CN202916286U - Latex enhanced turbidimetric immunoassay kit for quantitatively detecting procalcitonin (PCT) - Google Patents

Abstract

The utility model relates to a kit for quantitatively detecting procalcitonin (PCT) in human blood through a latex enhanced turbidimetric immunoassay method, wherein the kit comprises a kit body, a reagent bottle R1, a reagent bottle R2 and a calibrator solution bottle, and the reagent bottle R1, the reagent bottle R2 and the calibrator solution bottle are arranged in the kit body. The reagent bottle R1 contains reagents R1 which are respectively 1-10 mg/ml of protective agent, 1-5 percent of reaction enhancer, 0.03-0.2 percent of preservative and 20-200 mM of buffer solution; the reagent bottle R2 contains reagent R2 which are respectively 1-10 mg/ml of protective agent, 0.03-0.2 percent of preservative, 20-200 mM of buffer solution and 0.04-0.16 percent of polystyrene latex-streptavidin-biotin-anti-human PCT antibody complex; the PCT calibrator solution bottle contains PCT calibrators which are respectively 0.5-8 mg/ml of protective agent, 0.03-0.2 percent of preservative, 20-200 mM of buffer solution and 0.2-100 ng/ml of PCT recombinant protein.

Description

The latex enhancing immune of a kind of quantitative detection Procalcitonin PCT is than turbid kit

Technical field

The utility model relates to technical field of biological, is specifically related to a kind of kit that adopts Procalcitonin PCT in the quantitative human body blood of latex enhancing immune turbidimetry.

Background technology

Procalcitonin (procalcitonin, PCT) be the precursor substance of calcitonin C T, formed by 116 amino acid, molecular weight 13kD, be comprised of terminal three parts of N end-calcitonin-C, its Stability Analysis of Structures is not subjected to the impact of hormone in vivo level, half life period in human body is 25-30 hour, has good stability.The main position that produces PCT in the body is at liver, and other neuroendocrine cells such as peripheral blood lymphocytes, spleen, lung or small intestine also are the important places that produces PCT.The release that endotoxin, cell factor can stimulate PCT.

PCT has unique advantage as a kind of clinical marker thing.PCT content in the healthy human blood extremely low (＜0.05ng/ml), 0.5ng/ml is considered to the cut off value of systemic infection (pyemia) diagnosis.PCT concentration in sepsis patient significantly increases, and can reach 1000ng/ml.PCT is directly proportional with the order of severity of inflammation, at SIRS(system inflammation response syndrome), PCT concentration has notable difference in pyemia (septicemia), severe sepsis (septicemia) and pyemia (septicemia) shock patient.PCT can detect after infecting 2 hours, peaks in 12-24 hour after infection, recovers normal after inflammation disappears.PCT in vivo, external stability is good, not affected by chronic inflammation or autoimmune disease, is not subjected to clinical application to affect (except the OKT3), infects and pyemia etc. has high susceptibility and specificity for systemic bacterium.

Clinical research shows, PCT detects at different medical domains has very high value to diagnosis and the treatment of various diseases, can be applicable to numerous section office such as ICU, hematology, oncology, paediatrics, premature and neonate's ICU, surgery, internal medicine, organ transplant section, emergency department and treatment laboratory.Compare with present applied diagnosis index, PCT provides extra information at antidiastole and infection control and serious aspect of inflammation, relates to diagnosis, the treatment project of following various diseases: septicemia, systemic severe bacterial infections (peritonitis or soft tissue infection etc.) are done early diagnosis; Septicemia and SIRS are made a differential diagnosis; the antidiastole of bacterium infection and non-bacteria inflammation reaction (autoimmune disease etc.); the antidiastole (encephalomyelitis etc.) of bacterium infection and virus infections; (bacterium infects the antidiastole of organ transplant postoperative; virus infections; rejection; fungal infection etc.); to unknown cause fever (UFO) diagnosis and to special infection high-risk patient (intensive care unit; the organ transplant postoperative; the immunosupress phase) monitoring; the antidiastole of septic shock and non-septic shock.Traditional diagnosis index that PCT and other bacteriums infect, as: white blood cell count(WBC), erythrocyte sedimentation rate, c reactive protein, microbe growth etc. relatively, can realize in early days, fast, higher sensitivity and specificity; More help the doctor promptly and accurately to understand patient's disease condition as the reliability index of judging the state of an illness and prognosis and observation of curative effect, the process of follow-up disease, accurately prognosis and methods for the treatment of instructed, provide foundation for formulating rational microbiotic application scheme, reduce the unnecessary medical expense of patient and prevent antibiotic abuse.

