CN102928606A - Multi-antibody marked quick procalcitonin detecting kit - Google Patents
Multi-antibody marked quick procalcitonin detecting kit Download PDFInfo
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Abstract
The invention relates to a multi-antibody marked quick procalcitonin detecting kit in the technical field of detection. The kit comprises a common carrier, a monoclonal or polyclonal capturing body which is fixed on the common carrier and corresponding to different procalcitonin areas, and a monoclonal or polyclonal capturing body which is compatible with the capturing body and is suitable for a plurality of same or different areas for the procalcitonin marked by a marker. According to the multi-antibody marked quick procalcitonin detecting kit, different distinguishing areas with multi-antibody pair coated are marked and the spaces are subjected to compatibility of medicines, the procalcitonin in a sample can be captured to the greatest extent, and the sensitivity in detection can be improved; the detection sensitivity of PCT (procalcitonin) is improved to 0.1ng/ml by the kit, so that the application value and the application range of a quick PCT detecting agent in diagnosis of bacterial infection, differential diagnosis and treatment can be increased and expanded; and the kit can be applied to diagnosis of bacterial infection, differential diagnosis, treatment effect monitoring and post-curing assessment.
Description
Technical field
The invention belongs to the detection technique field, be specifically related to a kind of Procalcitonin quick detection kit of multispecific antibody mark.
Background technology
1993, Lancet reported first Procalcitonin (PCT, PCT) level significantly raises when septicemia, time prompting septicemia may greater than 0.5ng/ml for blood PCT concentration, existence greater than 2ng/ml prompting septicemia, then point out serious pyaemia septica or shock greater than 10ng/ml, PCT can be used as the specific index of diagnosis of sepsis septicemia.
In recent years, along with going deep into of research, people find gradually, and bacterium infects low-level rising that PCT has often been arranged in early days (0.1ng/ml～0.5ng/ml), the PCT elevated levels is relevant with the bacterial infection order of severity and scope, and the PCT level advises using antibiotic therapy during greater than 0.25ng/ml.Normal health crowd PCT level extremely low (less than 0.05ng/ml), non-bacterial infection or inflammation are (such as viral infection, fungal infection or autoimmune inflammation etc.) often do not follow rising or slight rising of PCT, therefore PCT can not only be as the diagnosis index of septicemia, broader applications are in early diagnosis and the antidiastole of bacterial infection, instruct clinical antibiotics to use, and be used for the treatment of curative effect and Prognosis scoveillance.Adopt the Procalcitonin detection kit of colloid gold label that detection characteristics quick, easy and simple to handle are arranged, but the Procalcitonin colloidal gold immunochromatographimethod reagent detection sensitivity under the routine techniques is difficult to reach 0.25ng/ml.
Application number is open day of 93118343.X(: patent document 1993.8.19) has disclosed a kind of medicine box of the method for realizing the early detection septicemia and treatment being followed the tracks of, it is 0.1～500ng/ml serum or blood plasma that this medicine box can be measured PCT content, and then judge the existence of septicemia, the order of severity and/or to the result for the treatment of of septicemia; This medicine box specifically comprises two kinds of antibody: (1) one or more the first monoclonal or polyclonal antibodies of being combined in order to the PCT in sample on the carrier that are fixed on; (2) another antibody that carries mark and combine in another zone of the calmodulin binding domain CaM that is different from first antibody with PCT.The technique effect that this medicine box can be realized is: the content of the non-complete calcitonin fragments by measuring peptide PCT in patient's humoral sample and/or its formation, whether exist by measuring this definite peptide, what of content, draw relevant whether have septicemia, the order of severity of septicemia and/or the conclusion of result for the treatment of.
Yet, there are many technological deficiencies in the technical scheme that the aforementioned patent document discloses, those skilled in the art clearly can know by general knowledge, in the aforementioned patent so-called " one or more be fixed on the carrier in order to in the sample before the first monoclonal or the polyclonal antibody that calcium is combined falls ", its essence is the capture antibody of PCT, but in this technical scheme, corresponding labelled antibody only is a kind of.Patent documentation is pointed out in the 4th page in its instructions: " PCT " on the meaning of the present invention represents one or more peptides; All these peptides all comprise the peptide sequence that forms with upper amino acid by 57, particularly resemble 116 amino acid sequences the complete PCT, they or consistent with known sequence, perhaps part is consistent, just may there are differences at the 108th to 116 amino acid region corresponding to PCT.Obviously, only a kind of labelled antibody described in the patent documentation can't be realized multiple " PCT " fragment is carried out significant notation, " PCT " thereby that can't guarantee as far as possible multidigit point combination is detected, if adopt the developers such as collaurum, the colored magnetic bead single antibody of substance markers that serves as a mark in this patented technology, to cause the sensitivity that detects on the low side, the application in practice effect is had a greatly reduced quality.
