CN102183635A - In vitro detection kit for TAFI content - Google Patents
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- CN102183635A CN102183635A CN2010106127760A CN201010612776A CN102183635A CN 102183635 A CN102183635 A CN 102183635A CN 2010106127760 A CN2010106127760 A CN 2010106127760A CN 201010612776 A CN201010612776 A CN 201010612776A CN 102183635 A CN102183635 A CN 102183635A
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Abstract
The invention discloses an in vitro detection kit for thrombin-activatable fibrinolysis inhibitor (TAFI) content. The kit is used for the in vitro detection of the TAFI content by using the biological property that a polyamidoamine (PAMAM) dendritic polymer can be combined with an antibody and combining the traditional enzyme-linked immuno sorbent assay (ELISA) method, and the kit develops a new in vitro diagnostics method for the TAFI and improves the sensitivity of the TAFI in vitro detection. The kit comprises an antibody coated ELISA plate, a sample diluent, a TAFI standard, a detergent, a PAMAM-labeled horse radish peroxidase-linked antibody, an antibody diluent, color development buffer solution, 30 percent hydrogen peroxide, o-phenylenediamine and stop solution. The kit overcomes the defects of expensive equipment, long detection time and the like in the prior art; compared with other detection kits, the kit is simple, convenient and fast in operation, low in cost, sensitive in diagnosis and has low detection limit and the like; the kit for auxiliary diagnosis improves the detection rate and the accuracy of related diseases, and fulfills the purposes of early discovery and early treatment; and the kit reduces unnecessary pain and medicinal expense of examined persons, and improves the quality of life of the examined persons.
Description
Technical field
The present invention relates to a kind of inhibitors of fibrinolysis (thrombin-activatable fibrinolysis inhibitor that is used for the detection thrombin activation of possibility occurrences such as dlinial prediction cardiovascular and cerebrovascular disease, abbreviation TAFI) kit of content, especially for detecting the individual human biological sample, as the vitro detection kit of the TAFI content of blood, serum, blood plasma, urine, mucus, ight soil, cerebrospinal fluid, pleural effusion, ascites, saliva, tissue or cell etc.In medical test, it is mainly by the external test to the TAFI content in the individual human biological sample, with the diagnosis and the extensive clinical generaI investigation of relevant diseases such as auxiliary cardiovascular and cerebrovascular disease, acute promyelocytic leukemia, intrinsic coagulation system deficiency disorders, inflammation.
Background technology
As everyone knows, thrombotic diseases is that a class has a strong impact on the first reason that healthy disease, especially cardiovascular and cerebrovascular embolism class diseases have become China human mortality.Because the clinical manifestation of thrombotic diseases varies, diagnosis and treatment are often very complicated, and therefore, early detection, early treatment are particularly important.At present, the inspection method of thrombotic diseases mainly contains cardiogram, echocardiogram, chest X ray, monitoring of blood pressure, head X ray, computed tomography brain scan, brain MRI inspection, DSA, MRA, TCD inspection etc.Ray class inspection meeting is to human body generation ionising radiation to a certain degree, and the functional imaging inspection method of tissue blood flow's perfusion is more, and these checkout facilities are relatively more expensive usually, increased cost, what have also needs special equipment, and imaging time is longer, can't short-term obtain the result.In medical test, still lack a kind of detection method that is used for possibility occurrences such as dlinial prediction cardiovascular and cerebrovascular disease simply, fast, accurately, be unfavorable for early detection, early treatment to disease.
According to the pertinent literature report, TAFI has by the function of downward modulation fibrinolytic behind the thrombin activation, is a kind of carboxypeptidase precursor that is present in the containing metal Zn in the blood plasma, the blood plasma procarboxypeptidase B that is otherwise known as, blood plasma procarboxypeptidase U, blood plasma procarboxypeptidase R.The TAFI precursor is synthetic by liver, is one and comprises 423 amino acid whose strand glycoprotein, in the process in being circulated to blood, cuts away 22 amino acid whose signal peptides of N end, forms the TAFI of 401 amino acid whose 56kDa; TAFI also is present in the blood platelet, is released into blood during platelet activation.Proteolytic enzymes such as fibrin ferment, trypsase, fibrinolysin can act on the Arg92 of TAFI, are cut to the activating peptide and the TAFIa with 36kDa of carboxypeptidase activity of 20kDa.TAFIa has the fibrin C end Arg of cut-out degraded or the ability of Lys, the fibrinolytic protein proenzyme can not be activated, thereby have the function of downward modulation fibrinolytic system, but its conformation has the height instability.