At present, the method that detects PCT has a lot, not only can be qualitative, can also be quantitative, method commonly used has: gel chromatography and efficient liquid phase chromatographic analysis, enzyme linked immunosorbent assay (ELISA) (Enzyme-Linked Immunosorbent Assay, ELISA), radiommunoassay (Radioimmunoassay, RIA), immunoluminescence method and colloidal gold chromatography.Wherein, gel chromatography and efficient liquid phase chromatographic analysis are time-consuming and be difficult for robotization; ELISA standard measure poor accuracy, the running time is long, automaticity is low, and is multiplex in qualitative detection; The RIA method can detect free PCT, can detect mating type PCT again, also can detect calcitonin gene associated precursors (Pro-CGRP), the credible susceptibility of this method is 4pg/ml, can detect normal human serum PCT, and is responsive than the double antibodies sandwich method, the RIA shortcoming is that required time is longer, testing result is unstable, and repeatability is poorer than ELISA, and exists radioactive contamination dangerous.Though gold mark method stability better, but sensitivity is lower, generally can only be qualitative, and can not be quantitative, particularly this drawbacks limit of poor repeatability its application clinically, especially be not suitable for the quantitative detection that needs to help by accurate quantitative analysis body fluid marker protein that disease is diagnosed.The immunoluminescence method adopts two antigen-specific antibodies to be combined in respectively the different loci of PCT.One strain antibody is signal (tracer), and another strain then is incorporated on the encrusting substance of inboard wall of test tube.In the process of hatching, the Procalcitonin molecule of antibody in sample is combined and formed sandwich complex, and cursor antibody then is incorporated into the encrusting substance on test tube surface.After reaction is finished, clean, the tracer of surplus is discarded, use suitable photometer and luminous detection reagent measuring light signal, the amount of the residue tracer of calculations incorporated to the tube wall encrusting substance.Light signal strength is directly proportional with PCT concentration.Use known antigen concentration Criterion curve, just can try to achieve PCT concentration unknown in serum or the blood plasma by it.The method high specificity, susceptibility is high, but needs expensive instrument and equipment and veteran operating personnel, general how the use in specific medical mechanism.

The ultimate principle of immunoturbidimetry is when antigen and antibody reacts in special dilution system and during ratio suitable (general provision antibody excess), under short poly-agent (polyglycol etc.) effect of the soluble immune complex that forms in dilution system, separate out from liquid phase, form particulate, make reactant liquor turbidity occur.When antibody concentration fixedly the time, the amount of the immune complex of formation increases along with the increase of antigen amount in the sample, and the turbidity of reactant liquor also increases thereupon.By turbidity and the contrast of series of standards product of assaying reaction liquid, can calculate the content of antigen in the sample.Latex particle strengthens immunoturbidimetry (particle-enhanced turbidimetric immunoassay, PETIA) be occur in recent years a kind of comparatively stable, body fluid albumen homogeneous phase immunoturbidimetry detection method accurately, surface-crosslinked specific antibody at the polymer latex microballoon, the microballoon of antibody is arranged after antigen is combined when crosslinked, can flock together rapidly at short notice, astigmatic performance or the light transmission of reactant liquor have been changed, reactant liquor astigmatism performance or the change of light transmission (being absorbance) and the concentration of tested antigen have stronger correlativity, can reflect within the specific limits the concentration of tested antigen.It is the mensuration of carrying out antigen, antibody response and result in homogeneous reaction system that PETIA detects, the direct absorbance of assaying reaction liquid after the antigen-antibody reaction, saved the ELISA method and repeatedly hatched and wash the loaded down with trivial details operation stepss such as plate, a few minutes just can obtain the result, and are time saving and energy saving.Although the sensitivity of immunoturbidimetry is more weaker than ELISA method, is enough to detect the lower limit of many marker proteins among the healthy human blood, and can be used for the self-reacting device detection.At present commercially available various latex immunoturbidimetry testing products detect lower bound and are limited to 0.1 μ g/ml more, far from being enough and this detection lower bound is applied to clinical for Procalcitonin than turbid product, Procalcitonin 0.5ng/ml is the cut off value of systemic infection (pyemia) diagnosis, low respiratory tract bacterial infection patients PCT concentration may also be lower than this critical value (〉=0.25ng/ml and＜0.5ng/ml), therefore it is higher to develop sensitivity, specificity is stronger, stability is better, the PCT immunoturbidimetry product that antijamming capability is stronger is still the major issue that solution is needed in clinical diagnosis product research field badly.

