CN101833010B - Kit for detecting fibrin (ogen) degradation products (FDP) (by using emulsion immunoturbidimetry) - Google Patents
Kit for detecting fibrin (ogen) degradation products (FDP) (by using emulsion immunoturbidimetry) Download PDFInfo
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Abstract
The invention relates to the field of biotechnology and discloses a kit for detecting fibrin (ogen) degradation products (FDP) by using emulsion immunoturbidimetry. The kit comprises an anti-human FDP antibody emulsion which is formed by performing chemical crosslinking of acidulated anti-human FDP antibodies and polyvinyl benzyl chloride emulsion particles and sealing the reaction product. The kit of the invention has the advantages that: the emulsion immunoturbidimetry is adopted to detect the FDP content, so the sensitivity is high and can reach 0.10 mu g/ml; the stability is high, the operation is simple and quick and it only takes 5 to 10 minutes to obtain a detection result; the specificity is high and the detection avoids interference effectively; and the quantification is accurate and the application range is wide.
Description
Technical field
The present invention relates to biological technical field, be specifically related to the kit of a kind of employing latex immunoturbidimetry detection fibers albumen (former) catabolite (FDP) content.
Background technology
Fibrinolytic system, be called for short fibrinolytic system, it is the most important anticoagulation system of human body, to keeping the normal permeability of vascular wall, flow state and the organization restoration of keeping blood play an important role, and are comprised of 4 kinds of major parts: plasminogen, plasminogen activator (such as t-PA, u-PA), fibrinolysin, plasmin inhibitor.Fibrin (former) catabolite (Fibrin (ogen) Degradation Products, FDP) be under the effect of the fibrinolysin of generation after fibrinolytic system is activated, fibrinogen and fibrin in the human body are degraded, the general designation of various dimers, polymer and the compound that produces, the aggregate level of the FDP reflection body fibrinolytic of generation.
The activation of fibrinolytic system has number of ways, mainly is divided into interior activation, outer activation and exogenous activated pathway.Interior activated pathway is that Hageman factor a makes kallikreinogen change kallikrein in the intrinsic coagulation approach, kallikrein makes single chain urokinase type plasminogen activator type activator of plasminogen change double chain urokinase type activator of plasminogen into, thereby plasminogen activation becomes fibrinolysin, shows as secondary increased fibrinolytic activity; And outer activated pathway is to discharge tissue plasminogen activator (t-PA) by vascular endothelial cell, and the plasminogen cracking is become fibrinolysin, shows as the primary hyperfibrinolysis.In addition, exogenous activated pathway is to enter thrombolytic drug such as SK, UK and t-PA etc. in the body by the external world, and Plasminogen activation is become fibrinolysin.In the presence of fibrinolysin, fibrinogen becomes various solvable fragments with fibrin degradation, forms fibrin degradation product (FDP) (FDP).This shows that the FDP level all can raise in primary and secondary increased fibrinolytic activity in the body, is the sensitive indicator of concentrated expression hyperfibrinolysis, the simultaneously detection of FDP also can reflect the treatment curative effect of thrombolytic drug.
In recent years, the detection of FDP content is widely used in clinical diagnosis, examination, its content provable hyperfibrinolysis that has that raises, such as Secondary cases and primary hyperfibrinolysis due to the diseases such as the rejection of malignant tumour, disseminated intravascular coagulation, DVT formation, pulmonary embolism, severe bacterial infections, chronic liver disease, organ transplant, pregnancy-hypertension syndrome, raising all appears in FDP content.In the therapeutic process of thrombolytic drug, the dissolving of thrombus is accelerated in the generation of a large amount of fibrinolysins, and FDP raises in the blood, to the detection of serum or plasma F DP concentration, can monitor the result for the treatment of of thrombolytic drug.In addition because intravascular coagulation, fibrinolytic is unusual, a large amount of FDP through kidney discharge or since ephritis cause fibrin the renal tubule deposition simultaneously secondary activation fiber protein degradation system generate FDP and from urine, discharge, so occur FDP in the urine and also mean in the body or kidney has intravascular coagulation and fibrinolytic phenomenon.Dynamically observe the variation of urine FDP, significant to the diagnosis of rejection after the kidney transplant.Therefore, detect content that sample comprises FDP in serum, blood plasma, the urine to the fibrinolytic system disease early stage, fast, Accurate Diagnosis and the monitoring course for the treatment of and the efficacy assessment of thrombolytic drug therapy had important using value.
