CN106771152A - A kind of kit of quick detection calprotectin - Google Patents
A kind of kit of quick detection calprotectin Download PDFInfo
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- CN106771152A CN106771152A CN201710042911.4A CN201710042911A CN106771152A CN 106771152 A CN106771152 A CN 106771152A CN 201710042911 A CN201710042911 A CN 201710042911A CN 106771152 A CN106771152 A CN 106771152A
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- G—PHYSICS
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- 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
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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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
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention provides a kind of kit, including following component：Buffer solution containing polymer；And combine the polystyrene colloidal milk solution of anti-human calprotectin antibody.When detecting calprotectin using the kit, have the advantage that：Need to be only separately added at two time points when in use, it is quick that operation is very easy；Sensitivity is very high, it is ensured that be able to detect that the calprotectin of extremely low level；High specificity, reproducible and low cost；Strong antijamming capability, even if there is certain density chaff interference in detection sample including hemoglobin, bilirubin, Vc, triglycerides etc., the testing result on kit of the present invention is influenceed very little.Kit of the invention adapts to single part, quantitative determination that is quick and going out result immediately because having above advantage, with clinical value very high.
Description
Technical field
The present invention relates to medical immunology in-vitro diagnosis field, more particularly to a kind of calprotectin detection kit.
Background technology
Calprotectin is calcium, the zinc-binding protein that a molecular weight is 36kD.By two molecular weight for 14kD heavy chain and
One molecular weight is constituted for the light chain of 8kD with the calcium-binding protein heterotrimer that covalent bond is connected.Every chain can combine two
Ca2+, so that with heat resistance and the water-disintegrable characteristic of enhancing.
Calprotectin is mainly derived from neutrophil leucocyte and monocyte, with various biological function, in vitro study table
It is bright it there is bacteriostasis property, fungistatic effect can be mentioned in the same breath with antibiotic.Calprotectin is the macrophage of neutrophil leucocyte and activation
Important protein in cytoplasm, content increases under many inflammatory conditions, can be used as acute inflammatory mark.
Although current calprotectin many places in human biological object are found, including：Serum, saliva, cerebrospinal fluid and urine.
But easily measured in excrement, and its stable components, at room temperature can in stool stable existence 7d or so, and be difficult by bacterium and
Various enzyme destructions, are not influenceed by diet.The detection of the calprotectin of excrement is that one kind is simple, rapid, sensitivity is high, spy
The inflammatory bowel disease detection method of strong, cheap, the non-invasion and attack of the opposite sex.This detection method can be in the mucous membrane of normal person and ill trouble
Accurate testing result can be obtained in the Mucositis of person.
Calprotectin is considered as reliable inflammation index in various diseases, and it can clearly distinguish organic disease such as
Diseases associated with inflammation IBD and functional disease such as IBS.This in-vitro diagnosis instrument can avoid the expense and Sigmoidoscope of costliness from invading
The inspection of property.Gastroenterology association of Britain (BSG) annual meeting for being held in Birmingham for the 14-17 of in March, 2005 days, seminar inflammation
Disease property enteropathy (IBD)-calprotectin-irritable bowel syndrome (IBS), using calprotectin as a new useful diagnosis reference
Mark, is used for：Difference inflammatory bowel disease and irritable bowel syndrome；Assessment inflammatory bowel disease inflammation activity；Inflammatory bowel disease mucous membrane is evaluated to control
More situation.
For the detection of calprotectin, current clinical method includes：The side such as ELISA (ELISA), colloidal gold method
Method, but these methods are the characteristics of have respective and deficiency.ELISA method is widely used in hospital laboratory due to quantitatively accurate, but
Detection time is long, not convenient；Colloidal gold method is although convenient, fast, but it is a kind of qualitative method, and sensitivity is relatively low；Therefore, face
A kind of quick, convenient, economic calprotectin quantitative detecting method is set up in active demand on bed.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of kit for detecting calprotectin, is detected using the kit
During calprotectin, easy to operate, high specificity, sensitivity high, reproducible, low cost, testing result are accurate and anti-dry
Disturb ability strong, be suitable to clinical quick detection.
In order to solve the above-mentioned technical problem, the present invention is achieved through the following technical solutions:
First aspect present invention provides composition, including following component：Buffer solution containing polymer；And combine anti-human
The polystyrene colloidal milk solution of anti-calprotectin antibody.The polymer is Triton series of surfactants, Tween series surface
In activating agent, bay ether, polyoxyethylene alkyl phenyl ether, ethylene nonyl phenyl ether and polyoxethylene octylphenyl phenylate at least
It is a kind of.The particle diameter of the polystyrene latex is 100-300nm.