For example: the achieved detection lower limit of Procalcitonin colloidal gold immunochromatographimethod reagent PCT-Q that comes from this patented technology only is 0.5ng/ml, this patent is that German B.R.A.H.M.S is used, only satisfying the sieving and diagnosis of septicemia, and can not reach for the bacterium Infect And Diagnose and instruct the detection sensitivity demand of the 0.1ng/ml that microbiotic uses; Analyze its reason; pass through collaurum; the labelled antibodies such as colored magnetic bead are realized detecting; be based on collaurum; colored magnetic bead has colour rendering; when a certain amount of collaurum or colored magnetic bead by being labeled antibody after determined antigen is combined; combination by determined antigen and capture antibody so that colloid gold particle or colored magnetic bead particles flock together; then form macroscopic lines; when determined antigen concentration higher; the collaurum or the magnetic bead that then flock together are more; the intensity of lines is just stronger; analyze interpretation by supporting instrument, just can measure the concentration of determined antigen.Take collaurum as example, routine is many at 20～40nm for the diameter of the colloid gold particle of antibody labeling, Existential Space steric hindrance between the colloid gold particle, if just mark is for an antibody in the some zones of Procalcitonin, the quantity that can be combined in so the collaurum on the antibody is limited, when the concentration of Procalcitonin to be measured is very low, can in conjunction with the quantity of this labelled antibody also be seldom, even if therefore adopt a plurality of antibody as capture antibody, can be also less by the be hunted down quantity of the collaurum assembled of labelled antibody, be difficult to form macroscopic lines, therefore be difficult to the detection sensitivity that reaches higher.And adopt the technical program to carry out the multispecific antibody mark of zones of different, Procalcitonin to be measured can be in conjunction with the labelled antibody of zones of different, the colloid gold particle that can be combined on the Procalcitonin so is more, even if when the very low Procalcitonin of detectable concentration, its colloid gold particle that can assemble is more more easily to produce analyzable lines, thus the detection sensitivity of significantly improving.
Open day of Chinese invention patent application CN101029897A(: 2007.9.5) disclosed a kind of Procalcitonin test kit and method of testing thereof, the method adopts nano immune magnetic microballon as separation agent, luminous marker adopts the different Derivative of Luminol of anti-PCT labeling of monoclonal antibody, adopt PCT kit of the present invention and method of testing thereof, can reach other characteristics of pg/ml level based on the chemoluminescence method detection sensitivity, although its labelled antibody only is a kind of, but it still can possess higher sensitivity, can realize that PCT is in the application in bacterium Infect And Diagnose field, but, chemical illuminating reagent needs supporting large-scale chemical illumination instrument, and can not the direct-detection whole blood, need specimen centrifuge during detection, dilution, the multiple steps such as calibration, operating process is complicated, detection time is long, is not suitable for quick diagnosis.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of Procalcitonin quick detection kit of multispecific antibody mark is provided, the PCT that can realize 0.1～200ng/ml concentration range detects, and is used for diagnosis, antidiastole, treatment curative effect monitoring and the prognosis evaluation of bacterial infection.With the antibody of tense marker for the Procalcitonin different loci, can overcome better the sterically hindered impact of the labels such as collaurum, so that be combined with Procalcitonin in the labelled antibody maximizing ground of zones of different, improve sensitivity, and because the antibody centering of compatibility is contained the antibody for Procalcitonin specific peptide fragment, but the specific recognition Procalcitonin, and itself and calcitonin made a distinction, thereby realize the performance that high sensitivity and high specific detect.
The present invention realizes by following technical scheme,
The present invention relates to a kind of Procalcitonin quick detection kit of multispecific antibody mark; Described kit comprise conventional carrier and:
(1) is fixed on monoclonal or the polyclonal capture antibody of the corresponding Procalcitonin zones of different on the described conventional carrier;
And (2) and (1) described capture antibody compatibility, with a plurality of monoclonal or the polyclonal antibodies for the identical or different zone of Procalcitonin of appropriate flags substance markers.
Preferably, the corresponding Procalcitonin zones of different of described capture antibody refers to N end regions or the C end regions of Procalcitonin.
Preferably, the list of described and capture antibody compatibility or polyclonal antibody for the identical or different zone of Procalcitonin refer to: calcitonin zone and C end regions, or calcitonin is regional and the N end regions.
