CN110488025A - A kind of chemiluminescence quantitative detection excrement calprotectin and its detection method and its intestinal health detection purposes - Google Patents
A kind of chemiluminescence quantitative detection excrement calprotectin and its detection method and its intestinal health detection purposes Download PDFInfo
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- CN110488025A CN110488025A CN201910903795.XA CN201910903795A CN110488025A CN 110488025 A CN110488025 A CN 110488025A CN 201910903795 A CN201910903795 A CN 201910903795A CN 110488025 A CN110488025 A CN 110488025A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
Abstract
The invention discloses a kind of chemiluminescence quantitative detection excrement calprotectin and its detection method and its purposes in intestinal health detection, detection reagents, comprising: immunocomplex, antibody test system；Immunocomplex includes: calprotectin calibration object, calprotectin quality-control product, by the first specific antibodies after marking, the second specific antibodies after label and the compound rear labeled calprotectin complex antibody formed of sample to be tested；First specific antibodies, the combination of second specific antibodies and calprotectin are as follows: the amino acid epitope in a kind of antibody and calprotectin S100A8 chain amino acids sequence is specifically bound, and the amino acid epitope in another antibody and calprotectin S100A9 chain amino acids sequence is specifically bound；The present invention can calprotectin content in accurate, quantitative detection human faecal mass sample, have the advantages that high sensitivity, stability are good, clinical auxiliary with higher to intestinal health detection is worth.
Description
Technical field
The present invention relates to field of biological detection, especially a kind of chemiluminescence quantitative detection excrement calprotectin and its detection
Method and its purposes detected in intestinal health.
Background technique
Inflammatory bowel disease (inflammatory bowel disease, IBD) is to involve one kind of ileum, rectum, colon
Chronic, idiopathic bowl inflammatory diseases.Clinical manifestation diarrhea, abdominal pain, or even can have bloody stool.This disease includes ulcerative colitis
(ulcerative colitis, UC) and Crohn disease (Crohn ' s disease, CD).Ulcerative colitis is colonic mucosa
Layer and submucosa continuity inflammation, disease usually first involve rectum, gradually spread to total colectomy.Crohn disease, which can be involved, totally disappeared
Change road, be noncontinuity holostrome inflammation, most often involving position is terminal ileum, colon and crissum.The IBD cause of disease and pathogenesis are still
It is completely not clear, it is known that inflammatory reaction caused by the reaction of Intestinal Mucosal Immunity system exception plays an important role in IBD morbidity,
It is considered caused by multifactor interaction, mainly includes environment, heredity, infection and immune factor.
IBD has chronic and recurrent exerbation clinical characters, therefore over the course for the treatment of, needs repeatedly to treat disease
The assessment of effect, to judge therapeutic effect or adjust therapeutic scheme in time；For the patient of paracmasis, it is also desirable to long-term close follow-up
Check, to monitor whether it recurs.Judge that the most accurate method of IBD mobility is to carry out Sigmoidoscope joint biopsy at present
It looks into.But Sigmoidoscope and tissue biopsy have the following problems: belonging to invasive inspection, patient has the experience of pain；Review time
Relatively long, whole process needs the positive cooperation of patient；In clinical position, some patientss are difficult to receive, and compliance is poor；For
For children, it is not resistant to colonoscopy bring pain more, is difficult to mate to its inspection；Vigilance should be maintained to the fact that severe or outburst
Type patient, row colonoscopy have the risk for inducing toxic megacolon, for this kind of patient, repeated multiple times row colonoscopy
Belong to taboo；In addition, when merge narrow, shank cannot by when, Sigmoidoscope can not evaluate the inflammatory conditions of the narrow above enteric cavity.
In conclusion Sigmoidoscope and tissue biopsy cannot fully meet clinical needs.Therefore, it is necessary to a kind of energy effecting reaction enteron aisle is viscous
Film inflammation, easy, noninvasive Index for examination.
Inflammatory bowel disease common experimental room inspection method mainly have erythrocyte sedimentation rate, C reactive protein (C-reactive protein,
CRP) and total white blood cells (white blood cells, WBC), blood platelet (platelet, PLT), hemoglobin, albumin,
Erythrocyte sedimentation rate etc., but the sensibility and specificity of these indexs is still undesirable, and the influence vulnerable to other inflammation of whole body, clinical application
It is worth limited.The leucocyte excretion rate of excrement indium label is the goldstandard for reflecting IBD disease activity, but it samples time-consuming, behaviour
Make complexity, and need to inject nucleic, affects, limit its application to patient health.
Market need it is a kind of can noninvasive, easy, high specificity, sensibility is high, the enteron aisle that avoids systemic inflammatorome from influencing is strong
Health detection method.
Detection inflammatory bowel disease is assisted with excrement calprotectin, has been written to the recommendation of domestic and international guide.Such as China
Diagnosis status of inflammatory bowel disease and the National Consensus for the treatment of point out that " conditional unit can do the inspections such as excrement calprotectin as auxiliary
Index for examination ", World Gastroenterology Organization in 2010 is about diagnosis status of inflammatory bowel disease and the practice guideline for the treatment of also by fecal calprotectin
Albumen is defended as one of the auxiliary examination recommended.Excrement calprotectin finds that earliest, research is most wide in diagnosis status of inflammatory bowel disease
It is general, it is most representative.