In immunoassay technology, the coating technique that adopt to improve, improve detect connect on the carrier in the reagent the concentration of immunoreactive composition occurs with sample to be checked, be the important channel of improving the sensitivity of clinical detection product.Biotin-avidin system is a kind of the connection in the panimmunity detection technique to detect carrier and the coat system that detects antibody (or antigen) in the reagent.Biotin molecule has two ring texturees, and wherein the I ring is the main position of being combined with Avidin for the imidazolone ring; II ring is thiphene ring, and a valeric acid side chain is arranged on the C2, and its terminal carboxyl group is the macromolecular unique structure of binding antibody and other biological, and after chemical modification, biotin can become derivant with the various active group-activation biotin.Biotin very easy with protein (such as antibody etc.) with covalent bonds.Avidin mainly comprises white of an egg Avidin, streptavidin, yolk Avidin and class Avidin etc.Rear two kinds because its specificity affinity is low, research is few, furthers investigate at present and is used widely for front two kinds.Avidin (avidin, AV) and streptavidin (streptavidin, SA) are the natural specificity junction mixtures of biotin, and the two is high molecular weight protein.Avidin also claims avidin, avidin, is a kind of alkaline glycoprotein that is comprised of 4 same subunit that extracts from ovalbumin, and molecular weight is 68kD, and isoelectric point pI is 10 ~ 10.5, the effect of heat-resisting and tolerance multiple protein hydrolytic enzyme.Each Avidin can be in conjunction with the biotin of 4 molecules, and Ka is up to 10 for its affinity costant ¹⁵Mol/L.Streptavidin and Avidin have similar biological characteristics, secreted in incubation by Streptomyces avidin streptomycete, molecular weight is 65kD, and each SA molecule also has 4 sites that can be combined with biotin molecule, its affinity costant Ka is identical with AV, is about Ka(10 between antigen-antibody ⁵～10 ¹¹Mol/L) more than 10,000 times.Although SA is identical with the ka of AV, its detection sensitivity obviously is better than AV in actual applications.The high pI of AV and height contain sugared structure and cause on polystyrene board and the nitrocellulose filter or when histocyte DNA is combined, and easily produce non-specific binding to a certain degree, cause higher colour developing background, and SA have obviously overcome this shortcoming than AV in application.Biotin-avidin (streptavidin) system has following distinguishing feature: high specific, high sensitivity, simple and easy, quick, safe, stable and applicability.The association reaction of biotin Avidin presents the height selectivity because of affinity.Biotin-avidin system (BAS) can be by 4 biotinylated macromolecular derivatives of binding site multivalence bridging and label, the sensitivity that greatly improves detection method.BAS can unite for various biological reaction systems such as antigen, antibody, albumen, hormone, acceptor, nucleic acid systems with multiple Mk system such as enzyme, fluorescein, ferritin, agglutinin, SPA, radioactive nuclide etc., also can be used as separation, purifying that the affinity medium is used for above-mentioned all kinds of reaction system reactants.Therefore, BAS has extremely wide research and using value.In addition, the materials such as biotinylated high molecular weight protein, nucleic acid, enzyme not only keep its activity unaffected, more form the multivalence preparation of many " tentacles " because of biotinylation, make whole reaction system multistage enlarge-effect occur, thereby give the high sensitivity of BAS.Simultaneously, the high degree of specificity of BAS and sensitivity make its consumption in reaction system is used atomic, so with low cost.Biotin-avidin (streptavidin) system becomes one of new technology system that is widely used in trace antigen, antibody qualitative and quantitative analysis and position observation research in immunology, the biology field.In the immunodiagnosis field, biotin-avidin (streptavidin) system applies has been developed the multiple testing product that helps sensitivity to improve in ELISA, SABC and radio-immunity, golden mark technology.

Summary of the invention

The purpose of this utility model is to provide the latex enhancing immune of a kind of quantitative detection Procalcitonin PCT than turbid kit.

The utility model comprises box body, R1 reagent bottle, R2 reagent bottle and calibration object solution bottle.Described R1 reagent bottle, R2 reagent bottle and Procalcitonin calibration object solution bottle are positioned at box body, in the described R1 reagent bottle R1 reagent is housed, consist of 1 ~ 10mg/ml protective agent, mass volume ratio of described R1 reagent are that 1 ~ 5% increased response agent, mass volume ratio are 0.03 ~ 0.2% antiseptic and 20 ~ 200mM damping fluid; In the described R2 reagent bottle R2 reagent is housed, consist of 1 ~ 10mg/ml protective agent, mass volume ratio of described R2 reagent are that 0.03 ~ 0.2% antiseptic, 20 ~ 200mM damping fluid and mass volume ratio are polystyrene latex-streptavidin of 0.04 ~ 0.16%-biotin-anti-human Procalcitonin antibody complex; Described Procalcitonin calibration object solution bottle is equipped with the Procalcitonin calibration object, and consist of 0.5 ~ 8mg/ml protective agent, mass volume ratio of described Procalcitonin calibration object are 0.03 ~ 0.2% antiseptic, 20 ~ 200mM damping fluid and 0.2-100ng/ml Procalcitonin recombinant protein.

Protective agent in the described R1 reagent can be selected from bovine serum albumin(BSA) (BSA), ovalbumin (OVA) and gelatin.

