CN102359958A - Kit and method for detecting procalcitonin - Google Patents
Kit and method for detecting procalcitonin Download PDFInfo
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- CN102359958A CN102359958A CN2011102014575A CN201110201457A CN102359958A CN 102359958 A CN102359958 A CN 102359958A CN 2011102014575 A CN2011102014575 A CN 2011102014575A CN 201110201457 A CN201110201457 A CN 201110201457A CN 102359958 A CN102359958 A CN 102359958A
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Abstract
The invention discloses a kit for detecting procalcitonin. The kit comprises a luminous substrate, a magnetic particle coupling antibody, a biotinylation antibody and an enzyme marker, wherein the enzyme marker is horseradish peroxidase labeled streptavidin; and the antibody is a procalcitonin monoclonal antibody. In the kit, the magnetic particle coupling antibody is used as a fixed separation phase, a biotin-streptavidin multistage amplification system is adopted, the luminous substrate is catalyzed by using horseradish peroxidase serving as catalytic enzyme to emit light, and the detection sensitivity and accuracy of reagents are improved.
Description
Technical field
The present invention relates to the immunoassay field, particularly relate to a kind of detection method that detects the kit and the Procalcitonin of Procalcitonin.
Background technology
In the last few years; Pathology, physiology course to systemic infection have had more understandings; Support that intensive treatment also obtains remarkable improvement; But patient's the first cause of the death remains systemic infection and complication thereof in non-heart CICU, the annual nearly 500000 routine systemic infection patients of the U.S., and about 40% is final dead.Therefore, need a kind of label more effectively to reflect and infect the inflammation damnification degree that causes.Procalcitonin (procalcitonin; Initialism is PCT) be to have high sensitivity, specific New Set; It significantly raises when bacterium or fungal infection merge serious whole body system response or the low perfusion of organ; And level is normal or slight raises in virus or during focal infections, and this characteristic makes PCT be used for identification system infection and non-infectious inflammation or focal infection.
PCT is the precursor of human calcitonin, and from the single copy gene that is positioned (11p15,4) on the o.11 chromosome, this gene is made up of 2800 base-pairs, contains 6 extrons and 5 intrones, full length gene M
rAbout 7600, transcribe the back and in the parafollicular cell rough surfaced endoplasmic reticulum (RER), translate and form, PCT is made up of 116 amino acid residues; Relative molecular weight is 13KD; Comprise N end 84 amino acid, calcitonin (CT, 32 peptides) and katacalein (katacalcin, 21 peptides) three parts with hormonal activity; PCT is as a kind of glycoprotein but there is not hormonal activity; Under normal circumstances, the calcitonin with hormonal activity is to produce and secretion at parafollicular cell, and becomes calcitonin by special proteinase decomposed P CT in the cell.
PCT level very low (<0.05ng/mL) in the normal health human body, the risk that severe septicemia and/or septic shock appear in blood-serum P CT concentration≤0.5ng/mL is lower; It is higher that the risk of severe septicemia and/or septic shock appears in blood-serum P CT concentration>=2ng/mL.The horizontal Chang Xianzhu of blood-serum P CT increases when the systemic inflammatory response that bacterial infection (particularly septicopyemia, gram negative bacilli) causes; It increases degree and is proportionate with the infection order of severity and mortality ratio; It is more Zao than C-reactive protein (CRP) and the appearance of other inflammatory factor to raise in systemic bacterial patients serum PCT concentration; Infecting 2h appearance rising, 6h sharply rises, and 8～24h keeps high level; And the discriminating to whole body and local bacterial infection has special value, and blood-serum P CT concentration does not raise when non-bacterial infection.PCT half life period in blood is 22～29h; Detection is not influenced by clinical application, and aspect whole body severe bacterial infections, septicemia and the syndromic auxiliary diagnosis of multiple dysfunction of organ, PCT is the New Set of a tool high sensitivity, high specific; Can not only differentiate bacterium and non-bacterial infection in early days; The early warning of septicemia and diagnosis index especially, and PCT becomes positive correlation with the degree of bacterial infection, therefore more and more receives clinical attention.
