CN109142335A - The kit and its detection method of spatial neighbor chemoluminescence method detection Procalcitonin - Google Patents
The kit and its detection method of spatial neighbor chemoluminescence method detection Procalcitonin Download PDFInfo
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- CN109142335A CN109142335A CN201811115054.7A CN201811115054A CN109142335A CN 109142335 A CN109142335 A CN 109142335A CN 201811115054 A CN201811115054 A CN 201811115054A CN 109142335 A CN109142335 A CN 109142335A
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- 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
-
- 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
- G01N33/53—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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/585—Calcitonins
Abstract
The present invention relates to the kits and its detection method of a kind of spatial neighbor chemoluminescence method detection Procalcitonin.Kit includes: enzyme marker, luminous marker, adjuvant, triggering agent and calibration object；Wherein, calibration object includes the calibration object and 0.1M phosphate dilution of various concentration PCT antigen；The component of enzyme marker includes the PCT detection antibody and 0.05M phosphate buffer of peroxidase labelling；The component of luminous marker includes the PCT capture antibody and 0.05M Tris buffer of acridan label；The component of adjuvant includes shine adjuvant and citrate buffer；It triggers agent and selects 0.05M Tris buffer.Detection method provided by the invention is not necessarily to carrier, is not coated with, washing process as a kind of homogeneous chemistry luminescence technology truly, and reagent constituents are few, and high sensitivity, reproducible, easy to operate.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of examination of spatial neighbor chemoluminescence method detection Procalcitonin
Agent box and its detection method.
Background technique
Procalcitonin (PCT) is made of amino acid, is belonged to peptide material before the calcitonin of no hormonal activity, is had good stability,
Long half time is up to 25~30h.PCT generally results from thyroxine cell, forms calcitonin by the proteolysis of specificity.Pathology
Under state, PCT is stimulated through inflammatory cytokine and bacteriotoxin and induction, and secretion results from the outer amount tissue of thyroxine and device
Official.It is reported that its level will not generally rise in non-infectious inflammation reaction.A large amount of scholars unanimously think: PCT level is thin
It is in rise shape when bacterium infection, low-level shape is maintained in local inflammation reaction and virus infection, this variation is stable and rapid,
It is one of the optimal parameter of discriminating bacteria infection and virus infection.
Procalcitonin content in the serum of Healthy People is extremely low, but when bacterium infection, in addition to thyroid follicular cells, monokaryon
Cell, macrophage, lymphocyte, Studies of Endocrine-like Cells etc. can secrete PCT.PCT can obviously rise after infecting 2h
Height, the PCT after antibiotic is effectively treated in blood can decline, and immunocompromised patients are equally applicable；Therefore, PCT is that monitoring is thin
Bacterium infection and one of therapeutic effect and the important symbol object of Index for diagnosis.
Pyemia latest definition is the caused mortality organ dysfunction of host response imbalance for infection, morbidity
Mechanism is related to complicated systemic inflammatorome network effects, immune dysfunction, dysfunction of blood coagulation, gene pleiomorphism, tissue damage
And host is to many aspects such as the reactions of infection, but its specific mechanism is not yet completely clear.Procalcitonin is in pyemia and seriously
Its horizontal significant raising when infection, and it has significant correlation with blood culture result, septic shock, it is considered to be pyemia
Best clinical assistant diagnosis index.
Summary of the invention
For the defects in the prior art, it is an object of that present invention to provide a kind of spatial neighbor chemoluminescence methods to detect drop calcium
Plain former kit and its detection method.The method is not necessarily to carrier, does not have as a kind of homogeneous chemistry luminescence technology truly
There are coating, washing process, reagent constituents are few, and high sensitivity, reproducible, easy to operate.
To achieve the above object, technical solution provided by the invention are as follows:
In a first aspect, the present invention provides a kind of detection kit of Procalcitonin, kit includes: enzyme marker, shines
Marker, adjuvant, triggering agent and calibration object；Wherein, the raw material components of enzyme marker include the PCT inspection of peroxidase labelling
Antibody is surveyed, the raw material components of luminous marker include that the PCT of 9,10- acridan label captures antibody.
