, tween 80, CHAPS, triton X-100, alkoxypolyethylene hydroxy ethanol, octyl glucoside, dodecyl maltoside, N-octyl alpha-D-glucoside, N-octanoyl-N-methyl glucamine, N-nonanoyl-N-methyl glucamine, N-decanoyl-N-methyl glucamine, nonyl-beta-D-glucopyranoside, dodecyl-beta-D-maltoside, N-dimethyl dodecylamine-N-oxide, decyl glucopyranoside, 1-O-decyl-beta-D-maltoside, deox-Bigchap, ground saponin, triton X-114, nonyl phenol polyoxyethylene ether, tetraethylene glycol monolodecyl ether, tetramethyl ammonium hydroxide pentahydrate, polysorbate-85, tetrabutylphenol, undecyl-beta-D-maltoside, octyl-beta-D-thioglucopyranoside, N-octyl-beta-D-glucopyranoside, N-glucopyranoside, decyl-beta-D-maltoside, 1-O-decyl-beta-D-maltoside, deoxymaltoside, deoxyd-glucopyranoside, N-methyl-beta-D-glucopyranoside, N-methyl-dodecyl-6-D-glucopyranoside, N-D-methyl-D-glucopyranoside, N-D, N-methyl-D.
According to an embodiment of the present invention, the nonionic surfactant is preferably tween 20. Through adding Tween 20, nonspecifically adsorbed impurity proteins on the antigen-antibody-magnetic bead complex and other impurities in the sample can be efficiently removed. Therefore, the kit can be used for further detecting the accuracy and consistency of the result.
According to an embodiment of the invention, the second buffer is selected from one of PBS buffer, MES buffer, tris buffer, DPBS buffer, CBS buffer, BBS buffer, HEPES buffer, TBS buffer, BES buffer, TEA buffer, MOPS buffer, AMPD buffer, EPPS buffer, MOPSO buffer, AMPSO buffer, DIPSO buffer, TAPSO buffer.
According to an embodiment of the invention, the second buffer is preferably a PBS buffer. The antigen-antibody-magnetic bead complex is maintained stable by providing stable pH conditions with PBS buffer. Therefore, the accuracy and consistency of the detection result can be further improved by using the kit.
Further, the concentration of tween 20 is 0.1 percent by mass volume, and the unit of mass volume is mg/mL; the concentration of the PBS buffer was 10mM.
According to an embodiment of the invention, the second buffer is a PBS buffer containing 0.1% tween 20. According to an embodiment of the invention, the second buffer used for the first washing is a PBS buffer containing 0.1% Tween 20. Therefore, the nonspecifically adsorbed impurity protein on the antigen-antibody-magnetic bead complex and other impurities in the sample can be effectively removed.
According to an embodiment of the present invention, the elution solution contains a matrix comprising at least one selected from the group consisting of sinapic acid, α -cyano-4-hydroxycinnamic acid, 2, 5-dihydroxybenzoic acid, 2-mercaptobenzothiazole, 2,4, 6-trihydroxyacetophenone, anthratriphenol, 3-indoleacrylic acid, preferably sinapic acid, α -cyano-4-hydroxycinnamic acid, 2, 5-dihydroxybenzoic acid, or 2-mercaptobenzothiazole, more preferably sinapic acid, and an antigen antibody-magnetic bead complex dissociation reagent.
According to an embodiment of the invention, the concentration of the sinapic acid is 5-10 mg/mL.
The invention preferably uses sinapic acid as a matrix of a MALDI-TOF MS platform, and can further improve the peak value number and intensity of a mass spectrum.
According to an embodiment of the present invention, the antigen-antibody-magnetic bead complex dissociation reagent includes at least one selected from an organic acid solution, a mineral acid solution, and an alkali solution;
The organic acid solution comprises at least one selected from formic acid solution, acetic acid solution, trifluoroacetic acid solution, propionic acid solution, glycolic acid solution, amino acid solution, butyric acid solution, citric acid, succinic acid, ascorbic acid, lactic acid, malic acid and tartaric acid;
the inorganic acid solution comprises at least one selected from hydrochloric acid solution, sulfuric acid solution and phosphoric acid solution;
the alkali solution comprises at least one selected from sodium hydroxide solution, potassium hydroxide solution, tris (hydroxymethyl) aminomethane solution, tetraethylammonium bromide solution, sodium carbonate solution, sodium bicarbonate solution, ammonium bicarbonate solution, ammonia water solution and urea solution.
According to an embodiment of the invention, the antigen-antibody complex dissociation reagent is a trifluoroacetic acid solution. Wherein, the matrix (such as sinapic acid) used for MALDI-TOF MS platform in the eluting solution does not influence the eluting effect of the antigen-antibody complex dissociating reagent.
According to an embodiment of the present invention, the concentration of the trifluoroacetic acid solution is 0.07 to 7%, preferably 0.1 to 0.7% by volume. The inventor researches find that the trifluoroacetic acid solution with the concentration is used as an antigen-antibody complex dissociation reagent, so that the dissociation efficiency of Cys C protein and Cys C specific antibody can be further improved, and the antigen-antibody is completely dissociated, thereby further improving the accuracy of a detection result.