The FDP detection method of using clinically at present has: ethanol gel test, protamine paracoagulation test, latex agglutination, Enzyme-linked Immunosorbent Assay (ELISA) method and latex immunoturbidimetry etc.But all there is certain limitation.
Ethanol gel test, protamine paracoagulation test be can only by with the FDP product in X, Y fragment produce grumeleuse, FDP is carried out qualitative detection; Though latex agglutination has fast, easy and simple to handle, expense is advantages of higher not, but its sensitivity is low, by visual inspection, only can detect the FDP of 5 μ g/ml levels, be usually used in qualitative or semiquantitative determination as examination; Enzyme-linked Immunosorbent Assay (ELISA) is though that method has is highly sensitive, and testing result is a kind of method of classics accurately and reliably, but operation requirements is strictly time-consuming, testing cost is high, is subject to various factors, is not suitable for the needs of in time diagnosis of clinical patient and emergency case and treatment.
The advantages such as that the latex immunoturbidimetry has is easy and simple to handle, quick, detection sensitivity is high, special, quantitatively accurate are extensively approved in clinical practice.But adopt the kit that this method measures fibrin (former) catabolite (FDP) content generally can not accurate quantitative analysis when sample FDP concentration is lower than 2.5 μ g/ml on the existing market, detection sensitivity be not good enough.
Summary of the invention
The objective of the invention is to detect for existing latex immunoturbidimetry the low defective of method susceptibility of FDP, FDP content in a kind of quantitative detection blood plasma, serum, the urine equal samples is provided, and diagnostic kit simple to operate, quick, quantitatively accurate, that susceptibility is higher.
In order to solve the problems of the technologies described above, the present invention is achieved in that
A kind of kit that adopts the latex immunoturbidimetry to detect FDP content comprises reagent R1, reagent R2, dilution, FDP calibration object.
Reagent R2 of the present invention is anti-human FDP antibody latex reagent, and the preparation of described anti-human FDP antibody latex reagent comprises following steps:
Step 1: anti-human FDP antibody is carried out acidifying in the pH value in the solution of 2.5-3.0;
Step 2: step 1 gained solution is regulated pH to 7.5-8.0 with aqueous slkali, and adding particle diameter is the polyvinyl benzyl chloride latex solution of 100-200nm, stirs 0.5-2 hour at 20-25 ℃, hatches 2-4 hour at 37 ℃ again;
Step 3: be that the confining liquid of 7.5-8.5 was 37 ℃ of sealings 1-8 hour with the pH value with step 2 gained solution;
Step 4: with step 3 gained solution after centrifugal, get precipitation disperse with the damping fluid washing that contains stabilizing agent and antiseptic after and get final product.
As preferably, the described solution of step 1 is a kind of in glycocoll, hydrochloric acid or the phosphoric acid solution.In embodiment, glycine solution is selected in the described acidifying of step 1, and the pH value of regulator solution is 2.5-3.0, and keeps 30-40min at 20-25 ℃.
As preferably, the described aqueous slkali of step 2 is tris solution.Step 2 with the acidifying of anti-human FDP antibody after, regulate pH to neutral with tris solution, within 10min, be the polyvinyl benzyl chloride latex particle solution chemical crosslinking sensitization of 100-200nm with particle diameter, form covalent antibody-latex compound, otherwise the space three-dimensional structure of antibody in neutral solution is returned to easily and do not have activated natural conformation after the acidifying.
Step 3 is the covalent antibody that forms-latex compound solution, is that the glycocoll-sodium hydrate buffer solution of the 7.5-8.5 0.1mol/L that contains BSA seals the not surface active groups methyl chloride of binding antibody of latex particle with pH value.
More preferably, the BSA concentration quality percent by volume in the described damping fluid of step 3 is counted 0.1-0.5% with g/ml.
