CN204287192U - Myeloperoxidase immunochromatographiassay assay quantitative detection test paper - Google Patents
Myeloperoxidase immunochromatographiassay assay quantitative detection test paper Download PDFInfo
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- CN204287192U CN204287192U CN201420645293.4U CN201420645293U CN204287192U CN 204287192 U CN204287192 U CN 204287192U CN 201420645293 U CN201420645293 U CN 201420645293U CN 204287192 U CN204287192 U CN 204287192U
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Abstract
The utility model discloses a kind of myeloperoxidase immunochromatographiassay assay quantitative detection test paper, comprise base plate, sample pad, fluorescent marker pad, cellulose nitrate and adsorptive pads; Described test strips is overlapped successively to be pasted onto on base plate formed by sample pad, fluorescent marker pad, cellulose nitrate, adsorptive pads.Described fluorescent marker pad contains marked by streptavidin fluorescin and biotin labeled myeloperoxidase monoclonal antibody; Described nitrocellulose membrane has detection line and nature controlling line, detection line is by myeloperoxidase monoclonal antibody, and it has different identification epi-positions from above-mentioned biotin labeled myeloperoxidase monoclonal antibody.The utility model is (as: chemoluminescence method/Immune-enhancing effect turbidimetry/colloidal gold method) compared with the method for detection myeloperoxidase common at present, not only substantially reduces detection time, also improves detection sensitivity simultaneously.
Description
Technical field
The utility model belongs to field of immunodetection, is specifically related to a kind of high sensitivity myeloperoxidase immunochromatographiassay assay quantitative detection test paper.
Background technology
Myeloperoxidase (MPO) detection kit (fluorescence immune chromatography method), for quantitatively detecting myeloperoxidase (Myeloperoxidase, MPO) in serum, blood plasma or whole blood.Myeloperoxidase is also called peroxidase, and being a kind of important iron content lysosome, being present in the azurophilic granule of myeloid cell (mainly neutrophil leucocyte and monocyte), is myelocytic specificity marker.
Along with going deep into of studying MPO, it is found that MPO gene pleiomorphism causes the individual difference to some diseases neurological susceptibility, with mankind's various diseases have close contacting, such as angiocardiopathy, tumour, leukaemia, endemic arsenic poisoning etc.Research shows, MPO is as a kind of Inflammation Marker, both relevant with innate immune defence also relevant with tissue damage, angiocardiopathy their early stage and develop into the atherosclerotic acute compound pathology phase, the pathways of inflammation of MPO and mediation thereof facilitates atherosclerotic formation, therefore, MPO can as the index of the developing of predicting cardiovascular disease, prognosis and assessment risk stratification.
The common method of current detection myeloperoxidase has:
1, colourimetry:
The method is under the condition of acidity, and with tetramethyl benzidine, o-methoxyphenol or 3,3-dimethylbenzidine for substrate, the MPO measured in blood or tissue is active.General under the condition of PH=6.0, with H2O2 and 3,3-dimethoxy benzidine hydrochloride for substrate, MPO catalytic substrate becomes crocus product, and at 460nm wavelength, place monitors continuously, and the rate that increases of absorbance is directly proportional to the activity of MPO.This method has the features such as quick, sensitive, cost is low, but is subject to other peroxidase (such as eosinophile peroxidase), and the interference of some protoheme (such as haemoglobin, myoglobins).
2, enzyme linked immunosorbent assay (ELISA):
Enzyme-linked immunosorbent assay is based on immunological response, antigen and antibody specific is combined the experimental technique that a kind of susceptibility of combining with the efficient catalytic product effect of zymolyte is very high.Utilize MPO antigenicity to prepare specific antibody, directly measured the protein content of enzyme in blood by immunological method.This method is highly sensitive, can reach the level of ng/ml; Specificity is high, hardly by the impact of activator, inhibitor in body fluid, does not also disturb by peroxidase and haemoglobin.Due to needs preparation high price antibody, cost is higher, and minute is longer, and therefore, this fado is used for scientific research field at present.