TAFI was found to have inherent instability after the activation first in 1988, hatched 2h usually under 37 ℃ of conditions and lost activity, and can decompose arginine on the substrate specifically.After the multidigit scientific worker separates its cDNA and measures, and the method for utilizing plasminogen-sepharose column chromatography its zymogen protein pro-pCPB that from blood plasma, separates, purifies, further analyze and find to have carboxypeptidase activity after TAFI is by thrombin activation, performance fibrinolytic inhibiting effect.
TAFI and cardiovascular and cerebrovascular disease have certain relation.Atherosclerotic (atherosclerosis is called for short AS) is the common pathologic basis of ischemic angiocardiopathy and cerebrovascular disease (as angina pectoris, myocardial infarction, cerebral apoplexy etc.), also is to cause thrombotic main cause in the blood vessel.Studies show that in recent years, the generation development of AS and inflammation, blood coagulation-fibrinolytic regulating system are closely related.Studies show that at present TAFI can promote venothrombotic formation by reducing fibrinolytic, the high TAFI level of blood plasma can increase the phlebothrombosis incident.
At present, the method that is used for detecting the TAFI level mainly contains two classes, and a class is by measuring the method indirect determination TAFI content of enzyme activity, promptly utilize fibrin ferment and fibrin ferment to regulate albumen and activate TAFI, measuring the enzyme activity of TAFIa; Another kind of is immunological method, comprise enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA) and rocket immunoelectrophoresis.Wherein, it is ripe relatively at present detection method that the sandwich ELISA method detects TAFI antigen, the detection sensitivity height, and high specificity, accuracy is good, and detection method safety is easily gone.Occurred two kinds of monoclonal antibody specifics in 2005, successfully used the ELISA method to detect the TAFI of TAFI, TAFIa and inactivation at TAFI.But these methods all do not become internationally recognized TAFI reference standard method because of self there being certain defective.
The inventor was in " the Her-2 immunohistochemical diagnosis kit of breast cancer " of CN200410020546.X in the patent No. once, utilization contains the biological nature labelled antibody of ammonia functional group macromolecule PAMAM tree ridge portion, carry out the immunohistochemical in-vitro diagnosis of Her-2 of breast cancer, can make detection breast cancer obtain judged result accurately.Thus, opened up new in-vitro diagnosis method.PAMAM (polyamide-amide) is a kind of nanoscale dendron shape polymer, and PAMAM dendrimer (PAMAM Dendrimer) is a class new polymers synthetic in recent years and that develop rapidly.This polymkeric substance and traditional polymer phase ratio, can be on nanometer level strict controlling Design molecular size, structure and surface group, thereby have accurate molecular structure, fine and close spherical shapes, monodispersity, fabulous water-soluble.Show after deliberation, carry few nuclear acid anhydride acid gene fragment with PAMAM and carry out in the body and experiment in vitro, prove that it has extraordinary propagation function; Can use PAMAM binding antibody fragment magnetic target therapy prostate cancer.The patent No. be US2003/0044407A1's " transmission or diagnostic reagent that the immune lipid of labelled antibody or antibody fragment or polymer are used for systematic treating ", also disclose a kind of application PAMAM mark rare metal and antibody and injected in the body, carried out the method for imaging diagnosis by nuclear magnetic resonance measuring.But because of the toxic action that it decomposes in vivo, metabolism produced it be unclear that, have animal experiment study to show, certain PAMAM dosage can make mouse produce the hemolytic variation.Therefore, experiment is difficult to widespread use in the body.
Summary of the invention
The vitro detection kit that the purpose of this invention is to provide a kind of TAFI content, it has solved the apparatus expensive that prior art exists, defectives such as detection time is long are compared with other detection kit, have easy and simple to handle, quick, with low cost, diagnosis is responsive, advantages such as detection limit is low as the auxiliary diagnosis that is used for the dlinial prediction cardiovascular and cerebrovascular disease, can significantly improve the recall rate and the accuracy rate of disease, help the early detection of disease, early treatment; Reduce unnecessary misery and the health care expenditures of examinee, improve examinee's life quality.