In a preference, in the buffer solution containing polymer, the quality of polymer accounts for its total volume percent and is
0.1%-5.0%.
In a preference, with reference to anti-human calprotectin antibody polystyrene colloidal milk solution solid content w/v be 0.5-
5.0%.
In a preference, the polystyrene colloidal in the polystyrene colloidal milk solution of the combination anti-human calprotectin antibody
Breast with the coating of anti-human calprotectin antibody is prepared using chemical crosslink technique；
In a preference, the chemical crosslinking buffer solution that the chemical crosslink technique is used is selected from 2- (N- morpholines) ethyl sulfonic acid
MES buffer solutions, 3- morpholine -2s-hydroxy-propanesulfonic acid MOPSO buffer solutions, 3- (N- morpholines) propane sulfonic acid MOPS buffer solutions, 4- ethoxys
At least one in piperazine ethanesulfonic acid HEPES buffer solution and phosphate buffer.
In a preference, the chemical cross-linking agent that the chemical crosslink technique is used is selected from carbonization imines, N- hydroxysuccinimidyl acyls
At least one in imines, N- hydroxy thiosuccinimides, hydrazides and isocyanates.
In a preference, the buffer solution containing polymer includes following component：Trishydroxymethylaminomethane, chlorination
Sodium, Triton X-100, bovine serum albumin, ethylene polyethenoxy ether and Sodium azide；Or the buffer solution bag containing polymer
Include following component：Trishydroxymethylaminomethane, sodium chloride, Tw een-20, bovine serum albumin, ethylene polyethenoxy ether and folded
Nitrogen sodium；Or the buffer solution containing polymer includes following component：Phosphate, sodium chloride, Triton X-100, cow's serum egg
In vain, ethylene polyethenoxy ether and Sodium azide；The polystyrene colloidal milk solution of the combination anti-human calprotectin antibody is included such as
Lower component：Phosphate, anti-human calprotectin antibody's coating latex particle, sodium chloride, bovine serum albumin and Sodium azide.
In a preference, the buffer solution containing polymer includes each component of following concentration：
Trishydroxymethylaminomethane or phosphate 20-100mmol/L
Sodium chloride 7-10g/L
Triton X-100 or Tween-20 4-12g/L
Bovine serum albumin 4-7g/L
Ethylene polyethenoxy ether 40-60g/L
Sodium azide 3-6g/L.
In a preference, the pH value of the buffer solution containing polymer is 6.5-7.5；
In a preference, the polystyrene colloidal milk solution of the combination anti-human calprotectin antibody includes following concentration
Each component：
Phosphate 80-120mmol/L
Anti-human calprotectin antibody's coating latex particle 10-35%
Sodium chloride 7-10g/L
Bovine serum albumin 4-7g/L
Sodium azide 4-6g/L.
In a preference, the pH value of the polystyrene colloidal milk solution of the combination anti-human calprotectin antibody is 7-8.
In a preference, the buffer solution containing polymer includes each component of following concentration：
Trishydroxymethylaminomethane 25mmol/L
Sodium chloride 8.5g/L
Triton X-100 5g/L
Bovine serum albumin 5g/L
Ethylene polyethenoxy ether 50g/L
Sodium azide 4g/L.
In a preference, the pH value of the buffer solution containing polymer is 7.0.
In a preference, the polystyrene colloidal milk solution of the combination anti-human calprotectin antibody includes following concentration
Each component：
Phosphate 100mmol/L
Anti-human calprotectin antibody is coated with latex particle 15%
Sodium chloride 8.5g/L
Bovine serum albumin 5g/L
Sodium azide 5g/L；
In a preference, the pH value of the polystyrene colloidal milk solution of the combination anti-human calprotectin antibody is 7.5.
In a preference, also including calibration object.
In a preference, the calibration object is calprotectin calibration object, and the concentration of the calprotectin calibration object is distinguished
It is 10,30,100,300,600ug/g.
In a preference, the polystyrene colloidal milk solution of the combination anti-human calprotectin antibody uses following methods system
It is standby to form：
Take during polystyrene latex adds MES buffer solutions, while add EDC and NHS, it is to be dissolved it is well mixed after, activation
Latex, after the completion of latex activation, two parts is divided into by latex, and a copy of it latex adds anti-human calprotectin's monoclonal antibody
A, another latex adds anti-human calprotectin monoclonal antibody B, and both of which is crosslinked, carried out after the completion of crosslinking from
Be dissolved in both precipitations in MES buffer solutions respectively by the heart, abandoning supernatant, obtains both calprotectin monoclonal antibody marks
Note liquid, is then mixed both calprotectin labeling of monoclonal antibody liquid, that is, be obtained and combine anti-human calprotectin antibody
Polystyrene colloidal milk solution.