3, cells were tested by flow cytometry method:
Measure the content of MPO in neutrophil leucocyte slurry, whole blood is added in blood cytolysate, rear use immobile liquid and membrane permeability agent process cell, finally add fluorescein-labeled antibody MPO monoclonal antibody, antigen-antibody reaction is utilized to dye to MPO, flow cytometry is carried out to specific cells Colony Design, analyzes ratio and the average fluorescent strength of MPO positive cell.This method high specificity, highly sensitive, but technical program is complicated, needs to use specific apparatus.
4, alkaline phosphatase-alkali resistance phosphatase bridging enzyme decoration method (APAAP method)
Alkaline phosphatase-alkali resistance phosphatase bridging enzyme decoration method (APAAP method) is a kind of immunohistochemistry technique.Utilize how anti-Ab to play bridging effect, one of them Fab section connects McAb, and another Fab section connects APAAP compound, makes it the compound becoming Ag-Ab1-Ab2-anti-AP-AP.The existence of antigen molecule is judged again by the alkaline phosphatase substrate for enzymatic activity colour developing in compound, also anti-APAAP compound is repeated to stack up by two, thus the antigenic site that multiple alkali phosphatase enzyme mark first antibody on histocyte is identified, improve susceptibility.The method is owing to using immune bridging technology, therefore its susceptibility is high, and result is easy to judge, additionally reduces the impact of endogenous enzyme, high specificity simultaneously.Be widely used in the detection etc. of lymphocyte differentiation antigen, active antigen, MHC-I, II class antigen presentation at present.
Biotin-avidin system (biotinavidin system, BAS) is a kind of new bio reaction amplification system.Along with the appearance of various biotin derivative, BAS is widely used in each field of medical science very soon.This system is applied to SABC, enzyme linked immunological, fluorescence immunoassay, in the detection techniques such as radio-immunity, the susceptibility of above technical method, specificity and stability can be improved significantly, make method easier, contribute to clinical quick diagnosis, and be beneficial to and carry out extensive epidemiology survey, become the immunoreactive powerful of research.Because BAS detection system economy is quick, pollute without radiating matter again, do not need complex instrument, fully show the great potential of this system and the possibility of application.
Biotin-avidin system Cleaning Principle: BAS is on the basis of conventional ELISA principle, in conjunction with the height amplification between biotin and Avidin, and a kind of detection system set up.Avidin is a kind of alkaline glycoprotein extracted in ovalbumin, and molecular weight is 68kDa, is made up of 4 subunits, have very high affinity (binding constant is up to 1015M-1) to biology.Biotin is very easily combined with covalent bond with protein (as antibody etc.).Like this, the Avidin molecule combining enzyme and the biotin molecule being combined with specific antibody produce and react, both served multistage amplification, the colour generation due to the catalytic action of enzyme when running into corresponding substrate, reaches the object detecting unknown antigen (or antibody) molecule again.Streptavidin is and the affine a kind of protein have similar biological properties, in Streptavidin molecule the same as Avidin, every bar peptide chain can in conjunction with a biotin, because nearly all material for marking all can combine with Avidin or streptavidin.Utilize biotin-avidin system to develop myeloperoxidase detection examination fast diagnosis reagent and there is no report.
Utility model content
The purpose of this utility model, be to provide a kind of myeloperoxidase immunochromatographiassay assay quantitative detection test paper, it provides high sensitivity myeloperoxidase immunochromatographiassay assay quantitative detection test paper based on double antibody sandwich method, fluorescence immunoassay lateral chromatography and biotin-Streptavidin amplification system.When using the utility model, improve detection sensitivity and detect stability, reducing non-specific binding, be not more than 10 minutes detection time, substantially increase diagnosis efficiency.
The solution that the utility model solves its technical matters is: myeloperoxidase immunochromatographiassay assay quantitative detection test paper, it comprises base plate, described base plate is provided with sample pad successively, fluorescent marker pad, nitrocellulose filter and adsorptive pads, described adsorptive pads and fluorescent marker pad are overlapping to be respectively pressed in behind the two ends of nitrocellulose filter in the formation detection zone, surface of nitrocellulose filter, described sample pad is overlapping to be pressed on fluorescent marker pad, described fluorescent marker pad is fixed with the fluorescin of biotin labeled myeloperoxidase monoclonal antibody and marked by streptavidin, nitrocellulose filter in described detection zone is fixed with the nature controlling line of detection line and the sheep anti-mouse igg polyclonal antibody formation identifying that the monoclonal antibody of myeloperoxidase another one epi-position is formed.