Calprotectin belongs to S100 albumen large family, is secreted by positive bone marrow-derived cells such as neutrophil leucocyte, macrophages, is
Neutrophil leucocyte and protein important in the macrophage matter of activation, account for about neutrophil leucocyte cytoplasmic protein matter 60% are left
It is right.It is played in the important pathology such as inflammation, tumour, infection, immunological regulation, Apoptosis, aging and physiology course important
Effect.Calprotectin is widely distributed in the intracorporal cell of people, tissue and body fluid, can blood plasma, excrement, urine, saliva,
It is detected in synovial fluid, cerebrospinal fluid and colon mucosa tissues biopsy.Calprotectin, can be with as a kind of intestinal inflammation marker
Organic diseases associated with inflammation IBD and functional disease IBS is clearly distinguished, can be used as a kind of activity for evaluating inflammatory bowel disease
Property index, assess inflammatory bowel disease mucous membrane, evaluation drug therapy effect, cure and predictive disease recurrence.
Exactly because calprotectin is widely distributed in vivo, many important physiological functions, it once had more in history
Kind name: for example taken the lead in 1980 by Fagerhol et al. the L1 protein separated from the cytoplasm of neutrophil leucocyte；
The MRP-14 albumen found in the macrophage that rheumatoid arthritis infiltrates by Odink et al. in 1987；1988 by
The calgranulin of Wilkinsoin et al. discovery；And tumor metastasis suppressor gene GAP-associated protein GAP, the related antigen of cystic fibrosis,
P14 substance etc. finally confirms that above-mentioned albumen is same protein through Anderson et al., by further confirming this albumen
Matter has a structure feature of typical case S-100 albumen large family, thus by it is this in conjunction with calcium, it is multi-functional, with protective egg
It is white to be named as calprotectin.
Fecal calprotectin is defended property of protein and is stablized, and storage is conducive to.The physicochemical property of calprotectin is extremely stable, on the one hand with calcium knot
On the other hand resistance to enzyme after conjunction itself has antimicrobial bioactivity, these characteristics make calprotectin in enteric cavity and the external world
In environment can long-term retention properties it is stable and be conducive to storage.At room temperature, calprotectin keeps stability property can in stool
Up to 7 days.
Calprotectin being evenly distributed in stool, therefore single fecal calprotectin defends the favorable repeatability of Protein Detection.Research
The calprotectin levels of the excrement indium mark leucocyte excretion rate and primary sample that confirm IBD patient have good correlation,
Reliability for clinically random unitary determination calprotectin provides foundation.
Currently, the method for detection calprotectin content mainly has immunochromatographic method and enzyme-linked immunization.Wherein, using colloid
The listing representative products of golden chromatography are that Xiamen is positive the calprotectin detection kit of Biotechnology Co., Ltd, due to colloid
Gold and labeled protein be by electrostatic interaction in conjunction with, in conjunction with unstable, transaction is by external interferences such as soda acid factors, therefore
Its detection sensitivity is lower, is usually used in qualitative or half-quantitative detection, can not accurate quantitative detection；Using immunofluorescence chromatography
The calprotectin assay kit that representative products are Guangzhou Feng Run Biotechnology Co., Ltd is listed, because fluorescence radiation needs are additional
Light source detects on the direction of perpendicular light source, and the molecules such as protein, amino acid in biological sample can also generate background fluorescence,
Easily lead to measurement in background it is higher, generate disturbing factor the problems such as.Furthermore fluorescent immunological technology is for needing when quantitative determining
The processing method of suitable fluorescent reagent and sample is selected to reduce the influence of non-specific adsorption albumen, technology is complicated, and
Higher cost；The commercialized product of enzyme-linked immunization is used to detect for the calprotectin of Germany Buhlmann Laboratories AG
Kit can quantitative determine the content of calprotectin in excrement, but enzyme-linked immunization test is used to need to carry out board-washing, incubate
It educates, add the tedious steps such as chromogenic substrate, and test process and be affected by environment temperature etc., need professional to operate, test
Time is longer.Market needs one kind that can replace general immunological method, calcium can defend in accurate, quantitative detection human faecal mass sample
The detection reagent of protein content, the present invention solve such problems.
Summary of the invention
To solve the deficiencies in the prior art, the purpose of the present invention is to provide a kind of chemiluminescence quantitative detection fecal calprotectins to defend
Albumen and its detection method and its in purposes of intestinal health detection, egg can be defended by calcium in accurate, quantitative detection human faecal mass sample
Bai Hanliang has the advantages that high sensitivity, stability are good, clinical auxiliary value with higher to intestinal health detection.
In order to achieve the above objectives, the present invention adopts the following technical scheme that:
A kind of chemiluminescence quantitative detection excrement calprotectin, comprising: immunocomplex, magnetic bead chemiluminescence detection body
System, fixedly separated phase are magnetic particle；
Immunocomplex includes: calprotectin calibration object, calprotectin quality-control product, by after marking the first specific antibodies,
Compound rear " the first specific antibodies-calprotectin-mark after label formed of the second specific antibodies and sample to be tested after label
The labeled calprotectin complex antibody of the second specific antibodies after note "；
A kind of chemiluminescence quantitative detection excrement calprotectin above-mentioned, the first specific antibodies, the second specific antibodies and calcium
Defend the combination of albumen are as follows: a kind of antibody and the amino acid epitope in calprotectin S100A8 chain amino acids sequence are specific
In conjunction with the amino acid epitope in another antibody and calprotectin S100A9 chain amino acids sequence is specifically bound.
A kind of chemiluminescence quantitative detection excrement calprotectin above-mentioned, the first specific antibodies, the second specific antibodies and calcium
Defend the combination of albumen are as follows: a kind of antibody and the amino acid epitope in calprotectin S100A8 chain amino acids sequence are specific
In conjunction with the amino acid epitope in another antibody and calprotectin S100A8 chain amino acids sequence is specifically bound.
A kind of chemiluminescence quantitative detection excrement calprotectin above-mentioned, the first specific antibodies, the second specific antibodies and calcium
Defend the combination of albumen are as follows: a kind of antibody and the amino acid epitope in calprotectin S100A9 chain amino acids sequence are specific
In conjunction with the amino acid epitope in another antibody and calprotectin S100A9 chain amino acids sequence is specifically bound.