PCT is as a kind of experimental index of diseases such as serious bacterial infection new, that have innovative significance; Improved the accuracy of clinical diagnosis, for Intensive Care Therapy, chemicotherapy, take patients such as immunodepressant or organ transplant and merge the clinical foundation that adstante febre provides extremely important diagnosis, further checked and treat: PCT and can be widely used in ICU ward, hematology, oncology, paediatrics, premature and neonate's ICU, surgery, internal medicine, organ transplant section, emergency department, intervention diagnosis and treatment laboratory etc.
The method that is used to detect PCT at present mainly contains radioactive immunoassay (RIA), EUSA (ELISA), colloidal gold immunity chromatography (GICA) and chemiluminescence immunoassay (CLIA).RIA has very high detection sensitivity, but because label has radiological hazard, label poor stability, discarded object are difficult to shortcomings such as processing, withdraw from the clinical examination field gradually; The ELISA method adopts horseradish peroxidase (horseradish peroxidase; Initialism is HRP) or alkali phosphatase enzyme mark antibody; And catalytic substrate produces change color, have simple to operate, the characteristics that the stable reagent phase is long; But the detection sensitivity of ELISA method is lower, is mainly used in communicable disease examination etc. at present detection sensitivity is required lower project; Advantages such as GICA has simple to operate, and detection speed is fast, but also exist sensitivity low, reagent is unstable, and repeatability is relatively poor, is difficult to carry out quantitative shortcoming.
See that from the development of immune diagnostic technique CLIA will be the main flow analytical technology that replaces the ELISA method.CLIA utilizes chemiluminescent substance through the catalysis of catalyzer and the oxidation of oxygenant, forms the intermedium of an excited state, when this excited state intermedium is got back to stable ground state, launches photon, utilizes luminous signal surveying instrument measuring light subnumber.The CLIA that is used for the PCT detection that existing market occurs mainly contains following two kinds: the one, and the direct mark luminescence method of acridinium ester; Utilize double antibody sandwich method to detect the content of the PCT in serum or the blood plasma, two strains are to the mouse monoclonal antibody of PCT, and wherein a strain is to antibody sandwich reaction tube or the plate hole of PCT; Another strain antibody mark luminescent substance; Form sandwich complex with the PCT in the sample, luminescent substance is luminous under certain pH value, and device to be detected detects luminous signal; The 2nd, disclosed a kind of kit and the method for testing thereof that detects Procalcitonin of one Chinese patent application CN 101029897A, this method is the basis with the monoclonal antibody that two strains are directed against PCT, wherein the different luminol derivant of the direct mark luminescent substance of a strain monoclonal antibody; Another strain antibody flag F ITC (Fluorescein Isothiocyanate; The abbreviation of fluorescein isothiocynate), magnetic particle coupling goat-anti FITC antibody is through immune response; Form magnetic particle-goat-anti FITC antibody-FITC antibody-PCT-luminescent marking antibody complex; Under alkaline environment, luminous marker sends photon, detects luminous signal by detecting device.More than the common ground of two kinds of methods all be to adopt the antibody labeling luminescent substance directly luminous, the key distinction is, a kind of common tubular type or plate hole formula direct coated of adopting, the another kind of magnetic particle that adopts is as fixedly separated phase.
Biotin-avidin system is applied to the chemical illuminating reagent field, has significantly improved the detection sensitivity level of reagent.Biotin (claiming biotin again) relative molecular mass is 244.31, and structure is divided into I ring and II ring ringwise, and the I ring is the main position that Avidin combines, and the II ring is unique structure of labelled antibody and enzyme, can effectively combine albumen such as different antigens or antibody.Streptavidin (Streptavidin, initialism are SA) is a kind of slightly slant acidity albumen, and its relative molecular mass is 65000; Be made up of 4 identical peptide chains, each peptide chain can combine 1 biotin molecule, and promptly 1 SA molecule can combine 4 biotin molecules; So adopt biotin to combine to play effective biological magnification with multiple label; The multivalence reagent of many " feelers " that its biotinylation forms makes the entire reaction system multistage amplification reaction occur, and the detection that detects thing is had high sensitivity; And reaction velocity has shown splendid performance than very fast.At present, for the detection of PCT, the electrochemiluminescence product of applicating biotin-Avidin system has appearred on the market; This product adopts SA mark magnetic particle, and two strains are to the monoclonal antibody of PCT, and wherein a strain mark biotin combines with SA; Another strain mark tris (bipyridine) ruthenium is as luminous marker; Tris (bipyridine) ruthenium adds at electric field to be depressed, and the electrochemiluminescence reaction takes place, and sends the photon of 620nm.
