CN103592430A - ELISA kit for detecting 11-dehydro thromboxane B2 - Google Patents
ELISA kit for detecting 11-dehydro thromboxane B2 Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/22—Haematology
- G01N2800/226—Thrombotic disorders, i.e. thrombo-embolism irrespective of location/organ involved, e.g. renal vein thrombosis, venous thrombosis
Abstract
The invention provides an ELISA kit for detecting 11-dehydro thromboxane B2. The ELISA kit uses a double antibody sandwich method. A capture antibody of the ELISA kit is an anti-11-dehydro thromboxane B2 monoclonal antibody; a detection antibody is an anti-11-dehydro thromboxane B2 polyclonal antibody. The anti-11-dehydrogen thromboxane B2 polyclonal antibody and the anti-11-dehydro thromboxane B2 monoclonal antibody have different species sources. The ELISA kit provided by the invention has the following advantages and positive effects: the ELISA kit employs two antibodies to recognize different antigenic determinants and has higher specificity of detection; an antibody-antigen-antibody immune complex is more stable; and the detection results are more accurate. A detected antigen does not need to be highly purified, and can be used directly for detection.
Description
Technical field
The present invention relates to a kind of ELISA detection kit, in particular to a kind of ELISA kit for detection of 11-dehydrogenation thromboxane element B2.
Background technology
Cardiovascular and cerebrovascular disease has formed fashion trend in the world, greatly threatens public health, and brings serious burden on society.In recent years, China's cardiovascular and cerebrovascular disease incidence of disease rises rapidly, and cardiovascular and cerebrovascular disease is positioned at the front three of China's spectrum of disease always.The medical expense causing thus every year is according to estimates tens billion of, and is the trend of rapid growth always.Therefore, actively controlling hazards, particularly select safe, effective, economic medicine, is to reduce dead, to reduce medical expense key.Wherein, simple and effective prophylactic treatment method comprises application aspirin, and it can control for 50% clinical disabling or fatal rate.
Aspirin (acetylsalicylic acid) has been applied a century, and the foundation stone status of aspirin in cardiovascular disease prevention established in a large amount of research, becomes one of three large classical medicines in medical history, is described as " eighth wonder of the world ".In John's Thelma Hopkins medical college (John Hopkins University) prevention myocardial infarction that the U.S. is the most authoritative and cerebral apoplexy guide, all aspirin for treatment is gived top priority.Recently anti-bolt test cooperative groups (ATC) Macro or mass analysis demonstration, aspirin makes the thrombotic vascular events of the high-risk patients such as myocardial infarction, cerebral apoplexy reduce 1/4th, the dead sixth that reduces of thrombotic vascular conditions.Also demonstration of Retrospective review research, it is the twice that ester medicine falls in his tincture class that aspirin prevents the thrombotic vascular events of myocardial infarction and cerebral apoplexy efficient.
The anti-bolt effect of aspirin is apparent, yet still has the patient of part Aspirin to there will be thrombotic vascular events.For this phenomenon, some scholars have proposed the concept of aspirin " opposing " (Aspirin Resistance).According to the extensive clinical investigation of the U.S. repeatedly, in the middle of the patient of Aspirin, there is about 25% patient seldom or not to reach result for the treatment of.Further result of study shows, the probability that has the patient of aspirin resistance that myocardial infarction and cerebral apoplexy occur be aspirin sensitive patient 2-3 doubly.In recent years, for the extensive clinical investigation research of asian population, confirmed equally the extensive existence of Aspirin Resistance.Cause the reason of aspirin resistance to have a lot, comprise whether genes of individuals difference, environmental factor, taking dose and patient follow the doctor's advice etc.
Medical development has entered the Personalized medicine epoch, and the treatment application of aspirin is exactly Typical Representative.For the detection of aspirin for treatment effect, not only can be used for confirming aspirin resistance patient's existence, simultaneously can quantize patient for the reaction of aspirin, the therapeutic dose that regulates aspirin with this, reaches the object of individualized treatment.This detection method, by measuring aspirin for treatment effect, is gone back predicts cardiovascular events odds.Therefore, the result for the treatment of of measurement and monitoring aspirin is for normal and ill crowd, and especially cardio-cerebral vascular disease patient is of crucial importance.
What conventionally adopt at present is the index that the curative effect of aspirin is analyzed in 11-dehydrogenation thromboxane element B2 (11-dehydro-thromboxane B2) conduct, and its reason is as follows: the blood platelet in active state can be secreted the potent thromboxane element A2 (Thromboxane A2) that makes vessel retraction and platelet aggregation.Thromboxane element A2 is transformed by epoxidase-1 (Cyclooxygenase-1, COX-1), thromboxane synthetase (Thromboxane synthase) by arachidonic acid (Arachidonic acid).Thromboxane element A2 half life period in blood plasma is short, therefore can be become thromboxane element B2 by fast hydrolyzing.Thromboxane element B2 is converted to 11-dehydrogenation thromboxane element B2 by 11-dehydrogenasa (11-dehydrogenase) afterwards.And 11-dehydrogenation thromboxane element B2 is difficult to be degraded in blood plasma, and pass through renal excretion.Therefore, 11-dehydrogenation thromboxane element B2 can be used as the index of the generation of thrombus in vivo alkane element and platelet activation.And in not having the patient of aspirin resistance, aspirin can be by by epoxidase-1 acidylate irreversibly, and suppress the synthetic of thromboxane element.Therefore, measure thrombus in vivo alkane element metabolic product, such as 11-dehydrogenation thromboxane element B2, can be for the generation of judgement thrombus in vivo alkane element and the curative effect of analyzing aspirin.
In currently available technology, there is the diagnostic kit for detection of the response difference of accepting aspirin for treatment of Different Individual, for example, the detection kit (Aspirin Effect Detection Kit) that Cayman company etc. produce.The detection principle that such kit adopts is: adopt competitiveness enzyme-linked immunological technique to detect 11-dehydrogenation thromboxane element B2 in urine sample (in normal population urine, the concentration of 11-dehydrogenation thromboxane element B2 is at 0.4-4ng/ml), thereby the response difference of accepting aspirin for treatment of confirming Different Individual, wherein the principle of its competitiveness enzyme-linked immunological technique adopting is referring to Figure 1B.The existing defect of this technology is: the spectrophotometric value reading of its detection is on the low side, between reading, differs greatly, and therefore easily causes the error rate of result higher, and between result, difference maximum can reach 27%.
Summary of the invention
For solving above-mentioned problems of the prior art, the invention provides a kind of ELISA (enzyme-linked immunosorbent assay, enzyme linked immunosorbent assay) kit for detection of 11-dehydrogenation thromboxane element B2.