Preferably, the raw material components of enzyme marker further include 0.05M phosphate buffer；It is highly preferred that preparation method packet
It includes: weighing 5mg HRP and be dissolved in 1mL distilled water, the 0.1M sodium periodate solution that 0.2mL newly matches is added later, keeps away at room temperature
Light stirs 20min, and above-mentioned solution is fitted into bag filter, dialyses to the sodium-acetate buffer of 1mM pH4.4, and 4 DEG C overnight；Add 20
9.5 carbonate buffer solution of μ L 0.2M pH, the addition anti-PCT monoclonal antibody room temperature of 1mg, which is protected from light, immediately after is gently mixed 2h,
Add the 4mg/mL sodium borohydride liquid newly matched, mixes, in 4 DEG C of placement 2h, above-mentioned liquid is fitted into bag filter, to 0.15M pH
7.4PBS dialysis, 4 DEG C overnight；Dialysate is taken out, adds qs glycerin to set -20 DEG C of refrigerators and saves.
Preferably, the raw material components of luminous marker further include 0.05M Tris buffer；It is highly preferred that preparation method packet
Include: the luminous substrate Acridan DMF of 500 μ L dissolves；The Acridan41.3 μ L of dissolution is drawn, it is slow that 0.05M Boratex is added
708.7 μ L of fliud flushing adds the anti-PCT monoclonal antibody of 250 μ L, and overturning mixes 4~5 times, stands 30min at room temperature；It will mark
Note reaction tube is placed on shaking table, is mixed at 2~8 DEG C overnight, and taking-up addition qs glycerin is set -20 DEG C of refrigerators and saved.
Preferably, the component of adjuvant includes 6.0 citrate buffer of adjuvant and pH that shines；It is highly preferred that preparation
Method includes: to weigh citric acid 1.82g, and sodium citrate 10.45g adds pure water to dissolve and is settled to 1000mL, slow in citrate
Suitable luminous adjuvant is added in fliud flushing, is dispensed after mixing, every bottle of 10mL is placed in 4 DEG C of refrigerators and saves backup.
Preferably, 0.05M pH 8.0Tris-HCl buffer is selected in triggering agent；It is highly preferred that preparation method includes: to claim
Tris 6.06g, sodium chloride 9g are taken, appropriate pure water is added to dissolve, dense HCl 2.1mL is added and mixes.It is added and spits in above-mentioned solution
- 20 2mL of temperature are settled to 1000mL after mixing, and dispense after mixing, every bottle of 200mL is placed in 4 DEG C of refrigerators and saves backup.
Preferably, calibration object includes the calibration object and 0.1M phosphate buffer of various concentration PCT antigen；It is highly preferred that
Preparation method includes: to prepare calibration object dilution: weighing potassium dihydrogen phosphate 14.1g, sodium dihydrogen phosphate (2H2O) 3.0g adds appropriate
Ultrapure water dissolution, 0.5~1mL of Proclin-300 after mixing, add ultrapure water to be settled to 1000mL, as calibration object dilution,
2~8 DEG C store for future use；Prepare calibration object: the concentration of calibration object be 0,10ng/mL, 50ng/mL, 500ng/mL, 1000ng/mL,
10000ng/mL is diluted to respective concentration for Procalcitonin is purified with calibration object dilution, and 2~8 DEG C store for future use.
Kit of the present invention detects the content of Procalcitonin (PCT) in human serum using double antibody sandwich method.By sample, peppery
The anti-PCT Dan Ke that the anti-PCT monoclonal antibody of root peroxidase (HRP) label, acridan (Acridan) mark
Grand antibody one reacts, and antigen-antibody sandwich complex is formed, so that horseradish peroxidase and 9,10- acridan
(Acridan) close to each other, the luminous adjuvant of addition and triggering agent, generation flash type chemiluminescence are spatially able to；And not
In conjunction with free HRP labelled antibody and Acridan labelled antibody do not shine then.PCT content in sample is higher, the hair of measurement
Light value (RLU) is bigger.So luminous value is positively correlated with concentration of specimens within the scope of a certain concentration, pass through the school of known concentration
Quasi- product and its luminous value draw working curve, and the content of PCT in sample can be calculated according to the luminous value of sample.
Second aspect, detection kit provided by the invention answering in spatial neighbor chemoluminescence method detection Procalcitonin
With.