There is following shortcoming in the method that more than detects PCT: 1) adopt the luminescence efficiency of acridinium ester or the direct labelled antibody of different luminol (or antigen) low; Label is unstable; This direct labelling method all belongs to moment light emitting-type (being that whole luminescence process is accomplished) in less than 1s; Be difficult to guarantee result's repeatability, and need complicacy, accurate in situ detection mechanism, increased the design difficulty of detecting instrument; 2) reaction tube or luminous plate hole direct coated are as stationary phase; It is limited to have internal surface area; Can't reach higher antibody sandwich density, limit the raising of sensitivity, whole immunoreaction process is approximate from the Liquid Phase Diffusion to the solid phase in addition; Cause whole immunologic process very slow, prolonged the reaction time; 3) applicating biotin-Avidin system detects the electrochemiluminescence product of PCT; All belong to external imported product at present; This product belongs to the reaction of electrode stimulated luminescence; Electrode excites and the detection module complex design, and kit adopts closed fully automatic electric chemical luminous system, the fully automatic electric chemiluminescence detector device that needs are expensive.
Summary of the invention
Technical matters to be solved by this invention is: the deficiency that remedies above-mentioned prior art; Propose a kind of detection method that detects the kit and the Procalcitonin of Procalcitonin, have the detection sensitivity height, specificity is good; Testing process is simpler, does not need advantages such as complex instrument.
Technical matters of the present invention solves through following technical scheme:
A kind of kit that detects Procalcitonin comprises luminous substrate, magnetic particle coupling antibody, biotinylated antibody and enzyme labeling thing, and said enzyme labeling thing is the horseradish peroxidase-labeled Streptavidin, and said antibody is the Procalcitonin monoclonal antibody.
Preferably, said biotinylated antibody is the coupled product of biotin N maloyl imines ester and Procalcitonin monoclonal antibody.
Preferably, said magnetic particle size is 200nm～5 μ m, the reactive group on magnetic particle surface is-COOH or-NH
2
Preferably, the surperficial reactive group of said magnetic particle is-COOH.
Preferably, the concentration that is used for the magnetic particle of coupling in the said magnetic particle coupling antibody is 1 ~ 10mg/mL, and the concentration that is used for the antibody of coupling is 1～5mg/mL.
Preferably, the preservation liquid of said magnetic particle coupling antibody is the mixed liquor of PBS damping fluid and bovine serum albumin(BSA).
Preferably, said luminous substrate comprises A liquid and B liquid, and said A liquid mainly is luminol and reinforcing agent, or different luminol and reinforcing agent, and said B liquid is mainly oxygenant.
Preferably, said kit also comprises the dilution that is used to dilute said biotinylated antibody and enzyme labeling thing; Or said biotinylated antibody and enzyme labeling thing are dissolved in the said dilution and are mixed into working fluid; In the said working fluid; The volume ratio of said enzyme labeling thing and said working fluid is 1:2500～1:5000, and the volume ratio of said biotinylated antibody and said working fluid is 1:1500～1:3500.
Preferably, said kit also comprises at least a in Procalcitonin standard items and the cleansing solution.
A kind of method of using above-mentioned any one kit to carry out the Procalcitonin detection comprises the steps:
(1) respectively said enzyme labeling thing and biotinylated antibody dilution back mixing are obtained working fluid; In the said working fluid; The volume ratio of said enzyme labeling thing and said working fluid is 1:2500～1:5000, and the volume ratio of said biotinylated antibody and said working fluid is 1:1500～1:3500;
(2) respectively with said working fluid, magnetic particle coupling antibody-solutions and luminous substrate balance 30min at least at room temperature;
(3) in reaction vessel, add 50 μ L test serums and the said working fluid of 100 μ L, 37 ℃ of reaction 20min continued following steps behind the mixing, perhaps directly carry out following steps behind the mixing:
(4) the said magnetic particle coupling antibody-solutions of adding 50 μ L in the mixed solution of step (3), mixing is in 37 ℃ of reaction 10 ~ 30min; Form magnetic particle coupling antibody-Procalcitonin-biotinylated antibody-enzyme labeling thing compound, magnetic resolution discards the unreacted liquid in upper strata; Obtain the said compound of lower floor, wash mixing behind the said compound, place magnetic separator; Separate 3min; Discard the cleansing solution supernatant, repeated washing is removed clean up to unreacted liquid, to remove unconjugated reactant;
(5) A liquid in the said luminous substrate and B liquid equal-volume are mixed in the said compound in the back adding step (4), mixing is put into chemiluminescence detector, detects luminous intensity values;
(6) proportionate relationship according to the concentration of said luminous intensity values and Procalcitonin calculates the Procalcitonin concentration value in the said test serum.