In a preference, both calprotectin labeling of monoclonal antibody liquid is by 1：1 volume ratio is mixed
Close.
Second aspect present invention provides application of the described composition in the product for preparing detection calprotectin.
In a preference, the product is kit.
The present invention is the antibody using the coated anti-calprotectin of polystyrene latex, in sample to be tested such as excrement etc.
Calprotectin occur association reaction, immune complex is formed in homogeneous system, when transmitted light pass through reaction system when occur
Intensity variation, the luminous intensity according to measurement calprotectin calibration object draws calibration curve, the luminous intensity in measurement sample to be tested
Change, the concentration of calprotectin in detection sample is calculated according to calibration curve analysis meter.Key of the invention has following two sides
Face：First it is the suitable polystyrene latex of selection, its size (diameter) will be slightly smaller than wavelength, and the particle of polystyrene latex is straight
Footpath is 100-300nm, it is furthermore preferred that the particle diameter of polystyrene latex is 150-250nm；Next to that using suitable method
Polystyrene latex is coated with purpose antibody, conventional method for coating has physisorphtion and chemical crosslink technique, two kinds of the present invention
Method can be used, but more preferably select chemical crosslink technique.
" Triton X-100 " of the present invention is Triton X-100.
" Sodium azide " of the present invention is sodium azide.
" disodium hydrogen phosphate dodecahydrate " of the present invention, also known as " disodium hydrogen phosphate dodecahydrate ", No. CAS is
10039-32-4。
" two Heshui sodium dihydrogen phosphates " of the present invention, also known as " sodium dihydrogen phosphate dihydrate ", No. CAS is 13472-
35-0。
" ethylene polyethenoxy ether " of the present invention, also known as " polyethylene glycol ", No. CAS is 25322-68-3.
The compound method of " MES buffer solutions (25mmol/L, pH5.0) " of the present invention is：Weigh 2- (N- morpholines) second
Sulfonic acid monohydrate 5.33g is dissolved in 950ml water, is mended after regulation pH=5.0 and is settled to 1000ml, and room temperature preservation is standby.
The compound method of " MOPSO buffer solutions (30mmol/L, pH6.5) " of the present invention is：Weigh MOPSO 6.76g
It is dissolved in 950ml water, is mended after regulation pH=6.5 and be settled to 1000ml, room temperature preservation is standby.
The compound method of " HEPES buffer solution (50mmol/L, pH8.0) " of the present invention is：Weigh HEPES
13.42g is dissolved in 950ml water, is mended after regulation pH=8.0 and is settled to 1000ml, and room temperature preservation is standby.
The full name of " NHS " of the present invention is N-hydroxy-succinamide, and molecular formula is C4H5NO3, No. CAS is 6066-
82-6。
The full name of " EDC " of the present invention is 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochloride, molecule
Formula is C8H17N3HCl, No. CAS is 25952-53-8.
It is of the present invention " containing 1%BSA PBS (0.01M, pH7.0) " compound method be：Weigh 10gBSA
(bovine serum albumin(BSA)), 8g NaCl, 0.2g KCl, 1.44g Na2HPO4With 0.24g KH2PO4, it is dissolved in 800ml distilled water,
The pH value of solution is adjusted to 7.0 with HCl, finally adds distilled water to be settled to 1L.
The compound method of " Extraction buffer " of the present invention is：Weigh 150.15g urea, 2.78g CaCl2、
48.04g citric acids, 5g Sodium azides and 12.5g BSA, add 0.25M pH8.0Tris buffer solutions to be settled to 1L, obtain final product extraction slow
Fliud flushing, room temperature preservation is standby.
The invention has the advantages that：
(1) in the kit that the present invention is provided, using the buffer solution containing polymer for having prepared as reagent R1, matched somebody with somebody
The polystyrene colloidal milk solution of the combination anti-human calprotectin antibody for making, when in use only need to be in two times used as reagent R2
Point is separately added into, and it is quick that operation is very easy.
(2) sensitivity is very high during the kit detection calprotectin that the present invention is provided, it is ensured that be able to detect that extremely low level
Calprotectin.
(3) each component in the kit that the present invention is provided passes through accurate collocation design, when detecting calprotectin with it
High specificity, reproducible and low cost.