The technical solution adopted in the present invention is: comprise in the vitro detection kit content of this TAFI content: catch sample TAFI antigen to be checked and form the ELISA Plate of the coated antibody of antigen antibody complex on solid phase, be used to dilute the sample diluting liquid of sample to be checked, be used for making the TAFI standard items of the typical curve of the TAFI antigenic content that calculates sample to be checked, flush away is not combined in the detergent remover of the molecule on the ELISA Plate, be used for making proportional horseradish peroxidase len antibody and the antibody diluent that is combined in the PAMAM mark on the antigen antibody complex of enzyme labelled antibody, as 30% hydrogen peroxide and the o-phenylenediamine and the substrate colour developing damping fluid of zymolyte, the stop buffer that the substrate chromogenic reaction is stopped; During detecting operation, the 300 times of dilutions of antibody diluent of the horseradish peroxidase len antibody of PAMAM mark, the TAFI standard items add the dissolving of 1ml sample diluting liquid, and the amount ratio of each component is in the zymolyte: o-phenylenediamine 13mg: colour developing damping fluid volume 13ml: 30% hydrogen peroxide volume, 6.5 μ l.
Described coated antibody and enzyme labelled antibody are the monoclonal antibodies at the prepared mouse-anti people of recombined human TAFI antigen, and described mouse-anti human monoclonal antibodies comprises 3B1 and 4E7, and described 3B1 is the TAFI of specificity in conjunction with TAFI and inactivation, and described 4E7 only combines with TAFI.
The horseradish peroxidase len antibody of described PAMAM mark is to be mixed with positively charged PAMAM dendron shape polymer by the antibody of horseradish peroxidase-labeled, react at ambient temperature and made in 10～15 minutes, the melting concn of the two is 0.1～10: 1 mol ratio.
The preparation of the ELISA Plate of described coated antibody, with phosphate buffer dilution antibody 4E7 to 15 μ g/ml, every hole 100 μ l coated elisa plates use 1% gelatin solution that contains 10mM disodium ethylene diamine tetraacetate (pH7.3) as confining liquid, every hole 200 μ l sealase targets.
Being prepared as of described TAFI standard items, with markization TAFI as standard items, doubling dilution is done typical curve, the ELISA method is measured people's pooled plasma concentration, measurement result is 19200ng/ml, get this blood plasma 0.3ml, the disodium ethylene diamine tetra-acetic acid solution 240 μ l that add 100mM pH7.3, distilled water 3.3ml, 100mM chitosan solution 0.96ml, abundant mixing, making final concentration is the solution of TAFI 2.4 μ g/ml, disodium ethylene diamine tetraacetate 5mM, shitosan 20mM, get this solution 20 μ l branch and be filled to the frozen pipe bottom of sterilizing, 4 ℃ of preservations after the freeze drying.
Being prepared as of described sample diluting liquid got the 1g bovine serum albumin(BSA), adds the disodium ethylene diamine tetra-acetic acid solution 100ml of 100mM pH7.3,10 times of phosphorus concentration phthalate buffer 100ml and distilled water 800ml, fully mixing.
The application of the vitro detection kit of described TAFI content, its technical essential is: the blood, serum, blood plasma, urine, mucus, ight soil, cerebrospinal fluid, pleural effusion, ascites, saliva, tissue or the cell that are used for the human body biological sample.
The application of the vitro detection kit of described TAFI content, its technical essential are the diagnosis that is used for the disease that is associated with TAFI content of auxiliary cardiovascular and cerebrovascular disease, acute promyelocytic leukemia, intrinsic coagulation system deficiency disorders, inflammation.
Advantage and good effect that the present invention has are: owing to the monoclonal antibody specific and the required reagent of enzyme-linked immunoassay that comprise in the content of vitro detection kit of the present invention at TAFI, and according to the biological nature that contains ammonia functional group macromolecule PAMAM tree ridge portion, method with the PAMAM labelled antibody, combine with existing ripe ELISA detection method, the vitro detection that is used for the TAFI content of human body biological sample, opened up the new in-vitro diagnosis method of TAFI, defectives such as so it has solved the apparatus expensive that prior art exists, and detection time is long.This in-vitro diagnosis method is used for the possibility of dlinial prediction cardiovascular and cerebrovascular disease, and it is easy and simple to handle, quick, with low cost, and diagnosis is responsive, and detection limit is low.Because of PAMAM is the nanoscale dendron shape polymer a kind of commonly used that is synthesized, the placement of its size, shape and functional group can accurately be controlled, so present the monodispersity that can better be controlled on diameter.Simultaneously, it also has better water solubility, through polyreaction repeatedly, formed highdensity amino group on its surface, positively charged when the pH of physiological environment, can with electronegative enzyme len antibody protein combination, TAFI antigen in the sample and mark the enzyme connection specific antibody of PAMAM carry out immunological response, amplify reaction by enzymatic cascade, under the 492nm wavelength, detect light absorption value, the proportional relation of light absorption value and antigen concentration, thus reach the purpose of quantitative measurement antigenic content.PAMAM is applied to the ELISA detection can obviously improves the sensitivity and the specificity of detection, this in-vitro diagnosis method is better than other commonsense method of available technology adopting.Therefore, vitro detection kit of the present invention can be applicable to the external test of TAFI content in the individual human sample, according to the relation of TAFI and relevant disease, as auxiliary diagnosis, improve the recall rate and the accuracy rate of relevant disease, reached early detection, the purpose of early treatment; Reduce unnecessary misery and the health care expenditures of examinee, improve examinee's life quality.