The described damping fluid of step 4 is preferably the Tris/HCl damping fluid, and the solution after the sealing is disperseed with the Tris/HCl damping fluid washing that contains stabilizing agent and antiseptic, namely obtains stable reagent R2.
Polyvinyl benzyl chloride latex particle is a kind of latex particle of nucleocapsid form, and its latex nuclear is the vinyltoluene polymkeric substance, and the latex shell is the polymkeric substance of vinyl chloride, is a kind of hydrophobicity latex.Have highdensity chloro-methyl group on the surface of shell, these active surface functional groups can react by amino group direct and antibody, antigen or other part in the aqueous solution of gentleness, produce a kind of stable covalent compound by single step reaction.By adding suitable surfactant, the surface of this latex particle forms mechanicalness or electrical diaphragm, can suppress the flocculation of latex particle; According to the proportion of latex particle, regulate damping fluid proportion and viscosity by suitable suspending agent, can make the latex particle stable suspersion and can sedimentation.
Anti-human FDP antibody by suitable acid treatment after, the hinge area three-D space structure of antibody has carried out certain modification and has changed, cause the hinge area hydrophobicity FC fragment of embedding to expose, so that easilier be combined with the hydrophobicity latex particle, the site that FC is special and latex surface active groups methyl chloride generation covalent bonds form stable antibody-latex particle.FC fragment avtive spot is adsorbed on the surface of latex particle, because the impact of stereoeffect, the FC fragment can not be reacted with interfering material in the blood such as rheumatoid factor Rf and complement C1Q, thereby has reduced the impact of interfering material in the blood.Antibody because how available FDP antigen binding site exposes, improves the reactivity of latex antibody after acid treatment, thereby can reduce the consumption of antibody in the emulsion reagent preparation, reduces cost, also improves sensitivity for analysis simultaneously.
Stabilizing agent is selected from one or more in protein, amino acid, inorganic salts, surfactant, suspending agent, the antioxidant.
Those skilled in the art can be according to stabilization principle and the demand that stabilizing agent rose, select following multiple the combination: bovine serum albumin(BSA) and gelatin, can play good colloid protection stabilization to the surface-crosslinked antibody of latex particle, preferred BSA, its final concentration quality percent by volume is counted 0.1-0.5% with g/ml; Ethylene glycol, glycerine, maltose etc. are as a kind of suspending agent, can improve liquid density and viscosity, latex particle can be suspended in the solution not for a long time can sedimentation and play good stabilization, preferred glycerine, and its final concentration percent by volume is 1-10%; The amino acid such as glycocoll, serine, arginine can form stable complex compound with metallic ion, can prevent from entering in the solution metal ion disturbance, preferred serine, and its final concentration quality percent by volume is counted 4-6% with g/ml; Surfactant comprises cationic surfactant, anionic surfactant, non-ionics selection, can be adsorbed on the surface of latex particle, reduce the latex particle surface tension, make latex particle be difficult for assembling, effectively improve latex particle stability in solution, preferred polysorbate40, its final concentration percent by volume is 0.1-0.6%; Antioxidant is selected BHT, and final concentration quality percent by volume counts 0.01% with g/ml; Select the inorganic salts of debita spissitudo, count 0.80% sodium chloride such as the quality percent by volume with g/ml.
More preferably, described stabilizing agent is: BSA 0.1-0.5% (g/ml), the combination of serine 4-6% (g/ml), suspending agent glycerine 1-10%, surfactant polysorbate40 0.1-0.6%, antioxidant BHT (BHT) 0.01% (g/ml) and sodium chloride 0.7-0.9% (g/ml).
Described antiseptic is selected from suitable antiseptic well known by persons skilled in the art, such as the Sodium azide of 0.1% (g/ml).
Reagent R1 of the present invention is the damping fluid of pH value 7.0-9.0.
As preferably, described damping fluid is selected from one or more the combination in Tris/HCl damping fluid, phosphate buffer, HEPES damping fluid, glycine buffer, barbitol buffer solution, MOPSO damping fluid, DIPSO damping fluid, the HEPPS damping fluid.