Increased response agent in the described R1 reagent is optional from polyglycol 4000, Macrogol 6000, PEG 8000 and PEG 20000.

Antiseptic in the described R1 reagent can be selected from Sodium azide, thimerosal and Proclin 300.

Damping fluid in the described R1 reagent can be selected from borate buffer solution, carbonate buffer solution, Tris-HCl damping fluid, phosphate buffer and glycine buffer.

Protective agent in the described R2 reagent can be selected from BSA, OVA and gelatin.

Antiseptic in the described R2 reagent can be selected from Sodium azide, thimerosal and Proclin 300.

Damping fluid in the described R2 reagent can be selected from borate buffer solution, carbonate buffer solution, Tris-HCl damping fluid, phosphate buffer and glycine buffer.

Protective agent in the described Procalcitonin calibration object solution is bovine serum albumin(BSA) (BSA).

Antiseptic in the described Procalcitonin calibration object solution can be selected from Sodium azide and Proclin 300.

Damping fluid in the described Procalcitonin calibration object solution can be selected from Tris-HCl damping fluid, phosphate buffer and HEPES damping fluid.

The utility model can adopt the following methods preparation:

1, preparation R1 reagent

Protective agent, increased response agent and antiseptic are dissolved in the damping fluid, make R1 reagent.

Described protective agent is selected from one or more in BSA, OVA and the gelatin, preferred BSA; Described protectant concentration is 1 ~ 10mg/ml, preferred 2 ~ 5mg/ml;

Described increased response agent is selected from one or more among PEG4000, PEG6000, PEG8000 and the PEG20000, preferred PEG6000; The mass volume ratio of described increased response agent is 1 ~ 5%, preferred 5%;

Described antiseptic is selected from one or more among Sodium azide, thimerosal and the Proclin 300, preferred Sodium azide, and the mass volume ratio of described antiseptic is 0.03 ~ 0.2%, preferred 0.1%;

Described damping fluid is selected from one or more in borate buffer solution, carbonate buffer solution, Tris-HCl damping fluid, phosphate buffer and the glycine buffer, preferred Tris-HCl damping fluid or borate buffer solution; The concentration of described damping fluid is 20 ~ 200mM, and preferred 50 ~ 100mM, the pH of described damping fluid are 6 ~ 10, preferred 8 ~ 8.5.

2, preparation R2 reagent

1) activation of latex particle, washing

With centrifugal after the commercial polystyrene latex solution activation, abandon supernatant, precipitation lavation buffer solution repeated washing 3 times; The last precipitation is resuspended in the described lavation buffer solution, and the mass volume ratio final concentration that makes latex particle is 1%;

Described lavation buffer solution is selected from a kind of in PBS damping fluid and the MES damping fluid;

Described polystyrene latex particle can be carboxylated or amidized polystyrene latex particle, preferred carboxylated polystyrene latex particle.Carboxylated latex particle carries out streptavidin again through carbodiimides (EDAC) activation or amination latex particle after the glutaraldehyde activation coated, and the particle size range of latex particle is 40-500nm.

2) the coated latex particle of streptavidin

Streptavidin and polystyrene latex particle is crosslinked, obtain streptavidin-polystyrene latex compound.

Described streptavidin and polystyrene latex particle be crosslinked to adopt following concrete grammar:

1. get the latex solution after activation is washed, add streptavidin, making the streptavidin final concentration is 0.1 ~ 1mg/ml, behind the mixing, puts 4 ~ 37 ℃ of shaking tables with the coated 4 ~ 18h of 220rpm rotating speed;

The streptavidin of 2. not being combined with latex particle with the washing of PBS damping fluid repeats 3 times;

3. sealing: the latex particle of the coated streptavidin after will washing is dissolved in and comprises in protectant R2 reagent damping fluid, obtains the coated latex particle solution of streptavidin, and the mass volume ratio final concentration of described latex particle is 0.1 ~ 0.3%;

Described protective agent is selected from one or both in bovine serum albumin(BSA) and the ovalbumin, preferred bovine serum albumin(BSA), and described protectant concentration is 1 ~ 10mg/ml, preferred 4 ~ 8mg/ml;

Described damping fluid is selected from one or more in borate buffer solution, carbonate buffer solution, Tris-HCl damping fluid, phosphate buffer and the glycine buffer, preferred glycine buffer or Tris-HCl damping fluid; The concentration of described damping fluid is 20 ~ 200mM, and preferred 50 ~ 100mM, pH are 7 ~ 9, preferred 8 ~ 8.5.

3) biotinylation of anti-human Procalcitonin antibody

Biotin mixed with anti-human Procalcitonin antibody hatch, centrifugal, remove wherein a small amount of sediment, free biotin is removed in the supernatant dialysis.