Particularly, the invention provides:
(1) an ELISA kit of 11-dehydrogenation thromboxane element B2, what described ELISA kit adopted is double antibody sandwich method, wherein,
The capture antibody of described ELISA kit is anti-11-dehydrogenation thromboxane element B2 monoclonal antibody, and wherein said anti-11-dehydrogenation thromboxane element B2 monoclonal antibody is selected from the anti-11-dehydrogenation thromboxane element B2 monoclonal antibody of mouse, rabbit, chicken, dog or monkey source property;
The detection antibody of described ELISA kit is anti-11-dehydrogenation thromboxane element B2 polyclonal antibody, wherein said anti-11-dehydrogenation thromboxane element B2 polyclonal antibody is selected from the anti-11-dehydrogenation thromboxane element B2 polyclonal antibody of mouse, rabbit, chicken, dog or monkey source property, and described anti-11-dehydrogenation thromboxane element B2 polyclonal antibody is non-homology with the described plain B2 monoclonal antibody of anti-11-dehydrogenation thromboxane.
(2) according to the ELISA kit (1) described, wherein, described ELISA kit has the porous plate that is coated with anti-IgG antibody, described anti-11-dehydrogenation thromboxane element B2 monoclonal antibody is to be independently present in described ELISA kit as independent reagent, or is coated on described porous plate.
(3) according to the ELISA kit (2) described, wherein, when described anti-11-dehydrogenation thromboxane element B2 monoclonal antibody is independently present in described ELISA kit as independent reagent, the working concentration of described anti-11-dehydrogenation thromboxane element B2 monoclonal antibody is 0.2-2 μ g/ml, is preferably 1 μ g/ml.
(4), according to the ELISA kit (1) described, wherein, described anti-11-dehydrogenation thromboxane element B2 monoclonal antibody is mouse-anti people 11-dehydrogenation thromboxane element B2 monoclonal antibody.
(5), according to the ELISA kit (1) described, wherein, the working concentration of described anti-11-dehydrogenation thromboxane element B2 polyclonal antibody is 0.5-2 μ g/ml, is preferably 1 μ g/ml.
(6), according to the ELISA kit (1) described, wherein, described anti-11-dehydrogenation thromboxane element B2 polyclonal antibody is the anti-human 11-dehydrogenation of rabbit thromboxane element B2 polyclonal antibody.
(7) according to the ELISA kit (1) described, wherein, the marker enzyme of described ELISA kit is horseradish peroxidase or alkaline phosphatase, and corresponding chromogenic substrate is respectively tetramethyl benzidine or nitrobenzophenone disodium hydrogen phosphate.
(8), according to the ELISA kit (1) described, wherein, described marker enzyme is attached on anti-11-dehydrogenation thromboxane element B2 polyclonal antibody; The working concentration that is combined with the anti-11-dehydrogenation thromboxane element B2 polyclonal antibody of marker enzyme is 0.5-2 μ g/ml, is preferably l μ g/ml.
(9) according to the ELISA kit (1) described, wherein, described ELISA kit also comprises secondary antibody, and described marker enzyme is to be attached in described secondary antibody, wherein said secondary antibody is the anti-IgG antibody of the described anti-11-dehydrogenation thromboxane element B2 polyclonal antibody of identification specifically; The working concentration of the described secondary antibody that is combined with marker enzyme is 0.1-2.5 μ g/ml, is preferably 1.25 μ g/ml.
(10) according to the ELISA kit (9) described, wherein, described secondary antibody is the anti-IgG antibody of sheep, chicken, dog or monkey source property.
(11) according to the ELISA kit described in any one in (1)-(10), wherein, described ELISA kit also comprises: dilution, cleansing solution and/or 11-dehydrogenation thromboxane element B2 standard items.
(12) according to the ELISA kit (11) described, wherein, described dilution comprises: 100mM NaCl, 10mM EDTA and 1% (weight/volume) bovine serum albumin(BSA); And the pH of described dilution is 7.0-10.0, be preferably 9.6.
(13) the ELISA kit according to (11), wherein, described cleansing solution is the phosphate buffer of the Tween-20 that comprises 0.05 volume %, the pH of described phosphate buffer is 6.0-8.0, is preferably 7.4.
ELISA kit of the present invention compared with prior art has the following advantages and good effect:
1. two antigenic determinants that antibody recognition is different of ELISA kit application of the present invention, so the specific binding of enhancing and detected antigen, so the specificity detecting is higher, strengthen 2-3 times than the susceptibility of other enzyme-linked immunoassay method conventionally; Antibody-Ag-Ab immune complex is more stable, increases the repeatability of the method.Thereby the spectrophotometric sample reading that has correspondingly overcome traditional competitiveness enzyme linked immune detection method is on the low side, differs greatly and cause the error rate of result to increase between reading.
2. the accuracy of ELISA kit testing result of the present invention is higher, and variance rate is lower.
3. when application ELISA kit of the present invention, detected antigen does not need to carry out highly purified, can be directly used in detection.
4. each component preparation adopting of ELISA kit of the present invention is simple, is conducive to large-scale production.For example, a preferred embodiment of the present invention can adopt the anti-11-dehydrogenation thromboxane element B2 antibody test that is combined with marker enzyme, has reduced and has detected the difficulty that antibody connects with marker enzyme.
Accompanying drawing explanation
Fig. 1 is the principle schematic that two kinds of methods detect 11-dehydrogenation thromboxane element B2; Wherein A is according to double antibody enzyme-linked immunoassay method of the present invention, and B is competitiveness enzyme linked immunoassay method of the prior art.
Fig. 2 is the spectrophotometric value distribution schematic diagram that two kinds of methods detect 11-dehydrogenation thromboxane element B2 standard items; Wherein A is competitiveness enzyme linked immunoassay method of the prior art, and B is according to double antibody enzyme-linked immunoassay method of the present invention.
Embodiment
The below description by embodiment the invention will be further described with reference to accompanying drawing, but this is not limitation of the present invention, those skilled in the art are according to basic thought of the present invention, can make various modifications or improvement, but only otherwise depart from basic thought of the present invention, all within the scope of the present invention.
Capture antibody described in the present invention refer in DAS-ELISA kit be connected with solid phase carrier can specific recognition determined antigen antibody, after capture antibody is connected with solid phase carrier, also can be described as insolubilized antibody.
Detection antibody described in the present invention refers to the antibody that another in DAS-ELISA kit can specific recognition determined antigen, and it identifies respectively the different antigenic determinant on determined antigen molecule from capture antibody.
Secondary antibody described in the present invention refers to the antibody that can specific recognition detects antibody in DAS-ELISA kit, conventionally can be connected with marker enzyme (also can be described as ELIAS secondary antibody) on it.The amount that described secondary antibody can detect antibody by mensuration realizes the mensuration to determined antigen.