A kind of chemiluminescence quantitative detection excrement calprotectin above-mentioned, fixed separation are mutually micro- for the coated magnetic of Streptavidin
Grain.
A kind of chemiluminescence quantitative detection excrement calprotectin above-mentioned, fixedly separated phase are calprotectin monoclonal or more
The coated magnetic particle of clonal antibody.
A kind of chemiluminescence quantitative detection excrement calprotectin above-mentioned is labeled as luminescent label, luminescent marking
Object includes: acridinium ester, HRP, alkaline phosphatase or Rumi sodium.
A kind of chemiluminescence quantitative detection excrement calprotectin above-mentioned, the first specific antibodies, the second specific antibodies are more
Clonal antibody or monoclonal antibody, the monoclonal antibody derives from the hybridoma of mouse, rat, rabbit, described polyclonal
Antibody sources are in rabbit, sheep, chicken.
A kind of detection method of chemiluminescence quantitative detection excrement calprotectin, collects and extracts fecal sample to be measured, add
Enter in the reaction cup of Full-automatic chemiluminescence apparatus, the first labeled anti-calprotectin antibody is added in each reaction cup, is wrapped
The magnetic particle solution of quilt, the second labeled anti-calprotectin antibody, the anti-calprotectin antibody preincubation being labeled in addition second,
Cleaning, and secondary incubation, cleaning after the anti-calprotectin antibody that addition second is labeled, or defended in the calcium that addition second is labeled
It is once incubated for, cleans after protein antibodies, be eventually adding substrate solution or exciting liquid excitation is carried out to reaction system and shine, according to having determined
Target calibration curve information carries out data result processing；
First labeled anti-calprotectin antibody, the second labeled anti-calprotectin antibody are as follows: a kind of antibody and calcium defend egg
Amino acid epitope specific binding in white S100A8 or S100A9 chain amino acids sequence, another antibody and calprotectin
Amino acid epitope specific binding in S100A8 or S100A9 chain amino acids sequence.
A kind of purposes that chemiluminescence quantitative detection excrement calprotectin is detected in intestinal health, for detecting intestinal health
Situation.
The invention has the beneficial effects that:
The present invention uses " double-antibody sandwich " technology of two specific antibody combination calprotectin difference antigenic determinants,
Obtain the immunocomplex of " the second specific antibodies after the first specific antibodies-calprotectin-label after label ", Neng Goute
Calprotectin in opposite sex detection human faecal mass sample, high sensitivity, stability are good；
The detection method that the present invention uses Full-automatic magnetic beads chemiluminescence platform development to go out, detecting step is simple, and the time is short；
The result and clinical detection result obtained with present invention detection has good consistency, can reflect enteritis very well
Disease property disease symptoms evaluate the activity index of inflammatory bowel disease, assess inflammatory bowel disease mucous membrane, and evaluation drug therapy acts on,
It cures and predictive disease recurrence, clinical auxiliary value with higher to intestinal health detection.
Detailed description of the invention
Fig. 1 is the different tetramer structure schematic diagram of calprotectin of the present invention (S100A8/S100A9).
Specific embodiment
Specific introduce is made to the present invention below in conjunction with the drawings and specific embodiments.
A kind of chemiluminescence quantitative detection excrement calprotectin of △, comprising: immunocomplex, antibody test system；
Immunocomplex includes: calprotectin calibration object, calprotectin quality-control product, by after marking the first specific antibodies,
Compound rear " the first specific antibodies-calprotectin-mark after label formed of the second specific antibodies and sample to be tested after label
The labeled calprotectin complex antibody of the second specific antibodies after note "；
First specific antibodies, the combination of the second specific antibodies and calprotectin are as follows: a kind of antibody and calprotectin
Amino acid epitope specific binding in S100A8 chain amino acids sequence, another antibody and calprotectin S100A9 peptide chain ammonia
Amino acid epitope specific binding in base acid sequence.
Calprotectin is calcium, the zinc ion binding protein that a molecular weight is 36kD.The S100A9 for being 14kD by molecular weight
Peptide chain connects to form dimer or tetramer with non-covalent bond with the S100A8 peptide chain that molecular weight is 8kD.Every peptide chain can
In conjunction with multiple Ca2+ and Zn2+, to heat resistance and enhance water-disintegrable characteristic.
Shown in the structural schematic diagram of calprotectin (S100A8/S100A9) different tetramer as shown in Figure 1, label 1 and mark
Note 2 is S100A8 dimer, and label 3 and label 4 are S100A9 dimer, and it is poly- that the two specific hybrid forms calprotectin different four
Body, label C is calcium binding site in figure.
S100A8 chain amino acids sequence:
1 mltelekaln siidvyhkys likgnfhavy rddlkkllet ecpqyirkkg advwfkeldi
61 ntdgavnfqe flilvikmgv aahkkshees hke；
S100A9 chain amino acids sequence:
1 mtckmsqler nietiintfh qysvklghpd tlnqgefkel vrkdlqnflk kenknekvie
61 himedldtna dkqlsfeefi mlmarltwas hekmhegdeg pghhhkpglg egtp；
Wherein S100A9 is in conjunction with calcium, and the functional domain of S100A9 subunit is EF hand domain, i.e., by conveyor screw E,
The structure of conveyor screw F and the finger sample of hinge area composition between the two, can be in conjunction with calcium, and is activated.Activation
S100A9 can be combined in turn with Advanced Glycation End Product Receptors, the Toll-like receptor etc. on cell membrane, in active cell
NF- κ B stimulates endonuclear S100A9 promoter, transcribes S100A9 mRNA, more S100A9 albumen is expressed as, thin
It is intracellular in conjunction with calcium after, on the one hand in conjunction with arachidonic acid, and the compound precursor reactant of nadph oxidase on cell membrane, stimulation
Reactive oxygen species access, the signal transduction of transmitting inflammation access form the water fall effect of inflammation；In addition to this, intracellular activation
Calprotectin can also be in conjunction with cytoskeletal elements such as keratin intermediate filament, and transmitting inflammation cell is migrated.It is noted that
Calprotectin can also be realized such as nitrosylation, S- glutathione, methionine oxidation effect to inflammation by the modification of itself
The regulation of disease magnitude and inflammation range.