Biotin is selected the commercial product that activated described in the biotinylation reaction;

Described PCT antibody is the anti-human PCT antibody of commercial goodsization, is selected from a kind of in polyclonal antibody and the monoclonal antibody, preferred polyclonal antibody.Described Procalcitonin antibody can be mouse source, rabbit source or sheep source antibody, preferred sheep source antibody;

The mol ratio of described anti-human Procalcitonin antibody and biotin is 1:1 ~ 1:100, preferred 1:10;

Described hatching preferably at room temperature carried out; Incubation time is 1-4h, preferred 1h;

Described dialysis is for placing PBS damping fluid dialysed overnight under 0-4 ℃.

4) biotinylation Procalcitonin antibody is coated with being connected of latex particle with streptavidin

In step 2) in add the anti-human Procalcitonin antibody of biotinylation of gained in the step 3) in the coated latex particle solution of the streptavidin of gained, hatch behind the mixing, form latex particle-streptavidin-biotin-anti-human Procalcitonin antibody complex;

Described incubation temperature is 37 ℃; Described incubation time is 0.5 ~ 2h.

5) clean

With latex particle-streptavidin of forming in the step 4)-biotin-anti-human Procalcitonin antibody complex solution centrifugal, abandon supernatant, repeated washing 3 times; The last precipitation is dissolved in the R2 reagent damping fluid that contains protective agent and antiseptic, and the mass volume ratio final concentration that makes described latex particle is 0.04 ~ 0.16%.

Described protective agent is selected from one or both in bovine serum albumin(BSA) and the ovalbumin, preferred bovine serum albumin(BSA), and described protectant concentration is 1 ~ 10mg/ml, preferred 4 ~ 8mg/ml;

Described antiseptic is selected from one or more among Sodium azide, thimerosal and the Proclin 300, preferred Sodium azide, and the mass volume ratio of described antiseptic is 0.03 ~ 0.2%, preferred 0.1%;

Described damping fluid is selected from one or more in borate buffer solution, carbonate buffer solution, Tris-HCl damping fluid, phosphate buffer and the glycine buffer, preferred glycine buffer or Tris-HCl damping fluid; The concentration of described damping fluid is 20 ~ 200mM, and preferred 50 ~ 100mM, pH are 7 ~ 9, preferred 8 ~ 8.5.

3, preparation Procalcitonin calibration object solution

Select the former recombinant protein of commercialization HCT, carry out serial dilution with the solution of similar human serum matrix, the calibration object of preparation variable concentrations.Take Roche Holding Ag's Procalcitonin calibration object of import as primary standard, adopt its Procalcitonin kit that the calibration object of variable concentrations is detected respectively 20 times, obtain average, obtain the concentration of Procalcitonin calibration object.

The solution of described similar human serum matrix is formulated with protective agent, antiseptic and damping fluid;

Described protective agent is bovine serum albumin(BSA) (BSA), and concentration is 0.5 ~ 8mg/ml, preferred 1 ~ 5mg/ml;

Antiseptic in the described Procalcitonin calibration object solution can be selected from one or both among Sodium azide and the Proclin 300, and the mass volume ratio of described antiseptic is 0.03 ~ 0.2%, and preferred 0.1%;

Damping fluid in the described Procalcitonin calibration object solution can be selected from one or more in Tris-HCl damping fluid, phosphate buffer and the HEPES damping fluid, preferred Tris-HCl or PBS damping fluid.

Below provide the immunity of adopting described detection Procalcitonin and strengthen the actual conditions that carries out the Procalcitonin detection than turbid kit at Biochemical Analyzer:

Measure wavelength: 600nm;

Measure temperature: 37 ℃;

Analytical approach: 2 end-point methods;

Sample size/R1 reagent/R2 reagent (latex-streptavidin-biotin-anti-human PCT antibody complex suspension): 20 μ l/240 μ l/80 μ l.

The utility model adopts the coated polystyrene latex particle of biotinylated Procalcitonin antibody sensitized streptavidin, by biotin-streptavidin system and latex intensified mode, amplified antigen-antibody reaction, overcome the not high deficiency of common immunoturbidimetry sensitivity, realized that Procalcitonin robotization on Biochemical Analyzer and scattering turbidimetry analyser detects the target of Procalcitonin content in the blood.The utility model has been realized following technical advantage:

1) further adopted streptavidin-biotin amplification system at latex enhancing immune on than turbid principle basis, greatly improved the detection sensitivity than turbid reagent, lowest detectable limit can reach 0.2ng/ml, has satisfied the demand of clinical practice.

2) can realize the flexible Quantitative detection of multisample type at the scattering turbidimetry analyser.Commercially available Procalcitonin PCT detects serum or the blood plasma of adopting more and detects, can not satisfy clinical fast detecting demand, the utility model kit comprises whole blood, serum or blood plasma when adoptable sample type when the scattering turbidimetry analyser uses, and can use in clinical many section office.Commercially available PCT detects the time that needs approximately about half an hour, and the utility model kit needs 2-3 minute from sample collection to providing the fastest of testing result, for the patient has striven for the more quality time, can carry out detection of dynamic to conditions of patients.