The inventor found through experiments: although double antibody Enzyme-multiplied immune technique (double-antibody sandwich elisa) is widely applied in other detects, but in being applied to the process that detects 11-dehydrogenation thromboxane element B2, but exist due to problems such as various X factors cause the resolution of antibody and determined antigen not high, incomplete antigen antibody non-specific binding, thereby the detection error rate causing is high.Therefore in currently available technology, cannot effectively utilize the content that double antibody Enzyme-multiplied immune technique accurately detects 11-dehydrogenation thromboxane element B2 in testing sample.Therefore, the inventor to (such as) aspects such as selection of double antibody conduct in-depth research, and finally successfully developed the kit that utilizes quantitative Enzyme-multiplied immune technique to detect 11-dehydrogenation thromboxane element B2 content in urine, thereby realized, utilize double antibody Enzyme-multiplied immune technique accurately to detect 11-dehydrogenation thromboxane element B2.
Particularly, the invention provides a kind of ELISA kit for detection of 11-dehydrogenation thromboxane element B2, what described ELISA kit adopted is double antibody sandwich method, wherein, the capture antibody of described ELISA kit (capturing antibody) is anti-11-dehydrogenation thromboxane element B2 monoclonal antibody, and wherein said anti-11-dehydrogenation thromboxane element B2 monoclonal antibody is selected from the anti-11-dehydrogenation thromboxane element B2 monoclonal antibody of mouse, rabbit, chicken, dog or monkey source property; The detection antibody of described ELISA kit (detecting antibody) is anti-11-dehydrogenation thromboxane element B2 polyclonal antibody, wherein said anti-11-dehydrogenation thromboxane element B2 polyclonal antibody is selected from the anti-11-dehydrogenation thromboxane element B2 polyclonal antibody of mouse, rabbit, chicken, dog or monkey source property, and described anti-11-dehydrogenation thromboxane element B2 polyclonal antibody is different with the kind source property of the described plain B2 monoclonal antibody of anti-11-dehydrogenation thromboxane.
Preferably, the ELISA kit for detection of 11-dehydrogenation thromboxane element B2 of the present invention, it comprises:
Porous plate, is coated with anti-IgG antibody on described porous plate;
Capture antibody: anti-11-dehydrogenation thromboxane element B2 monoclonal antibody, the described anti-IgG antibody of wherein said anti-11-dehydrogenation thromboxane element B2 monoclonal antibody specificity ground identification;
Detect antibody: anti-11-dehydrogenation thromboxane element B2 polyclonal antibody; And
Marker enzyme and corresponding chromogenic substrate;
Wherein, described anti-11-dehydrogenation thromboxane element B2 monoclonal antibody is to be independently present in described ELISA kit as independent reagent, or is coated on described porous plate;
Described anti-11-dehydrogenation thromboxane element B2 monoclonal antibody is selected from the anti-11-dehydrogenation thromboxane element B2 monoclonal antibody of mouse, rabbit, chicken, dog or monkey source property;
Described anti-11-dehydrogenation thromboxane element B2 polyclonal antibody is selected from the anti-11-dehydrogenation thromboxane element B2 polyclonal antibody of mouse, rabbit, chicken, dog or monkey source property, and described anti-11-dehydrogenation thromboxane element B2 polyclonal antibody is different with the kind source property of the described plain B2 monoclonal antibody of anti-11-dehydrogenation thromboxane; And
Described anti-11-dehydrogenation thromboxane element B2 monoclonal antibody and described anti-11-dehydrogenation thromboxane element B2 polyclonal antibody are respectively for the antigenic determinant of the plain B2 of different 11-dehydrogenation thromboxanes.
Preferably, described anti-11-dehydrogenation thromboxane element B2 monoclonal antibody is coated on described porous plate, and wherein said anti-11-dehydrogenation thromboxane element B2 monoclonal antibody is by being combined specifically and being coated on described porous plate with described anti-IgG antibody.And when described anti-11-dehydrogenation thromboxane element B2 monoclonal antibody is independently present in described ELISA kit as independent reagent, the working concentration of described anti-11-dehydrogenation thromboxane element B2 monoclonal antibody solution is 0.2-2 μ g/ml, is preferably 1 μ g/ml.
Preferably, described anti-11-dehydrogenation thromboxane element B2 monoclonal antibody is mouse-anti people 11-dehydrogenation thromboxane element B2 monoclonal antibody.
Preferably, described anti-IgG antibody is sheep anti-mouse igg antibody.
Preferably, the working concentration of described anti-11-dehydrogenation thromboxane element B2 polyclonal antibody is 0.5-2 μ g/ml, is preferably 1 μ g/ml; Described anti-11-dehydrogenation thromboxane element B2 polyclonal antibody is preferably the anti-human 11-dehydrogenation of rabbit thromboxane element B2 polyclonal antibody.
Preferably, described marker enzyme is horseradish peroxidase or alkaline phosphatase, and described chromogenic substrate is respectively tetramethyl benzidine or nitrobenzophenone disodium hydrogen phosphate.
Preferably, described marker enzyme is attached on anti-11-dehydrogenation thromboxane element B2 polyclonal antibody; The working concentration that is combined with the anti-11-dehydrogenation thromboxane element B2 polyclonal antibody of marker enzyme is 0.5-2 μ g/ml, is preferably 1 μ g/ml.
Preferably, described ELISA kit also comprises secondary antibody, and described marker enzyme is to be attached in described secondary antibody, wherein said secondary antibody is the anti-IgG antibody of the described anti-11-dehydrogenation thromboxane element B2 polyclonal antibody of identification specifically; The working concentration of the described secondary antibody that is combined with marker enzyme is 0.2-2.5 μ g/ml, is preferably 1 μ g/ml.
Preferably, described secondary antibody is the anti-IgG antibody of sheep, chicken, dog or monkey kind source property.
Preferably, described ELISA kit also comprises: dilution, cleansing solution and/or 11-dehydrogenation thromboxane element B2 standard items.
Preferably, described dilution comprises: 100mM NaCl, 10mM EDTA and 1% (weight/volume) bovine serum albumin(BSA); And described dilution pH is 7.0-10.0, be preferably 9.6.
Preferably, described cleansing solution is the phosphate buffer of the Tween-20 that comprises 0.05 volume %, and the pH of described phosphate buffer is 6.0-8.0, is preferably 7.4.