More preferably, described damping fluid is the Tris/HCl damping fluid of 10-70mmol/L.
As preferably, contain set accelerator PEG-6000 in the described damping fluid, the quality percent by volume is counted 2-6% with g/ml, can accelerate the immune response speed of antigen-antibody, shortens detection time; Contain inorganic salts sodium chloride, the quality percent by volume is counted 0.7-0.9% with g/ml.
Dilution of the present invention is selected from a kind of in physiological saline, the phosphate buffered saline.
As preferably, described dilution is selected the phosphate buffered saline of pH7.4.
FDP calibration object of the present invention can be buied or be passed through self-control by market and obtain.In embodiment, a kind of method of the FDP of preparation calibration object is disclosed: the human fibrinogen is added the fibrinolysin degraded in the Tris/HCl damping fluid, it is 27-33 μ g/ml that fibrinogen degradation product (FDP) (FDP) solution that obtains is diluted to FDP concentration with the Tris/HCl damping fluid, makes by freeze-drying after adding stabilizing agent BSA, excipient sweet mellow wine and antiseptic again.The concentration 30-70mmol/L of Tris/HCl damping fluid wherein, pH is 7.0-8.0; The final concentration of BSA is 20-60mg/ml; The final concentration of sweet mellow wine is 10-30mg/ml.
The ultimate principle of latex immunoturbidimetry be FDP antibody is adsorbed in be of moderate size, uniformly on the latex particle, during FDP antigen in running into sample, antigen is combined with antibody latex, latex particle is assembled, the particle diameter of latex changes, thereby causes the change of turbidity, and the transmittance of reaction system reduces, namely linearity is during the phase when reaching stable fall off rate, and transmittance rate of change and FDP concentration are proportional.Adopt coaglation analyzer or Biochemical Analyzer to be determined at that reaction system can calculate the content of FDP in the sample at the rate of change of the transmittance of linear phase under certain wavelength by drawing calibration curve.
Adopt the latex immunoturbidimetry that the FDP calibration object is carried out the FDP content detection with kit of the present invention, set up calibration curve in rate of change and the FDP concentration of coaglation analyzer or its transmittance of Biochemical Analyzer mensuration.Get sample to be tested and measure the rate of change of its transmittance with method, can try to achieve at calibration curve the content of FDP in the sample to be tested.
Kit of the present invention compared with prior art has following features:
1, highly sensitive, can reach 0.10 μ g/ml;
2,0.50 μ g/ml～30.0 μ g/ml range of linearity inner heights are relevant, good reproducibility;
3, high specificity, and be difficult for being disturbed;
4, kit good stability of the present invention can be preserved 18 months for 2-8 ℃, can preserve at least 15 days behind each reagent.
5, simple to operate, quick, only need 5-10 minute from detecting out the result.
Description of drawings
Fig. 1 is the calibration curve of kit of the present invention;
Fig. 2 shows kit range of linearity correlativity of the present invention;
Fig. 3 is that kit of the present invention and commercially available import reagent box testing result correlativity compare.
Embodiment:
The invention discloses the kit of a kind of detection fibers albumen (former) catabolite (FDP) content, those skilled in the art can use for reference this paper content, suitably improve technological parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are deemed to be included in the present invention.Product of the present invention and application are described by preferred embodiment, the related personnel obviously can change or suitably change and combination methods and applications as herein described within not breaking away from content of the present invention, spirit and scope, realizes and use the technology of the present invention.
The invention will be further described below in conjunction with specific embodiment, but not as a limitation of the invention.
Embodiment 1: the composition of kit of the present invention and preparation method
Reagent R2 is the preparation of anti-human FDP antibody latex reagent: will contain 500 μ g mouse-anti people FDP monoclonal antibodies (available from American Diagnostica Inc., product article No. ADINo.313) dried frozen aquatic products, adding the 0.5ml deionized-distilled water redissolves, getting 50 μ l solution dilutes with 3ml 0.1M sodium chloride solution (containing Sodium azide 0.1% (g/ml)), in solution, add 2.0ml0.05M glycine buffer (pH2.3,0.1M sodium chloride), the pH value of solution is 2.8.The antibody-solutions of acidifying at room temperature (20-25 ℃) keeps 30-40min, then adds the tris solution of 100 μ l 1M in the solution, and the pH value is adjusted between the 7.5-8.0.