The third aspect, above-mentioned detection kit provided by the invention is when spatial neighbor chemoluminescence method detects Procalcitonin
Method, comprising steps of S1: be separately added into 25 μ L standard items, 25 μ L peroxidase labellings PCT detection antibody and 25 μ L hair
Signal object is to reaction tube；S2: 15min is reacted in 37 DEG C of insulating boxs；S3: 5 μ L adjuvants are separately added into reaction tube, concussion is mixed
It is even, stand 1~2min；It is separately added into triggering 75 μ L of agent later, concussion is mixed, detected immediately, reads signal value；S4: to calibration
The concentration and luminous value of product carry out tetra- parameter fitting of logistic, calculate concentration of specimens by sample luminous value.
Technical solution provided by the invention, have it is following the utility model has the advantages that
(1) it is needed with traditional chemiluminescence using microwell plate or magnetic particle as carrier coated antibody or antigen phase
Than, carrier is not necessarily in kit provided by the invention and detection method, is not coated with, washing process, be it is a kind of truly
Homogeneous chemistry luminescence technology.
(2) traditional enzyme-catalyzed chemical luminescence technology is in the test analyte successively detection antibody with capture antibody and enzyme label
It must be cleaned after combining 2~3 times, to remove the unstable non-specific substance of be not associated with or combination；And the present invention is
After the combination of test analyte and two specific antibodies, the interference that shines is eliminated by adjuvant effect, triggering agent is added and generates
Luminous signal, whole process is without washing.
(3) traditional enzyme-catalyzed chemical luminescence technology needs substrate for enzymatic activity, 5~10 minutes or so detection luminous values；And this
Invention is flash type chemiluminescence, generates luminous signal immediately after triggering agent is added.
(4) reagent constituents provided by the invention are few, and production process is simple, and production cost reduces, and are easy to amplify production；And
Detection process is convenient, it is easy to accomplish full-automatic.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical solution of the present invention is clearly and completely described.Implement below
Example is only used for clearly illustrating technical solution of the present invention, therefore is intended only as example, and cannot be used as a limitation and limit this hair
Bright protection scope.
Experimental method in following embodiments is unless otherwise specified conventional method.Examination as used in the following examples
Material is tested, is to be commercially available from routine biochemistry reagent shop unless otherwise specified.Quantitative test in following embodiment,
Three repeated experiments are respectively provided with, data are the average value or mean+SD of three repeated experiments.
The present invention provides a kind of detection kit of Procalcitonin, and kit includes: enzyme marker, luminous marker, auxiliary
Auxiliary agent, triggering agent and calibration object；Wherein, the component of enzyme marker includes the PCT detection antibody and 0.05M of peroxidase labelling
Phosphate buffer；The component of luminous marker includes that the PCT capture antibody of acridan label and 0.05M Tris delay
Fliud flushing；The component of adjuvant includes 6.0 citrate buffer of adjuvant and pH that shines；It triggers agent and selects 0.05M pH
8.0Tris-HCl buffer；Calibration object includes the calibration object and 0.1M calibration object dilution of various concentration PCT antigen.
One, the preparation of calibration object
(1) it prepares calibration object dilution: weighing potassium dihydrogen phosphate 14.1g, sodium dihydrogen phosphate (2H2O) 3.0g adds ultrapure water
Dissolution, 0.5~1mL of Proclin-300 after mixing, adds ultrapure water to be settled to 1000mL, obtains calibration object dilution, and 2~8 DEG C
It stores for future use.
(2) prepare calibration object: the concentration of calibration object be 0,10ng/mL, 50ng/mL, 500ng/mL, 1000ng/mL,
10000ng/mL is diluted to respective concentration for Procalcitonin is purified with calibration object dilution, and 2~8 DEG C store for future use.
Two, the preparation of enzyme marker
(1) it weighs 5mg HRP to be dissolved in 1mL distilled water, it is molten that the 0.1M sodium periodate that 0.2mL newly matches is added in upper liquid
Liquid is protected from light stirring 20min at room temperature, above-mentioned solution is fitted into bag filter, dialyses to the sodium-acetate buffer of 1mMpH 4.4, and 4
DEG C overnight.
(2) add 20 μ L 0.2M pH, 9.5 carbonate buffer solution, the anti-PCT monoclonal antibody room temperature of 1mg is added immediately after
It is protected from light and is gently mixed 2h, add the 4mg/mL sodium borohydride liquid newly matched, mix, in 4 DEG C of placement 2h, above-mentioned liquid is packed into bag filter
In, it dialyses to 0.15M pH 7.4PBS, 4 DEG C are overnight.