Calprotectin is a kind of transition metal complex albumen, and the performance of function relies on and the metal ions such as calcium, zinc, manganese
In conjunction with.In addition to it is above-mentioned in conjunction with calcium other than, two manganese binding sites can be collectively formed in two subunits of calprotectin.And
The peptide chain end of S100A9, there are zinc binding sites, in conjunction with zinc after, have antimicrobial activity.
Antibody test system is magnetic bead chemiluminescence detection system, and stationary phase is magnetic particle.There is the separation of following two solid phase
Embodiment:
Example scheme one, " Streptavidin-biotin " system；
A kind of calprotectin detection reagent, comprising:
The calprotectin monoclonal or polyclonal antibody of biotin labeling；
Mark the calprotectin monoclonal or polyclonal antibody of substance markers, as one embodiment, luminous signal marker
It can be acridine rouge, HRP, alkaline phosphatase or Rumi sodium；
Calprotectin calibrates product, and ingredient includes: human faecal mass calprotectin antigen；
Calprotectin quality-control product, ingredient include: human faecal mass calprotectin antigen；
Sample to be tested；
The coated magnetic particle of Streptavidin；
Calprotectin calibration card: burning calprotectin calibrates product relevant information in blocking；
Calprotectin reagent card: burning calprotectin calibration curve relevant information in blocking；
Cleaning solution, the ingredient of cleaning solution include: 0.01mol/L-0.2mol/L phosphate, 0.5mol/L-2mol/L chlorination
Sodium, 0.05%-0.5% polysorbas20；
Specimen extraction liquid, the ingredient of specimen extraction liquid include: calcium chloride, polysorbas20, Sodium azide；
Full-automatic immunity inspection system substrate solution A liquid, ingredient includes: hydrogen peroxide；
Full-automatic immunity inspection system substrate solution B liquid, ingredient includes: sodium hydroxide.
Example scheme two, the magnetic particle system of antibody direct coated；
A kind of chemiluminescence quantitative detection excrement calprotectin, comprising:
Calprotectin monoclonal or the coated magnetic particle of polyclonal antibody,
The calprotectin monoclonal or polyclonal antibody antibody of substance markers are marked, as one embodiment, marker can be with
It is acridine rouge, HRP, alkaline phosphatase or Rumi sodium；
Calprotectin calibrates product, and ingredient includes: human faecal mass calprotectin antigen；
Calprotectin quality-control product, ingredient include: human faecal mass calprotectin antigen；
Sample to be tested；
Calprotectin calibration card: burning calprotectin calibrates product relevant information in blocking；
Calprotectin reagent card: burning calprotectin calibration curve relevant information in blocking；
Cleaning solution, the ingredient of cleaning solution include: 0.01mol/L-0.2mol/L phosphate, 0.5mol/L-2mol/L chlorination
Sodium, 0.05%-0.5% polysorbas20；
Specimen extraction liquid, the ingredient of specimen extraction liquid include: calcium chloride, polysorbas20, Sodium azide；
Full-automatic immunity inspection system substrate solution A liquid, ingredient includes: hydrogen peroxide；
Full-automatic immunity inspection system substrate solution B liquid, ingredient includes: sodium hydroxide.
Detection method includes the following steps for a kind of chemiluminescence quantitative detection excrement calprotectin of △:
(1) Freshman fecal sample is collected and is extracted with specimen extraction liquid, obtain clarification sample to be tested；
(2) protein quantification detection is defended to fecal calprotectin with calibration product by calibration program to calibrate；
(3) quality-control product and sample for respectively adding 75uL are into specified reaction cup；
(4) 50uL biotin labeling anti-calprotectin antibody is added in each reaction cup；
(5) the coated magnetic particle solution of 20uL Streptavidin is added in each reaction cup；
(6) 37 DEG C of incubation 15min；
(7) each reaction cup is cleaned 3 times with cleaning solution；
(8) 100uL acridinium ester label anti-calprotectin antibody is added in each reaction cup；
(9) 37 DEG C of incubation 15min；
(10) each reaction cup is cleaned 3 times with cleaning solution；
(11) 200uL full-automatic immunity inspection system substrate solution A liquid is added in each reaction cup；
(12) 200uL full-automatic immunity inspection system substrate solution B liquid is added in each reaction cup；
(13) it is sequentially measured on Full-automatic chemiluminescence apparatus, the calibration curve information according to the burning of calprotectin reagent card
Carry out data result processing.
The calibration curve of calprotectin reagent card burning is as follows:
The purposes of reagent of the invention is: detection intestinal health situation.Ulcerative colitis and Crohn disease etc. are a series of
The auxiliary diagnosis of agnogenic enteron aisle nonspecific inflammation disease, disease are in active period or the interpretation of dormant period, inflammation
The direction of medication usage and curative effect evaluation of disease property bowel disease is applied to physical examination section, with being in reagent screening Check-up crowd of the present invention
It is no to have patients with inflammatory bowel disease, its intestinal health situation is detected, holds treatment best period in time；Other departments, such as internal medicine
Or Gastroenterology dept., there are the patient with sympotoms such as the diarrhea, abdominal pain, nausea and vomiting of unknown cause, is not suitable for carrying out the directly inspection such as colonoscopy
Patient is looked into, auxiliary diagnosis can be carried out by reagent through the invention；For having made a definite diagnosis patients with inflammatory bowel disease, tried with the present invention
Whether agent detects its severity, effect after clinical application, and fully recovers.