Get 1.25ml 4% (g/ml) polyvinyl benzyl chloride latex particle suspension (American I NVITROGEN company; the product article No. is C37345; mean grain size is 200nm); add the dilution of 3.75ml deionized-distilled water; the antibody-solutions that is adjusted to the acid activation after the neutrality added under the 300rpm magnetic agitation in the latex suspension after the dilution immediately fully mix; stirred 45 minutes lower continuation of room temperature (20-25 ℃); hatched 3 hours at 37 ℃ again, form stable latex particle-antibody complex.Then add 2.8ml confining liquid (the 0.1mol/L glycine buffer that contains 0.2% (g/ml) BSA, pH 8.0) 37 ℃ of sealings 1 hour, the centrifugal supernatant that inclines, (contain 0.2%BSA, 6% glycerine, 5% serine, 0.1% polysorbate40,0.8% sodium chloride, 0.01%BHT, 0.1% Sodium azide with 20ml 30mmol/L Tris/HCl damping fluid, pH is 8.0) wash 1 time, be dispersed into the milky suspension with same 20mlTris damping fluid again, the antibody latex granule density that makes sensitization is 2.5mg/ml.
Reagent R1 is damping fluid, its compound method: trishydroxymethylaminomethane (Tris) concentration is 50mmol/L, and the pH value is 8.0 after regulating with hydrochloric acid.Add again sodium chloride, PEG-6000, Sodium azide, its final concentration difference: sodium chloride 0.85%, PEG-60005%, Sodium azide 0.1%.
Those skilled in the art also can select other conventional damping fluids, such as in phosphate buffer, HEPES damping fluid, glycine buffer, barbitol buffer solution, MOPSO damping fluid, DIPSO damping fluid, the HEPPS damping fluid one or more.
Dilution: add the phosphate buffered saline (pH=7.4) of antiseptic, it consists of Na
2HPO
410mmol/L, NaH
2PO
42mmol/L, NaCl 135mmol/L, KCl 4.7mmol/L, Sodium azide 0.1%.
The preparation of FDP calibration object: commercially available human fibrinogen is joined 50mmol/LTris/HCl buffer solution (Tris 50mM, NaCl 0.15mM, pH value 7.4), make concentration reach 5.0mg/ml, in solution, add fibrinolysin (final concentration is 10mU/ml), stirred 6 hours at 37 ℃.Again 56 ℃ of water-baths 2 hours, with fibrinolysin deactivation cessation reaction.Left the heart 10 minutes with per minute 3000, the supernatant that obtains is fibrinogen degradation product (FDP) (FDP) solution.Japanese Sekisui Medical Treatment Co., Ltd take import produces the FDP calibration object as primary standard, adopts its FDP detection kit (latex immunoturbidimetry) to measure 20 times, obtains average.
The FDP solution that obtains is diluted to the solution that FDP concentration is 27-33 μ g/ml with above-mentioned 50mmo l/L Tris/HCl damping fluid, and then adds final concentration and be respectively 50mg/ml bovine serum albumin(BSA) (BSA), 20mg/ml sweet mellow wine, 0.1% Sodium azide and mix.To carry out freeze drying after the packing of 0.5ml/ bottle, can obtain the freeze-drying porous solid of FDP calibration object.Get 10 bottles of calibration objects, redissolve with 0.5ml distilled water respectively, with every bottle of replication of the Japanese FDP of Sekisui Medical Treatment Co., Ltd kit 2 times, the calculating grand mean is the sign value as sample, and concentration is 30.640 μ g/ml.