The beneficial effect of the present invention and prior art contrast is: method of the present invention belongs to magnetic particle enzyme-catalyzed chemical luminescence method; With magnetic particle coupling antibody as fixedly separated phase; The multistage amplification system of biotin-Streptavidin; Use horseradish peroxidase luminous, improved the detection sensitivity and the accuracy of reagent as catalyzing enzyme catalytic luminescence substrate; Specifically have following advantage: 1) adopt the multistage amplification system of biotin-Streptavidin, Streptavidin combines with biotin, though do not belong to immune response; But high specificity, affinity is big, and both are just very stable once combining; Because 1 Streptavidin molecule has the binding site of 4 biotin molecules; Can connect more biotinylated molecule, form the complex of a kind of type of quasi-lattice, therefore combine biotin-Streptavidin system and highly sensitive chemoluminescence method; The susceptibility of chemical illuminating reagent product be can improve greatly, pg rank even lower level detection sensitivity realized; 2) magnetic particle coupling antibody is as fixedly separated phase; Have bigger antibody sandwich surface area, increased the contact probability with detected material, whole immune response is all carried out in approximate liquid state; Can shorten the reaction time greatly, can shorten to board-like or tubular type reagent place takes time 1/5～1/2; 3) use horseradish peroxidase luminous as catalyzing enzyme catalytic luminescence substrate, compare with the direct labelled antibody of luminous substrate and have the luminescence efficiency height, fluorescent lifetime is long; Luminous plateau, is more than 30 minutes; Avoided direct mark luminescence efficiency to reduce, the problem of poor stability, and the requirement of detecting instrument reduced; Can be applicable to automanual detecting instrument, can realize that also full-automatic instrument detects; The fixedly separated system of chemoluminescence method binding immunoassay magnetic particle that experiment showed, biotin-Streptavidin is the effective ways of a kind of PCT of detection, can realize the detection to PCT concentration in the serum.
Description of drawings
Typical curve and curvilinear equation when Fig. 1 is the embodiment of the invention two employing two step methods detection PCT;
Fig. 2 is that the testing result correlativity of kit of the present invention and Biom é rieux PCT kit compares.
Embodiment
Contrast accompanying drawing below and combine preferred embodiment that the present invention is carried out detailed elaboration.
In one embodiment; Detect the kit of Procalcitonin; Comprise luminous substrate, magnetic particle coupling antibody, biotinylated antibody and enzyme labeling thing, said enzyme labeling thing is the horseradish peroxidase-labeled Streptavidin, and said antibody is the Procalcitonin monoclonal antibody.
Below be elaborated through preferred embodiment, the number percent among the present invention is volume fraction if no special instructions.
Implement one:
1. the preparation of magnetic particle coupling antibody
Magnetic particle coupling antibody in the kit is as fixedly separated phase; Antibody in the magnetic particle coupling antibody is the Procalcitonin monoclonal antibody; The concentration that is used for the magnetic particle of coupling is 1 ~ 10mg/mL; The concentration that is used for the antibody of coupling is 1～5mg/mL, and the magnetic particle size is 200nm～5 μ m, the reactive group on magnetic particle surface is-COOH or-NH
2, through chemical crosslinking reagent activation magnetic particle, make it respectively with the Procalcitonin monoclonal antibody-NH
2Or-COOH group covalent bond, form firm magnetic particle bond, the first-selected reactive group of present embodiment be-magnetic particle of COOH, the magnetic particle of-COOH group under chemical cross-linking agent EDC (carbodiimide) acts on, with the Procalcitonin monoclonal antibody-NH
2Group forms amido link and covalent bond, and magnetic particle coupling antibody is kept in the mixed liquor of PBS damping fluid and bovine serum albumin(BSA), after the participation immune response, under the effect in magnetic field, separates with unconjugated other components.