△ applies detection method of the invention
Step 1, physical examination sample collection and pre-treatment；Inspection body carries out medical examiner using specimen extraction liquid regular
The acquisition and extraction of fecal sample, extraction step are carried out in strict accordance with specimen extraction liquid operation instruction.Sample to be tested through extracting
It can be reserved at 15-25 DEG C 3 days, 2-8 DEG C can be reserved for 5 days, and -20 DEG C can be reserved for 3 months, only limit freeze thawing 3 times.Sample is answered in transport
Avoid strenuous vibration.
Detection reagent pre-treatment: step 2 before use, fecal calprotectin, which is defended protein assay reagent, is placed in room temperature rewarming, prepares
First labeled anti-calprotectin antibody, the second labeled anti-calprotectin antibody, calprotectin calibrate product, calprotectin Quality Control
Product, sample to be tested, coated magnetic particle, cleaning solution, full-automatic immunity inspection system substrate solution A liquid, full-automatic immune inspection
Check system substrate solution B liquid；As one embodiment, the first labeled anti-calprotectin antibody is biotin labeling calprotectin
Antibody, the second labeled anti-calprotectin antibody are acridinium ester label anti-calprotectin antibody, and coated magnetic particle is strepto- parent
With the coated magnetic particle solution of element.
Step 3 calibrates product, quality-control product pre-treatment: the distilled water or deionization of the every pipe addition 1.0mL of calibration product, quality-control product
Water dissolution, stands 5 minutes, mixing of turning upside down, it is ensured that all solids dissolutions are completely and bubble-free generates.After use
- 20 DEG C of preservations should be immediately placed on, are only limited freeze thawing 3 times.
Step 4, calibration: being put into reagent bottle suit for antibody and magnetic particle solution, and instrument is swept into calprotectin reagent card,
Reagent bottle suit is put into instrument designated position；Then, calprotectin calibration card is swept into instrument, and calibration product is put into finger
In fixed reaction frame, calprotectin quantitative detection is calibrated；
Step 5 detects on Full-automatic chemiluminescence analyzer, it presses list procedure and is loaded and is operated (unit: μ
L):
Step 6 calculates: standard curve and measurement of the chemiluminescent analyzer by burning in calprotectin reagent card
The concentration value of relative light intensity (RLU) calculating sample and quality-control product.
Step 7, Quality Control: according to the measurement concentration of quality-control product, being compared with sign value range, if the matter beyond mark
Range is controlled, illustrates that this detection is unqualified, detection need to be re-started.
Step 8 determines health condition according to term of reference and testing result.Term of reference is as follows:
Normal health crowd's defines value lower than 200 μ g/g；
Positive Populations define value greater than 50 μ g/g；
The value that defines of strong positive crowd is greater than 500 μ g/g.
The application of term of reference and the explanation of testing result:
Fecal calprotectin defends protein assay reagent being explained as follows for inspection result:
The detection of healthy population defines value lower than 200 μ g/g, is because defining value in different antibody and reaction system
Understand difference, and when the crowd to different regions detects, value is defined in the detection of normal population can have centainly
Fluctuation, for example, can by being detected to local certain amount healthy population, by healthy population detection define value position
For 180 μ g/g, or 150 μ g/g or 100 μ g/g, but its value of defining upper limit has to be lower than 200 μ g/g.Therefore when detection
When sample results are higher than 200 μ g/g, it can be determined that this sample is the positive, and clinical interpretation is bowl inflammatory diseases symptom occur,
Sigmoidoscope joint biopsy can be carried out as further making a definite diagnosis, need to be treated in the case where medical practitioner instructs；
Positive Populations define value greater than 50 μ g/g, are by a certain number of hemorrhage of lower digestive tract patient fecal samples
It is detected, will test the lower limit value that result is obtained by statistical method, the different crowd of different regions can be according to locality
Positive sample result determines positive value, for example, can by local a certain number of hemorrhage of lower digestive tract patient fecal samples into
Positive sample detection is defined value and is positioned as 60 μ g/g by row detection, or 70 μ g/g or 80 μ g/g, but under its value of defining
Limit has to be larger than 50 μ g/g.Therefore when detecting sample results lower than 50 μ g/g, it can be determined that this sample is feminine gender, clinical interpretation
Not occur bowl inflammatory diseases symptom；
The value that defines of strong positive crowd is greater than 500 μ g/g, is according to the Severe intestinal diseases associated with inflammation patient made a definite diagnosis
Excrement calprotectin content determine.When pattern detection result is more than 500 μ g/g, it is judged as strong positive, clinical interpretation is
More serious bowl inflammatory diseases symptom needs timely to be intervened and treated in the case where medical practitioner instructs.
A human faecal mass calprotectin concentration research is carried out with this reagent, then sample results are asked using method of percentiles
Term of reference is obtained, this term of reference can be good at assisting the diagnosis of personal bowl inflammatory diseases symptom, in experiment four
It is verified.
It should be understood that the present invention is a kind of testing product, and the method for the diagnosing and treating of non-disease, only assist
The tool of clinical judgment.
The value that defines of 50 μ g/g, 200 μ g/g and 500 μ g/g are illustrated below.