(4) strong antijamming capability during the kit detection calprotectin that the present invention is provided, even if having in detection sample
The chaff interference of concentration is determined including hemoglobin, bilirubin, Vc, triglycerides etc., and the testing result on kit of the present invention influences
Very little.
To sum up, the present invention is because having above advantage, and adapts to single part, quantitative determination that is quick and going out result immediately,
With very big clinical value.
Brief description of the drawings
Fig. 1 is the standard curve detected for the calprotectin content of the calibration object in embodiments of the invention 1.
Fig. 2 is using detection calprotectin kit of the invention and Correlation to That Abroad kit in embodiments of the invention 5
Comparative result schematic diagram.Abscissa is the result that the method for the present invention is determined in figure, and ordinate is Switzerland Buhlman
The fecal calprotectin of Laboratories AG companies defends the result of protein detection kit measure.
Specific embodiment
Unless specifically indicated, term used herein has the general sense in art of the present invention.
Below with reference to specific embodiments and the drawings, the present invention will be described, it is necessary to explanation, these embodiments are only
It is illustrative, and is not considered as limiting the invention.Unreceipted particular technique or condition in embodiment, according to ability
Technology described by document or condition in domain are carried out according to product description.Agents useful for same or the unreceipted factory of instrument
Shang Zhe, be can by city available from conventional products；Agents useful for same refers both to meet corresponding state when unreceipted other are required
The AR of family's standard；During the unreceipted compound method of agents useful for same, refer both to be prepared according to this area conventional method.
Embodiment 1
Present embodiments provide a kind of kit, including reagent R1, reagent R2 and calibration object.
The reagent R1 includes each component of following concentration：
Trishydroxymethylaminomethane 25mmol/L
Sodium chloride 8.5g/L
Triton X-100 5g/L
Bovine serum albumin 5g/L
Ethylene polyethenoxy ether 50g/L
Sodium azide 4g/L
The pH value of the reagent R1 is 7.0.
The reagent R1 is adopted and is prepared from the following method：
Accurately weigh 3.03g trishydroxymethylaminomethanes, 8.5g sodium chloride, 5g Triton X-100 (Aladdin, article No.：
T109026, lot number：G1604036), 5g bovine serum albumins (Proliant, article No.：68100, lot number：BB52650101)、50g
Ethylene polyethenoxy ether, 4g Sodium azides are dissolved in 900ml water, are stirred to after being completely dissolved, and pH=7.0 is adjusted with hydrochloric acid, then
1000ml is settled to, is saved backup.
The reagent R2 includes each component of following concentration：
Phosphate 100mmol/L
Anti-human calprotectin antibody is coated with latex particle 15% (W/V)
Sodium chloride 8.5g/L
Bovine serum albumin 5g/L
Sodium azide 5g/L
The pH value of reagent R2 is 7.5.
The reagent R2 is adopted and is prepared from the following method：
Take 5ml polystyrene latexs (Millipore, particle diameter 150nm, 5%w/v) and add 45mlMES buffer solutions
In (25mmol/L, pH5.0), while adding 0.05g EDC (Thermo, article No.：22981, lot number：) and 0.05g RC233685
NHS (Thermo, article No.：24500, lot number：RC230146), it is to be dissolved it is well mixed after, be placed in 37 ± 1 DEG C of environment and be incubated
Carry out activation latex within 1 hour, after the completion of latex activation, latex is divided into two parts, every part of 25ml, a copy of it latex is added
2.5mg anti-human calprotectin's monoclonal antibodies A (Anti-h Calprotectin 3402SPTN-5, article No.：10045, brand：
MedixBiochemica), another latex adds 2.5mg anti-human calprotectin monoclonal antibody B (Anti-h
Calprotectin 3404SPTN-5, article No.：100468, brand：MedixBiochemica 37 ± 1), and by both are placed in
DEG C environment in be incubated and be crosslinked for 3 hours, be centrifuged in the case where temperature is 4.0 DEG C and rotating speed is 12000rpm after the completion of crosslinking
Be dissolved in both precipitations in the MES buffer solutions (25mmol/L, pH5.0) of 25ml respectively by 60min, abandoning supernatant, obtains two
The calprotectin labeling of monoclonal antibody liquid of person, then presses 1 by both calprotectin labeling of monoclonal antibody liquid：1 volume
Than being mixed, that is, reagent R2 is obtained, is positioned over 2~8 DEG C and saves backup.