Embodiment 2: the assay method of kit of the present invention
With FDP calibration object in the kit, accurately add the 0.5ml dissolved in distilled water, become the calibration object solution of 6 variable concentrations with diluent preparing, concentration is respectively 0.958,1.915,3.830,7.660,15.320,30.640 μ g/ml.Be operating as example take Japanese East Asia CA550 coaglation analyzer: temperature of reaction is as 37 ℃, the mensuration wavelength is 575nm, get respectively the calibration object solution 10 μ l of variable concentrations, add dilution 10 μ l, react and add reagent R1100 μ l after 30 seconds, react again the reagent R2100 μ l that added afterwards embodiment 1 preparation in 60 seconds, the 50th second, the 300th second absorbance (A of assaying reaction
1, A
2), calculate absorbance difference Δ A=A
2-A
1, every pipe replication 3 times, the average of the absorbance difference Δ A that each calibration tube is recorded for 3 times is ordinate, corresponding concentration is horizontal ordinate, make " concentration-absorbance difference " calibration curve: y=0.01252x-0.00249, Fig. 1 is seen in correlation coefficient r=0.9995.
Get sample to be tested, the same method is measured the absorbance difference of sample, and the substitution calibration curve namely can calculate the content of FDP in the sample to be tested.If the detectable concentration of FDP exceeds the calibration curve scope in the sample, for the accuracy that guarantees to detect, again row detection after need to suitably diluting with the FDP dilution.
This kit is not only applicable to Japanese East Asia CA550 coagulo meter, but also is applicable to coaglation analyzer and the Biochemical Analyzer of other brand, model, and design parameter can suitably be adjusted according to the instrument difference.
Embodiment 3: the analytical performance assessment of kit of the present invention
1. the range of linearity
With the high concentration sample (30.42 μ g/ml) near the FDP of the range of linearity upper limit, with the FDP dilution it is pressed 1/2,1/10,1/15,1/30,1/60 dilution, be mixed with altogether the solution of 6 variable concentrations, detect each concentration with embodiment 2 described methods, every concentration replication 3 times carries out linear regression analysis with mean value and the theoretical concentration of measuring concentration, the calculating regression equation is Y=1.03917X-0.08011, related coefficient is r=0.9996, shows kit of the present invention good relationship in 0.50 μ g/ml～30.0 μ g/ml ranges of linearity, sees Fig. 2.
2. sensitivity is lowest detectable limit
Take 5% human serum albumins as dummy, measure by embodiment 2 described methods, replication 20 times, the result of calculation average is 0.0008, standard deviation SD is 0.00025, calculate the absorbance variable quantity as 0.0013 according to add twice standard deviation method for reporting take blank average, concentration is 0.05 μ g/ml with diluting afterwards, 0.10 the FDP calibration object measured in solution of μ g/ml and 0.15 μ g/ml, the absorbance changing value is respectively 0.0007,0.0014,0.0020, wherein concentration is that 0.10 μ g/mlFDP calibration object Δ A is higher than+calculated value of 2SD, so the sensitivity that kit of the present invention detects FDP is 0.10 μ g/ml.
3. repeatability and accuracy
The FDP calibration object that is respectively 4.5 μ g/ml and 28.6 μ g/ml with Japanese Sekisui Medical Treatment Co., Ltd sign value is pressed embodiment 2 described methods and is measured as sample, and each concentration is replication 10 times respectively, calculates respectively and measures average
And standard deviation (S).Respectively with
Calculate the coefficient of variation and carry out the repeatability investigation, the result shows that the coefficient of variation is respectively 1.62% and 1.21%; With
Calculate relative deviation and carry out the accuracy investigation, its relative deviation is respectively 1.71% and 1.13%.
The repeatability of table 1 kit of the present invention and accuracy are investigated
4. betweenrun precision (difference between batch)
The kit that forms with the embodiment of the invention 1 of 3 lot numbers is measured respectively with a plasma sample each 3 times, calculates respectively the mensuration average of each lot number 3 times
Grand mean with 3 lot numbers
With
Carry out betweenrun precision and calculate, the result shows that betweenrun precision is 2.38%.