The basic application principle of ELISA kit of the present invention is specific antibody enzyme linked immunosorbent assay, what ultimate principle adopted is double antibody sandwich ELISA, the method utilizes two antigenic determinants of double antibody on detected antigen molecule to be combined, and forms solid matrix antibody-antigen-enzyme labelled antibody immune complex.Double antibody be take respectively anti-11-dehydrogenation thromboxane element B2 monoclonal antibody as capture antibody and be take anti-11-dehydrogenation thromboxane element B2 polyclonal antibody as detecting antibody.Described anti-11-dehydrogenation thromboxane element B2 monoclonal antibody is selected from the anti-11-dehydrogenation thromboxane element B2 monoclonal antibody of mouse, rabbit, chicken, dog, monkey kind source property, described anti-11-dehydrogenation thromboxane element B2 polyclonal antibody is selected from the anti-11-dehydrogenation thromboxane element B2 polyclonal antibody of mouse, rabbit, chicken, dog, monkey kind source property, and described anti-11-dehydrogenation thromboxane element B2 monoclonal antibody is different with the kind source property of described anti-11-dehydrogenation thromboxane element B2 polyclonal antibody, and respectively for the antigenic determinant of the plain B2 of different 11-dehydrogenation thromboxanes.Wherein capture antibody is preferably used the anti-11-dehydrogenation of mouse-anti people thromboxane element B2 monoclonal antibody, and main cause is mouse antibody recognition single antigenic determinat, and specificity is high, is conducive to from sample in conjunction with antigen to be measured; Detect antibody and preferably use the anti-human anti-11-dehydrogenation thromboxane element B2 polyclonal antibody of rabbit, identify a plurality of antigenic determinants, combination that can enhancement antigen antibody, improves the efficiency detecting.After marker enzyme in measurement compound acts on the substrate adding, generate colored substance quality, can determine determined antigen content.
For example, the inventor determines suitable enzyme linked immunoassay condition by groping following condition:
1. capture antibody
The capture antibody that the present invention adopts is anti-11-dehydrogenation thromboxane element B2 monoclonal antibody, the effect of capture antibody is (for example to catch testing sample, urine sample) the 11-dehydrogenation thromboxane element B2 in, it catches principle is the specific binding by antigen-antibody.The present invention to the requirement of capture antibody be can capture antigen molecule 1 1-dehydrogenation thromboxane antigenic determinant on element B2, thereby make the two effectively specific binding, therefore, meet can with the antibody of antigen molecule specific binding, comprise that the anti-11-dehydrogenation thromboxane element B2 monoclonal antibody of the kind source property such as mouse, rabbit, chicken, dog or monkey all can be adopted as capture antibody.Wherein, preferably use mouse-anti people 11-dehydrogenation thromboxane element B2 monoclonal antibody, main cause is that the specificity of mouse antibody recognition single antigenic determinat is high, is conducive to from sample in conjunction with antigen to be measured.
The preparation process of capture antibody can comprise: by the antigen molecule 11-dehydrogenation thromboxane element B2 to animal used as test injection purifying, make its sensitization produce the lymphocyte of the anti-11-dehydrogenation of specificity thromboxane element B2 antibody, from the spleen separation of animal used as test, obtain the lymphocyte of a kind of anti-11-dehydrogenation thromboxane element B2 antibody of generation (antibody with a kind of antigenic determinant) that above-mentioned steps obtains.Application cell hybridization technique makes to take from the myeloma cell of spleen and lymphocyte that above-mentioned steps obtains, and the two merges, and obtains the myeloma cell of hybrid.With this cell mass that derives from single fused cell cultivation propagation, can prepare the specific anti-11-dehydrogenation thromboxane element B2 monoclonal antibody of anti-a kind of antigenic determinant.Then by protein purification process, obtain the reagin of purifying.Capture antibody can obtain by being purchased approach, also can prepare voluntarily.
In kit of the present invention, the scope of the working concentration of capture antibody is preferably: 0.2-2 μ g/ml, wherein preferred concentration is 1 μ g/ml.In previous experiments, the inventor, by relatively measure 20ng/ml 11-dehydrogenation thromboxane element B2 standard items under same reaction conditions, gropes the preferred concentration of capture antibody.Concrete implementation step is: in the interval of capture antibody concentration 0.2-2 μ g/ml, choose 4 kinds of antibody concentration (the anti-11-dehydrogenation of the mouse-anti people thromboxane element B2 monoclonal antibody of take is example), be respectively 0.2 μ g/ml, 0.5 μ g/ml, 1 μ g/ml, 2 μ g/ml.The capture antibody of 4 kinds of concentration is added respectively in reacting hole and spent the night at 4 ℃.In all reacting holes, add 20ng/ml 11-dehydrogenation thromboxane element B2 standard items, unconjugated standard items are washed liquid wash-out.Next in all reacting holes, be sequentially added into detection antibody (1Bg/m1) and the colour developing enzyme of same concentration.Cleansing solution is by the detection antibody elution of non-specific binding.Finally by decomposing chromogenic substrate, under spectrophotometer, read the reading of chromogenic reaction.In the situation that other conditions are identical with step, use respectively the capture antibody of above 4 kinds of concentration to detect 20ng/ml11-dehydrogenation thromboxane element B2 standard items, the reading interval obtaining is respectively 0.894～0.965,1.132～1.196,1.303～1.385,1.318～1.394.By relatively above data, can find out, detect the 11-dehydrogenation thromboxane element B2 standard items (20ng/ml) of same concentration, in the situation that other condition steps are identical, when the reading of chromogenic reaction is 0.2 μ g/ml and 0.5 μ g/ml compared with concentration when capture antibody concentration is 1 μ g/ml high (difference > 0.15), can improve like this accuracy and sensitivity that testing sample detects.Meanwhile, relatively capture antibody concentration is 1 μ g/ml and 2 μ g/ml, and the reading of chromogenic reaction approaches (difference < 0.02).So in this case, choice for use concentration is the use amount that the capture antibody of 1 μ g/ml can be saved antibody greatly.
2. detect antibody
The detection antibody that the present invention adopts is anti-11-dehydrogenation thromboxane element B2 polyclonal antibody, and the effect that detects antibody is to combine with the antigen molecule 11-dehydrogenation thromboxane element B2 on capture antibody, thereby detects the 11-dehydrogenation thromboxane element B2 in testing sample.The present invention is can detect with on antigenic determinant on antigen molecule 11-dehydrogenation thromboxane element B2 to combine to detecting the requirement of antibody, therefore, meet can with the antibody of antigen molecule specific binding, comprise that the anti-11-dehydrogenation thromboxane B2 polyclonal antibody of the kind source property such as mouse, rabbit, chicken, dog, monkey all can be adopted as detection antibody.Wherein, preferably use the anti-human 11-dehydrogenation of rabbit thromboxane B2 polyclonal antibody, main cause is that rabbit endogenous antibody can be identified a plurality of antigenic determinants, and combination that can enhancement antigen antibody improves the efficiency detecting.