The calibration object is calprotectin calibration object, and concentration of the calprotectin calibration object from A to E is respectively 10,
30,100,300,600ug/g.
The source of the calprotectin calibration object：Using the calprotectin antigen purchased from MedixBiochemica, with containing
The PBS (0.01M, pH7.0) of 1%BSA is diluted to aimed concn A~E.
Mentioned reagent R1, reagent R2 and calibration object are packed respectively, the detection reagent of the calprotectin of the present embodiment is obtained final product
Box.
Embodiment 2
Present embodiments provide a kind of kit, including reagent R1, reagent R2 and calibration object.
The reagent R1 includes each component of following concentration：
Phosphate 80mmol/L
Sodium chloride 8.5g/L
Triton X-100 10g/L
Bovine serum albumin 6g/L
Ethylene polyethenoxy ether 50g/L
Sodium azide 5g/L
The pH value of the reagent R1 is 7.5.
The reagent R1 is adopted and is prepared from the following method：It is accurate weigh 28.65g disodium hydrogen phosphates dodecahydrate,
10.89g potassium dihydrogen phosphates, 8.5g sodium chloride, 10g Triton X-100 (Aladdin, article No.：T109026, lot number：
G1604036), 6g bovine serum albumins (Proliant, article No.：68100, lot number：BB52650101), 50g ethylene glycol polyoxyethylene
Ether, 5g Sodium azides are dissolved in 900ml water, are stirred to after being completely dissolved, and pH=7.5 is adjusted with hydrochloric acid, then are settled to 1000ml, are protected
Deposit standby.
The reagent R2 includes each component of following concentration：
Phosphate 100mmol/L
Anti-human calprotectin antibody is coated with latex particle 30% (w/v)
Sodium chloride 8.5g/L
Bovine serum albumin 6g/L
Sodium azide 5g/L
The pH value of reagent R2 is 8.0.
The reagent R2 is adopted and is prepared from the following method：
Take 10ml polystyrene latexs (Millipore, particle diameter 200nm, 5%w/v) and add 90mlMOPSO buffer solutions
In (30mmol/L, pH6.5), while adding 0.1g EDC (Thermo, article No.：22981, lot number：) and 0.1g RC233685
NHS (Thermo, article No.：24500, lot number：RC230146), it is to be dissolved it is well mixed after, be placed in 37 ± 1 DEG C of environment and be incubated
Carry out activation latex within 2 hours, after the completion of latex activation, latex is divided into two parts, every part of 50ml, a copy of it latex is added
5.0mg anti-human calprotectin's monoclonal antibodies A (Anti-h Calprotectin 3402SPTN-5, article No.：100459, brand：
MedixBiochemica), another latex adds 5.0mg anti-human calprotectin monoclonal antibody B (Anti-h
Calprotectin 3404SPTN-5, article No.：100468, brand：MedixBiochemica 37 ± 1), and by both are placed in
DEG C environment in be incubated and be crosslinked for 5 hours, be centrifuged in the case where temperature is 4.0 DEG C and rotating speed is 12000rpm after the completion of crosslinking
Be dissolved in both precipitations in the MOPSO buffer solutions (30mmol/L, pH6.5) of 50ml respectively by 60min, abandoning supernatant, obtains
Both calprotectin labeling of monoclonal antibody liquid, then presses 1 by both calprotectin labeling of monoclonal antibody liquid：1 body
Product ratio is mixed, that is, reagent R2 is obtained, and is positioned over 2~8 DEG C and is saved backup.
The calibration object is with embodiment 1.
Mentioned reagent R1, reagent R2 and calibration object are packed respectively, the detection reagent of the calprotectin of the present embodiment is obtained final product
Box.
Embodiment 3
Present embodiments provide a kind of kit, including reagent R1, reagent R2 and calibration object.
The reagent R1 includes each component of following concentration：
The pH value of the reagent R1 is 7.0.
The reagent R1 is adopted and is prepared from the following method：
It is accurate weigh the Heshui sodium dihydrogen phosphates of 1.56g bis-, 3.58g disodium hydrogen phosphates dodecahydrate, 8.5g sodium chloride,
4.0g Tween-20,5g bovine serum albumin (Proliant, article No.：68100, lot number：BB52650101), 4g Sodium azides are dissolved in
In 900ml water, stir to after being completely dissolved, pH=7.0 is adjusted with hydrochloric acid, then be settled to 1000ml, save backup.
The reagent R2 includes each component of following concentration：
The pH value of reagent R2 is 7.5.