Inhibitors of fibrinolysis (TAFI) the enzyme-linked immuno assay kit of vitro detection kit of the present invention and people's fibrinolytic inhibiting factor/thrombin activation compares experiment.Heart and brain infarct patient blood plasma 58 examples of experiment material for having made a definite diagnosis, use TAFI enzyme-linked immuno assay kit and the used vitro detection kit of the present invention to carry out enzyme-linked immunoassay simultaneously, the positive rate of vitro detection kit of the present invention and TAFI enzyme-linked immuno assay kit measurement is respectively 36.2% and 20.6%, this result shows, the present invention is higher, more accurate to TAFI detection of antigens specificity.
Description of drawings
Below in conjunction with accompanying drawing the present invention is further described.
Fig. 1 is monoclonal antibody 3B1 and TAFI specificity binding immunoassay trace figure.
Sequence number explanation among the figure: 1. Marker; 2. the TAFI of purifying, concentration is 0.252 μ g; 3. blank; 4. human plasma.
Fig. 2 is that monoclonal antibody 4E7 combines the ELISA curve with the TAFI specificity.
Fig. 3 is the TAFI typical curve.
Fig. 4 is heart and brain infarct patient and healthy person blood plasma TAFI content contrast figure.
Embodiment
Below in conjunction with Fig. 1～4 and embodiment the present invention is further described.Comprise in the vitro detection kit content of this TAFI content: catch sample TAFI antigen to be checked and on solid phase, form one of the ELISA Plate (96 hole) of the coated antibody of antigen antibody complex; Be used to dilute the sample diluting liquid of sample to be checked, 2 * 100ml/ bottle; Be used for making the TAFI standard items of the typical curve of the TAFI antigenic content that calculates sample to be checked, 1 * 48ng/ manages (dried frozen aquatic products); Flush away is not combined in the detergent remover of the molecule on the ELISA Plate, 2 * 100ml/ bottle; Be used for making the proportional horseradish peroxidase len antibody that is combined in the PAMAM mark on the antigen antibody complex of enzyme labelled antibody, 1 * 30 μ l/ pipe; Antibody diluent, 1 * 10ml/ bottle; As 30% hydrogen peroxide of zymolyte, 1 * 30 μ l/ pipe, o-phenylenediamine, 1 * 13mg/ sheet, and substrate colour developing damping fluid, 1 * 30ml/ bottle; The stop buffer that the substrate chromogenic reaction is stopped, 1 * 10ml/ bottle; During detecting operation, the 300 times of dilutions of antibody diluent of the horseradish peroxidase len antibody of PAMAM mark, the TAFI standard items add the dissolving of 1ml sample diluting liquid, and the amount ratio of each component is in the zymolyte: o-phenylenediamine 13mg: colour developing damping fluid volume 13ml: 30% hydrogen peroxide volume, 6.5 μ l.By colorimetric, predict the amount of TAFI antigen in the sample.The dosage of each reagent can be according to the needs preparation of actual detected sample to be checked in the box.
The preparation and the using method of the vitro detection kit content of TAFI content of the present invention are as follows:
1, PAMAM Dendrimer's is synthetic
PAMAM Dendrimer is by 0 generation in generation to 10, and its molecular diameter exists
Extremely
Between, belong to the molecule of nanoscale.It is synthetic to be to adopt branch method, and promptly molecule is begun to outgrowth by the core.According to the rule of trees branch ready made monomer is met branch again by the branch of radial order, branch, repeated combination is got up successively.With ethylenediamine (EDA) is initiated core, and the 1st step reaction (a) generates 1 quaternary ester [being called 0.5 generation (G)] with methyl acrylate (MA) by the Michael addition reaction; The 2nd step reaction (b) quaternary ester and excessive ethylenediamine generation ammonolysis reaction generation 1 quaternary amide compound (being called for 1 generation).Repeat the reactions steps that (a) and (b) Michael addition and ammonia are separated, can obtain the PAMAM dendrimer compound of different algebraically.Synthetic product all carries out structural characterization with FT2IR, FAB2MS, 1H NMR and 13C NMR.It below is PAMAM synthetic reaction route.