As the further improvement of technique scheme, described fluorescent marker pad comprises the first stacked fluorescent marker pad and the second fluorescent marker pad, one end pad of described first fluorescent marker pad in the below of sample pad, described second overlapping one end being pressed in nitrocellulose filter of fluorescent marker pad.
As the further improvement of technique scheme, in described first fluorescent marker pad, the one end of inserting below sample pad is provided with positioning strip, and the lower surface of described sample pad is provided with the locating slot that can hold positioning strip.
As the further improvement of technique scheme, also comprise and getting stuck, in described base plate, sample pad, fluorescent marker pad, nitrocellulose filter and adsorptive pads are all placed in and get stuck, the position described shell face of getting stuck corresponding to nitrocellulose membrane is provided with view window, and position shell face of getting stuck corresponding to sample pad is provided with well.
As the further improvement of technique scheme, described view window is slot.
As the further improvement of technique scheme, described fluorescin can be the one in green fluorescent protein, phycobniliprotein.
As the further improvement of technique scheme, described sample pad is glass fibre component dry after surfactant damping fluid immersion treatment.
As the further improvement of technique scheme, described base plate is polystyrene component or tygon component.
As the further improvement of technique scheme, described in get stuck and comprise plastics upper casing and plastics lower casing, described plastics upper casing fastens to be formed after on plastics lower casing and gets stuck.
Compared with prior art, tool has the following advantages the utility model:
(1) the utility model is using fluorescin as label, and this label has good stability, and is conducive to improving detecting stability.
(2) the utility model is by utilizing " Streptavidin-biotin amplification system ", improves detection sensitivity, reduces non-specific binding, is conducive to improving kit performance.
(3) detection time utilizing the utility model to carry out Procalcitonin is not more than 10 minutes, and the detection range of linearity is 0.10ng/ml ~ 100.00ng/ml, drastically increases detection efficiency.
(4) the utility model carries out interpretation by fluorescence immunity analyzer to result, can realize robotization, reduces the impact of subjective factor, provides convenient, quick, reliable diagnostic result.
(5) novel the getting stuck of this use is provided with the well that sample enters and the view window supplying observed result, according to analytical instrument result of determination, accurately and reliably.
(6) the utility model is easy to make, and volume is little, be easy to carry.
(7) the utility model testing cost is lower.
(8) the utility model can be mass, and is applicable to clinical quick diagnosis and on-the-spot quick diagnosis; Be easy to preserve, be conducive to grass-roots unit and promote.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the utility model embodiment, below the accompanying drawing used required in describing embodiment is briefly described.Obviously, described accompanying drawing is a part of embodiment of the present utility model, instead of whole embodiment, and those skilled in the art, under the prerequisite not paying creative work, can also obtain other design proposals and accompanying drawing according to these accompanying drawings.
Fig. 1 is the structural representation of the utility model embodiment one;
Fig. 2 is the structural representation of the utility model embodiment two;
Fig. 3 is the structural representation of the utility model embodiment three;
Fig. 4 is the structural representation of the utility model embodiment four.
Embodiment
Be clearly and completely described below with reference to embodiment and the accompanying drawing technique effect to design of the present utility model, concrete structure and generation, to understand the purpose of this utility model, characteristic sum effect fully.Obviously; described embodiment is a part of embodiment of the present utility model, instead of whole embodiment, based on embodiment of the present utility model; other embodiments that those skilled in the art obtains under the prerequisite not paying creative work, all belong to the scope of the utility model protection.In addition, all connection/annexations mentioned in literary composition, not singly refer to that component directly connects, and refer to and according to concrete performance, can connect auxiliary by adding or reducing, and form more excellent draw bail.