(3) dialysate is taken out, adds qs glycerin to set -20 DEG C of refrigerators and saves.
Three, the preparation of luminous marker
(1) the luminous substrate Acridan DMF of 500 μ L is dissolved.
(2) the 41.3 μ L of Acridan for drawing dissolution, is added 708.7 μ L of 0.05M sodium borate buffer liquid, adds 250 μ L
Anti- PCT monoclonal antibody, overturning mix 4~5 times, stand 30min at room temperature.
(3) label reaction tube is placed on shaking table, is mixed at 2~8 DEG C overnight, taken out addition qs glycerin and set -20 DEG C
Refrigerator saves.
Four, the preparation of adjuvant
Citric acid 1.82g, sodium citrate 10.45g are weighed, pure water is added to dissolve and is settled to 1000mL, it is slow in citrate
The adjuvant that shines is added in fliud flushing, is dispensed after mixing, is placed in 4 DEG C of refrigerators and saves backup；Wherein, more preferably per bottled 10mL.
Five, the preparation of agent is triggered
Tris 6.06g, sodium chloride 9g are weighed, appropriate pure water is added to dissolve, dense HCl 2.1mL is added and mixes；It is added later
Tween-20 2mL is settled to 1000mL after mixing, packing is placed in 4 DEG C of refrigerators and saves backup；Wherein, more preferably per bottled
200mL。
Drop calcium is detected in spatial neighbor chemoluminescence method using Procalcitonin detection kit the present invention also provides a kind of
Method when plain former, comprising steps of
S1: the PCT detection antibody and 25 μ L luminous markers of 25 μ L standard items, 25 μ L peroxidase labellings are separately added into
To chemiluminescence reaction pipe.
S2: 15min is reacted in 37 DEG C of insulating boxs.
S3: being separately added into 5 μ L adjuvants to chemiluminescence reaction pipe, and concussion mixes, and stands 1~2min；Add respectively later
Enter to trigger 75 μ L of agent, concussion is mixed, detected immediately, reads signal value.
S4: concentration and luminous value to calibration object carry out tetra- parameter fitting of logistic, calculate sample by sample luminous value
This concentration.
Technical solution provided by the invention is described further combined with specific embodiments below.
Embodiment one
The present embodiment provides a kind of detection kits of Procalcitonin, comprising: enzyme marker, luminous marker, adjuvant,
Trigger agent and calibration object；Wherein, calibration object includes that the calibration object of no less than 6 various concentration PCT antigens and 0.1M phosphate delay
Fliud flushing；Enzyme marker includes the PCT detection antibody and 0.05M phosphate buffer of peroxidase labelling；Luminous marker packet
Include the PCT capture antibody and 0.05M Tris buffer of acridan label；The component of adjuvant includes the adjuvant that shines
With 6.0 citrate buffer of pH；It triggers agent and selects 0.05M pH 8.0Tris-HCl buffer.
One, the preparation of calibration object
(1) it prepares calibration object dilution: weighing potassium dihydrogen phosphate 14.1g, sodium dihydrogen phosphate (2H2O) 3.0g adds ultrapure water
Dissolution, Proclin-300 0.8mL after mixing, add ultrapure water to be settled to 1000mL, obtain calibration object dilution, and 4 DEG C of storages are standby
With.
(2) prepare calibration object: the concentration of calibration object be 0,10ng/mL, 50ng/mL, 500ng/mL, 1000ng/mL,
10000ng/mL is diluted to respective concentration for Procalcitonin is purified with calibration object dilution, and 4 DEG C store for future use.
Two, the preparation of enzyme marker
(1) it weighs 5mg HRP to be dissolved in 1mL distilled water, it is molten that the 0.1M sodium periodate that 0.2mL newly matches is added in upper liquid
Liquid is protected from light stirring 20min at room temperature, above-mentioned solution is fitted into bag filter, dialyses to the sodium-acetate buffer of 1mMpH 4.4, and 4
DEG C overnight；
(2) add 20 μ L 0.2M pH, 9.5 carbonate buffer solution, the anti-PCT monoclonal antibody room temperature of 1mg is added immediately after
It is protected from light and is gently mixed 2h, add the 4mg/mL sodium borohydride liquid newly matched, mix, in 4 DEG C of placement 2h, above-mentioned liquid is packed into bag filter
In, it dialyses to 0.15M pH 7.4PBS, 4 DEG C are overnight；
(3) dialysate is taken out, adds qs glycerin to set -20 DEG C of refrigerators and saves.