Preferably, in the described kit, every kind of consumption that is labeled antibody is during all less than this antibody separate marking and minimum amount that can stable labelling thing solution, and then guarantees that every kind of antibody can be labeled the thing significant notation.
Preferably, described label is collaurum, colored magnetic bead or latex particle.
Compared with prior art, the present invention has following beneficial effect:
The Procalcitonin quick detection kit of multispecific antibody mark of the present invention by mark and coated multispecific antibody right different identified regions and space compatibility, can catch Procalcitonin in the sample to greatest extent, improves detection sensitivity;
Kit of the present invention improves 0.1ng/ml with the detection sensitivity of PCT by 0.5ng/ml, can realize the PCT of 0.1～200ng/ml, using value and the scope of PCT quick detection reagent in bacterial infection diagnosis, antidiastole and treatment expanded in detection, can be used for diagnosis, antidiastole, treatment curative effect monitoring and the prognosis evaluation of bacterial infection;
Simultaneously because the antibody centering of compatibility is contained the antibody for Procalcitonin specific peptide fragment, but the specific recognition Procalcitonin and its and calcitonin made a distinction, thereby realize the performance of high sensitivity and high specific detection;
Kit of the present invention only can be realized whole testing processes with 15 minutes in use, and efficient is greatly enhanced;
Kit of the present invention can be used for the direct-detection whole blood, really realizes the single step operation;
Therefore, adopt PCT of the present invention to detect reagent, both guaranteed the easy, quick of detection, realized the unapproachable high sensitivity of conventional quick detection reagent by the multispecific antibody mark again, use for the promptly and accurately diagnosis that advances bacterium to infect and antibiotic standard, have important social effect.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are not used in for explanation the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor LaboratoryPress, 1989) condition described in, or the condition of advising according to manufacturer.
The present inventor has overcome many technical matterss by a large amount of research, and then obtains technical scheme of the present invention.The present inventor carries out the compatibility selection of capture antibody and labelled antibody and realizes that the technical barrier such as effective multispecific antibody mark paid performing creative labour for solving for Procalcitonin or its peptide.
The present invention is when selecting best compatibility antibody, at first carry out single antibody labeling, as: capture antibody be N end regions monoclonal and (or) polyclonal antibody, then after the antibody separate marking to calcitonin zone and C end regions, with the capture antibody combined test, choose the highest labelled antibody of sensitivity respectively; Capture antibody be C end regions monoclonal and (or) polyclonal antibody, then after the antibody separate marking to calcitonin zone and N end regions, with the capture antibody combined test, choose the highest labelled antibody of sensitivity respectively.
The labelled antibody of zones of different is then determined label concentration and the mark pH value of multiple labelled antibody by little sample testing after determining.At first find out the minimum amount of every kind of labelled antibody energy stable labelling thing solution.Two kinds of antibody are during with tense marker, and the consumption of every kind of antibody is minimum amount 80%, and three or more antibody is during with tense marker, and then the consumption of every kind of antibody is 40% of minimum amount.After determining the label concentration of antibody, mixed antibody then, determine that according to routine the method for mark pH value is determined the pH value after, carry out afterwards the batch mark.
In the kit of the present invention, the a plurality of antibody that are labeled need according to screening with the sensitivity of capture antibody compatibility rather than random compatibility, with corresponding capture antibody compatibility, the antibody that detection sensitivity is the highest in the same area is defined as final labelled antibody behind the alternative antibody single marking; Simultaneously, best pH value is determined by the sample test of gradient pH value during the multispecific antibody mark, and adopting the pH value of the highest group of 525nm wavelength place optical density value is optimum mark PH.