The value upper limit is defined using 200 μ g/g as what fecal calprotectin defended protein assay reagent normal population, is by using this hair
Fecal calprotectin described in bright patent defends protein assay reagent to 575 fresh excreta samples (wherein male 288, average ages
± standard deviation 18.86 years old 35.53 years old；Women 287,36.52 years old ± standard deviation of average age 17.12 years old) it is detected, it will examine
Survey what result was obtained with spss software statistics.Its statistical result shows that the detection range of subject is 0-460.2ng/mL, average
Value is 110.25ng/mL, and 95% confidence interval is 21.34-402.38ng/mL.By the 95% confidence interval upper limit of result
What 402.38ng/mL was set to excrement calprotectin in normal population defines the value upper limit, according to the extension rate of excrement, is converted into every
The value upper limit that defines of calprotectin content in gram excrement, normal population is 200 μ g/g.
Value lower limit is defined using 50 μ g/g as what fecal calprotectin defended protein assay reagent Positive Populations.Described in the invention patent
Fecal calprotectin defend protein assay reagent to 141 bowl inflammatory diseases patients (wherein 63 make a definite diagnosis patients of ulcerative colitis,
58 are made a definite diagnosis cd patient and 20 other bowl inflammatory diseases patients) fresh excreta sample detect, detect
As a result spss software statistics are used.Its statistical result shows that the detection range of subject is 92.34-1010.2ng/mL, average value
For 490.25ng/mL, 95% confidence interval is 100.60-858.38ng/mL.By 95% lower limit of confidence interval of result
What 100.60ng/mL was set to excrement calprotectin in Positive Populations defines value lower limit, according to the extension rate of excrement, is converted into every
The value lower limit that defines of calprotectin content in gram excrement, Positive Populations is 50 μ g/g.
Value is defined using 500 μ g/g as what fecal calprotectin defended protein assay reagent strong positive.The excrement described in the invention patent
Just calprotectin detection reagent (wherein makes a definite diagnosis patients of ulcerative colitis, 58 for 63 to 141 bowl inflammatory diseases patients
Make a definite diagnosis cd patient and 20 other bowl inflammatory diseases patients) fresh excreta sample detect, testing result
With spss software statistics.Its statistical result shows that the detection range of subject is 92.34-1060.2ng/mL, and average value is
490.25ng/mL, 95% confidence interval are 100.60-858.38ng/mL.By the detection range upper limit 1010.2ng/ of subject
ML is set to strong positive and defines value, according to the extension rate of excrement, is converted into calprotectin content in every gram of excrement, the boundary of strong positive
Definite value is 500 μ g/g.
It should be understood that the present invention is a kind of testing product, and the method for the diagnosing and treating of non-disease, only assist
The tool of clinical judgment.
Effect △ of the invention below by way of experimental verification；
Experiment one, sensitivity experiment:
It takes zero-dose calibration object as its relative light intensity angle value (xi) of sample measures, replication 20 times, obtains 20 surveys
The relative light intensity angle value (xi) for measuring result, finds out average valueStandard deviation (SD) is calculated by formula (1), is obtainedInstitute
Corresponding relative light intensity angle value.It, will according to the calibrating curve equation of reagent card burning in kitCorresponding is opposite
Light intensity value substitutes into above-mentioned equation, corresponding concentration value, as sensitivity is asked, according to professional standard and design requirement, sensitivity
1.0 μ g/g should be not more than.
According to the above method, this experiment carries out the compound analyte detection of Hemoglobin-haptoglobin with zero-dose calibration object L1 and tries
The sensitivity experiment of agent.Experimental result is as follows:
The sensitivity that the above results show that fecal calprotectin defends protein assay reagent is 0.83ng/mL, the mark lower than 1.0ng/mL
Standard illustrates that the detection sensitivity of kit is higher.
Experiment two, accuracy experiment:
The working calibration product an of concentration level are taken, detection 3 times is repeated, calculate relative deviation (B%) by formula (2), root
It is judged to close if the relative deviation (B%) of 3 results is all not more than 15.0% requirement according to professional standard and design requirement
Lattice.Result if it is larger than or equal to 2 times is not met, that is, is judged to unqualified.Result is not met if there is 1 time, then should be again continuous
Test 20 times, and relative deviation is calculated according to formula (3) respectively, the relative deviation for the result tested if it is larger than or equal to 19 times
(B%) all it is not more than 15.0% requirement, then accuracy meets the requirements.
B%=(M-T)/T × 100% ... ... ... ... ... (2)
B%: relative deviation；
M: calibration object test value；
T: calibration object target value.
According to the above method, this experiment defends protein assay reagent working calibration product L4 with fecal calprotectin and carries out excrement calprotectin
The accuracy of detection reagent is tested.Experimental result is as follows:
The above results show that fecal calprotectin is defended the relative deviation (B%) of protein assay reagent working calibration product L4 and is below
15.0% standard, illustrate fecal calprotectin defend protein assay reagent accuracy it is preferable.
Experiment three, range of linearity experiment:
It configures 6 diluted concentrations (xi) and close to the sample of range of linearity upper and lower bound, measures relative light intensity angle value, often
A concentration determination 3 times finds out the mean value (yi) of measurement result respectively.With concentration (xi) for independent variable, with measurement result mean value
(yi) equation of linear regression y=a+bx is found out for dependent variable.The related coefficient (r) that linear regression is calculated by formula (3), according to row
Industry standard and design requirement, related coefficient (r) should be not less than 0.990.