The reagent R2 is adopted and is prepared from the following method：
Take 5ml polystyrene latexs (Millipore, particle diameter 250nm, 3%w/v) and add 25mlHEPES buffer solutions
In (50mmol/L, pH8.0), while adding 0.03g EDC (Thermo, article No.：22981, lot number：) and 0.03g RC233685
NHS (Thermo, article No.：24500, lot number：RC230146), it is to be dissolved it is well mixed after, be placed in 37 ± 1 DEG C of environment and be incubated
Carry out activation latex within 1.5 hours, after the completion of latex activation, latex is divided into two parts, every part of 15ml, a copy of it latex is added
1.5mg anti-human calprotectin's monoclonal antibodies A (Anti-h Calprotectin 3402SPTN-5, article No.：100459, brand：
MedixBiochemica), another latex adds 1.5mg anti-human calprotectin monoclonal antibody B (Anti-h
Calprotectin 3404SPTN-5, article No.：100468, brand：MedixBiochemica 37 ± 1), and by both are placed in
DEG C environment in be incubated and be crosslinked for 3 hours, be centrifuged in the case where temperature is 4.0 DEG C and rotating speed is 12000rpm after the completion of crosslinking
Be dissolved in both precipitations in the HEPES buffer solution of 15ml (50mmol/L, pH8.0) respectively by 60min, abandoning supernatant, obtains
Both calprotectin labeling of monoclonal antibody liquid, then presses 1 by both calprotectin labeling of monoclonal antibody liquid：1 body
Product ratio is mixed, that is, reagent R2 is obtained, and is positioned over 2~8 DEG C and is saved backup.
The calibration object is with embodiment 1.
Mentioned reagent R1, reagent R2 and calibration object are packed respectively, the detection reagent of the calprotectin of the present embodiment is obtained final product
Box.
Embodiment 4
The method for present embodiments providing detection calprotectin, specially using latex enhancing immune turbidimetry quantitative determination
Calprotectin concentration in excrement, the method use the kit of embodiment 1 or embodiment 2.
The above method comprises the following steps：
First, sample extraction
50-100mg fecal samples are put into test tube, add 49 times of fecal sample net weight of Extraction buffer (to contain
2.5M urea, 0.025M CaCl2, 0.25M citric acids, 0.5% Sodium azide, the pH8.0 of 1.25%BSA 0.25M Tris delay
Fliud flushing), mixing is mixed, and room temperature is placed 10 minutes, and 3000g is centrifuged 5 minutes, is taken supernatant as sample extraction thing and (namely is treated test sample
This).
2nd, kit detection
Comprise the following steps：
(1) degree of reaction is determined on automatic clinical chemistry analyzer (MINDRAY, BS-380) using calprotectin calibration object
(absorbance difference) and fit standard curve.As shown in figure 1, for the calprotectin of the calibration object in embodiments of the invention 1 contains
Measure the standard curve of detection.
(2) 10ul samples to be tested are taken, is added in 250ul reagents R1, obtain the first mixed liquor, and 5 are reacted in 37 DEG C
Minute, read the absorbance OD1 of the first mixed liquor.
(3) to 50ul reagent R2 are added in the first mixed liquor, the second mixed liquor, and reaction 5 minutes in 37 DEG C are obtained, so
The absorbance OD2 of the second mixed liquor is read afterwards.
The dominant wavelength of above-mentioned detection is 546nm.
3rd, interpretation of result
The degree of reaction of computation and measurement sample：Degree of reaction (absorbance difference)=OD1-OD2, then according to degree of reaction and calibration
The regression equation calculation analysis of curve obtains the content of the calprotectin of testing sample.
Embodiment 5
The method of the detection calprotectin that the present embodiment is set up to embodiment 4 has carried out specificity and accuracy validation.
Using the kit of embodiments of the invention 2, and according to embodiment 4 detection method have detected 40 samples (come
From institute of traditional Chinese medicine of Shenzhen)；Meanwhile, the use of the article No. of Buhlman Laboratories AG companies of Switzerland is the excrement of HR0593
Calprotectin detection kit, 40 above-mentioned samples are detected using the method (ELISA) of its kit specification.
Testing result is as shown in table 1.
Table 1
(come with the measured value that the fecal calprotectin of Buhlman Laboratories AG companies of Switzerland defends protein detection kit
From table 1) it is ordinate, with kit measurement value of the invention (coming from table 1) as abscissa, correlation curve is drawn, such as Fig. 2 institutes
Show, the regression equation that linear regression is obtained is y=0.9917x+0.5498, coefficient R2=0.9889.Result shows the two
Correlation very well so that illustrate kit of the present invention and detection method have to detection excrement calprotectin specificity well and
The degree of accuracy.