It should be noted that, although capture antibody and detection antibody are all anti-11-dehydrogenation thromboxane element B2 antibody, can not adopt the anti-11-dehydrogenation thromboxane element B2 antibody of same kind.Reason is: adopting the anti-11-dehydrogenation thromboxane element B2 antibody of different genera is can be attached on the antigen site different from capture antibody in order to make to detect antibody, thereby detect different epitopes, and when adopting the antibody of same kind, with the antibody of chromogenic reaction enzyme can be simultaneously with catch and detect antibody and combine, cause chromogenic reaction not there is specificity, cannot determine the amount of determined antigen.Therefore,, when selecting capture antibody and detecting antibody, need to choose the anti-11-dehydrogenation thromboxane element B2 antibody that comes from different genera.
The preparation process that detects antibody is as follows: by the antigen molecule 11-dehydrogenation thromboxane element B2 to animal used as test injection purifying, the specific reagin of original molecule that makes to create antagonism in blood plasma after its sensitization then obtains the reagin of purifying from serum by protein purification process.Resulting anti-11-dehydrogenation thromboxane B2 polyclonal antibody, can identify a plurality of antigenic determinants, combination that can enhancement antigen antibody.Detecting antibody can obtain by being purchased approach, also can prepare voluntarily.
In kit of the present invention, the concentration range that detects antibody is: 0.5-2 μ g/ml, wherein preferred concentration is 1 μ g/ml.The concrete method of groping is similar to capture antibody, and difference is only to change the concrete concentration that detects antibody.
3. secondary antibody
Kit of the present invention also can comprise secondary antibody, meets the antibody of recognition detection antibody specifically and all can be used as secondary antibody in the present invention.
Wherein, secondary antibody can adopt anti-immunoglobulin IgG polyclonal antibody, and this anti-IgG antibody can the FC section on detecting antibody be combined, therefore can recognition detection antibody, and the antigen recognition site that detects antibody is positioned at variable region, therefore above-mentioned two recognition sites are nonoverlapping.The anti-immunoglobulin IgG antibody that comprises the kind source property such as sheep, chicken, dog, monkey is all applicable.
4. chromogenic reaction
The principle of chromogenic reaction of the present invention is to react by marker enzyme and chromogenic substrate, thereby by the variation of color, measures the amount of 11-dehydrogenation thromboxane element B2 antigen molecule in testing sample.For the enzyme for chromogenic reaction, wish that it can effectively decompose corresponding chromogenic substrate.Wherein, preferably use (for example) horseradish peroxidase (Horse peroxidase, HRP) or alkaline phosphatase (Akaline phophatase, AP), its corresponding chromogenic substrate can be respectively: tetramethyl benzidine (tetramethylbenzidine, TMB) or nitrobenzophenone disodium hydrogen phosphate (p-Nitrophenyl phosphate, disodium salt, pNPP).It will be appreciated by those skilled in the art that the chromogenic substrate meeting with horseradish peroxidase or alkaline phosphatase reaction conditions all may be used in the present invention simultaneously.
Marker enzyme can be combined in and detect in antibody or secondary antibody.Wherein, marker enzyme can adopt the method for sodium cyanoborohydride (Sodium cyanoborohydride) to be attached to and detect in antibody or secondary antibody, for example can for example, referring to the chromogenic reaction elisa kit of () Thermo company.The concentration range of chromogenic substrate can be 0.1-1g/L, and wherein preferred concentration can be 0.4g/L.Chromogenic reaction condition is under conventional room temperature, to be placed on to shake bed reaction 15-30 minute.
In kit of the present invention, with the anti-11-dehydrogenation thromboxane element B2 polyclonal antibody of marker enzyme, concentration range can be 0.5-2 μ g/ml, and wherein preferred concentration can be 1 μ g/ml.
In kit of the present invention, with the secondary antibody of marker enzyme, concentration range is 0.1-2.5 μ g/ml, and wherein preferred concentration is 1 μ g/ml.And can obtain by being purchased approach (for example can purchased from Thermo company) with the secondary antibody of marker enzyme, also can prepare voluntarily.Wherein the step of associated methods can for example, referring to the chromogenic reaction elisa kit of () Thermo company.
5. can identify specifically the anti-immunoglobulin IgG antibody of capture antibody
The effect of the antibody of the anti-immunoglobulin IgG that can identify specifically capture antibody used in the present invention is to be connected with capture antibody with ELISA Plate respectively, thereby capture antibody is fixed in porous plate or ELISA Plate, and the principle adopting is the combination between antibody and antibody.In the present invention, meet the anti-immunoglobulin IgG antibody that can identify specifically capture antibody, comprise that the AIA of the kind source property such as sheep, chicken, dog, monkey is all applicable.
The preparation process of anti-IgG polyclonal antibody can comprise: by the immunoglobulin (Ig) to animal used as test injection purifying, its sensitization is produced the specific antibody of immunoglobulin (Ig).After the blood plasma of the animal used as test of collection sensitization, then by antibody purification process, obtain the immune globulin antibody of purifying.Anti-IgG antibody can obtain by being purchased approach, also can prepare voluntarily.
In kit of the present invention, adopt be coated with the anti-immunoglobulin IgG antibody that can identify specifically capture antibody porous plate (for example, be coated with the ELISA Plate of the anti-immunoglobulin IgG antibody that can identify specifically capture antibody), above-mentioned porous plate can obtain by being purchased approach, also can be coated with voluntarily, method for coating is this area common method, for example it is positioned in the reacting hole on porous plate and is spent the night at 4 ℃, will can not pass through cleansing solution wash-out with the antibody of the effective combination of porous plate afterwards.Wherein, in coated process, the concentration range of described anti-IgG antibody is 0.2-2 μ g/ml, and preferred concentration is 0.4 μ g/ml.What preferably adopt is sheep anti mouse anti-immunoglobulin IgG antibody.
6. dilution
In kit of the present invention, the effect of dilution is for dilution standard tester, testing sample and reagin.In kit of the present invention, due to needs antigen and antibody specific association reaction, wish that the ion concentration of dilution and potential of hydrogen are moderate, wherein acidity-basicity ph 7.0-10.0 all can.