In a second aspect of the invention, the invention proposes a sample processing kit for detecting Cys C using MALDI-TOF MS. The kit comprises the following reagents:
an internal standard Cys C solution;
a first buffer;
anti-Cys C antibodies-magnetic beads;
eluting the solution;
wherein,
the first buffer solution is Tris-HCl buffer solution, the pH of the Tris-HCl buffer solution is 10.6, and the concentration of the Tris-HCl buffer solution is 0.1M;
the internal standard Cys C solution contains BSA, wherein the concentration of the BSA is 1% in terms of mass volume percentage, and the mass volume unit is mg/mL;
the anti-Cys C antibody is a monoclonal antibody;
the elution solution adopted in the elution treatment contains a matrix and an antigen-antibody-magnetic bead complex dissociation reagent, wherein the matrix is sinapic acid of 5-10 mg/mL, the dissociation reagent is trifluoroacetic acid, and the concentration of the trifluoroacetic acid solution is 0.7% according to the volume ratio.
According to the embodiment of the invention, the sample to be detected is processed by the sample processing kit to obtain the solution to be detected containing Cys C, and the content of Cys C in the solution to be detected is detected by MALDI-TOF MS, so that qualitative and quantitative detection of wild Cys C and variants thereof can be realized simultaneously, the reproducibility and consistency of detection results are good, the methodology is stable, the detection results are accurate, the sensitivity is high, the detection range is large, the flux is high, the operation is simple and convenient, the cost is low, and the application value is high. The sample treatment kit is used for treating clinical samples, and has the advantages of less reagent consumption and low cost.
In a third aspect of the invention, the invention provides a kit for detecting Cys C using MALDI-TOF MS. The kit comprises:
reagents in the aforementioned kit;
cys C standard.
According to the embodiment of the invention, the kit for detecting Cys C is utilized to process a sample to be detected and/or a standard sample, so as to obtain a solution to be detected of the sample to be detected and/or the standard sample, and the processing of the sample to be detected is realized. And then, the solution to be detected is spotted on a target plate for MALDI-TOF MS detection, after the solution to be detected is completely crystallized, data acquisition is carried out by using MALDI-TOF-MS mass spectrum, and the content of Cys C in the sample to be detected is determined based on the acquired data. Therefore, the qualitative and quantitative detection of the wild Cys C and the variant thereof can be realized simultaneously, the reproducibility and consistency of the detection result are good, the methodology is stable, the detection result is accurate, the sensitivity is high, the detection range is large, the flux is high, the operation is simple and convenient, the cost is low, and the application value is high. The sample treatment kit is used for treating clinical samples, has less reagent consumption and low cost, and is favorable for popularization.
According to an embodiment of the invention, the Cys C standard contains BSA.
According to an embodiment of the invention, the concentration of BSA is 0.5-5%, preferably 0.5-1.5%, more preferably 1% by mass volume in mg/mL. The inventors have performed a number of experiments to obtain the above-mentioned preferred BSA concentration. Therefore, by using the detection kit provided by the invention, the enrichment rate of the Cys C of the standard substance can be further improved, the linear range of the standard curve can be improved, and the detection sensitivity can be improved.
According to an embodiment of the invention, the concentration of Cys C standard is 0.5-1.5 mg/mL. The inventor obtains the concentration of the better standard substance through a large number of experiments, and the concentration is further used for gradient dilution of the standard substance, so that the dilution result is accurate, the accuracy of the detection result can be further improved, and the purpose of accurate detection is achieved.
The beneficial effects are that:
1. the kit can accurately detect the peak signal response value of the Cys C body (namely the wild type Cys C) and the variant thereof, and determine the concentration of the Cys C body and the variant thereof in the sample to be detected based on the detected signal value;
2. the method for detecting the Cys C content of the sample to be detected by using the kit has the advantages of good reproducibility and consistency of detection results, stable methodology and variation coefficient CV <4 percent (n=15);
3. the method for detecting the Cys C content of the sample to be detected by using the kit has high detection flux and high detection speed, hundreds of samples can be detected at one time, and each sample is detected for only about 1min;
4. the method for detecting the Cys C content of the sample to be detected by using the kit has low sample usage amount, particularly the serum sample usage amount is as low as 30 mu L, and the method for detecting the Cys C by using MALDI-TOF MS is beneficial to be popularized and applied clinically.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a map of MALDI-TOF mass spectra of 6 concentration gradients of Cys C standard curve of 8. Mu.g/mL, 6. Mu.g/mL, 2. Mu.g/mL, 1. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL, respectively, of Cys C standard curve of Cys C standard protein for serum sample according to the present invention;
FIG. 2 is a graph of the Cys C quantification standard of example 1 of the present invention, wherein the graph is applicable to serum samples;
FIG. 3 is a MALDI-TOF mass spectrum of sample number 108 of serum in example 1 of the present invention;
FIG. 4 is a map of MALDI-TOF mass spectrum of 5 concentration gradients of Cys C standard curve of 4. Mu.g/mL, 2. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL, 0.1. Mu.g/mL, respectively, for a urine sample according to the present invention;
FIG. 5 is a graph of a Cys C quantification standard in example 2 of the present invention, wherein the graph is applicable to urine samples;
FIG. 6 is a MALDI-TOF mass spectrum of urine sample number 201 in example 2 of the present invention.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
Terms and definitions
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.
In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
As used herein, the term "Cys C" is equivalent to "cysteine protease inhibitor 3", "cysteine protease inhibitor C", "serum Cystatin C", "Cystatin C", and is a low molecular weight, alkaline non-glycosylated protein consisting of 120 amino acids and having a molecular weight of 13.3kDa, which is produced by all nucleated cells of the body, and which has a Cys C content of 0.51-1.09 mg/L in normal human serum.
In this context, the term "anti-Cys C monoclonal antibody" is equivalent to "Cys C monoclonal antibody", typically labeled with biotin, for detection of Cys C.