Three, the preparation of luminous marker
(1) the luminous substrate Acridan DMF of 500 μ L is dissolved；
(2) the 41.3 μ L of Acridan for drawing dissolution, is added 708.7 μ L of 0.05M sodium borate buffer liquid, adds 250 μ L
Anti- PCT monoclonal antibody, overturning mix 5 times, stand 30min at room temperature；
(3) label reaction tube is placed on shaking table, is mixed at 4 DEG C overnight, taken out addition qs glycerin and set -20 DEG C of refrigerators
It saves.
Four, the preparation of adjuvant
Citric acid 1.82g, sodium citrate 10.45g are weighed, pure water is added to dissolve and is settled to 1000mL, it is slow in citrate
The adjuvant that shines is added in fliud flushing, is dispensed after mixing, every bottle of 10mL is placed in 4 DEG C of refrigerators and saves backup.
Five, the preparation of agent is triggered
Tris 6.06g, sodium chloride 9g are weighed, appropriate pure water is added to dissolve, dense HCl 2.1mL is added and mixes；It is added later
Tween-20 2mL is settled to 1000mL after mixing, packing, every bottle of 200mL is placed in 4 DEG C of refrigerators and saves backup.
Embodiment two
Calcitonin is detected in spatial neighbor chemoluminescence method using Procalcitonin detection kit the present embodiment provides a kind of
Method when former, comprising steps of
S1: the PCT detection antibody and 25 μ L luminous markers of 25 μ L standard items, 25 μ L peroxidase labellings are separately added into
To chemiluminescence reaction pipe.
S2: 15min is reacted in 37 DEG C of insulating boxs.
S3: being separately added into 5 μ L adjuvants to chemiluminescence reaction pipe, and concussion mixes, and stands 1~2min；Add respectively later
Enter to trigger 75 μ L of agent, concussion is mixed, detected immediately, reads signal value.
S4: concentration and luminous value to calibration object carry out tetra- parameter fitting of logistic, calculate sample by sample luminous value
This concentration.
The present invention detects Procalcitonin using spatial neighbor chemoluminescence method, and sensitivity is up to 0.1ng/mL.Blood-serum P CT liter
Height is to septicopyemia early diagnosis specificity 81.8%；When blood-serum P CT concentration is greater than 2ng/mL, pneumonia of newborn is diagnosed special
The opposite sex is up to 93.3%.
Certainly, the case where being enumerated in addition to embodiment one and embodiment two, other conditions and parameter in preparation process etc.
It is possible.
Applicant has found after creative work: detection method provided by the invention is as a kind of truly equal
Phase chemiluminescence is not necessarily to carrier, is not coated with, washing process, and reagent constituents are few, and high sensitivity, reproducible, behaviour
Make simple.
It should be noted that unless otherwise indicated, technical term or scientific term used in this application should be this hair
The ordinary meaning that bright one of ordinary skill in the art are understood.Unless specifically stated otherwise, it otherwise illustrates in these embodiments
Component and opposite step, numerical expression and the numerical value of step are not limit the scope of the invention.It is illustrated and described herein
In all examples, unless otherwise prescribed, any occurrence should be construed as merely illustratively, not as limitation, because
This, other examples of exemplary embodiment can have different values.
In the description of the present invention, it is to be understood that, term " first ", " second " are used for description purposes only, and cannot
It is interpreted as indication or suggestion relative importance or implicitly indicates the quantity of indicated technical characteristic.Define as a result, " the
One ", the feature of " second " can explicitly or implicitly include one or more of the features.In the description of the present invention,
The meaning of " plurality " is two or more, unless otherwise specifically defined.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme should all cover in protection scope of the present invention.
Claims (8)
1. a kind of detection kit of Procalcitonin, which is characterized in that the kit includes:
Enzyme marker, luminous marker, adjuvant, triggering agent and calibration object；
Wherein, the raw material components of the enzyme marker include the PCT detection antibody of peroxidase labelling, the luminous marker
Raw material components include acridan label PCT capture antibody.