Different pH value PAMAM are to the difference that influences of solubilising, and test also produces 5.0 generation PAMAM different quality concentration to the PAMAM solution of different quality concentration to solubilising.Prove by experiment in bigger pH value scope and descend stable existence, do not dissociate with different buffer conditions (water, phosphate buffer, 0.5mmol/LNaCl etc.).
2, the preparation of coated antibody and enzyme labelled antibody
Coated antibody and enzyme labelled antibody are that the mouse-anti human monoclonal antibodies comprises 3B1 and 4E7 at the prepared mouse-anti human monoclonal antibodies of recombined human TAFI antigen.
The hybridoma preparation: the human plasma TAFI with purifying is an immunogene, immune according to a conventional method BALB/c mouse; Getting its splenocyte and SP2/0 myeloma cell merges; Hybridoma is screened, and 3 limiting dilution assays of positive aperture clone obtains finally that 2 strains continue and the hybridoma cell strain of the anti-people TAFI of stably excreting monoclonal antibody, called after 3B1 and 4E7 respectively;
Antibody is identified: the type of measuring these two kinds of monoclonal antibodies according to Roche Holding Ag's antibody subclass kit instructions is IgG1, κ; With simulation blood plasma in contrast, Western blotting and elisa assay show that these two kinds of monoclonal antibodies of 3B1 and 4E7 all combine with people source TAFI antigentic specificity, wherein 3B1 can specificity in conjunction with the TAFI of TAFI and inactivation, as shown in Figure 1, the TAFI of 3B1 in can specific recognition blood plasma.Fig. 2 monoclonal antibody 4E7 combines the ELISA curve with the TAFI specificity.With 15 μ g/ml4E7 coated elisa plates, respectively as antigen, 3B1 carries out the ELISA reaction as the enzyme len antibody with TAFI, TAFI and 4E7 potpourri, TAFI and mouse IgG potpourri.From curve map as can be seen, the competitive TAFI that suppresses of free 4E7 combines with solid phase 4E7, illustrates that 4E7 only combines with the TAFI specificity.
Antibody Preparation: hybridoma extracorporeal culture-ing.
Antibody purification: hollow fiber filter post (AM General company) concentrates hybridoma culture supernatant, the monoclonal antibody in albumin A-agarose affinity chromatography post (AM General company) the separation and purification concentrate.
Antibody titer: 3B1 and 4E7 monoclonal antibody after the ELISA method is measured purifying, the difference 10 of tiring
5With 10
6
3, the preparation of the horseradish peroxidase len antibody of PAMAM mark
Connect the kit operation instructions by U.S. KPL company horseradish peroxidase, positively charged PAMAM Dendrimer with made in the antibody 3B1 of horseradish peroxidase-labeled and the above-mentioned synthesis step 1, with 0.1～10: the concentration of 1 molar ratio is mixed, room temperature condition reacted 10-15 minute down, made the horseradish peroxidase len antibody of PAMAM mark.This is fabulous ratio and order that an antibody that makes PAMAM and horseradish peroxidase-labeled mixes, be simple, effectively, stable, associated methods non-chemically.This method than chemistry or additive method effectively or more effective.
4, the preparation of TAFI standard items
From the TAFI of the purchaser of Haemanetics Corporation, USA blood plasma purifying, relative molecular mass 60000, specific activity 5.8Units/mg is as the TAFI of markization concentration.Utilize the TAFI of people's pooled plasma and markization concentration to compare, the dilution suitable multiple, acquisition contains the blood plasma of expection concentration TAFI, as the experiment standard items.This method prepares the TAFI standard items and need not purifying, and is simple to operate, with low cost, is fit to preparation in enormous quantities.The concrete operations step is as follows:
As standard items, doubling dilution is done typical curve (as shown in Figure 3) with markization TAFI, and the ELISA method is measured people's pooled plasma concentration, and method of operating is seen embodiment one.Measurement result is 19200ng/ml, get this blood plasma 0.3ml, the disodium ethylene diamine tetra-acetic acid solution 240 μ l that add 100mM pH7.3, distilled water 3.3ml, 100mM chitosan solution 0.96ml, abundant mixing, making final concentration is the solution of TAFI 2.4 μ g/ml, disodium ethylene diamine tetraacetate 5mM, shitosan 20mM, get this solution 20 μ l branch and be filled to the frozen pipe bottom of sterilizing, 4 ℃ of preservations after the freeze drying.