In this context, the term "SD" is equivalent to "standard deviation", "standard deviation", a form of quantization most commonly used to reflect a set of degrees of data dispersion, and is an important indicator of accuracy.
In this context, the term "CV" is equivalent to "Coefficient of Variation" and "coefficient of variation" and refers to the ratio of standard deviation to average value, expressed as a percentage (%), reflecting the absolute value of the degree of dispersion of data, and the data size thereof is affected not only by the degree of dispersion of variable values but also by the average level of the variable values.
As used herein, the term "LoD" is equivalent to "Limit of detection" and "limit of detection" and refers to the lowest value that can be measured under certain probability conditions for testing a low concentration analyte sample.
Herein, the term "LoQ" is equivalent to "Limit of quantitation" and "limit of quantitation" and refers to testing a sample of analyte at a low concentration to obtain a lower value of the exact value.
In this context, the antibody-magnetic beads may be obtained by any magnetic bead bonding method known to the person skilled in the art, for example: carboxyl magnetic bead bonding by a one-step method, carboxyl magnetic bead bonding by a two-step method, streptavidin magnetic bead bonding and the like.
According to some specific embodiments of the invention, the anti-Cys C antibody-beads are obtainable by a carboxyl bead bonding method.
In some specific embodiments of the invention, the concentration gradient of Cys C of the standard is between 10 and 0.1 μg/mL. The inventor obtains the Cys C standard substance with the preferable concentration gradient range through research and analysis, thereby further improving the correlation between the concentration of the standard substance and the fitting curve of the detection signal, improving the accuracy of the detection result and achieving the purpose of accurate detection.
In some embodiments of the invention, the content of Cys C in the test sample is the sum of the content of wild-type Cys C and/or the content of variants thereof and/or the content of wild-type Cys C and variants thereof in the test sample. By using the kit, the qualitative and quantitative detection of the wild type Cys C and the variant thereof can be simultaneously realized by detecting the Cys C content of the solution to be detected through MALDI-TOF MS. Therefore, the kit provided by the invention provides an accurate concentration quantitative reagent and method for the research on the effects of Cys C and the variants thereof in the occurrence and development of diseases, and provides a good technical basis for the research on Cys C related diseases.
In some specific embodiments of the invention, the wild-type Cys C variants comprise at least one of the following three:
a wild-type Cys C variant with hydroxylation of proline at the third N-terminal position;
a wild-type Cys C variant with a deletion of serine at the first position of the N-terminal and hydroxylation of proline at the third position of the N-terminal;
wild-type Cys C variants with deletions of the first serine, the second serine and the third proline at the N-terminus.
The inventor obtains the wild type Cys C variant with higher clinical value through a large amount of clinical data arrangement and experiments, and provides accurate concentration quantification reagent and method for further research of the wild type Cys C variant. The method for detecting the Cys C by using the kit can distinguish detection signals of the wild type Cys C and the variant thereof in the sample to be detected, and realizes simultaneous quantification of the wild type Cys C and the variant thereof. Therefore, the reagent and the method for accurately quantifying the concentration are provided for the research on the action of the wild Cys C and the variant thereof in the occurrence and development of diseases, and a good technical basis is provided for the research on Cys C related diseases.
In some specific embodiments of the invention, the kit of the invention is used to detect Cys C concentration, when data acquisition is performed using MALDI-TOF-MS mass spectrometry, the detected ion is 13 m/z when wild-type Cys C is detected, the detected ion is 13 m/z when wild-type Cys C variant is hydroxylated at the third proline at the N-terminus, the detected ion is 13 m/z when wild-type Cys C variant is hydroxylated at the first serine at the N-terminus and at the third proline at the N-terminus, the detected ion is 13 273 when wild-type Cys C variant is hydroxylated at the first serine at the N-terminus, the second serine at the second position and the third proline at the third position, and the detected ion is 13 073 when wild-type Cys C variant is hydroxylated at the N-terminus.
The conditions for data acquisition by MALDI-TOF MS mass spectrometry are not particularly limited, and conventional conditions for MALDI-TOF MS in the art can be employed, but preferable conditions are as follows:
a laser: a semiconductor laser;
laser frequency: 2000-5000 Hz;
two-dimensional platform movement speed: 0.5 to 3.0mm/sec;
focusing quality: 13000-26000 Da;
the acquisition mass range is as follows: 8-10 kDa.
When the detected ion of the wild type Cys C has one positive charge, m/z is 13344, when the detected ion has two positive charges, m/z is 6673, and when the detected ion has three positive charges, m/z is 4449; preferably, the detection ion of the wild type Cys C has a positive charge and m/z is 13344;
When the detection ion of the wild-type Cys C variant with the hydroxylation of the third proline at the N-terminal is positively charged, the m/z is 13 to 360, the m/z is 6680, the m/z is three positive charges, and the m/z is 4454; preferably the detection ion of the wild-type Cys C variant with hydroxylation of the proline at the third position of the N-terminal is positively charged with m/z being 13.360;
when the detection ion of the Cys C variant with the first serine at the N end deleted and the third proline at the N end hydroxylated carries one positive charge, m/z is 13 273, when carrying two positive charges, m/z is 6637, and when carrying three positive charges, m/z is 4425; preferably, the detection ion of the wild-type Cys C variant with the N-terminal first serine deleted and the N-terminal third proline hydroxylated has a positive charge, and m/z is 13 273;
when the detection ion of the wild-type Cys C variant with the deletion of the first serine, the second serine and the third proline at the N-terminal is provided with one positive charge, m/z is 13 073, and when the detection ion is provided with two positive charges, m/z is 6537, and when the detection ion is provided with three positive charges, m/z is 4358; preferably the detection ion of the N-terminal first serine, second serine and third proline deleted wild type Cys C variant has a positive charge and m/z is 13 073.