2. the detection kit of Procalcitonin according to claim 1, it is characterised in that:
The raw material components of the enzyme marker further include 0.05M phosphate buffer；
Preferably, preparation method includes:
It weighs 5mg HRP to be dissolved in 1mL distilled water, the 0.1M sodium periodate solution that 0.2mL newly matches is added later, keeps away at room temperature
Light stirs 20min, and above-mentioned solution is fitted into bag filter, dialyses to the sodium-acetate buffer of 1mM pH 4.4, and 4 DEG C overnight；
20 μ L 0.2M pH, 9.5 carbonate buffer solution is added, the anti-PCT monoclonal antibody room temperature of 1mg is added immediately after and is protected from light gently
Light stirring 2h, adds the 4mg/mL sodium borohydride liquid newly matched, mixes, in 4 DEG C of placement 2h, above-mentioned liquid is fitted into bag filter, right
0.15M pH 7.4PBS dialysis, 4 DEG C overnight；
Dialysate is taken out, adds qs glycerin to be placed on -20 DEG C of refrigerators and saves.
3. the detection kit of Procalcitonin according to claim 1, it is characterised in that:
The raw material components of the luminous marker further include 0.05M Tris buffer；
Preferably, preparation method includes:
The luminous substrate Acridan DMF of 500 μ L is dissolved；
The 41.3 μ L of Acridan of dissolution is drawn, 708.7 μ L of 0.05M sodium borate buffer liquid is added, it is mono- to add the 250 anti-PCT of μ L
Clonal antibody, overturning mix 4~5 times, are stored at room temperature 30min；
Label reaction tube is placed on shaking table, is mixed at 2~8 DEG C overnight, addition qs glycerin is taken out and is placed on -20 DEG C of refrigerators
It saves.
4. the detection kit of Procalcitonin according to claim 1, it is characterised in that:
The component of the adjuvant includes the citrate buffer of luminous adjuvant and pH 6.0；
Preferably, preparation method includes:
Citric acid 1.82g, sodium citrate 10.45g are weighed, pure water is added to dissolve and is settled to 1000mL, in citrate buffer
It is middle that the adjuvant that shines is added, it is dispensed after mixing, is placed in 4 DEG C of refrigerators and saves backup；Wherein, more preferably per bottled 10mL.
5. the detection kit of Procalcitonin according to claim 1, it is characterised in that:
0.05M pH 8.0Tris-HCl buffer is selected in the triggering agent；
Preferably, preparation method includes:
Tris 6.06g, sodium chloride 9g are weighed, appropriate pure water is added to dissolve, dense HCl 2.1mL is added and mixes；It is added and spits later
- 20 2mL of temperature, are settled to 1000mL after mixing, packing is placed in 4 DEG C of refrigerators and saves backup；Wherein, more preferably per bottled 200mL.
6. the detection kit of Procalcitonin according to claim 1, it is characterised in that:
The calibration object includes the calibration object and 0.1M calibration object dilution of various concentration PCT antigen；
Preferably, preparation method includes:
It prepares calibration object dilution: weighing potassium dihydrogen phosphate 14.1g, NaH2PO4·2H2O 3.0g, adds ultrapure water to dissolve,
0.5~1mL of Proclin-300 after mixing, adds ultrapure water to be settled to 1000mL, obtains calibration object dilution, 2~8 DEG C of storages
It is spare；
Prepare calibration object: the concentration of calibration object be 0,10ng/mL, 50ng/mL, 500ng/mL, 1000ng/mL, 10000ng/mL,
It is diluted to respective concentration by Procalcitonin is purified with the calibration object dilution, 2~8 DEG C store for future use.
7. the described in any item detection kits of claim 1~6 are in spatial neighbor chemoluminescence method detection Procalcitonin
Using.