5, the preparation of the disodium ethylene diamine tetra-acetic acid solution of 100mM pH7.3
Get the 37.22g disodium ethylene diamine tetraacetate and be dissolved in the 800ml distilled water, 1mol/l NaOH regulates pH to 7.3, is settled to 1L.
6, the preparation of confining liquid
Get the 2g gelatin and be dissolved in the 800ml distilled water, 60 ℃ of water-bath dissolvings, the disodium ethylene diamine tetra-acetic acid solution 100ml of adding 100mM pH7.3, room temperature is settled to 1L.
7, the preparation of the ELISA Plate of coated antibody
Press catalogue preparation phosphate buffer.With phosphate buffer dilution antibody 4E7 to 15 μ g/ml, 100 μ l are added in every hole in ELISA Plate, and dark cold place spends the night; Remove coating buffer, every hole adds 300 μ l phosphate buffers, leaves standstill 10 minutes, discards; As confining liquid, 200 μ l confining liquids are added in every hole with 1% gelatin solution of the disodium ethylene diamine tetra-acetic acid solution that contains 10mM pH7.3, and dark cold place spends the night, and remove confining liquid, and every hole adds 300 μ l detergent removers, leaves standstill 10 minutes, discards lyophilized overnight.
8, the preparation of sample diluting liquid
Get the 1g bovine serum albumin(BSA), add the disodium ethylene diamine tetra-acetic acid solution 100ml of 100mM pH7.3,10 times of phosphorus concentration phthalate buffer 100ml, distilled water 800ml, fully mixing.
9, the preparation of detergent remover
In the 1L phosphate buffer, add the 0.5ml polysorbas20, fully mixing.
10, the preparation of antibody diluent
Get the 100ml phosphate buffer, add 1g bovine serum albumin(BSA) mixing.
11, the preparation of colour developing damping fluid
Get the 7.0g monohydrate potassium and the 23.9g disodium hydrogen phosphate dodecahydrate is dissolved in the 800ml distilled water, be settled to 1L.
12, the preparation of zymolyte
In 13ml colour developing damping fluid, add the o-phenylenediamine of a slice 13mg, add 6.5 μ l, 30% hydrogen peroxide again, mixing, lucifuge.
13, the preparation of stop buffer
Get 18mol/l concentrated sulphuric acid 20ml, slowly add in the 160ml distilled water, make 2mol/l sulfuric acid.
The application of the vitro detection kit of TAFI content of the present invention: it is mainly used in blood, serum, blood plasma, urine, mucus, ight soil, cerebrospinal fluid, pleural effusion, ascites, saliva, tissue or cell etc. in the human body biological sample.Will be to TAFI Determination on content in the individual human sample, according to the relation of TAFI and relevant disease, be applied to the diagnosis of the disease that is associated with TAFI content of auxiliary cardiovascular and cerebrovascular disease, acute promyelocytic leukemia, intrinsic coagulation system deficiency disorders, inflammation etc.
Embodiment one:
Now be example, patient's plasma sample and the 30 routine healthy plasma samples that 58 examples are diagnosed as the heart and brain infarct carried out the TAFI assay with the vitro detection kit of TAFI content of the present invention with the human plasma.The concrete operations step is as follows:
In 30 minutes, under 4 ℃ of conditions, centrifugal 10 minutes of 3000rpm gets supernatant as sample to be checked to step 1 collection anticoagulated blood ,-20 ℃ of preservations immediately.
Step 6 discards the liquid in the ELISA Plate hole, pats dry, and adds detergent remover 300 μ l, leaves standstill 10 minutes, discards to pat dry, and repeats the not bound substances in the flush away ELISA Plate hole 3 times.
Step 7 is got the enzyme len antibody 20 μ l of PAMAM mark, adds among the antibody diluent 6ml, makes enzyme len antibody working fluid.