With reference to Fig. 1, myeloperoxidase immunochromatographiassay assay quantitative detection test paper, it comprises base plate 5, described base plate 5 is provided with sample pad 1 successively, fluorescent marker pad 2, nitrocellulose filter 3 and adsorptive pads 4, described adsorptive pads 4 and fluorescent marker pad 2 are overlapping to be respectively pressed in behind the two ends of nitrocellulose filter 3 in the formation detection zone, surface of nitrocellulose filter 3, fluorescent marker pad 2 is glass fibre membrane, described sample pad 1 is overlapping to be pressed on fluorescent marker pad 2, the fluorescin described fluorescent marker pad 2 being fixed with biotin labeled myeloperoxidase monoclonal antibody (concentration 0.3 ~ 1.5mg/mL) and marked by streptavidin (or Avidin) (uses excitation wavelength 530nm, wavelength of transmitted light 570nm, concentration 0.1 ~ 1.0mg/mL), nitrocellulose filter 3 in described detection zone is fixed with the nature controlling line C (concentration 0.2 ~ 2.0mg/mL) of detection line T (concentration 0.5 ~ 3mg/mL) and the sheep anti-mouse igg polyclonal antibody formation identifying that the monoclonal antibody of myeloperoxidase another one epi-position is formed, nature controlling line C is used for the validity of test strip.
The fluorescin that biotin labeled myeloperoxidase monoclonal antibody and Streptavidin (or Avidin) mark is fixed on fluorescent marker pad 2, the monoclonal antibody and sheep anti-mouse igg polyclonal antibody that have identification myeloperoxidase another one epi-position are fixed on nitrocellulose filter as detection line T and nature controlling line C.When testing sample is added to after in sample pad 1, moved forward by chromatography effect, in sample, on myeloperoxidase and fluorescent marker pad 2, the myeloperoxidase enzyme antibody (Mab-MPO*Fluoro) of combined with fluorescent label (fluorescin) reacts and forms compound MPO-Mab-MPO*Fluoro, compound is reacted when continuing to be advanced past MPO antibody (detection line) nitrocellulose membrane wrapping quilt under chromatography effect, MPO antibody capture formation compound (Mab-MPO-MPO-Mab-MPO*Fluoro) (detection line) that reaction compound is coated, the reaction signal of detection line is read by fluorescence immunity analyzer, under the effect of excitation source, fluorescent material launches the fluorescence signal of specific wavelength, fluorescence immunity analyzer captures fluorescence signal, the typical curve transformed by signal and set is converted into quantitative value automatically, calculate the concentration of myeloperoxidase in sample, obtain myeloperoxidase testing result.
As the further improvement of technique scheme, with reference to Fig. 3, described fluorescent marker pad 2 comprises the first stacked fluorescent marker pad 20 and the second fluorescent marker pad 21, one end pad of described first fluorescent marker pad 20 in the below of sample pad 1, described second overlapping one end being pressed in nitrocellulose filter 3 of fluorescent marker pad 21.The layering of fluorescent marker pad 2 is arranged, and is convenient to fluorescent marker pad 2 to be arranged between sample pad 1 and nitrocellulose filter 3.
As the further improvement of technique scheme, be provided with positioning strip 22 with reference to one end of inserting below sample pad in the first fluorescent marker pad 20 described in Fig. 3, the lower surface of described sample pad 1 is provided with the locating slot that can hold positioning strip 22.The installation be convenient between fluorescent marker pad 2 and sample pad 1 by positioning strip 22 is fixed.
As the further improvement of technique scheme, with reference to Fig. 2 and Fig. 4, also comprise and getting stuck, described base plate 5, sample pad 1, fluorescent marker pad 2, nitrocellulose filter 3 and adsorptive pads 4 are all placed in and get stuck in 6, the position described 6 shell faces of getting stuck corresponding to nitrocellulose membrane 3 is provided with view window 8, and the position 6 shell faces of getting stuck corresponding to sample pad 1 is provided with well 7.
As the further improvement of technique scheme, described view window 8 is slot.
As the further improvement of technique scheme, described fluorescin can be the one in green fluorescent protein, phycobniliprotein.
As the further improvement of technique scheme, described sample pad 1 is glass fibre component dry after surfactant damping fluid immersion treatment.Sample pad is made up of glass fibre, through surfactant damping fluid immersion treatment, uses after dry.The cutting width of described sample pad 1 is 0.3 ~ 0.5cm.
As the further improvement of technique scheme, described base plate 5 is polystyrene component or tygon component.