Get-the magnetic particle 5mg of COOH reactive group, place 4min on the magnetic separator, abandoning supernatant; Add 0.1mol/mL PBS damping fluid 3mL, piping and druming evenly places and separates 4min on the magnetic separator, abandoning supernatant, repeated washing 2 times.After cleaning finishes, add 10mg/mL EDC solution (pH 7.0) 1500 μ L activation magnetic particles, after the piping and druming evenly, place on the oscillator, reaction 30min ~ 1h.Add the PCT monoclonal antibody then and carry out coupling, the dissolving of PCT monoclonal anti body and function 10mmol PBS damping fluid adds 1 mg/mL PCT monoclonal anti liquid solution, 500 μ L in the magnetic particle after activation, place and react 3～6 h on the oscillator; After coupling is accomplished, add 1 mol/L glycocoll 3mL sealing magnetic particle surface-COOH, behind the mixing, place reaction 30 min on the oscillator; Add 0.1mol/mL PBST damping fluid 3mL, mix, place on the magnetic separator and separate, abandoning supernatant, repeated washing 2 times; The preservation liquid that adds 0.1mol/mL PBS and 0.5% bovine serum albumin(BSA) (Bovine Serum Albumin, initialism are BSA) at last redissolves, and making the concentration of magnetic particle coupling antibody is 0.5～2mg/mL, place 4 ℃ subsequent use.
2. the preparation of biotinylated antibody
Because biotin is a small-molecule substance; Can not be directly and antibody carry out coupling; Need through after modifying, ability and albumen coupling form biotin N maloyl imines ester (biotin-hydroxysuccinimide behind the preferred N-maloyl of the present embodiment imines ester modified biological element; Initialism is BNHS), again with the coupling of PCT monoclonal antibody.The PCT monoclonal antibody that is used for the coupling biotin is the antibody to the different epi-positions of PCT with the PCT monoclonal antibody of coupling magnetic particle.
(1) preparation of BNHS: get biotin, each 1mmol of N-maloyl imines and dicyclohexylcarbodiimide adds among the dimethyl formamide solution 6mL in the lump; Room temperature (18～25 ℃) stirs 18h, filters, and filtrate decompression is drained; Dry thing is repeatedly with redissolving in isopropyl alcohol after the ether washing, drains method with vacuum again and makes and be condensed into half volume of commercial weight, places the low temperature refrigerator crystallization; Filter paper filtering is collected crystal, promptly obtains BNHS after washing with ether.
(2) biotinylation of antibody: use 0.1mol/L NaHCO
3Solution is diluted to the PCT monoclonal antibody antibody-solutions of 1mg/mL; Add BNHS (20mg/mL) the 10 μ L that dimethyl formamide dissolves in every milliliter of antibody-solutions; Mixing in room temperature (22 ℃) reaction 1h, places the 0.1mM PBS damping fluid 24h that dialyses in 4 ℃ of environment.
3. HRP mark SA
Adopt improved sodium periodate oxidizing process preparation to obtain HRP mark SA: to get HRP 5mg and be dissolved among the distilled water 0.5mL, add 60mmol/L NaIO
40.5mL, place 4 ℃, mix, oxidation 30min adds 0.16mol/L monoethylene glycol 0.5mL then, puts 4 ℃ again and stops 30min; Add SA 5mg, with the dialysis of 0.05mol/L carbonate buffer solution, 4 ℃ are spent the night, add KBH
4(5mg/mL) 0.3mL puts 4 ℃ and stops 3h, add and the isometric saturated ammonium sulfate of above-mentioned mixed solution, behind 4 ℃ of deposition 30min with the centrifugal 15min of 4000r/min.Sediment is with pH 7.4, and 0.02mol/L PBS damping fluid 0.5mL dissolving removes ammonium with the dialysis of PBS damping fluid ,-20 ℃ of preservations after accomplishing.