For example, the inventor has carried out contrast experiment for following 4 kinds of integrated enzyme reaction dilute solutions:
(1) dilution 1:100mM NaCl, 10mM EDTA, 1% (weight/volume) bovine serum albumin(BSA), pH7.4;
(2) dilution 2:100mM NaCl, 10mM EDTA, 1% (weight/volume) bovine serum albumin(BSA), pH9.6;
(3) dilution 3:50mM Tris, 150mM NaCl, pH7.4;
(4) dilution 4:50mM Tris, 150mM NaCl, pH9.6;
In experiment, the inventor, by relatively measure 20ng/ml 11-dehydrogenation thromboxane element B2 standard items under same reaction conditions, gropes preferred dilution.Concrete implementation step is: the capture antibody (1 μ g/ml) that is diluted in 4 kinds of dilutions is inserted respectively in reacting hole and spent the night at 4 ℃.Then in all reacting holes, add 20ng/ml 11-dehydrogenation thromboxane element B2 standard items, unconjugated standard items are washed liquid wash-out.Next in all reacting holes, be sequentially added into the detection antibody (1 μ g/ml) and the colour developing enzyme that are diluted in 4 kinds of dilutions.Cleansing solution is by the detection antibody elution of non-specific binding.Finally, by decomposing chromogenic substrate, under spectrophotometer, read the reading of chromogenic reaction.In the situation that other conditions are identical with step, use respectively above 4 kinds of different dilutions to detect 20ng/ml 11-dehydrogenation thromboxane B2 standard items, its reading interval of detecting 20ng/ml 11-dehydrogenation thromboxane element B2 standard items is respectively 1.152～1.236,1.297～1.365,0.953～1.042,1.035～1.128.From the above results, can find out, adopt the second dilution, for measuring same concentration (20ng/ml) 11-dehydrogenation thromboxane element B2 standard items, compared with its excess-three kind dilution reacting hole reading the highest (difference > 0.10), can improve like this accuracy and sensitivity that testing sample detects.Experimental result illustrates simultaneously, the combination between alkaline solution environment (pH9.6) energy enhancement antigen antibody, thus improve the identification of antibody to antigen molecule to be measured.
7. cleansing solution
In kit of the present invention, cleansing solution is for the unconjugated sample in elution of reactive hole, standard control thing or antibody.In kit of the present invention, owing to there being incomplete antigen antibody non-specific binding, to the requirement of cleansing solution, be to reduce antigen-antibody non-specific binding, improve antibody specific recognition capability.Wherein, the phosphate buffer that preferably use contains 0.05 volume %Tween-20 is as the cleansing solution of kit of the present invention, and the pH of described phosphate buffer is 6.0-8.0, is preferably 7.4.
Mode by the following examples further explains and describes content of the present invention, but these embodiment are not to be construed as limiting the scope of the invention.
In following examples, what capture antibody adopted is the anti-11-dehydrogenation of mouse anti human thromboxane element B2 monoclonal antibody; What detect antibody employing is the anti-human anti-11-dehydrogenation thromboxane element B2 polyclonal antibody of rabbit; But the invention is not restricted to this, what also can adopt other cited kind source property of the present invention catches or detects antibody.
In following examples, through the testing sample of standard control or dilution process, be placed in the reacting hole that absorption has 11-dehydrogenation thromboxane element B2 antibody.Experiment carrys out the content of 11-dehydrogenation thromboxane element B2 in calculation sample by measuring the binding capacity of reagin: the numerical value drawing according to measurement variable concentrations 11-dehydrogenation thromboxane element B2 reference material, can extrapolate the computing formula of calculating unknown sample concentration.
1.1 experiment kits
The described ELISA kit for detection of 11-dehydrogenation thromboxane element B2 comprises:
Be coated with the porous plate of anti-IgG antibody: employing be the 96 hole ELISA Plate that are coated with sheep anti-mouse igg antibody.Can be coated voluntarily by experimenter.Method for coating for example, spends the night during can be () be positioned over reacting hole by sheep anti-mouse igg antibody (concentration is 0.4 μ g/ml, can purchased from Thermo company) at 4 ℃, and every hole adds 100 μ l.By (filling a prescription as follows: 8gNaCl, 0.2g KCl, 1.44g Na with PBS solution
2hPO
4, 0.24g KH
2pO
4, 1000ml water, pH7.4) washs 3 times, thereby removes the unnecessary sheep anti-mouse igg antibody not being connected with ELISA Plate.
Capture antibody: employing be mouse-anti people 11-dehydrogenation thromboxane element B2 monoclonal antibody, can the preparation of YouCD Bioscience company, final concentration is 1 μ g/ml.
Detect antibody: employing be the anti-human 11-dehydrogenation of rabbit thromboxane element B2 polyclonal antibody, can the preparation of YouCD Bioscience company, final concentration is 1 μ g/ml.
Marker enzyme: employing be horseradish peroxidase, described horseradish peroxidase is combined on the anti-rabbit anti-immunoglobulin of secondary antibody IgG antibody, can for example, purchased from () Thermo company, concentration is 1mg/ml.
Chromogenic substrate: employing be tetramethyl benzidine (TMB), concentration is 0.3mg/ml, can for example, purchased from () Sigma company.
Cleansing solution: employing be 10mM K
2hPO
4, 2mM KH
2pO
4, 150mM NaCl, 0.05% (volume %) Tween-20, pH7.6.
Dilution: employing be 100mM NaCl, 10mM EDTA, 1% (weight/volume) bovine serum albumin(BSA), pH9.6.
11-dehydrogenation thromboxane B2 standard items: concentration is 200ng/ml, can for example, purchased from () Enzo company.
Wherein, described cleansing solution, dilution, 11-dehydrogenation thromboxane B2 standard items can directly be included in described kit, also can be prepared voluntarily by experimenter.
1.2 experimental procedure
1.2.1 the preparation of testing sample
Get each 1ml of urine sample of person to be measured, centrifugal 5 minutes of 3000rpm, to remove insoluble sediment.Get supernatant, add dilution to dilute, wherein the volume ratio of urine sample and dilution is 1: 5.Urine sample after dilution can directly be measured, or is placed in-20 ℃ of refrigerator-freezers, can stablize and preserve three months.
1.2.2 the preparation of standard control
Take out 8 sample hoses.First sample hose adds 900 μ l dilutions and 200ng/ml11-dehydrogenation thromboxane B2 standard items 100 μ l, is diluted to 20ng/ml, mixes.All the other 7 sample hoses add 500 μ l dilutions.From first sample hose, take out 500 μ l, join in second sample hose, mix.Configure in the same way the 3rd to the 8th sample hose, so that 11-dehydrogenation thromboxane B2 standard items are carried out to serial dilution, (final concentration is respectively 20ng/ml; 10ng/ml; 5ng/ml; 2.5ng/ml; 1.25ng/ml; 0.625ng/ml; 0.3125ng/ml; 0.15625ng/ml).