The type of instrument for data acquisition by MALDI-TOF MS mass spectrometry is not particularly limited, and a conventional instrument for MALDI-TOF MS in the art can be used, but the following type of instrument is preferable:
a QuanPro type mass spectrum or a quanpof type I mass spectrum.
The inventor obtains the MALDI-TOF-MS mass spectrum data acquisition condition suitable for the kit through a large number of experimental tests, so that the detection flux and the detection speed can be further improved. According to an example of the present invention, the method of the present invention may detect hundreds of samples at a time, each sample detecting only about 1 minute. Therefore, the method has the advantages of high flux, high detection speed, simple and convenient operation and low cost, and is suitable for clinical popularization and application.
The partial reagent used in the embodiment of the invention and the preparation method thereof are as follows:
1) Preparation of 0.1% Tween 20 in PBS (PBST): adding 500 mu L of nonionic surfactant Tween 20 into 500mL of 10mM PBS solution, and uniformly mixing by ultrasonic assistance.
2) Preparation of 0.1M MES aqueous solution: 21.32g of MES is accurately weighed, 1L of deionized water is added, and the MES is completely dissolved for later use.
3) Preparation of 0.1M MES pH5.0 and 0.1M MES pH6.0 aqueous solution: 40mL of 0.1M MES aqueous solution was taken and pH was adjusted to 5.0 and 6.0, respectively, using standard NaOH solution for further use.
4) Preparing EDC solution: accurately weighing 10mg of EDC powder, and dissolving with 1mL of pre-chilled 0.1M MES pH6.0 stored at 4deg.C.
5) Preparation of 1% bsa solution: accurately weighing 0.5mg of bovine serum albumin, adding 50mL of 10mM PBS solution, and completely dissolving for standby.
6) Preparing Cys C internal standard solution: cys C recombinant protein (product No. 10439-H08H, yinqiao, shen) with 6 XHis-Tag attached was formulated with 1% BSA solution as Cys C internal standard solution of the desired concentration.
7) Preparing Sinapic Acid (SA) matrix solution: accurately weighing 0.75g sinapic acid solid powder in a 100mL reagent bottle, adding 30mL acetonitrile and 630 mu L trifluoroacetic acid solution, uniformly mixing, and using deionized water to fix the volume to 90mL to serve as SA substrate liquid, and preserving in a dark place for later use.
The antibody-magnetic beads used in the examples of the present invention were prepared as follows:
200 μg Cys C monoclonal antibody (10439-MM 06, yiqiaoshen) was placed in a 30kDa ultrafilter tube, and washed 3 times with 0.1MMES (pH 5.0) buffer solution to remove antibody protectant. 20mg of carboxyl magnetic beads were placed in a centrifuge tube, and after washing 3 times with 0.1M MES buffer solution (pH 5.0), the magnetic beads were resuspended with 0.1M MES buffer solution (pH 5.0). All antibodies in the ultrafiltration tube were transferred to the activated carboxyl magnetic bead solution and flipped for 30min at room temperature. 200. Mu.L of the now prepared EDC solution was added, vortexed and mixed well and coupled for 3h at room temperature with inversion. 200 μL of CE510 was added and blocked by inversion at room temperature for 3h. After 3 washes with TBE, the wash was performed twice with PBST. After being resuspended in 1000. Mu.L of PBST, the mixture was kept in a refrigerator at 4℃for use (antibody concentration 0.2 mg/mL).
In the embodiment of the invention, the immune enrichment and elution method of Cys C in the sample to be detected is as follows:
1) Immune enrichment of Cys C in serum samples
Accurately transferring 30 mu L of serum sample or 30 mu L of Cys C standard protein solution sample with different concentrations into a 0.6mL centrifuge tube, adding 140 mu L of 0.1M Tris pH 10.59 solution and 30 mu L of Cys C internal standard (2.4 mu g/mL) using liquid vortex for uniform mixing, adding 2 mu g of magnetic bead antibody, vortex for uniform mixing, and turning over for 20min. After the reaction, the liquid in the tube was discarded, and the PBST solution was added for washing 3 times. The supernatant was discarded, and deionized water was added to wash for 2 times. The supernatant was discarded.
2) Immune enrichment of Cys C in urine samples
Accurately transferring 100 mu L of urine sample or 100 mu L of Cys C standard protein solution sample with different concentrations into a 0.6mL centrifuge tube, adding 70 mu L of 0.1M Tris pH 10.59 solution and 30 mu L of Cys C internal standard (1.2 mu g/mL) using liquid vortex for uniform mixing, adding 2 mu g of magnetic bead antibody, vortex for uniform mixing, and turning over for 20min. After the reaction, the liquid in the tube was discarded, and the PBST solution was added for washing 3 times. The supernatant was discarded, and deionized water was added to wash for 2 times. The supernatant was discarded.
3) Elution of Cys C on enriched magnetic beads
And accurately adding 10 mu L of SA matrix solution into the magnetic beads enriched with Cys C and washed for multiple times for eluting, immediately swirling, and then placing the centrifuge tube on a magnetic rack for magnetic dissociation.