The Procalcitonin quick detection kit of embodiment, the multispecific antibody mark take collaurum as label
Step 1 is determined capture antibody and labelled antibody
(1) selects two monoclonal antibodies for Procalcitonin N end regions, perhaps choose 1 monoclonal antibody and 1 polyclonal antibody, be fixed on after it is mixed on the cellulose membrane as capture antibody, select the monoclonals of a plurality of and C end regions regional for calcitonin or the polyclonal antibody antibody that serves as a mark; The aforementioned monoclonal antibody that relates to, polyclonal antibody all can obtain by open channel;
(2) use respectively colloid gold label for monoclonal or the polyclonal antibody of calcitonin zone and C end regions, and spray to the gold mark with the concentration of OD25 respectively and fill up 37 ℃ of oven dry;
(3) carry out combined test with capture antibody, adopt routine techniques to filter out for calcitonin zone and the highest monoclonal or the polyclonal antibody of C end regions detection sensitivity, as final labelled antibody;
Step 2 is determined optimum mark concentration and pH value
(1) determines the minimum mark amount of each antibody;
1. according to the isoelectric point of antibody to be marked, collaurum is mixed up after the pH packing 10 pipes, every pipe 1ml;
2. labelled protein is done serial dilution as 5 μ g/ml～40 μ g/ml take the mark damping fluid, get respectively 1ml, add in the above-listed gold size solution, mixing, control tube only adds the 1ml dilution;
3. behind the 5min, in above-mentioned each pipe, add the NaCl solution of 0.1ml 10%, leave standstill 2h behind the mixing, observe;
4. the result observes, and the quantity not sufficient of control tube (not adding protein) and adding protein all presents the coagulation phenomenon by red stain indigo plant with each pipe of stable colloid gold; Still maintenance is red constant and the adding protein content meets or exceeds each quantitative pipe of minimum steady; To stablize the red constant minimum antibody consumption of 1ml colloidal gold solution, be the minimum mark amount of this antibody; Antibody minimum mark amount through experiment calcitonin zone is the 10ug/ml colloidal gold solution, and the antibody minimum mark amount of C end regions is the 12ug/ml colloidal gold solution;
(2) antibody of determining the calcitonin zone adds in the 1ml colloidal gold solution as final label concentration, with this concentration searching optimum mark pH value take 9.6ug with the antibody of 8ug, C end regions;
1. adopt the antibody in 16ug calcitonin zone and the antibody of 19.2ugC end regions/1.8ml collaurum ratio to determine optimal pH;
2. pH gradient: pH6.0 is set, 6.5,7.0,7.5,8.0,8.5,9.0,9.5 mark damping fluids;
3. get 8 test tubes, in order, add respectively 400ul ddH2O, the mark damping fluid of the different pH values of 200ul, mixing treat labeling antibody, colloidal gold solution 1.8ml leaves standstill 5～10min after mixing, the NaCl solution 200ul of adding 10%;
If the at first removal by the coagulation phenomenon of red stain indigo plant 4. occurs; If the need that range estimation can't be clear and definite detect the OD value at 525nm place, the highest pH value of selection OD525, result prove that the pH value is 7.5 the most suitable;
Step 3 is according to antibody concentration and the best pH value labelled antibody determined
(1) basis goes out the total amount of needed antibody to be marked in order to the calculation of total of the collaurum of mark;
(2) under electromagnetic agitation, it is in 7.5 the colloidal gold solution that antibody-solutions is added the modulated pH of deciding value, should dropwise add when adding protein, and the about 5min of the protein of 1mg adds;
(3) adding 5% bovine serum albumin(BSA) (BSA) under magnetic stirring, to make its final concentration be 1%, stirred 15 minutes;
(4) during the collaurum that mark is good was packed bag filter into, tightened at two, puts into sucrose or silica gel concentrated, is concentrated to 1/10 amount of original volume, concentrated complete rear purifying;
Step 4, the collaurum that mark is good sprays to formation gold mark pad on the glass fibre, 37 ℃ of oven dry with the concentration of OD25;
Step 5, capture antibody (mouse) has been determined to form detection line (T line) on the position to nitrocellulose filter with the suitable concn specking, and another determines to form on the position control line (C line) to nitrocellulose filter with the suitable concn specking with sheep anti-mouse igg antibody;
Step 6 is marked pad, nitrocellulose filter, sample pad, hemofiltration film and thieving paper opsition dependent with gold and is pieced into agent plate together, and slitting dress shell namely forms Procalcitonin and detects reagent card;
Step 7, Procalcitonin standard items with concentration 0.1～200ng/ml are tested, and put into the necessary instrument interpretation, and its detection sensitivity can reach 0.1ng/ml, and the detection reagent of monoclonal antibody body tag before this, its sensitivity of maximum sensitivity group only can reach 0.4ng/ml.
Those skilled in the art know the following fact, when label was selected colored magnetic bead or latex particle, kit of the present invention also can be realized same implementation result, realized the performance that high sensitivity and high specific detect, and this moment, those skilled in the art need not to pay performing creative labour.