1.2.3 response procedures
Mouse-anti people 11-dehydrogenation thromboxane element B2 monoclonal antibody and dilution are mixed, wherein the volume ratio of mouse-anti people 11-dehydrogenation thromboxane element B2 monoclonal antibody and dilution is 1: 500, add afterwards in the reacting hole of the 96 hole ELISA Plate that are coated with sheep anti-mouse igg antibody, every hole adds 100 μ l, spends the night at 4 ℃.By (filling a prescription as follows: 8g NaCl, 0.2g KCl, 1.44g Na with PBS solution
2hPO
4, 0.24g KH
2pO
4, 1000ml water, pH7.4) washs 3 times, thereby removes the unnecessary mouse-anti people 11-dehydrogenation thromboxane element B2 monoclonal antibody not being connected with ELISA Plate.
The standard control that 100 μ l have been diluted or testing sample join respectively in the respective reaction hole of 96 orifice plates, and each standard control or testing sample need add two parts.Cover plastics epiphragma, at room temperature react, on shaking table, slightly mix sample, continue one hour.The sample of falling dereaction flicks off residual sample in reacting hole on clean paper handkerchief.Add 300 μ l cleansing solutions, wash away standard control and the testing sample of not absorption, repeat three to five times.
The anti-human 11-dehydrogenation of the rabbit thromboxane element B2 polyclonal antibody that adds 100 μ l to dilute in each reacting hole, on shaking table, room temperature reaction continues one hour.The sample of falling dereaction flicks off residual sample in reacting hole on clean paper handkerchief.Add 300 μ l cleansing solutions, wash away the not reagin of absorption, repeat three to five times.
The anti-rabbit immunoglobulin IgG antibody that adds 100 μ l horseradish peroxidases (HRP) to connect in each reacting hole, on shaking table, room temperature reaction continues one hour.
Add 100 μ l chromogenic substrate tetramethyl benzidines (TMB), at room temperature reaction continues 30 minutes.Before measurement data, slightly shake up sample, so that the uniformity that develops the color in each reacting hole.With spectrum, read instrument (can derive from Thermo Scientific company) in wavelength 460nm reading out data.
1.3 experimental result
1.3.1 the drafting of typical curve
Reading out data according to standard control, draws typical curve, and result is referring to Fig. 2 B.
1.3.2 the measurement result of testing sample
The equation of typical curve of drawing according to 1.3.1 and the OD value of testing sample calculate the amount of 11-dehydrogenation thromboxane element B2 in testing sample.
Adopt this kit to measure 14 persons' to be measured urine sample, the numeric distribution scope of testing result is 0.21-2.89ng/ml.
1.4 accuracy measurement
The reference material that adopts this detection method measurement to have different nominal concentration (is respectively 0.1ng/ml; 0.5ng/ml; 2ng/ml; 5ng/ml; 10ng/ml), each nominal concentration detects three times, obtain detectable concentration, calculate the difference of this detectable concentration and nominal concentration, wherein the computing formula of the difference of detectable concentration and nominal concentration is: ((detectable concentration-nominal concentration)/nominal concentration) * 100%.Result demonstration, the difference of testing result and actual concentrations is only between 2-9.2%.
2.1 experiment kits
The described ELISA kit for detection of 11-dehydrogenation thromboxane element B2 comprises:
Be coated with the porous plate of anti-IgG antibody: employing be the 96 hole ELISA Plate that are coated with sheep anti-mouse igg antibody.Can be coated voluntarily by experimenter, method for coating is as described in example 1 above.
Capture antibody: employing be mouse-anti people 11-dehydrogenation thromboxane element B2 monoclonal antibody, can the preparation of YouCD Bioscience company, final concentration is 1 μ g/ml.
Detect antibody: employing be the anti-human 11-dehydrogenation of rabbit thromboxane element B2 polyclonal antibody, can the preparation of YouCD Bioscience company, final concentration is 1 μ g/ml.
Marker enzyme: employing be horseradish peroxidase, can be purchased from Thermo company, and described horseradish peroxidase is to be combined on the described anti-human 11-dehydrogenation of rabbit thromboxane element B2 polyclonal antibody, and the chromogenic reaction enzyme that is (for example) Thermo company in conjunction with the method adopting by the two connects kit.
Chromogenic substrate: employing be tetramethyl benzidine (TMB), concentration is 0.3mg/ml, can for example, purchased from () Sigma company.
Cleansing solution: employing be 10mM K
2hPO
4, 2mM KH
2pO
4, 150mM NaCl, 0.05% (volume %) Tween-20, pH7.6.
Dilution: employing be 100mM NaCl, 10mM EDTA, 1% (weight/volume) bovine serum albumin(BSA), pH9.6.
11-dehydrogenation thromboxane B2 standard items: concentration is 200ng/ml, can for example, purchased from () Enzo company.
Wherein, described cleansing solution, dilution, 11-dehydrogenation thromboxane B2 standard items can directly be included in described kit, also can be prepared voluntarily by experimenter.
2.2 experimental procedure
2.2.1 the preparation of testing sample
Get each 1ml of urine sample of person to be measured, centrifugal 5 minutes of 3000rpm, to remove insoluble sediment.Get supernatant, add dilution to dilute, wherein the volume ratio of urine sample and dilution is 1: 5.Urine sample after dilution can directly be measured, or is placed in-20 ℃ of refrigerator-freezers, can stablize and preserve three months.
2.2.2 the preparation of standard control
Take out 8 sample hoses.First sample hose adds 900 μ l dilutions and 200ng/ml11-dehydrogenation thromboxane B2 standard items 100 μ l to be diluted to 20ng/ml, mixes.All the other 7 sample hoses add 500 μ l dilutions.From first sample hose, take out 500 μ l, join in second sample hose, mix.Configure in the same way the 3rd to the 8th sample hose, so that 11-dehydrogenation thromboxane B2 standard items are carried out to serial dilution.
2.2.3 response procedures
Mouse-anti people 11-dehydrogenation thromboxane element B2 monoclonal antibody and dilution are mixed, wherein the volume ratio of mouse-anti people 11-dehydrogenation thromboxane element B2 monoclonal antibody and dilution is 1: 500, add afterwards in the reacting hole of the 96 hole ELISA Plate that are coated with goat-anti mouse-anti IgG antibody, every hole adds 100 μ l, spends the night at 4 ℃.By (filling a prescription as follows: 8g NaCl, 0.2g KCl, 1.44g Na with PBS solution
2hPO
4, 0.24g KH
2pO
4, 1000ml water, pH7.4) washs 3 times, thereby removes the unnecessary mouse-anti people 11-dehydrogenation thromboxane element B2 monoclonal antibody not being connected with ELISA Plate.