1 concentration of specimens of table dilution preparation example
| Sample number | 1 | 2 | 3 | 4 | 5 | 6 |
| High level sample (mL) | 0 | 0.2 | 0.4 | 0.6 | 0.8 | 1.0 |
| Low value sample (mL) | 1.0 | 0.8 | 0.6 | 0.4 | 0.2 | 0 |
According to the above method, the high level sample of this experiment chooses fecal calprotectin and defends protein assay reagent working calibration product L7, low
Value sample chooses fecal calprotectin and defends protein assay reagent working calibration product L1, according to the dilution ratio of table 1, carries out range of linearity survey
It is fixed.Experimental result is as follows:
Interpretation of result: the linearly dependent coefficient (r) that the above results show that fecal calprotectin defends protein assay reagent is 0.992, symbol
Close the range of linearity regulation, illustrate fecal calprotectin defend protein assay reagent within the measurement range it is linear preferably.
Experiment four, the comparative test of detection method of the invention and clinical detection:
Tested fecal sample is provided by Jiaxing physical examination mechanism.Sample is Freshman fecal sample, tested sample totally 120
Example, wherein male 55, women 65, for the range of age at 10-84 year old, average age was 44 years old.It is strong for Jiaxing row to test place
The level-one biology laboratory of Biotechnology Co., Ltd.
The calprotectin detection kit that contrast agents choose Switzerland Buhlmann Laboratories AG company is (enzyme-linked
Immunization).The reagent is the import reagent of domestic registered listing, and registration certificate number is that state's tool is infused into 20172400529.Control examination
Agent desired use is the calprotectin content in Quantitative in vitro detection stool extract, the sample type with reagent of the present invention
And desired use is consistent.The packing specification of contrast agents is 96 person-portions/box.Contrast agents quality is preferable, and clinical application is extensive.
It is required according to contrast agents specification, the survey of excrement calprotectin content is carried out to above-mentioned tested fecal sample using contrast agents
It is fixed.Obtain the testing result of calprotectin content in tested fecal sample.
It is examination reagent with a kind of calprotectin detection reagent described in the invention patent, mating detecting instrument is Nanjing enlightening
Gneuss ACL2800 Full-automatic chemiluminescence analyzer carries out the survey of excrement calprotectin content to above-mentioned tested fecal sample
It is fixed.Tested fecal sample first passes through the extraction of specimen extraction liquid in advance, and extraction process can be carried out according to specimen extraction liquid specification,
Fecal sample can be sampled and weighed according to balance weighing method, according to the Sample Dilution ratio in specimen extraction liquid specification,
Fecal sample is extracted.Then upper machine is detected, detecting step and process in strict accordance with examination reagent specification and its
Mating Full-automatic chemiluminescence analyzer specification carries out, and obtains the testing result of calprotectin content in tested fecal sample.
The following are a kind of testing results of chemiluminescence quantitative detection excrement calprotectin clinical comparison test:
Result treatment 1:
Contrast agents and examination Reagent Protocol are subjected to consistency analysis: with Analyse-it data processing software to above-mentioned
Data do Passing&Bablok regression analysis, show that Passing&Bablok regression equation is Y=-0.45+0.96X, section
Data away from (A) and slope (B) are as shown in the table:
Interpretation of result:
Passing&Bablok regression analysis criterion: (1) intercept A is the degree of systematic divergence between two methods
Amount.95% confidence interval of intercept A can be used for examining the hypothesis of A=0.If the confidence interval of A includes value 0, illustrate the two
Between be not present systematic divergence.If it is assumed that being rejected, it is concluded that conclusion: A from 0 that there are conspicuousnesses is different, and two methods
At least phase difference constant amount；(2) slope B is the measurement of proportional difference between two methods.95% confidence interval of slope B can be used for
Examine the hypothesis of B=1.If the confidence interval of B includes value 1, illustrate that there is no proportional differences between the two.If it is assumed that by refusing
Absolutely, it is concluded that conclusion: B from 1 that there are conspicuousnesses is different, and at least there is proportional difference between two methods.
According to criterion, the intercept (A) of this clinical test and 95% confidence interval of slope (B) include assumption value,
Illustrate examine reagent measurement result and contrast agents measurement fecal sample calprotectin content there is no systematic divergence with
And proportional difference, there is good consistency.
Result treatment 2:
The paired data of contrast agents and examination Reagent Protocol is subjected to consistency analysis: using spss data processing software pair
Above-mentioned data do paired samples T inspection, as a result as follows:
Paired samples statistic:
Paired samples related coefficient:
Paired samples are examined:
Interpretation of result:
Paired samples T inspection principle: two different processing are received to two homogeneity samples respectively or the same sample is first
It is followed by by different processing, to judge whether processing has difference.Paired samples T examine according to data to sample from pairing it is total
Whether the mean value of body has significant difference to be inferred.
According to statistical " small probability principle ", level of significance α=0.05 is chosen in this clinical test, if P >=0.05,
Illustrate that difference is not significant；If P < 0.05, illustrates significant difference.
According to professional standard and clinical trial design requirement, if correlation coefficient r >=0.975, it is certain to illustrate that the two has
Correlation.
From paired samples correlation coefficient charts, show that the measurement result related coefficient of contrast agents and examination reagent is
0.989, greater than the 0.975 of professional standard and clinical trial design requirement, the good relationship that both illustrates；And related coefficient
Probability value (i.e. P value) is 0.000, is less than significance 0.05, illustrates contrast agents and examines survey of the reagent to same sample
Fixed number is according to significant linear dependence.
It is -0.777 that paired samples check table, which provides contrast agents and examines the T value of reagent measurement result, and freedom degree is
0.119, the two-sided significance index (P value) of the two is 0.439, is greater than significance 0.05, illustrates the two without conspicuousness
Difference, consistency are preferable.
The present invention provide a kind of chemiluminescence quantitative detection excrement calprotectin and its detection method and its in intestinal health
The purposes of detection, can calprotectin content in accurate, quantitative detection human faecal mass sample, have high sensitivity, stability good
Advantage, clinical auxiliary value with higher to intestinal health detection.