3) the utility model kit and import reagent box are to the PCT content detection of same sample statistical analysis as a result, and results relevance is good, and without significant difference, testing result can be used by import substitutes accurately and reliably clinically, significantly reduces testing cost.

Description of drawings

Fig. 1 is that the structure of the utility model embodiment forms front view synoptic diagram (1: box body; The 2:R1 reagent bottle; The 3:R2 reagent bottle; 4: Procalcitonin calibration object solution bottle);

Fig. 2 is that the structure of the utility model embodiment forms vertical view synoptic diagram (1: box body; The 2:R1 reagent bottle; The 3:R2 reagent bottle; 4: Procalcitonin calibration object solution bottle);

Fig. 3 is the utility model embodiment's " concentration-absorbance difference " calibration curve;

Fig. 4 is the range of linearity correlativity of the utility model embodiment;

Fig. 5 is that the correlativity of the utility model Procalcitonin kit embodiment and Luo Shi PCT kit testing result compares.

Embodiment

Referring to Fig. 1, the immunity enhancing of detection Procalcitonin described in the utility model comprises box body 1, R1 reagent bottle 2, R2 reagent bottle 3 and Procalcitonin calibration object solution bottle 4 than turbid kit embodiment, described R1 reagent bottle 2 and R2 reagent bottle 3 and Procalcitonin calibration object solution bottle 4 are positioned at box body 1, in the described R1 reagent bottle R1 reagent is housed, consist of 1 ~ 10mg/ml protective agent, mass volume ratio of described R1 reagent are that 1 ~ 5% increased response agent, mass volume ratio are 0.03 ~ 0.2% antiseptic and 20 ~ 200mM damping fluid; R2 reagent (polystyrene latex-streptavidin-biotin-anti-human Procalcitonin antibody complex suspension) is housed in the described R2 reagent bottle, and consist of 1 ~ 10mg/ml protective agent, mass volume ratio of described R2 reagent are that 0.03 ~ 0.2% antiseptic, 20 ~ 200mM damping fluid and mass volume ratio are polystyrene latex-streptavidin of 0.04 ~ 0.16%-biotin-anti-human Procalcitonin antibody complex; In the described Procalcitonin calibration object solution bottle Procalcitonin calibration object is housed, consist of 0.5 ~ 8mg/ml protective agent, mass volume ratio of described Procalcitonin calibration object are 0.03 ~ 0.2% antiseptic, 20 ~ 200mM damping fluid and 0.2-100ng/ml Procalcitonin recombinant protein.

One, the Procalcitonin immunity strengthens the preparation than turbid kit

1, Procalcitonin R1 reagent preparation

Trishydroxymethylaminomethane (Tris) concentration is 0.2M, is 8.0 with the salt acid for adjusting pH.Add again BSA, PEG6000 and Sodium azide, make final concentration be: BSA 5mg/ml, PEG60005%(w/v), Sodium azide 0.1%(w/v).

2, Procalcitonin R2 reagent preparation

1) activation of latex particle, washing

Get 10.1% carboxylic polystyrene latex particle solution (Bangs Laboratories, Inc., particle diameter are 200nm), 100 μ l, in latex solution, add 6mg/ml EDAC solution 1.68ml, place 37 ℃ of reciprocating type shaking tables to react 0.5h.The centrifugal 30min of 12000rpm abandons supernatant, adds 50mM pH 6.5MES damping fluid washing three times, and precipitation is scattered in the 50mM pH 6.5MES damping fluid, and making latex particle concentration is 1%(w/v).

2) the coated latex particle of streptavidin

Get the carboxylic polystyrene latex solution after the 1ml activation is washed, add 2mg/ml streptavidin solution 500 μ l, mixing is put 37 ℃ of reciprocating type shaking tables with the coated 16h of 220rpm rotating speed, with the centrifugal 20min of 12500rpm, abandons supernatant; Sediment is dissolved in 0.1MpH 7.4PBS damping fluid, and mixing with the centrifugal 20min of 12500rpm, is abandoned supernatant, the streptavidin that then twice removal of repeated washing is not combined with latex particle; The latex particle sediment that streptavidin is coated is dissolved in 20mM pH 8.0 glycine buffers that contain 4mg/mlBSA, and making the latex particle final concentration is 0.3%(w/v).

3) preparation of biotinylation Procalcitonin antibody

6mg/ml goat-anti people PCT polyclonal antibody (Shanghai Ye Min Bioisystech Co., Ltd) is diluted to the antibody-solutions of 2mg/ml with 0.1M pH 9.5 carbonate buffer solutions, add the BNHS(5mg/ml that dimethyl formamide dissolves in every milliliter of antibody-solutions) 10 μ l, mixing, in room temperature (22 ℃) reaction 1h, centrifugal, remove wherein a small amount of sediment, supernatant is packed in the bag filter, in 4 ℃ of environment, place 0.1M pH 7.4PBS damping fluid dialysis 24h.