4. the preparation of biotinylated antibody and Streptavidin enzyme labeling thing mixed liquor (hereinafter to be referred as working fluid)
(1) preparation of dilution
Dilution is used to dilute biotinylated antibody and enzyme labeling thing, and the composition of dilution comprises: 50mM PB damping fluid (pH7.4), and 150mM NaCl, 2.5% isinglass, 1%BSA, 0.1%Proclin 300.
Can be when using kit; Just dilution is used to dilute biotinylated antibody and enzyme labeling thing; And be mixed into working fluid by certain concentration requirement; Perhaps also can leave in the kit with diluted biotinylated antibody and enzyme labeling thing and after being mixed into working fluid by certain concentration requirement.
(2) preparation of working fluid
Get HRP mark SA that biotinylated antibody that step 2 prepares and step 3 prepare according to the chessboard titrimetry; Selected working concentration mixes, and obtains working fluid with mixing behind diluted biotinylated antibody and the HRP mark SA respectively, in the working fluid; The volume ratio of biotinylated antibody and working fluid can be 1:1500～1:3500; Be 1:2000 in the present embodiment, the volume ratio of HRP mark SA and working fluid can be 1:2500～1:5000, and present embodiment is 1:5000.
5. the preparation of PCT standard items
The PCT standard items are the former albumen of the HCT of genetic recombination (being the PCT recombinant protein), during use, with horse serum PCT recombinant protein (purity of protein>95%) are diluted to the PCT standard items; Dilute concentration is 0ng/mL, 0.2ng/mL, 0.8ng/mL; 6.25ng/mL; 25ng/mL, 50ng/mL is used for the production standard curve.
6. the configuration of cleansing solution
The cleansing solution of configuration 2L: disodium hydrogen phosphate 102g, sodium dihydrogen phosphate dihydrate 19.5, sodium chloride 310g, Tween-20 20mL, be settled to the 2L volume with distilled water, add 0.1% antiseptic Proclin300.Dilute 20 times of uses during use again.
7. luminous substrate
Luminous substrate comprises A liquid and B liquid, and wherein A liquid is mainly luminol and reinforcing agent, or different luminol and reinforcing agent, and B liquid is mainly oxidants hydrogen peroxide, and during use, with A, B liquid equal-volume mixes use.That present embodiment is used is commodity SuperStar by name
TMThe luminous substrate of ECL can certainly be used other commercially produced product.
When using kit of the present invention, can form a magnetic particle coupling antibody as solid phase, separation agent, the detection system of biotin-Streptavidin amplification system and HRP-luminol or different luminol luminescence system.
Embodiment two:
The detection method of Procalcitonin; Testing process can adopt single stage method or two step method reaction pattern: in the single stage method, working fluid, test serum and magnetic particle coupling antibody-solutions are joined in the reaction tube simultaneously, behind the reaction certain hour; Magnetic resolution and washing; Add luminous substrate, luminol or different luminol issue out photon in the catalysis of HRP, detect luminous signal by detecting device.In the two step method; Test serum and working fluid joined earlier react certain hour in the reaction tube; Add magnetic particle coupling antibody-solutions then, magnetic resolution and washing add luminous substrate behind the certain hour; The catalysis of luminol or different luminol HRP issues out photon, detects luminous signal by detecting device.Specifically comprise:
1) use before, with working fluid, magnetic particle coupling antibody-solutions, Procalcitonin standard items and luminous substrate balance 30min at least at room temperature.