Embodiment 6
The method of the detection calprotectin that the present embodiment is set up to embodiment 4 has carried out precision and repeatability is verified.
Using the kit of embodiment 1, and detection method according to embodiment 4 have detected 1 part of randomized clinical fecal sample,
Using BS-380 automatic clinical chemistry analyzers to same sample duplicate detection 10 times,
As shown in table 2, the coefficient of variation is 2.35% to testing result, less than 5%, shows that kit of the invention has higher
Precision and repeatability.
Table 2
Embodiment 7
The method of the detection calprotectin that the present embodiment is set up to embodiment 4 has carried out anti-interference checking.
Using the kit of embodiment 1, and after sample extraction according to embodiment 4, sample extraction thing is divided into some groups,
Different chaff interferences are added respectively, and sample extraction thing is pressed in every group of sample extraction thing：Chaff interference=99 of various concentrations：1
Volume ratio addition chaff interference, by the detection method in embodiment 4 to addition chaff interference after sample (i.e. in embodiment 4 detect
When sample extraction thing consumption be 10ul, be then that sample extraction thing+total consumption of chaff interference is 10ul in contrast to the present embodiment) carry out
Detection.
1 group：Sample extraction thing+triglycerides；Wherein addition triglyceride concentration be respectively 0g/dl, 1g/dl, 10g/dl,
20g/dl。
2 groups：Sample extraction thing+hemoglobin；Wherein addition HC is respectively 0g/dl, 10g/dl, 50g/
dl、100g/dl。
3 groups：Sample extraction thing+Vc；Wherein addition Vc concentration is respectively 0g/dl, 10g/dl, 20g/dl, 50g/dl.
4 groups：Sample extraction thing+bilirubin；Wherein addition bilirubin concentration be respectively 0mg/dl, 10mg/dl, 50mg/dl,
100mg/dl。
Testing result is as shown in table 3.After adding various chaff interferences respectively in the sample, entered successively using kit of the present invention
During row detection, as a result show, when there is 0.1g/dl triglycerides, 0.5g/dl hemoglobins, 0.2g/dl Vc, 0.5mg/dl courages
During red pigment, kit measurement result relative deviation of the present invention is within 10%；Even if this shows there is one in sample is detected
The chaff interference of concentration is determined including hemoglobin, bilirubin, Vc, triglycerides etc., and the testing result on kit of the present invention influences
Still very little.
Table 3
Above content is to combine specific preferred embodiment further description made for the present invention, it is impossible to assert
Specific implementation of the invention is confined to these explanations.For general technical staff of the technical field of the invention,
Some equivalent substitutes or obvious modification are made on the premise of not departing from present inventive concept, and performance or purposes are identical, all should wrap
Include within protection scope of the present invention.
Claims (10)
1. composition, it is characterised in that：Including following component：
Buffer solution containing polymer；
And combine the polystyrene colloidal milk solution of anti-human calprotectin antibody；
The polymer is Triton series of surfactants, Tween series of surfactants, bay ether, polyxyethylated
At least one in phenyl ether, ethylene nonyl phenyl ether and polyoxethylene octylphenyl phenylate；
The particle diameter of the polystyrene latex is 100-300nm.
2. composition according to claim 1, it is characterised in that：
In the buffer solution containing polymer, the quality of polymer accounts for its total volume percent for 0.1%-5.0%；
It is optional, it is 0.5-5.0% with reference to the solid content w/v of the polystyrene colloidal milk solution of anti-human calprotectin antibody.
3. composition according to claim 1, it is characterised in that：
Polystyrene latex and anti-human calprotectin in the polystyrene colloidal milk solution of the combination anti-human calprotectin antibody
The coating of antibody is prepared using chemical crosslink technique；
It is optional, the chemical crosslinking buffer solution that the chemical crosslink technique is used be selected from 2- (N- morpholines) ethyl sulfonic acid MES buffer solutions,
3- morpholine -2s-hydroxy-propanesulfonic acid MOPSO buffer solutions, 3- (N- morpholines) propane sulfonic acid MOPS buffer solutions, 4- HEPESs
At least one in HEPES buffer solution and phosphate buffer；
Optional, the chemical cross-linking agent that the chemical crosslink technique is used is selected from carbonization imines, N-hydroxy-succinamide, N- hydroxyls
At least one in thiosuccimide, hydrazides and isocyanates.