In the embodiment of the invention, a MALDI-TOF MS is adopted to measure a sample to be measured containing Cys C and an internal standard solution, and the signal acquisition conditions and the spectrogram processing method are as follows:
1) Point target
Firstly, placing a hydrophobic target plate with the aperture of 2.4mm on an electric heating plate at 39 ℃ for preheating for 5min, taking 2 mu L of SA matrix eluent for spot targeting, and carrying out data acquisition by using MALDI-TOF MS after all samples on the target plate are crystallized.
2) Serum sample MALDI-TOF MS acquisition condition and spectrogram processing method
The MALDI-TOF MS acquisition condition is a semiconductor laser; the laser frequency is 2000Hz; collecting line number 15rows; two-dimensional platform movement speed: 2mm/sec; focus Mass: 13 000Da; the acquisition mass range is as follows: 8-10 kDa.
Correcting the spectrogram after the data acquisition is completed, wherein the correction parameters are baseline correction: linear10k mode; smoothing mass spectrogram: setting 9 windows by adopting a Moving-Average mode; miniSNR 10; % Height to use 1; mass-to-charge ratio calibration: calibration was performed with m/z13 344 (mass spectrum peak of wild type Cys C) mass spectrum peak; the calibration mode is as follows: single point calibration; automatic internal calibration mass to charge ratio: m/z16 240 (mass spectrum peak of internal standard); automatic internal calibration limits: 1500in PPM.
3) Urine sample MALDI-TOF MS acquisition condition and spectrogram processing method
The MALDI-TOF MS acquisition condition is a semiconductor laser; the laser frequency is 1000Hz; collecting line number 15rows; two-dimensional platform movement speed: 1mm/sec; focus Mass: 13 000Da; the acquisition mass range is as follows: 8-10 kDa.
Correcting the spectrogram after the data acquisition is completed, wherein the correction parameters are baseline correction: linear10k mode; smoothing mass spectrogram: setting 9 windows by adopting a Moving-Average mode; miniSNR 10; % Height to use 1; mass-to-charge ratio calibration: calibration was performed with m/z13 344 (mass spectrum peak of Cys C) mass spectrum peak; the calibration mode is as follows: single point calibration; automatic internal calibration mass to charge ratio: m/z16 240 (mass spectrum peak of internal standard); automatic internal calibration limits: 1500in PPM.
The specific type of MALDI-TOF MS adopted in the embodiment of the invention is QuanPro type mass spectrum, and the MALDI-TOF MS can be purchased from the Izod biosciences of Iceland.
The quantitative result of the total concentration of Cys C is calculated according to the following method:
after spectrogram correction, the total Cys C content in the sample is calculated according to the following formula:
cys C total concentration (μg/mL) = (C) Cys C +C 3Pro-OH Cys C +C des-S3Pro-OHCys C +C des-SSP Cys C )×f
C Cys C Concentration of wild-type Cys C (m/z 13.344) in the reaction tube (. Mu.g/mL)
C 3Pro-OH Cys C Concentration of 3Pro-OH Cys C (m/z 13.360) in the reaction tube (. Mu.g/mL)
C des-S 3Pro-OH Cys C Concentration of des-S3Pro-OHCys C (m/z 13 273) in the reaction tube (. Mu.g/mL)
C des-SSP Cys C Concentration of des-SSP Cys C (m/z 13 073) in the reaction tube (. Mu.g/mL)
Dilution of f-sample
Remarks:
the total concentration of Cys C is the sum of the concentrations of wild-type Cys C and each Cys C variant;
C Cys C is wild Cys C;
3Pro-OH Cys C is a Cys C variant with hydroxylation of proline at the third position of the N-terminal;
des-S3 Pro-OH Cys C is a Cys C variant with the N-terminal serine at the first position deleted and the N-terminal proline at the third position hydroxylated;
des-SSP Cys C is a Cys C variant with N-terminal deletion of serine at the first position, serine at the second position and proline at the third position.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: quantification of Cys C in serum samples
In this example, 10 serum samples were randomly selected, and sample specific information is shown in table 1.
Table 110 serum sample numbers and designations
Sample numbering Sample name
1 108
2 109
3 110
4 111
5 112
6 113
7 114
8 115
9 116
10 117
1. Cys C standard curve preparation (for serum sample)
Cys C standard protein (Cys C recombinant protein) was formulated as a standard protein stock solution at 0.5mg/mL with 1% BSA solution. Cys C standard protein stock was then serially diluted to 6 concentration levels of 8. Mu.g/mL, 6. Mu.g/mL, 2. Mu.g/mL, 1. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL with 1% BSA solution. Accurately transferring 30 mu L of Cys C standard protein solution samples with different concentrations into a 0.6mL centrifuge tube, adding 140 mu L of 0.1M Tris pH 10.59 solution and 30 mu L of Cys C internal standard (Cys C recombinant protein connected with 6 XHis-Tag, 2.4 mu g/mL) solution, mixing uniformly by vortex, adding 2 mu g of magnetic bead antibody, mixing uniformly by vortex, and turning over for reaction for 20min. After the reaction, the liquid in the tube was discarded, and the PBST solution was added for washing 3 times. The supernatant was discarded, and deionized water was added to wash for 2 times. The supernatant was discarded.
And accurately adding 10 mu L of SA matrix liquid into the magnetic beads enriched with Cys C and washed for multiple times for eluting, immediately swirling, and then placing the centrifuge tube on a magnetic rack for magnetic dissociation. mu.L of SA matrix eluent was taken out for spotting, and after all the samples on the target plate were crystallized, data collection was performed by MALDI-TOF MS (FIG. 1).