Step 8 adds the enzyme len antibody 50 μ l of PAMAM mark in the ELISA Plate hole that is combined with antigen antibody complex, room temperature left standstill 1 hour, and the enzyme len antibody of PAMAM mark combines with TAFI antigen on the solid phase antigen antibody complex.
Step 9 discards the liquid in the ELISA Plate hole, pats dry, and adds detergent remover 300 μ l, left standstill 10 minutes, and discarded and pat dry, repeat 3 times, the amount of being examined antigen in the enzyme len antibody of unconjugated PAMAM mark in the flush away ELISA Plate hole, the enzyme amount that have on the solid phase carrier this moment and sample to be checked is directly proportional.
Step 11 adds zymolyte 100 μ l, lucifuge reaction 10 minutes, and the substrate for enzymatic activity on the solid phase becomes faint yellow product.
Step 12 adds stop buffer 100 μ l cessation reactions, and faint yellow product is converted into brown immediately, measures light absorption value in the 492nm place.
Step 13 is according to the relation of standard items light absorption value and concentration, the production standard curve, as shown in Figure 3.Calculate the content of TAFI antigen in the sample to be checked according to typical curve, as shown in Figure 4.
Through the T check, heart and brain infarct group blood plasma TAFI content and healthy P＜0.01 (P=0.0028) of organizing TAFI content, two groups of data have significant difference.With health group content mean value and 2 times of standard deviations and be reference upper level, the recall rate of heart and brain infarct can reach 36.2%.Therefore this method is as auxiliary diagnosis, and the prediction of cardiovascular and cerebrovascular disease is had meaning.
Embodiment two:
Kit of the present invention can also be measured the TAFI content in the human urine.Gather people's urina sanguinis, in 30 minutes, under 4 ℃ of conditions, centrifugal 5 minutes of 1500rpm gets supernatant as sample to be checked.Treat the sample product with the sample diluting liquid in the kit and carry out 100 times of dilutions.Subsequent step is with embodiment one.
Claims (8)
1. the vitro detection kit of a TAFI content, it is characterized in that: comprise the ELISA Plate of catching sample TAFI antigen to be checked and on solid phase, forming the coated antibody of antigen antibody complex in the box content, be used to dilute the sample diluting liquid of sample to be checked, be used for making the TAFI standard items of the typical curve of the TAFI antigenic content that calculates sample to be checked, flush away is not combined in the detergent remover of the molecule on the ELISA Plate, be used for making proportional horseradish peroxidase len antibody and the antibody diluent that is combined in the PAMAM mark on the antigen antibody complex of enzyme labelled antibody, as 30% hydrogen peroxide and the o-phenylenediamine and the substrate colour developing damping fluid of zymolyte, the stop buffer that the substrate chromogenic reaction is stopped; During detecting operation, the 300 times of dilutions of antibody diluent of the horseradish peroxidase len antibody of PAMAM mark, the TAFI standard items add the dissolving of 1ml sample diluting liquid, and the amount ratio of each component is in the zymolyte: o-phenylenediamine 13mg: colour developing damping fluid volume 13ml: 30% hydrogen peroxide volume, 6.5 μ l.
2. the vitro detection kit of TAFI content according to claim 1, it is characterized in that: described coated antibody and enzyme labelled antibody are at the prepared mouse-anti human monoclonal antibodies of recombined human TAFI antigen, described mouse-anti human monoclonal antibodies comprises 3B1 and 4E7, described 3B1 is the TAFI of specificity in conjunction with TAFI and inactivation, and described 4E7 only combines with TAFI.
3. the vitro detection kit of TAFI content according to claim 1, it is characterized in that: the horseradish peroxidase len antibody of described PAMAM mark is to be mixed with positively charged PAMAM dendron shape polymer by the antibody of horseradish peroxidase-labeled, react at ambient temperature and made in 10～15 minutes, the melting concn of the two is 0.1～10: 1 mol ratio.
4. the vitro detection kit of TAFI content according to claim 1, it is characterized in that: the preparation of the ELISA Plate of described coated antibody, with phosphate buffer dilution antibody 4E7 to 15 μ g/ml, every hole 100 μ l coated elisa plates, with 1% gelatin solution of the disodium ethylene diamine tetra-acetic acid solution that contains 10mM pH7.3 as confining liquid, every hole 200 μ l sealase targets.