8. method of the described in any item kits of claim 1~6 when spatial neighbor chemoluminescence method detects Procalcitonin,
Comprising steps of
S1: 25 μ L standard items, the PCT detection antibody of 25 μ L peroxidase labellings and 25 μ L luminous markers are separately added into change
Learn luminescence-producing reaction pipe；
S2: 15min is reacted in 37 DEG C of insulating boxs；
S3: being separately added into 5 μ L adjuvants to chemiluminescence reaction pipe, and concussion mixes, and stands 1~2min；It is separately added into touching later
75 μ L of agent is sent out, concussion is mixed, detected immediately, reads signal value；
S4: concentration and luminous value to calibration object carry out tetra- parameter fitting of logistic, and it is dense to calculate sample by sample luminous value
Degree.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811115054.7A CN109142335A (en) | 2018-09-25 | 2018-09-25 | The kit and its detection method of spatial neighbor chemoluminescence method detection Procalcitonin |
| CN202110503772.7A CN113238055A (en) | 2018-09-25 | 2018-09-25 | Kit for detecting procalcitonin by using space proximity chemiluminescence method, and detection method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811115054.7A CN109142335A (en) | 2018-09-25 | 2018-09-25 | The kit and its detection method of spatial neighbor chemoluminescence method detection Procalcitonin |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110503772.7A Division CN113238055A (en) | 2018-09-25 | 2018-09-25 | Kit for detecting procalcitonin by using space proximity chemiluminescence method, and detection method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109142335A true CN109142335A (en) | 2019-01-04 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811115054.7A Pending CN109142335A (en) | 2018-09-25 | 2018-09-25 | The kit and its detection method of spatial neighbor chemoluminescence method detection Procalcitonin |
| CN202110503772.7A Pending CN113238055A (en) | 2018-09-25 | 2018-09-25 | Kit for detecting procalcitonin by using space proximity chemiluminescence method, and detection method and application thereof |
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Cited By (2)
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| CN110441293A (en) * | 2019-08-05 | 2019-11-12 | 济南大学 | A kind of electrochemical luminescence sensor preparation method and application based on protein active protection |
| CN110779912A (en) * | 2019-11-22 | 2020-02-11 | 无锡壹闪生物科技有限公司 | Microsphere-free homogeneous chemiluminescence system of biotin-avidin or streptavidin |
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| CN101368969A (en) * | 2008-03-14 | 2009-02-18 | 北京科美东雅生物技术有限公司 | Chemical luminescence immune analysis quantitative measuring reagent kit for IV type collagen and preparation method thereof |
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| US7732153B2 (en) * | 2006-05-09 | 2010-06-08 | Beckman Coulter, Inc. | Nonseparation assay methods |
| CN101709091A (en) * | 2009-03-02 | 2010-05-19 | 广东虹业抗体科技有限公司 | Preparation of haplotype antibody of Ang2 and other polymer proteins and application of haplotype antibody in immunodetection method |
| CN107807240A (en) * | 2017-11-03 | 2018-03-16 | 太原瑞盛生物科技有限公司 | A kind of chemiluminescence detection kit of Procalcitonin and preparation method thereof |
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| CN101368969A (en) * | 2008-03-14 | 2009-02-18 | 北京科美东雅生物技术有限公司 | Chemical luminescence immune analysis quantitative measuring reagent kit for IV type collagen and preparation method thereof |
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| MARK J. CAMERON ET AL.: "Rapid and Cost Effective Immunoassay Development and Validation of a Biomarker Assay Using Spatial Proximity Analyte Reagent Capture Luminescence, SPARCLTM", 《POST NUMBER 4003》 * |
| PAUL HELD PH. D. ET AL: "Detection of Human IgG Using a Luminescence-based Proximity Assay，Using Spatial Proximity Analyte Reagent Capture Luminescence (SPARCL) Assays", 《ADME/TOX, BIOCHEMICAL ASSAYS, BIOMARKERS, CELL BIOLOGY》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110441293A (en) * | 2019-08-05 | 2019-11-12 | 济南大学 | A kind of electrochemical luminescence sensor preparation method and application based on protein active protection |
| CN110441293B (en) * | 2019-08-05 | 2021-09-24 | 济南大学 | Method for preparing electrochemical luminescence sensor based on protein activity protection |
| CN110779912A (en) * | 2019-11-22 | 2020-02-11 | 无锡壹闪生物科技有限公司 | Microsphere-free homogeneous chemiluminescence system of biotin-avidin or streptavidin |
| CN110779912B (en) * | 2019-11-22 | 2022-05-17 | 无锡壹闪生物科技有限公司 | Biotin-avidin or streptavidin microsphere-free homogeneous chemiluminescence system |
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