4) biotinylation Procalcitonin antibody is coated with being connected of latex particle with streptavidin

In 1ml step 2) add the biotinylated antibody solution of 400 μ l step 3) preparation in the coated latex particle solution of streptavidin of preparation, mixing is put 37 ℃ of shaking table 1h, forms latex particle-streptavidin-biotin-PCT polyclonal antibody and connects compound.The centrifugal 20min of 12500rpm speed abandons supernatant, and sediment is dissolved in 0.1M pH 7.4PBS damping fluid, mixing, and the centrifugal 20min of 12500rpm abandons supernatant, and then repeated washing is twice; Last is precipitated and dissolved in 50mM pH 8.0 glycine buffers that contain 4mg/ml BSA and 0.1% Sodium azide.

3, PCT calibration object preparation

The former recombinant protein of commercially available HCT is dissolved in the solution of similar human serum matrix, and (NaCl 0.8%, BSA0.2%, NaN ₃0.4%, Tris-HCl pH 8.0) in, makes the calibration object of variable concentrations.Take Roche Holding Ag's Procalcitonin calibration object of import as primary standard, adopt its Procalcitonin kit that the calibration object of variable concentrations is detected respectively 20 times, obtain average, obtain the concentration of Procalcitonin calibration object: 0.2,1,3,10,30,100ng/ml.

Two, the Procalcitonin immunity strengthens the detection method than turbid kit

1, Biochemical Analyzer detects

Be operating as example take Hitachi 7020: the mensuration wavelength is 600nm, get respectively the calibration object solution (20 μ l) of variable concentrations, add Procalcitonin R1 reagent (240 μ l), mixing, 37 ℃ hatch 5 minutes after, add Procalcitonin R2 reagent (80 μ l), mixing, 37 ℃ hatch 1 minute after, read and respectively manage absorbance A ₁, react after 4 ~ 5 minutes, read and respectively manage absorbance A ₂, calculate absorbance difference Δ A=A ₂-A ₁, every pipe replication 3 times, the mean value of the absorbance difference Δ A that records for 3 times take each calibration tube are as ordinate, and corresponding calibration object concentration is horizontal ordinate, draws " concentration-absorbance difference " calibration curve (see figure 3).

Get test serum or plasma sample, measure the absorbance difference of sample with method, the substitution calibration curve can calculate the content of Procalcitonin PCT in the sample to be tested.If the concentration of Procalcitonin exceeds the calibration curve scope in serum or the blood plasma, detect again to guarantee the accuracy of testing result after need diluting sample.

This kit is not only applicable to Hitachi 7020, also is applicable to semi-automatic, the automatic clinical chemistry analyzer of other brand and model, and design parameter can be adjusted according to instrument.

2, NORMAN series scattering turbidimetry analyser detects

With whole blood 35 μ l(serum or blood plasma 20 μ l) add in the example reaction cup, then add reagent R1240 μ l, mixing, etc. the scattered light intensity in the reaction cup stable after, add reagent R280 μ l, mixing, within 1-2 minute, measure the variation difference DELTA A of scattered light intensity, with Δ A substitution calibration curve, can calculate the content of Procalcitonin in the sample to be tested.

Three, the Procalcitonin immunity strengthens the analytical performance assessment than turbid kit

1, the range of linearity

With the Procalcitonin high concentration sample (100.20ng/ml) near the range of linearity upper limit, with physiological saline it is pressed 1/2,1/4,1/8,1/16,1/32,1/64 dilutes, and is mixed with altogether the solution of 6 dilute concentrations (xi), measures each diluted sample concentration with described Biochemical Analyzer detection method.Each concentration replication 3 times is obtained respectively the mean value (yi) of measurement result.Take dilute concentration (xi) as independent variable, obtain equation of linear regression take measurement result mean value (yi) as dependent variable.By formula (1) calculates the correlation coefficient r of linear regression, and the result shows that regression equation is y=1.0086x-0.1464, correlation coefficient r=0.9998, table

2, sensitivity (lowest detectable limit)

Take 5% bovine serum albumin solution as dummy, press described Biochemical Analyzer detection method replication 20 times, add twice standard deviation report lowest detectable limit with blank average, the result shows that the lowest detection of the utility model kit is limited to 0.2ng/ml.