5. the vitro detection kit of TAFI content according to claim 1, it is characterized in that: being prepared as of described TAFI standard items, with markization TAFI as standard items, doubling dilution is done typical curve, the ELISA method is measured people's pooled plasma concentration, measurement result is 19200ng/ml, get this blood plasma 0.3ml, the disodium ethylene diamine tetra-acetic acid solution 240 μ l that add 100mM pH7.3, distilled water 3.3ml, 100mM chitosan solution 0.96ml, abundant mixing, making final concentration is TAFI2.4 μ g/ml, disodium ethylene diamine tetraacetate 5mM, the solution of shitosan 20mM is got this solution 20 μ l branch and is filled to the frozen pipe bottom of sterilizing, 4 ℃ of preservations after the freeze drying.
6. the vitro detection kit of TAFI content according to claim 1, it is characterized in that: being prepared as of described sample diluting liquid, get the 1g bovine serum albumin(BSA), add the disodium ethylene diamine tetra-acetic acid solution 100ml of 100mM pH7.3,10 times of phosphorus concentration phthalate buffer 100ml and distilled water 800ml, fully mixing.
7. the application of the vitro detection kit of a TAFI content according to claim 1 is characterized in that: the blood, serum, blood plasma, urine, mucus, ight soil, cerebrospinal fluid, pleural effusion, ascites, saliva, tissue or the cell that are used for the human body biological sample.
8. the application of the vitro detection kit of TAFI content according to claim 7 is characterized in that: be used for the diagnosis of the disease that is associated with TAFI content of auxiliary cardiovascular and cerebrovascular disease, acute promyelocytic leukemia, intrinsic coagulation system deficiency disorders, inflammation.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103063847A (en) * | 2012-12-21 | 2013-04-24 | 杭州茂天赛科技有限公司 | Preparation method for enzyme-linked immunosorbent assay (ELISA) standard substances |
| CN103675288A (en) * | 2013-12-26 | 2014-03-26 | 泸州医学院 | Blood platelet marker and preparation method thereof |
| CN103728453A (en) * | 2014-01-26 | 2014-04-16 | 辽宁迈迪生物科技有限公司 | Extracorporal test kit for TAFI (Thrombin Activatable Fibrinolysis Inhibitor) content, and test method of extracorporal test kit |
| CN103760352A (en) * | 2014-01-26 | 2014-04-30 | 辽宁迈迪生物科技有限公司 | Kit and method for in-vitro detection of content of TAFI (Thrombin Activatable Fibrinolysis Inhibitor) |
| CN110632324A (en) * | 2019-09-25 | 2019-12-31 | 百盛(广州)生物制品有限公司 | Polyamide-amine structure polymer for secondary antibody detection system and preparation method thereof |
| CN112236677A (en) * | 2018-02-09 | 2021-01-15 | 贵州美鑫达医疗科技有限公司 | Direct immunohistochemical and immunocytochemical methods |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103063847A (en) * | 2012-12-21 | 2013-04-24 | 杭州茂天赛科技有限公司 | Preparation method for enzyme-linked immunosorbent assay (ELISA) standard substances |
| CN103675288A (en) * | 2013-12-26 | 2014-03-26 | 泸州医学院 | Blood platelet marker and preparation method thereof |
| CN103675288B (en) * | 2013-12-26 | 2015-10-28 | 泸州医学院 | A kind of blood platelet marker and preparation method thereof |
| CN103728453A (en) * | 2014-01-26 | 2014-04-16 | 辽宁迈迪生物科技有限公司 | Extracorporal test kit for TAFI (Thrombin Activatable Fibrinolysis Inhibitor) content, and test method of extracorporal test kit |
| CN103760352A (en) * | 2014-01-26 | 2014-04-30 | 辽宁迈迪生物科技有限公司 | Kit and method for in-vitro detection of content of TAFI (Thrombin Activatable Fibrinolysis Inhibitor) |
| CN103760352B (en) * | 2014-01-26 | 2015-09-30 | 辽宁迈迪生物科技有限公司 | A kind of kit for TAFI content vitro detection and external detection method thereof |
| CN103728453B (en) * | 2014-01-26 | 2016-05-25 | 辽宁迈迪生物科技有限公司 | A kind of vitro detection kit and detection method thereof of TAFI content |
| CN112236677A (en) * | 2018-02-09 | 2021-01-15 | 贵州美鑫达医疗科技有限公司 | Direct immunohistochemical and immunocytochemical methods |
| CN110632324A (en) * | 2019-09-25 | 2019-12-31 | 百盛(广州)生物制品有限公司 | Polyamide-amine structure polymer for secondary antibody detection system and preparation method thereof |
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