A quantitative standard curve was fitted with the concentration of Cys C as Y, with the ratio x of the sum of the peak signal response values of wild-type Cys C and its variants in the standard protein solution to the Y-value obtained on MALDI-TOF MS as internal standard (FIG. 2).
Cys C quantitative standard curve fitting results were:
y=kx ∈b (correlation coefficient r 2 ＝0.9958)，
Wherein: k=0.0036; b= 1.2049; y is the concentration μg/mL of Cys C; x is the ratio x of the sum of the peak signal response values of the wild Cys C and the variant thereof in the standard protein solution to the Y-value obtained by internal standard on MALDI-TOF MS; "x ∈b" represents the power of x to the power of b.
2. Quantification of Cys C in serum samples
Accurately transferring 30 mu L of serum sample into a 0.6mL centrifuge tube, adding 140 mu L of 0.1M Tris pH 10.59 solution and 30 mu L of Cys C internal standard (2.4 mu g/mL) using liquid, mixing uniformly by vortex, adding 2 mu g of magnetic bead antibody, mixing uniformly by vortex, and turning over for 20min. After the reaction, the liquid in the tube was discarded, and the PBST solution was added for washing 3 times. The supernatant was discarded, and deionized water was added to wash for 2 times. The supernatant was discarded. And accurately adding 10 mu L of SA matrix liquid into the magnetic beads enriched with Cys C and washed for multiple times for eluting, immediately swirling, and then placing the centrifuge tube on a magnetic rack for magnetic dissociation. And taking out 2 mu L of SA matrix eluent to spot the target, and carrying out data acquisition by using MALDI-TOF MS after all samples on the target plate crystallize.
Inputting the obtained ratio of the sum of the peak signal response values of the wild-type Cys C and the variant thereof of each sample to the Y-Value of the internal standard as an x Value, inputting the x Value into a linear equation, calculating the concentration of the wild-type Cys C and the variant thereof in the sample tube, and calculating the total concentration of the Cys C according to a calculation formula given in the specific embodiment, namely, the quantitative result (mug/mL) of the Cys C in the embodiment.
Cys C quantification results for randomly selected 10 serum samples in this example are shown in Table 2.
TABLE 2 Cys C quantification of 10 serum samples
Sample numbering Sample name Cys C quantification (μg/mL)
1 108 4.65
2 109 1.75
3 110 1.06
4 111 1.07
5 112 1.14
6 113 1.71
7 114 2.77
8 115 1.55
9 116 1.44
10 117 1.12
The experimental result shows that the method is used for measuring the Cys C of the serum sample, the LoD is 0.047 mug/mL, the LoQ is 0.16 mug/mL, and the detection range is 0.25 mug/mL to 10 mug/mL, so that the quantitative requirement of most clinical samples can be met.
Example 2: quantification of Cys C in urine samples
In this example, 10 urine samples were randomly selected, and sample specific information is shown in table 3.
Table 310 urine sample numbers and names
1. Cys C standard curve preparation (for urine sample)
Cys C standard protein was formulated as a standard protein stock solution at 0.5mg/mL with 1% BSA solution. Cys C standard protein stock was then serially diluted to 5 concentration levels of 4. Mu.g/mL, 2. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL, 0.1. Mu.g/mL, etc. with 1% BSA solution. Accurately transferring 100 mu L of Cys C standard protein solution samples with different concentrations into a 0.6mL centrifuge tube, adding 70 mu L of Tris solution with pH of 10.59 and 30 mu L of Cys C internal standard (1.2 mu g/mL) using liquid vortex for uniform mixing, adding 2 mu g of magnetic bead antibody for uniform vortex mixing, and turning over for reaction for 20min. After the reaction, the liquid in the tube was discarded, and the PBST solution was added for washing 3 times. The supernatant was discarded, and deionized water was added to wash for 2 times. The supernatant was discarded.
And accurately adding 10 mu L of SA matrix liquid into the magnetic beads enriched with Cys C and washed for multiple times for eluting, immediately swirling, and then placing the centrifuge tube on a magnetic rack for magnetic dissociation. mu.L of SA matrix solution is taken out for spotting, and after all samples on the target plate are crystallized, data acquisition is performed by MALDI-TOF MS (FIG. 4).
A quantitative standard curve was fitted with the concentration of Cys C as Y, with the ratio x of the sum of the peak signal response values of the wild-type Cys C and its variants in the standard solution to the Y-value obtained on MALDI-TOF MS as internal standard (FIG. 5).
y=kx ∈b (correlation coefficient r 2 ＝0.9971)，
Wherein: k=0.0004; b= 1.2636; y is the concentration μg/mL of Cys C; x is the ratio x of the sum of the peak signal response values of the wild Cys C and the variant thereof in the standard protein solution to the Y-value obtained by internal standard on MALDI-TOF MS; "x ∈b" represents the power of x to the power of b.
2. Quantification of Cys C in urine samples
Accurately transferring 100 mu L of urine sample or 100 mu L of Cys C standard protein solution sample with different concentrations into a 0.6mL centrifuge tube, adding 70 mu L of Tris solution with pH of 10.59 and 30 mu L of Cys C internal standard (1.2 mu g/mL) to be uniformly mixed by using liquid vortex, adding 2 mu g of magnetic bead antibody to be uniformly mixed by vortex, and turning over for 20min. After the reaction, the liquid in the tube was discarded, and the PBST solution was added for washing 3 times. The supernatant was discarded, and deionized water was added to wash for 2 times. The supernatant was discarded. And accurately adding 10 mu L of SA matrix liquid into the magnetic beads enriched with Cys C and washed for multiple times for eluting, immediately swirling, and then placing the centrifuge tube on a magnetic rack for magnetic dissociation. And taking out 2 mu L of SA matrix eluent to spot the target, and carrying out data acquisition by using MALDI-TOF MS after all samples on the target plate crystallize.