2) two step method detects
Need to be ready to the reaction tube of quantity, in a reaction tube, to add the test serum of 50 μ L, add PCT standard solution (adopting the concentration of PCT standard items in embodiment 1 step 5) in other reaction tubes respectively; In each reaction tube, add the working fluid of 100 μ L successively, vibration mixes, and places in 37 ℃ of water-baths and reacts 20 minutes; Form PCT-biotinylated antibody-HRP mark SA compound, add the magnetic particle coupling antibody-solutions of 50 μ L then, 37 ℃ of water-baths 10 minutes; Form magnetic particle coupling antibody-PCT-biotinylated antibody-HRP mark SA compound, adding under the action of a magnetic field, magnetic particle coupling antibody-PCT-biotinylated antibody-HRP mark SA compound is attracted to the reaction tube bottom; Magnetic resolution; Discard other compositions and unreacted composition in the upper serum, in reaction tube, add, fully mix with the 400 μ L of the cleansing solution behind the distilled water diluting 20; Place magnetic separator; Separate 3min, discard the cleansing solution supernatant, repeated washing is removed clean 5 times up to unreacted liquid.Add the mixed luminous substrate 100 μ L of A and B liquid equal-volume then, fully mix, put into chemiluminescence detector, detect the luminous intensity values (relative light unit, initialism RLU) of each pipe after 5 minutes.As shown in Figure 1, be horizontal ordinate with the variable concentrations value of PCT standard items, the RLU of corresponding concentration is an ordinate, sets up typical curve according to linear fit, draws curvilinear equation; Bring the RLU value of test serum into curvilinear equation, can calculate the concentration value of PCT in the test serum.
Perhaps
2) single stage method detects:
Need to be ready to the reaction tube of quantity; The blood serum sample that in a reaction tube, adds 50 μ L adds PCT standard solution (adopting the concentration of PCT standard items in embodiment 1 step 5) respectively in other reaction tubes, in each reaction tube, add the working fluid of 100 μ L successively; The magnetic particle coupling antibody-solutions that adds 50 μ L then; Mix,, form magnetic particle coupling antibody-PCT-biotinylated antibody-HRP mark SA compound in 37 ℃ of water-baths 30 minutes; Adding under the action of a magnetic field; Magnetic particle coupling antibody-PCT-biotinylated antibody-HRP mark SA compound is attracted to the reaction tube bottom, and magnetic resolution discards other compositions and unreacted composition in the upper serum; In reaction tube, add with the 400 μ L of the cleansing solution behind the distilled water diluting 20, fully mix, place magnetic separator, separate 3min, discard the cleansing solution supernatant, repeated washing is removed clean 5 times up to unreacted liquid.Add mixed reaction substrate A and B liquid 100uL then, fully mix, put into chemiluminescence detector, detect the RLU value of each pipe after 5 minutes.Value with the variable concentrations of standard items is a horizontal ordinate, and the RLU value of corresponding concentration is an ordinate, sets up typical curve according to linear fit, draws curvilinear equation; The RLU value of test serum is brought into curvilinear equation, can calculates the concentration value of PCT in the test serum.
Two step method is superior to single stage method.
Embodiment three: the evaluation of methodology of kit of the present invention
1. accuracy
The accuracy measurement is meant with known quantity PCT standard items and joins in normal person's the serum specimen, measures the theoretical value that adds back concentration value and adding and compares, and calculates the recovery of PCT.Testing result is following:
2. accuracy
Choose the sample of 3 parts of variable concentrations, distinguish duplicate measurements according to the method described in the present invention 20 times.According to 20 times measurement result, calculate mean deviation C.V.% value.
3. sensitivity for analysis
Being defined as of sensitivity for analysis: be meant on statistical significance can with the amount of zero-dose difference.Repeat 20 times and measure the zero-dose point, calculate its mean value (X) and standard deviation (SD), be the sensitivity for analysis of this kit with the concentration value of the calculating of X+2SD.The sensitivity for analysis of kit of the present invention is 0.05ng/mL.
4. specificity
With calcitonin (CT) and other be easy to generate cross reaction material reacting value all in allowed limits.
5. correlativity
As shown in Figure 2, with the correlativity of Biom é rieux (biological Mei Liai) PCT chemical luminescence reagent kit be:
Y=0.8623X+0.3673
R
2=0.9891
The present invention compares with product with existing method, has the detection sensitivity height, specificity is good, cost is lower, and detecting instrument is required low advantage.
Above content is to combine concrete preferred implementation to the further explain that the present invention did, and can not assert that practical implementation of the present invention is confined to these explanations.For the those of ordinary skill of technical field under the present invention, do not breaking away under the prerequisite of the present invention design, make some being equal to substitute or obvious modification, and performance or purposes are identical, all should be regarded as belonging to protection scope of the present invention.
Claims (10)
1. kit that detects Procalcitonin; It is characterized in that: comprise luminous substrate, magnetic particle coupling antibody, biotinylated antibody and enzyme labeling thing; Said enzyme labeling thing is the horseradish peroxidase-labeled Streptavidin, and said antibody is the Procalcitonin monoclonal antibody.