The standard control that 100 μ l have been diluted or testing sample join respectively in the respective reaction hole of 96 orifice plates, and each standard control or testing sample need add two parts.Cover plastics epiphragma, at room temperature react, on shaking table, slightly mix sample, continue one hour.The sample of falling dereaction flicks off residual sample in reacting hole on clean paper handkerchief.Add 300 μ l cleansing solutions, wash away standard control and the testing sample of not absorption, repeat three to five times.
The element of the anti-human 11-dehydrogenation of the rabbit thromboxane with horseradish peroxidase (HRP) the B2 polyclonal antibody that adds 100 μ l to dilute in each reacting hole, on shaking table, room temperature reaction continues one hour.The sample of falling dereaction flicks off residual sample in reacting hole on clean paper handkerchief.Add 300 μ l cleansing solutions, wash away the not reagin of absorption, repeat three to five times.
The anti-rabbit immunoglobulin IgG antibody that adds 100 μ l horseradish peroxidases (HRP) to connect in each reacting hole, on shaking table, room temperature reaction continues one hour.
Add 100 μ l chromogenic substrate tetramethyl benzidines (TMB), at room temperature reaction continues 30 minutes.Before measurement data, slightly shake up sample, so that the uniformity that develops the color in each reacting hole.With spectrum, read instrument (can derive from Thermo Scientific company) in wavelength 460nm reading out data.
2.3 experimental result
Reading out data according to standard control, draws typical curve.Can calculate according to the OD value of equation and testing sample the amount of 11-dehydrogenation thromboxane element B2 in testing sample afterwards.
2.4 accuracy measurement
Can apply this kit, according to method measurement standard thing identical in " 1.4 accuracy measurement " with embodiment 1.Result demonstration, the difference between testing result is only between 3.4-10.2%.
Comparative example 1
Use is positioned at the detection kit (Aspirin Effect-Detection Kit) of the Cayman Chemical company production in U.S.'s Ann Arbor (Ann Arbor) city, and article No. is 519510.Experimental procedure is according to this kit appendix.
Reading out data according to standard control, draws typical curve, and result is referring to Fig. 2 A.Can calculate according to the OD value of equation and testing sample the amount of 11-dehydrogenation thromboxane element B2 in testing sample afterwards.
According to finding out in Fig. 2 A, adopt traditional competitiveness enzyme linked immune detection method measurement standard thing, its spectrophotometric sample reading is on the low side, between reading, differs greatly, and often causes the error rate of result to increase.
Can apply this kit afterwards, according to method measurement standard thing identical in " 1.4 accuracy measurement " with embodiment 1, result demonstration, between result, difference maximum reaches 27%.
Claims (13)
1. for detection of an ELISA kit of 11-dehydrogenation thromboxane element B2, what described ELISA kit adopted is double antibody sandwich method, wherein,
The capture antibody of described ELISA kit is anti-11-dehydrogenation thromboxane element B2 monoclonal antibody, and wherein said anti-11-dehydrogenation thromboxane element B2 monoclonal antibody is selected from the anti-11-dehydrogenation thromboxane element B2 monoclonal antibody of mouse, rabbit, chicken, dog or monkey source property;
The detection antibody of described ELISA kit is anti-11-dehydrogenation thromboxane element B2 polyclonal antibody, wherein said anti-11-dehydrogenation thromboxane element B2 polyclonal antibody is selected from the anti-11-dehydrogenation thromboxane element B2 polyclonal antibody of mouse, rabbit, chicken, dog or monkey source property, and described anti-11-dehydrogenation thromboxane element B2 polyclonal antibody is non-homology with the described plain B2 monoclonal antibody of anti-11-dehydrogenation thromboxane.
2. ELISA kit according to claim 1, wherein, described ELISA kit has the porous plate that is coated with anti-IgG antibody, described anti-11-dehydrogenation thromboxane element B2 monoclonal antibody is to be independently present in described ELISA kit as independent reagent, or is coated on described porous plate.
3. ELISA kit according to claim 2, wherein, when described anti-11-dehydrogenation thromboxane element B2 monoclonal antibody is independently present in described ELISA kit as independent reagent, the working concentration of described anti-11-dehydrogenation thromboxane element B2 monoclonal antibody is 0.2-2 μ g/ml, is preferably 1 μ g/ml.
4. ELISA kit according to claim 1, wherein, described anti-11-dehydrogenation thromboxane element B2 monoclonal antibody is mouse-anti people 11-dehydrogenation thromboxane element B2 monoclonal antibody.
5. ELISA kit according to claim 1, wherein, the working concentration of described anti-11-dehydrogenation thromboxane element B2 polyclonal antibody is 0.5-2 μ g/ml, is preferably 1 μ g/ml.
6. ELISA kit according to claim 1, wherein, described anti-11-dehydrogenation thromboxane element B2 polyclonal antibody is the anti-human 11-dehydrogenation of rabbit thromboxane element B2 polyclonal antibody.
7. ELISA kit according to claim 1, wherein, the marker enzyme of described ELISA kit is horseradish peroxidase or alkaline phosphatase, corresponding chromogenic substrate is respectively tetramethyl benzidine or nitrobenzophenone disodium hydrogen phosphate.
8. ELISA kit according to claim 1, wherein, described marker enzyme is attached on anti-11-dehydrogenation thromboxane element B2 polyclonal antibody; The working concentration that is combined with the anti-11-dehydrogenation thromboxane element B2 polyclonal antibody of marker enzyme is 0.5-2 μ g/ml, is preferably 1 μ g/ml.
9. ELISA kit according to claim 1, wherein, described ELISA kit also comprises secondary antibody, and described marker enzyme is to be attached in described secondary antibody, wherein said secondary antibody is the anti-IgG antibody of the described anti-11-dehydrogenation thromboxane element B2 polyclonal antibody of identification specifically; The working concentration of the described secondary antibody that is combined with marker enzyme is 0.1-2.5 μ g/ml, is preferably 1.25 μ g/ml.
10. ELISA kit according to claim 9, wherein, described secondary antibody is the anti-IgG antibody of sheep, chicken, dog or monkey source property.
11. according to the ELISA kit described in any one in claim 1-10, and wherein, described ELISA kit also comprises: dilution, cleansing solution and/or 11-dehydrogenation thromboxane element B2 standard items.
12. ELISA kits according to claim 11, wherein, described dilution comprises: 100mM NaCl, 10mM EDTA and 1% (weight/volume) bovine serum albumin(BSA); And the pH of described dilution is 7.0-10.0, be preferably 9.6.
13. ELISA kits according to claim 11, wherein, described cleansing solution is the phosphate buffer of the Tween-20 that comprises 0.05 volume %, the pH of described phosphate buffer is 6.0-8.0, is preferably 7.4.
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