In sum, the Procalcitonin quick detection kit of multispecific antibody mark of the present invention by mark and coated multispecific antibody right different identified regions and space compatibility, can catch Procalcitonin in the sample to greatest extent, improves detection sensitivity; Kit of the present invention is increased to 0.1ng/ml with the detection sensitivity of PCT, the PCT that can realize 0.1～200ng/ml detects and to have expanded using value and the scope of PCT quick detection reagent in bacterial infection diagnosis, antidiastole and treatment, can be used for diagnosis, antidiastole, treatment curative effect monitoring and the prognosis evaluation of bacterial infection.
Above specific embodiments of the invention are described.It will be appreciated that, the present invention is not limited to above-mentioned specific implementations, and those skilled in the art can make various distortion or modification within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (5)
1. the Procalcitonin quick detection kit of a multispecific antibody mark is characterized in that, described kit comprise conventional carrier and:
(1) is fixed on monoclonal or the polyclonal capture antibody of the corresponding Procalcitonin zones of different on the described conventional carrier;
And (2) and (1) described capture antibody compatibility, with a plurality of monoclonal or the polyclonal antibodies for the identical or different zone of Procalcitonin of appropriate flags substance markers.
2. the Procalcitonin quick detection kit of multispecific antibody mark as claimed in claim 1 is characterized in that, the corresponding Procalcitonin zones of different of described capture antibody refers to N end regions or the C end regions of Procalcitonin.
3. the Procalcitonin quick detection kit of multispecific antibody mark as claimed in claim 1, it is characterized in that, the list of described and capture antibody compatibility or polyclonal antibody for the identical or different zone of Procalcitonin refer to: calcitonin zone and C end regions, or calcitonin is regional and the N end regions.
4. the Procalcitonin quick detection kit of multispecific antibody mark as claimed in claim 1, it is characterized in that, in the described kit, every kind of consumption that is labeled antibody is during all less than this antibody separate marking and minimum amount that can stable labelling thing solution, and then guarantees that every kind of antibody can be labeled the thing significant notation.
5. the Procalcitonin quick detection kit of multispecific antibody mark as claimed in claim 1 is characterized in that, described label is collaurum, colored magnetic bead or latex particle.
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| CN103645321A (en) * | 2013-12-03 | 2014-03-19 | 张超 | Test paper for screening procalcitonin and preparation method of test paper |
| CN104090109A (en) * | 2014-07-25 | 2014-10-08 | 胡晓武 | Colloidal gold immunochromatography test paper and colloidal gold immunochromatography test method for quickly detecting human blood procalcitonin |
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| CN104237525B (en) * | 2013-06-24 | 2016-04-13 | 北京美康生物技术研究中心有限责任公司 | A kind of latex enhancing immune for measuring Procalcitonin is than turbid kit and its preparation method and application |
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| CN104792997A (en) * | 2014-01-22 | 2015-07-22 | 天津汇滨生物科技有限公司 | Human procalcitonin immunodetection kit, and preparation method and application thereof |
| CN104090109A (en) * | 2014-07-25 | 2014-10-08 | 胡晓武 | Colloidal gold immunochromatography test paper and colloidal gold immunochromatography test method for quickly detecting human blood procalcitonin |
| CN104531715A (en) * | 2014-10-27 | 2015-04-22 | 西北工业大学 | Preparation of human procalcitonin recombination expression monoclonal and polyclonal antibody and ELISA detection method |
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| CN105866420A (en) * | 2015-01-22 | 2016-08-17 | 艾托金生物医药（苏州）有限公司 | Method and apparatus for detecting immunogen |
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| CN109734791A (en) * | 2019-01-17 | 2019-05-10 | 武汉明德生物科技股份有限公司 | People NF186 antigen, people's NF186 antibody assay kit and the preparation method and application thereof |
| CN109734790B (en) * | 2019-01-17 | 2022-05-24 | 武汉明德生物科技股份有限公司 | Human Agrin antigen, human Agrin antibody detection kit, preparation method and application thereof |
| CN109734792B (en) * | 2019-01-17 | 2022-05-24 | 武汉明德生物科技股份有限公司 | Human CNTN1 antigen, human CNTN1 antibody detection kit, preparation method and application thereof |
| CN109734791B (en) * | 2019-01-17 | 2022-07-12 | 武汉明德生物科技股份有限公司 | Human NF186 antigen, human NF186 antibody detection kit, preparation method and application thereof |
| CN109734795B (en) * | 2019-01-17 | 2022-07-12 | 武汉明德生物科技股份有限公司 | Human Lrp4 antigen, human Lrp4 antibody detection kit, preparation method and application thereof |
| CN113125741A (en) * | 2019-12-30 | 2021-07-16 | 深圳市帝迈生物技术有限公司 | Procalcitonin detection reagent, kit, system and detection method |
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