The difference between batch of table 2 kit of the present invention is investigated
5. the impact of interfering material
Be 16.1 μ g/mlFDP calibration object solution with concentration, each the interfering material solution that adds respectively equal volume, concentration is respectively cholerythrin (18mg/L), haemoglobin (470mg/L) after adding, chyle (formal hydrazine) turbidity be 2800 and rheumatoid factor (RF) (470IU/ml), each sample replication 3 times, average, compare with the distilled water that adds with volume, observe the relative error of FDP measured value after adding interfering material and adding distilled water.The result shows: the relative error with adding with measured value behind the volume distilled water that adds above concentration interfering material is no more than 3%, can think that the testing result of this assay method is substantially interference-free when mild or moderate haemolysis, jaundice or chyle.
The interfering material impact experiment of table 3 kit of the present invention
| The interfering material that adds | Measure average (μ g/ml) | Relative error |
| Distilled water | 5.82 | |
| Cholerythrin (18mg/L) | 5.67 | -2.58％ |
| Haemoglobin (470mg/L) | 5.72 | -1.72％ |
| Chyle (formal hydrazine) turbidity is 2800 | 5.85 | 0.52％ |
| Rheumatoid factor (RF) (470IU/ml) | 5.76 | -1.03％ |
Embodiment 4: the stability of detection kit
Kit uncork of the present invention was placed on 2-8 ℃ of preservation after 15 days, taking-up is measured the calibration object that Japanese Sekisui Medical Treatment Co., Ltd sign value is respectively 4.5 μ g/ml and two variable concentrations of 28.6 μ g/ml by embodiment 2 described methods, each replication 3 times, calculating mean value reaches the relative deviation with the sign value.Experimental result shows, the relative deviation of uncork kit detected value of the present invention and sign value after 15 days is all less than 3%, and stability better.
Place 2-8 ℃ of preservation after 18 months kit of the present invention, detect the calibration object that Japanese Sekisui Medical Treatment Co., Ltd sign value is respectively 4.6 μ g/ml and two variable concentrations of 29.8 μ g/ml, each replication 3 times, calculating mean value reaches the relative deviation with the sign value.Experimental result shows the relative deviation of detected value and sign value all less than 3%, shows that it is more stable that kit of the present invention is preserved 18 months at this 2-8 ℃.
The stability experiment data of table 4 kit of the present invention
Embodiment 5: the comparison of kit of the present invention and the product that gone on the market
1. the comparison of repeatability and accuracy
Measure simultaneously the FDP calibration object that Japanese Sekisui Medical Treatment Co., Ltd concentration is 4.5 μ g/ml with kit of the present invention and commercially available import FDP kit, replication is 10 times respectively, computation of mean values
Variance S
2, carry out statistical study, adopt relatively both difference of F check and t check.
Table 5 pair concentration is that 4.5 μ g/ml calibration object testing results compare
2 groups of population variance significant differences that detect data are carried out one-sided F check: the α that tables look-up=0.05, υ
1=9, υ
2=9, F
α (υ 1, and υ 2)=3.19; The F value of two groups of data variance ratios is 1.5423, and then the P value is greater than 0.05, and both population variances do not have significant difference.Detect data population mean significant difference to 2 groups and carry out the sided t check: as can be known α=0.1 of tabling look-up, υ=18, t
α (υ)=1.734; According to statistical calculations, the t=-0.6935 of two groups of data, then the P value is greater than 0.1, and both population means do not have significant difference.Show by comparative result: kit of the present invention and commercially available import FDP kit are basically identical at the aspect of performance of repeatability and accuracy.
2. the correlativity of hospital's normal population and the detection of patient's plasma sample compared
Be in hospital and normal population and patient's 70 parts of blood samples are randomly drawed in outpatient service male 42 examples wherein, women 28 examples totally from the Shanghai City Ruijin Hospital.Blood in 9: 1 ratio and 0.109mol/L sodium citrate anti-freezing liquid mixing after, centrifugal 15 minutes of 3000r/min separates to obtain blood plasma., calculate respectively and measure average respectively to sample replication 2 times with kit of the present invention and commercially available import FDP kit, calculate both related coefficients, the line linearity of going forward side by side returns.