Inputting the obtained ratio of the sum of the peak signal response values of the wild-type Cys C and the variant thereof of each sample to the Y-Value of the internal standard as an x Value, inputting the x Value into a linear equation, calculating the concentration of the wild-type Cys C and the variant thereof in the sample tube, and calculating the total concentration of the Cys C according to a calculation formula given in the specific embodiment, namely, the quantitative result (mug/mL) of the Cys C in the embodiment.
Cys C quantification results for 10 urine samples randomly selected in this example are shown in Table 4.
TABLE 4 Cys C quantification results for 10 urine samples
Sample numbering Sample name Cys C quantification (μg/mL)
1 86 0.129
2 87 <Lower limit of quantification
3 88 <Lower limit of quantification
4 89 0.118
5 90 <Lower limit of quantification
6 201 2.182
7 202 4.351
8 209 6.250
9 213 <Lower limit of quantification
10 214 <Lower limit of quantification
The experimental result shows that the method is used for measuring Cys C of a urine sample, the LoD is 0.026 mug/mL, the LoQ is 0.085 mug/mL, and the detection range is 0.1 mug/mL to 4 mug/mL, so that the quantitative requirement of most clinical samples can be met.
Example 3: quantitative reproducibility test of Cys C in serum samples
30. Mu.L of Cys C serum samples were accurately removed in 0.6mL centrifuge tubes and 15 sets of technical replicates were set. Calculating the ratio of the sum of peak signal response values of the wild Cys C and the variant thereof to the Y-Value of the internal standard, calculating the quantitative values of the wild Cys C and the variant thereof according to a Cys C quantitative standard curve applicable to serum samples, calculating the total concentration of Cys C according to a calculation formula given in a specific embodiment, namely the quantitative result (mug/mL) of Cys C in the embodiment, and further calculating the mean Value and standard deviation of the quantitative result Value of Cys C. The coefficient of variation (CV, expressed as a percentage) was determined based on the standard deviation and the mean.
The experimental results are shown in Table 5.
The results show that the method of the invention has good reproducibility and consistency, stable methodology and a coefficient of variation CV <4% (n=15).
TABLE 5 quantitative results reproducibility evaluation results of Cys C serum samples
Example 4: quantitative reproducibility test of Cys C in urine sample
Accurately remove 100 μl of Cys C urine sample in a 0.6mL centrifuge tube, and set 15 sets of technical replicates. Calculating the ratio of the sum of peak signal response values of the wild Cys C and the variant thereof to the Y-Value of the internal standard, calculating the quantitative values of the wild Cys C and the variant thereof according to a Cys C quantitative standard curve applicable to urine samples, calculating the total concentration of Cys C according to a calculation formula given in a specific embodiment, namely the quantitative result (mug/mL) of Cys C in the embodiment, and further calculating the mean Value and standard deviation of the quantitative result Value of Cys C. The coefficient of variation (CV, expressed as a percentage) was determined based on the standard deviation and the mean.
The experimental results are shown in Table 6.
The results show that the method of the invention has good reproducibility and consistency, stable methodology and a coefficient of variation CV <4% (n=15).
Table 6Cys C urine sample quantitative result reproducibility evaluation results
The experimental results show that the method comprises the following steps: (1) Good reproducibility and consistency, stable methodology, coefficient of variation CV <4% (n=15); (2) The detection result is accurate, the sensitivity is high, the detection range is large, the application value is high, the lowest LoD is 0.026 mug/mL, the lowest LoQ is 0.085 mug/mL, and the maximum detection range is 0.1-10 mug/mL aiming at different clinical samples, so that the quantitative requirement of most clinical samples can be met; (3) The sample using amount is low, especially the serum sample using amount can be as low as 30 mu L, which is beneficial to the popularization and application of the method for detecting Cys C by MALDI-TOF MS in clinic.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims (10)

1. A sample processing kit for detecting Cys C using MALDI-TOF MS, the kit comprising the following reagents:
An internal standard Cys C solution;
a first buffer;
anti-Cys C antibodies-magnetic beads;
eluting the solution.
2. The kit according to claim 1, wherein the first buffer is selected from one of MES buffer, PBS buffer, tris-HCl buffer, HEPES buffer, preferably Tris-HCl buffer;
optionally, the pH of the first buffer is 10.1 to 11.1, preferably 10.4 to 10.8;
optionally, the concentration of the first buffer is 0.05M to 0.15M, preferably 0.08M to 0.12M.
3. The kit of claim 1, wherein the internal standard Cys C solution comprises BSA;
optionally, the concentration of BSA is 0.5-5%, preferably 0.5-1.5%, more preferably 1% by mass volume in mg/mL.