The basic principles, main features and advantages of the invention have been shown and described above.The technical staff of the industry should
Understand, the above embodiments do not limit the invention in any form, all obtained by the way of equivalent substitution or equivalent transformation
Technical solution is fallen within the scope of protection of the present invention.
Sequence table
<110>Jiaxing Hang Jian Biotechnology Co., Ltd
<120>a kind of reagent of chemiluminescence quantitative detection excrement calprotectin and its detection method and purposes
<141> 2019-09-24
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Claims (10)
1. a kind of chemiluminescence quantitative detection excrement calprotectin characterized by comprising immunocomplex, magnetic bead chemistry hair
Light detection system, fixedly separated phase are magnetic particle；
Immunocomplex includes: calprotectin calibration object, calprotectin quality-control product, by the first specific antibodies after marking, label
" after the first specific antibodies-calprotectin-label after label of the compound rear formation of the second specific antibodies and sample to be tested afterwards
The second specific antibodies " labeled calprotectin complex antibody.
2. a kind of chemiluminescence quantitative detection excrement calprotectin according to claim 1, which is characterized in that described first
Specific antibodies, the combination of the second specific antibodies and calprotectin are as follows: a kind of antibody and calprotectin S100A8 peptide chain amino
Amino acid epitope specific binding in acid sequence, another antibody and the ammonia in calprotectin S100A9 chain amino acids sequence
Base acid epitope specificity combines.
3. a kind of chemiluminescence quantitative detection excrement calprotectin according to claim 1, which is characterized in that described first
Specific antibodies, the combination of the second specific antibodies and calprotectin are as follows: a kind of antibody and calprotectin S100A8 peptide chain amino
Amino acid epitope specific binding in acid sequence, another antibody and the ammonia in calprotectin S100A8 chain amino acids sequence
Base acid epitope specificity combines.
4. a kind of chemiluminescence quantitative detection excrement calprotectin according to claim 1, which is characterized in that described first
Specific antibodies, the combination of the second specific antibodies and calprotectin are as follows: a kind of antibody and calprotectin S100A9 peptide chain amino
Amino acid epitope specific binding in acid sequence, another antibody and the ammonia in calprotectin S100A9 chain amino acids sequence
Base acid epitope specificity combines.
5. a kind of chemiluminescence quantitative detection excrement calprotectin according to claim 1, which is characterized in that the fixation
Separation is mutually the coated magnetic particle of Streptavidin.
6. a kind of chemiluminescence quantitative detection excrement calprotectin according to claim 1, which is characterized in that the fixation
Separation is mutually calprotectin monoclonal or the coated magnetic particle of polyclonal antibody.
7. a kind of chemiluminescence quantitative detection excrement calprotectin according to claim 1, which is characterized in that the label
For luminescent label, the luminous marker includes: acridinium ester, HRP, alkaline phosphatase or Rumi sodium.
8. a kind of chemiluminescence quantitative detection excrement calprotectin according to claim 1, which is characterized in that described first
Specific antibodies, the second specific antibodies be polyclonal antibody or monoclonal antibody, the monoclonal antibody from mouse, rat,
The hybridoma of rabbit, the polyclonal antibody derive from rabbit, sheep, chicken.
9. a kind of detection method of chemiluminescence quantitative detection excrement calprotectin, which is characterized in that collect and extract excrement to be measured
Just sample is added in the reaction cup of Full-automatic chemiluminescence apparatus, and the first labeled calprotectin is added in each reaction cup
Antibody, coated magnetic particle solution, the second labeled anti-calprotectin antibody, it is anti-in the calprotectin that addition second is labeled
Body preincubation, cleaning, and secondary incubation, cleaning after the anti-calprotectin antibody that addition second is labeled, or the second quilt is being added
It is once incubated for, cleans after the anti-calprotectin antibody of label, be eventually adding substrate solution or exciting liquid and excitation hair is carried out to reaction system
Light carries out data result processing according to the calibration curve information calibrated；
Described first labeled anti-calprotectin antibody, the second labeled anti-calprotectin antibody are as follows: a kind of antibody and calcium defend egg
Amino acid epitope specific binding in white S100A8 or S100A9 chain amino acids sequence, another antibody and calprotectin
Amino acid epitope specific binding in S100A8 or S100A9 chain amino acids sequence.
10. a kind of purposes that chemiluminescence quantitative detection excrement calprotectin is detected in intestinal health, which is characterized in that for examining
Survey intestinal health situation.
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| CN111004324A (en) * | 2019-12-31 | 2020-04-14 | 苏州和锐生物科技有限公司 | Calprotectin monoclonal antibody and application thereof |
| CN111381046A (en) * | 2020-03-12 | 2020-07-07 | 迪瑞医疗科技股份有限公司 | Calprotectin chemiluminescence immunoassay kit and preparation method thereof |
| CN112578123A (en) * | 2019-09-27 | 2021-03-30 | 成都中医药大学 | Application of reagent for detecting content of calprotectin in preparation of uterine lesion screening kit |
| CN117430701A (en) * | 2023-12-20 | 2024-01-23 | 南京佰抗生物科技有限公司 | Monoclonal antibody composition of anti-human calprotectin and application |
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| WO2013132338A2 (en) * | 2012-03-06 | 2013-09-12 | Calpro As | Competitive immunoassay for calprotectin |
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| CN112578123A (en) * | 2019-09-27 | 2021-03-30 | 成都中医药大学 | Application of reagent for detecting content of calprotectin in preparation of uterine lesion screening kit |
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| CN117430701A (en) * | 2023-12-20 | 2024-01-23 | 南京佰抗生物科技有限公司 | Monoclonal antibody composition of anti-human calprotectin and application |
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Application publication date: 20191122 |