4. composition according to claim 1, it is characterised in that：
The buffer solution containing polymer includes following component：Trishydroxymethylaminomethane, sodium chloride, Triton X-100, ox
Haemocyanin, ethylene polyethenoxy ether and Sodium azide；
Or the buffer solution containing polymer includes following component：Trishydroxymethylaminomethane, sodium chloride, Tween-20, ox blood
Albumin, ethylene polyethenoxy ether and Sodium azide；
Or the buffer solution containing polymer includes following component：Phosphate, sodium chloride, Triton X-100, bovine serum albumin,
Ethylene polyethenoxy ether and Sodium azide；
The polystyrene colloidal milk solution of the combination anti-human calprotectin antibody includes following component：Phosphate, anti-human calcium defend egg
Bai Kangti coatings latex particle, sodium chloride, bovine serum albumin and Sodium azide.
5. composition according to claim 4, it is characterised in that：
The buffer solution containing polymer includes each component of following concentration：
Trishydroxymethylaminomethane or phosphate 20-100mmol/L
Sodium chloride 7-10g/L
Triton X-100 or Tween-20 4-12g/L
Bovine serum albumin 4-7g/L
Ethylene polyethenoxy ether 40-60g/L
Sodium azide 3-6g/L
Optional, the pH value of the buffer solution containing polymer is 6.5-7.5；
Optional, the polystyrene colloidal milk solution of the combination anti-human calprotectin antibody includes each component of following concentration：
Phosphate 80-120mmol/L
Anti-human calprotectin antibody's coating latex particle 10-35%
Sodium chloride 7-10g/L
Bovine serum albumin 4-7g/L
Sodium azide 4-6g/L
Optional, the pH value of the polystyrene colloidal milk solution of the combination anti-human calprotectin antibody is 7-8.
6. composition according to claim 5, it is characterised in that：
The buffer solution containing polymer includes each component of following concentration：
Trishydroxymethylaminomethane 25mmol/L
Sodium chloride 8.5g/L
Triton X-100 5g/L
Bovine serum albumin 5g/L
Ethylene polyethenoxy ether 50g/L
Sodium azide 4g/L
Optional, the pH value of the buffer solution containing polymer is 7.0；
Optional, the polystyrene colloidal milk solution of the combination anti-human calprotectin antibody includes each component of following concentration：
Phosphate 100mmol/L
Anti-human calprotectin antibody is coated with latex particle 15%
Sodium chloride 8.5g/L
Bovine serum albumin 5g/L
Sodium azide 5g/L；
Optional, the pH value of the polystyrene colloidal milk solution of the combination anti-human calprotectin antibody is 7.5.
7. composition according to claim 1, it is characterised in that：Also include calibration object；
Optional, the calibration object is calprotectin calibration object, and the concentration of the calprotectin calibration object is respectively 10,30,
100,300,600ug/g。
8. composition according to claim 7, it is characterised in that：The polystyrene of the combination anti-human calprotectin antibody
Latex solution is prepared from using following methods：
Take during polystyrene latex adds MES buffer solutions, while add EDC and NHS, it is to be dissolved it is well mixed after, activate latex,
After the completion of latex activation, latex is divided into two parts, a copy of it latex adds anti-human calprotectin monoclonal antibody A, separately
A latex adds anti-human calprotectin monoclonal antibody B, and both of which is crosslinked, and is centrifuged after the completion of crosslinking,
, be dissolved in both precipitations in MES buffer solutions respectively by abandoning supernatant, obtains both calprotectin labeling of monoclonal antibodies
Liquid, is then mixed both calprotectin labeling of monoclonal antibody liquid, that is, be obtained and combine anti-human calprotectin antibody's
Polystyrene colloidal milk solution.
9. composition according to claim 8, it is characterised in that：Both calprotectin labeling of monoclonal antibody liquid
It is by 1：1 volume ratio is mixed.
10. application of the composition described in any one of claim 1 to 9 in the product for preparing detection calprotectin；
Optional, the product is kit.
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| WO2019153630A1 (en) * | 2018-02-07 | 2019-08-15 | 深圳市伯劳特生物制品有限公司 | Kit for detecting calprotectin in human feces and preparation method |
| CN110133275A (en) * | 2019-04-01 | 2019-08-16 | 芜湖森爱驰生物科技有限公司 | A kind of calprotectin detection kit and preparation method thereof rapidly and efficiently |
| CN110161254A (en) * | 2019-05-16 | 2019-08-23 | 杭州博谱医药科技有限公司 | A kind of human serum calprotectin detection kit and preparation method thereof |
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