3, repeatability and accuracy

The Procalcitonin calibration object that is respectively 0.67ng/ml and 51.4ng/ml with AUDIT company sign value is measured by described Biochemical Analyzer detection method as sample, and each concentration is replication 10 times respectively, calculates respectively and measures average

And standard deviation (S), with

Calculate the coefficient of variation and carry out the repeatability investigation, the result shows that the coefficient of variation is respectively 2.86% and 1.38%; With

Calculate relative deviation and carry out the accuracy investigation, its relative deviation is respectively 1.49% and 0.79%.

The repeatability of table 1 Procalcitonin kit and accuracy are investigated

4, difference between batch

Prepare three batches of kits by the described method of the utility model embodiment, to carrying out replication with a serum sample, each lot number replication 3 times calculates respectively every batch of average of measuring for 3 times with three batches of kits

(i=1,2,3), by formula relative deviation (R) is calculated in (2), (3), and the result shows that relative deviation R is 2.68%.

$\stackrel{\‾}{x}=\frac{\stackrel{\‾}{x_1}+\stackrel{\‾}{x_2}+\stackrel{\‾}{x_3}}{3}...\left(2\right)$

$R=\frac{{\stackrel{\‾}{x}}_{\max }-{\stackrel{\‾}{x}}_{\min }}{\stackrel{\‾}{x}}\×100\%...\left(3\right)$

In the formula:

In maximal value;

In minimum value;

Three batches of reagent detect average.

The difference between batch of table 2 Procalcitonin kit is investigated

5, interference experiment

Getting concentration is the Procalcitonin calibration object solution of 0.67ng/ml, each the interfering material solution that adds respectively equal volume, make each the interfering material concentration after the adding be respectively haemoglobin 10g/L, cholerythrin 684 μ mol/L, triglyceride 20g/L, with the utility model kit each is prepared sample replication 3 times, average, compare with the sample that adds equal volume distilled water, observe the relative deviation of PCT measured value after adding interfering material and adding distilled water.The result shows, adds above concentration interfering material measured value and is no more than 4% with adding with measured value relative deviation behind the volume distilled water.In detecting sample, exist certain density interfering material to comprise haemoglobin, cholerythrin and triglyceride, very little on the measurement result impact of the utility model kit.

6, stability

The utility model kit is carried out uncork stability and long-time stability investigation.

Uncork stability: the uncork of the utility model kit was placed on 2 ℃-8 ℃ preservations after 30 days, taking-up is measured the Procalcitonin calibration object that AUDIT company sign value is respectively 0.67ng/ml and 51.4ng/ml by described Biochemical Analyzer detection method, each concentration replication 3 times, the relative deviation of calculating testing result and sign value.The result shows, uncork after 30 days the relative deviation of the utility model kit detected value and sign value be respectively 1.99% and 1.12%, uncork stability is better.

Long-time stability: place 2 ℃-8 ℃ preservations after 12 months the utility model kit, taking-up is measured the Procalcitonin calibration object that AUDIT company sign value is respectively 0.24ng/ml and 45.9ng/ml by described Biochemical Analyzer detection method, each concentration replication 3 times, the relative deviation of calculating testing result and sign value.The result shows that the relative deviation of the utility model kit detected value and sign value is respectively 2.78% and 1.32%, and it is more stable that kit is preserved 12 months at 2 ℃-8 ℃.

The uncork of table 3 Procalcitonin kit and long-time stability are investigated

Four, the comparison of the utility model kit and the product that gone on the market

The Nanjing Military Command hospital general provides 79 routine inpatient serum, the male sex's 45 examples wherein, women's 34 examples, 39 years old mean age.The patient who has 21 examples to pick up from the pathogenic microorganism examination positives such as blood culture in the 79 routine samples, front 3 of the pathogenic microorganism of separation is respectively: Escherichia coli, Klebsiella Pneumoniae, pseudomonas aeruginosa.With the utility model Procalcitonin Measurement kit and commercially available import Luo Shi Procalcitonin electrochemiluminescence kit respectively to sample replication 2 times, the difference calculating mean value, 79 routine pattern detection results are carried out linear regression analysis, calculate the related coefficient of two kinds of kit testing results.

The result shows, the correlation coefficient r of two kinds of kit testing results=0.9986, equation of linear regression is that y=0.9475x+0.1073(sees Fig. 5).According to Association for Standardization of U.S. clinical labororatory (CLSI) documentation requirements (r〉0.975), the utility model Procalcitonin Measurement kit and commercially available import Luo Shi Procalcitonin electrochemiluminescence kit have good consistance.This shows that the utility model Procalcitonin Measurement kit can replace the import reagent box to use clinically, reduce testing cost, alleviate patient's financial burden.

Claims (1)

1. a latex enhancing immune that quantitatively detects Procalcitonin PCT is than turbid kit, it is characterized in that comprising box body, R1 reagent bottle, R2 reagent bottle and Procalcitonin calibration object solution bottle, described R1 reagent bottle, R2 reagent bottle and Procalcitonin calibration object solution bottle place in the described box body.

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