4. The kit of claim 1, wherein the internal standard Cys C solution contains 1.0-3.0 μg/mL of internal standard Cys C;
optionally, the internal standard Cys C is selected from a Cys C recombinant protein having a tag sequence or an isotopically labeled Cys C protein;
optionally, the tag sequence comprises at least one selected from His tag, flag tag, GST tag, MBP tag, C-MYC tag, HA tag, FC tag, AVI tag, MBP tag, DDDDK tag, preferably His tag;
Optionally, the isotope is 13 C or 15 N。
5. The kit of claim 1, wherein the anti-Cys C antibody is a monoclonal antibody.
6. The kit of any one of claims 1 to 5, further comprising the following reagents: further comprising a second buffer comprising a nonionic surfactant, and/or deionized water;
optionally, the nonionic surfactant comprises a surfactant selected from the group consisting of tween 20, tween 60, tween 80, CHAPS, triton X-100, alkoxypolyethylene hydroxy ethanol, octyl glucoside, dodecyl maltoside, N-octyl alpha-D-glucoside, N-octanoyl-N-methyl glucamine, N-nonanoyl-N-methyl glucamine, N-decanoyl-N-methyl glucamine, nonyl-beta-D-glucopyranoside, dodecyl-beta-D-maltoside, N, at least one of N-dimethyldodecylamine-N-oxide, decylpyranoside, 1-O-decyl- β -D-maltoside, deoxy-Bigchap, ground saponin, triton X-114, nonylphenol polyoxyethylene ether, tetraethyleneglycol monolodecyl ether, tetramethylammonium hydroxide pentahydrate, polysorbate-85, tetrabutyl phenol, undecyl- β -D-maltoside, octyl- β -D-thiopyranoside, N-octyl- β -D-glucopyranoside, octaglycol monolodecyl ether, saponin, span, sucrose dodecanoate, glycerol monooleate, 6-O- (N-heptanoyl) -methyl- α -D-glucoside, polyethylene glycol monostearate, N-nonyl- β -D-thiomaltoside, N-dodecyl- β -D-maltoside, preferably tween 20;
Optionally, the second buffer is selected from one of PBS buffer, MES buffer, tris buffer, DPBS buffer, CBS buffer, BBS buffer, HEPES buffer, TBS buffer, BES buffer, TEA buffer, MOPS buffer, AMPD buffer, EPPS buffer, MOPSO buffer, AMPSO buffer, DIPSO buffer, tap buffer, preferably PBS buffer;
further, the concentration of tween 20 is 0.1 percent by mass volume, and the unit of mass volume is mg/mL; the concentration of the PBS buffer was 10mM.
7. Kit according to claim 1, wherein the elution solution contains a matrix comprising at least one selected from the group consisting of sinapic acid, α -cyano-4-hydroxycinnamic acid, 2, 5-dihydroxybenzoic acid, 2-mercaptobenzothiazole, 2,4, 6-trihydroxyacetophenone, anthratriphenol, 3-indoleacrylic acid, preferably sinapic acid, α -cyano-4-hydroxycinnamic acid, 2, 5-dihydroxybenzoic acid or 2-mercaptobenzothiazole, more preferably sinapic acid, and an antigen-antibody-bead complex dissociation reagent;
optionally, the concentration of the sinapic acid is 5-10 mg/mL;
optionally, the antigen-antibody-magnetic bead complex dissociation reagent includes at least one selected from an organic acid solution, an inorganic acid solution, and an alkali solution;
The organic acid solution comprises at least one selected from formic acid solution, acetic acid solution, trifluoroacetic acid solution, propionic acid solution, glycolic acid solution, amino acid solution, butyric acid solution, citric acid, succinic acid, ascorbic acid, lactic acid, malic acid and tartaric acid;
the inorganic acid solution comprises at least one selected from hydrochloric acid solution, sulfuric acid solution and phosphoric acid solution;
the alkali solution comprises at least one selected from sodium hydroxide solution, potassium hydroxide solution, tris (hydroxymethyl) aminomethane solution, tetraethylammonium bromide solution, sodium carbonate solution, sodium bicarbonate solution, ammonium bicarbonate solution, ammonia water solution and urea solution;
optionally, the antigen-antibody complex dissociation reagent is a trifluoroacetic acid solution;
optionally, the concentration of the trifluoroacetic acid solution is 0.07 to 7%, preferably 0.1 to 0.7% by volume.
8. A sample processing kit for detecting Cys C using MALDI-TOF MS, the kit comprising the following reagents:
an internal standard Cys C solution;
a first buffer;
anti-Cys C antibodies-magnetic beads;
eluting the solution;
wherein,
the first buffer solution is Tris-HCl buffer solution, the pH of the Tris-HCl buffer solution is 10.6, and the concentration of the Tris-HCl buffer solution is 0.1M;
The internal standard Cys C solution contains BSA, wherein the concentration of the BSA is 1% in terms of mass volume percentage, and the mass volume unit is mg/mL;
the anti-Cys C antibody is a monoclonal antibody;
the elution solution adopted in the elution treatment contains a matrix and an antigen-antibody-magnetic bead complex dissociation reagent, wherein the matrix is sinapic acid of 5-10 mg/mL, the dissociation reagent is trifluoroacetic acid, and the concentration of the trifluoroacetic acid solution is 0.7% according to the volume ratio.
9. A kit for detecting Cys C using MALDI-TOF MS, comprising:
a reagent in a kit according to any one of claims 1 to 8;
cys C standard.
10. The kit of claim 9, wherein the Cys C standard comprises BSA;
optionally, the concentration of BSA is 0.5-5%, preferably 0.5-1.5%, more preferably 1% by mass volume in mg/mL;
optionally, the concentration of the Cys C standard is 0.5-1.5 mg/mL.
CN202311207060.6A 2023-09-18 2023-09-18 Sample processing kit for detecting Cys C by MALDI-TOF MS and application thereof Pending CN117310182A (en)

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