As the further improvement of technique scheme, described in get stuck and 6 comprise plastics upper casing 60 and plastics lower casing 61, described plastics upper casing 61 fastens to be formed after on plastics lower casing 61 and gets stuck 6.
More than that better embodiment of the present utility model is illustrated, but the invention is not limited to described embodiment, those of ordinary skill in the art also can make all equivalent modifications or replacement under the prerequisite without prejudice to the utility model spirit, and these equivalent modification or replacement are all included in the application's claim limited range.
Claims (9)
1. myeloperoxidase immunochromatographiassay assay quantitative detection test paper, it is characterized in that: it comprises base plate, described base plate is provided with successively sample pad, fluorescent marker pad, nitrocellulose filter and adsorptive pads, described adsorptive pads and fluorescent marker pad are overlapping to be respectively pressed in behind the two ends of nitrocellulose filter in the formation detection zone, surface of nitrocellulose filter, described sample pad is overlapping to be pressed on fluorescent marker pad, described fluorescent marker pad is fixed with the fluorescin of biotin labeled myeloperoxidase monoclonal antibody and marked by streptavidin; Nitrocellulose filter in described detection zone is fixed with the nature controlling line of detection line and the sheep anti-mouse igg polyclonal antibody formation identifying that the monoclonal antibody of myeloperoxidase another one epi-position is formed.
2. myeloperoxidase immunochromatographiassay assay quantitative detection test paper according to claim 1, it is characterized in that: described fluorescent marker pad comprises the first stacked fluorescent marker pad and the second fluorescent marker pad, one end pad of described first fluorescent marker pad in the below of sample pad, described second overlapping one end being pressed in nitrocellulose filter of fluorescent marker pad.
3. myeloperoxidase immunochromatographiassay assay quantitative detection test paper according to claim 2, it is characterized in that: in described first fluorescent marker pad, the one end of inserting below sample pad is provided with positioning strip, and the lower surface of described sample pad is provided with the locating slot that can hold positioning strip.
4. the myeloperoxidase immunochromatographiassay assay quantitative detection test paper according to any one of claims 1 to 3, it is characterized in that: also comprise and getting stuck, in described base plate, sample pad, fluorescent marker pad, nitrocellulose filter and adsorptive pads are all placed in and get stuck, the position described shell face of getting stuck corresponding to nitrocellulose membrane is provided with view window, and position shell face of getting stuck corresponding to sample pad is provided with well.
5. myeloperoxidase immunochromatographiassay assay quantitative detection test paper according to claim 4, is characterized in that: described view window is slot.
6. myeloperoxidase immunochromatographiassay assay quantitative detection test paper according to claim 4, is characterized in that: described fluorescin can be the one in green fluorescent protein, phycobniliprotein.
7. myeloperoxidase immunochromatographiassay assay quantitative detection test paper according to claim 4, is characterized in that: described sample pad is glass fibre component dry after surfactant damping fluid immersion treatment.
8. myeloperoxidase immunochromatographiassay assay quantitative detection test paper according to claim 4, is characterized in that: described base plate is polystyrene component or tygon component.
9. myeloperoxidase immunochromatographiassay assay quantitative detection test paper according to claim 4, is characterized in that: described in get stuck and comprise plastics upper casing and plastics lower casing, described plastics upper casing fastens to be formed after on plastics lower casing and gets stuck.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107064490A (en) * | 2017-05-18 | 2017-08-18 | 中国科学院烟台海岸带研究所 | A kind of utilize recombinates detection reagent bar of fluorescence phycobniliprotein subunit and preparation method thereof |
| CN108333374A (en) * | 2018-04-08 | 2018-07-27 | 广州天宝颂原生物科技开发有限公司 | C reactive protein, serum amyloid A protein immunochromatography quantify combined detection test paper and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107064490A (en) * | 2017-05-18 | 2017-08-18 | 中国科学院烟台海岸带研究所 | A kind of utilize recombinates detection reagent bar of fluorescence phycobniliprotein subunit and preparation method thereof |
| CN108333374A (en) * | 2018-04-08 | 2018-07-27 | 广州天宝颂原生物科技开发有限公司 | C reactive protein, serum amyloid A protein immunochromatography quantify combined detection test paper and preparation method thereof |
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