The detectable concentration of plasma F DP is 12 parts less than the sample of 2.5 μ g/ml, and this concentration level should be normal person's blood plasma, has exceeded the sensing range of commercially available import FDP kit, quantitatively is not very accurate; Concentration is 58 parts at the sample of 2.5 μ g/ml-180 μ g/ml, the correlation coefficient r of two kinds of kits=0.9989, and equation of linear regression is y=1.011x-0.1238 (seeing Fig. 3).According to the requirement (r＞0.975) of standardization body of U.S. clinical labororatory (CLSI) file, the detection data of kit of the present invention and commercially available import reagent box have good consistance.This shows that two kinds of kits detect for the diagnosis of the diseases such as disseminated intravascular coagulation (DIC), DVT (DVT) and pulmonary embolism (PE) and the course of disease clinically and the same-action such as have.
The above only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (9)
1. a kit that adopts the latex immunoturbidimetry to detect FDP content is characterized in that, comprises reagent R1, reagent R2, dilution, FDP calibration object; Described reagent R1 is the damping fluid of pH value 7.0-9.0, and described reagent R2 is anti-human FDP antibody latex reagent, and anti-human FDP antibody latex reagent is prepared by following methods:
Step 1: anti-human FDP antibody is carried out acidifying in the pH value in the solution of 2.5-3.0;
Step 2: step 1 gained solution is regulated pH to 7.5-8.0 with aqueous slkali, and adding particle diameter is the polyvinyl benzyl chloride latex solution of 100-200nm, stirs 0.5-2 hour at 20-25 ℃, hatches 2-4 hour at 37 ℃ again;
Step 3: be that the confining liquid of 7.5-8.5 was 37 ℃ of sealings 1-8 hour with the pH value with step 2 gained solution;
Step 4: step 3 gained solution after centrifugal, got precipitation and disperseed with the damping fluid washing that contains stabilizing agent and antiseptic and get final product.
2. kit according to claim 1 is characterized in that, the described solution of step 1 is a kind of in glycocoll, hydrochloric acid or the phosphoric acid solution.
3. kit according to claim 1 is characterized in that, the described aqueous slkali of step 2 is tris solution.
4. kit according to claim 1 is characterized in that, the described confining liquid of step 3 is the glycocoll-sodium hydroxide buffer solution that contains the 0.1mol/L of BSA, and BSA concentration quality percent by volume is counted 0.1-0.5% with g/ml.
5. kit according to claim 1 is characterized in that, the described damping fluid of step 4 is the Tris/HCl damping fluid.
6. kit according to claim 1 is characterized in that, the described stabilizing agent of step 4 is selected from one or more in protein, amino acid, inorganic salts, surfactant, suspending agent, the antioxidant.
7. kit according to claim 6, it is characterized in that to be the quality percent by volume count sodium chloride, 0.01% antioxidant BHT and the percent by volume of serine, the 0.7-0.9% of BSA, the 4-6% of 0.1-0.5% as the surfactant polysorbate40 of 0.1-0.6%, the suspending agent glycerine of 1-10% take g/ml with described stabilizing agent.
8. kit according to claim 1, it is characterized in that described damping fluid is selected from one or more the combination in Tris/HCl damping fluid, phosphate buffer, HEPES damping fluid, glycine buffer, barbitol buffer solution, MOPSO damping fluid, DIPSO damping fluid, the HEPPS damping fluid.
9. kit according to claim 1 is characterized in that, described damping fluid is the Tris/HCl damping fluid of 10-70mmol/L, contains set accelerator PEG-6000, and the quality percent by volume is counted 2-6% with g/ml.
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| CN104630325B (en) * | 2015-02-04 | 2018-03-02 | 上海长岛生物技术有限公司 | A kind of liquid-type fibrinogen detection reagent and preparation method thereof |
| JP6640494B2 (en) * | 2015-08-28 | 2020-02-05 | シスメックス株式会社 | Blood sample analysis method, blood sample analyzer, and computer program |
| CN110531067B (en) * | 2019-08-30 | 2021-01-22 | 山东博科生物产业有限公司 | Detection kit for fibrin and fibrinogen degradation products |
| CN112730827A (en) * | 2020-12-11 | 2021-04-30 | 常州爱复康生物科技有限公司 | Novel coronavirus antibody detection kit and preparation method thereof |
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