2. kit as claimed in claim 1 is characterized in that: said biotinylated antibody is the coupled product of biotin N maloyl imines ester and Procalcitonin monoclonal antibody.
3. kit as claimed in claim 1 is characterized in that: said magnetic particle size is 200nm～5 μ m, the reactive group on magnetic particle surface is-COOH or-NH
2
4. kit as claimed in claim 3 is characterized in that: the reactive group on said magnetic particle surface is-COOH.
5. kit as claimed in claim 1 is characterized in that: the concentration that is used for the magnetic particle of coupling in the said magnetic particle coupling antibody is 1 ~ 10mg/mL, and the concentration that is used for the antibody of coupling is 1～5mg/mL.
6. kit as claimed in claim 1 is characterized in that: the preservation liquid of said magnetic particle coupling antibody is the mixed liquor of PBS damping fluid and bovine serum albumin(BSA).
7. kit as claimed in claim 1 is characterized in that: said luminous substrate comprises A liquid and B liquid, and said A liquid mainly is luminol and reinforcing agent, or different luminol and reinforcing agent, and said B liquid is mainly oxygenant.
8. like any described kit of claim 1-7, it is characterized in that: said kit also comprises the dilution that is used to dilute said biotinylated antibody and enzyme labeling thing; Or said biotinylated antibody and enzyme labeling thing are dissolved in the said dilution and are mixed into working fluid; In the said working fluid; The volume ratio of said enzyme labeling thing and said working fluid is 1:2500～1:5000, and the volume ratio of said biotinylated antibody and said working fluid is 1:1500～1:3500.
9. like any described kit of claim 1-7, it is characterized in that: said kit also comprises at least a in Procalcitonin standard items and the cleansing solution.
10. one kind is used any described kit of claim 1-9 to carry out the method that Procalcitonin detects, and it is characterized in that, comprises the steps:
(1) respectively said enzyme labeling thing and biotinylated antibody dilution back mixing are obtained working fluid; In the said working fluid; The volume ratio of said enzyme labeling thing and said working fluid is 1:2500～1:5000, and the volume ratio of said biotinylated antibody and said working fluid is 1:1500～1:3500;
(2) respectively with said working fluid, magnetic particle coupling antibody-solutions and luminous substrate balance 30min at least at room temperature;
(3) in reaction vessel, add 50 μ L test serums and the said working fluid of 100 μ L, 37 ℃ of reaction 20min continued following steps behind the mixing, perhaps directly carry out following steps behind the mixing:
(4) the said magnetic particle coupling antibody-solutions of adding 50 μ L in the mixed solution of step (3), mixing is in 37 ℃ of reaction 10 ~ 30min; Form magnetic particle coupling antibody-Procalcitonin-biotinylated antibody-enzyme labeling thing compound, magnetic resolution discards the unreacted liquid in upper strata; Obtain the said compound of lower floor, wash mixing behind the said compound, place magnetic separator; Separate 3min; Discard the cleansing solution supernatant, repeated washing is removed clean up to unreacted liquid, to remove unconjugated reactant;
(5) A liquid in the said luminous substrate and B liquid equal-volume are mixed in the said compound in the back adding step (4), mixing is put into chemiluminescence detector, detects luminous intensity values;
(6) proportionate relationship according to the concentration of said luminous intensity values and Procalcitonin calculates the Procalcitonin concentration value in the said test serum.
Priority Applications (1)
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| CN105606597A (en) * | 2016-01-28 | 2016-05-25 | 武汉华美生物工程有限公司 | Chemiluminiscent substrate solution and procalcitonin detection kit utilizing same |
| CN105891194A (en) * | 2016-06-30 | 2016-08-24 | 深圳市亚辉龙生物科技股份有限公司 | Anticardiolipin antibody IgG chemiluminescence immunoassay kit and preparation method thereof |
| CN105928928A (en) * | 2016-06-30 | 2016-09-07 | 深圳市亚辉龙生物科技股份有限公司 | Chemiluminescence immune detection kit of anti-trophoblast cell membrane antibody and preparation method of chemiluminescence immune detection kit |
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