CN103344771A - Immunofluorescent chromatographic test strip and detection method for HIT (Heparin-induced thrombocytopenia) antibodies - Google Patents
Immunofluorescent chromatographic test strip and detection method for HIT (Heparin-induced thrombocytopenia) antibodies Download PDFInfo
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Abstract
The invention discloses an immunofluorescent chromatographic test strip and a detection method for HIT (Heparin-induced thrombocytopenia) antibodies. The test strip comprises a sample pad, a reaction membrane and an adsorption pad which are overlapped with one another on a liner plate, wherein the two ends of the sample pad are respectively provided with a first sample adding point and a second sample adding point, and the reaction membrane is fixedly provided with an internal quality control belt and at least one antibody detection belt. The detection method comprises the following steps of: adding a sample to be detected into the sample pad, and then adding a fluorescently labeled secondary antibody to be detected by a fluorescence detection instrument. Through the mode, according to the immunofluorescent chromatographic test strip and the detection method for the HIT antibodies, provided by the invention, the detection process is simple, efficient, accurate, cheap and practical, and the semi-quantitative or quantitative detection can be realized. The invention provides a relativley reliable and practical method for diagnosis of clinical heparin-induced thrombocytopenia and related complications in China.
Description
Technical field
The present invention relates to the medical science detection range, particularly relate to a kind of immunofluorescence chromatograph test strip and be used for HIT detection of antibodies method.
Background technology
Heparin is one of anticoagulant that is most widely used clinically, its major side effects is hemorrhage, decrease of platelet and follows thromboembolism, and allergic reaction and injection site cutaneous necrosis etc., (the heparin-induced thrombocytopenia of the thrombopenia due to the heparin wherein, HIT) and thromboembolic complication (heart embolism and myocardial infarction, extremity vascular embolism and limb necrosis) the most serious, the incidence of disease is greater than 5%, and wherein mortality ratio is about 20-30%.
Because HIT and thrombus complication clinical manifestation diversity, and lack specific auxiliary examination method, accomplish that early diagnosis has any problem.Patient usually is that the multiple life-threatening vein and the arterial thrombus that are difficult to reverse taken place by clinical definite diagnosis the time; The treatment of enforcement platelet transfusion is led in this disease mistaken diagnosis constant error again, and then increases the weight of the thrombus syndrome and increase mortality ratio.Spell out " immune thrombocytopenia that heparin causes is blood platelet infusion contraindication " in Britain's platelet transfusion guide, yet the diagnosis of whole world HIT so far and treatment be not standardization as yet all, particularly clinical in China, generally the decrease of platelet patient not being carried out associated antibodies detects, absolutely wrong its carries out the antibody evaluation and analyzes, cause a large amount of HIT patients to be difficult to accurate diagnosis, the mistake platelet transfusion causes serious arteriovenous thrombus even death.Early diagnosis HIT also takes the treatment measure significant for the generation that reduces severe complications such as thromboembolism, except close observation clinical manifestation, dynamic monitoring platelet counts, patient heparin therapy or that highly suspect HIT is carried out the HIT antibody test to support diagnosis necessary, but HIT antibody test is not at present popularized as yet, its diagnosis is mainly according to clinical manifestation, although external existing HIT antibody test technology and method are more, but there are many defectives, still lack at present a kind of practicality, easy, quick and detection method that specificity is high.
Studies show that, the HIT antibody test is the necessary condition of diagnosis HIT, Korea S scholar Lee etc. detect 91 routine Maintenance Hemodialysis Patients serum HIT antibody, the result shows that the positive rate of hemodialysis patients HIT antibody reaches 34%, be 10 times of normal healthy people, the incidence that its vascular access of the hemodialysis patients of HIT antibody positive stops up is significantly higher than the negative patient.The probability that HIT antibody produces is along with the treatment duration of using heparin prolongs rising gradually., produce HIT antibody person and can reach 30%-60% because of the long-time heparin that uses as the operation on vessels of heart patient.Usually, the HIT antibody inspection patient that is strong positive is easy to develop into HIT.CarrierM etc. discover, the generation of the interior HIT antibody of body is relevant with mortality in said patients, further studies show that, the generation of HIT antibody can significantly increase the cardiovascular death rate in patient's body of MHD treatment.U.S.'s pathology can advise that the patient who occurs decrease of platelet or new thrombus during accepting heparin therapy should detect HIT antibody.The diagnosis of HIT at present is mainly according to clinical manifestation, and the laboratory that HIT antibody is relevant is detected and be can be HIT and make a definite diagnosis important evidence is provided, and can predict the generation of the bad cardiac event of non-HIT patient.
The diagnosis of domestic present HIT and treatment be not standardization as yet all, alternative anticoagulant therapy seldom is employed especially, even the especially non-blood specialist of clinician is not enough to the awareness of HIT, in clinical position, need to strengthen watching out for and understanding HIT, thereby find treatment in time early, avoid forming serious thrombus complication as far as possible.Although existing HIT antibody test technology and method are a lot, but domestic easy, the quick and very high detection method of specificity that does not also have practical value clinically, therefore to HIT accurately, the further research of detection method easily, will help this sick early diagnosis and control.
Countries such as the U.S. have developed relevant detection reagent, but specificity is relatively poor, expensive in addition, buy and loaded down with trivial detailsly also limited its application at home, and wherein the predicted value of yin and yang attribute is to come according to national colony statistics, directly judges the testing result of domestic individuality with the negative and positive predicted value of external product, difference because of colony, may cause the generation of spurious results, therefore should there be the predicted value of the yin and yang attribute of oneself in Chinese colony, and the accuracy of testing result is improved.
Summary of the invention
The technical matters that the present invention mainly solves provides a kind of immunofluorescence chromatograph test strip and is used for HIT detection of antibodies method, and this method is simple, efficient, accurate, inexpensive and practical.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is: a kind of immunofluorescence chromatograph test strip is provided, comprise reaction film, sample pad, absorption pad and base plate, described reaction film is positioned on the described base plate, the band of detection and quality control band are fixedly arranged on the described reaction film, described sample pad and described absorption pad are overlapping with the two side portions of described reaction film respectively, and be positioned on described reaction film and the described base plate, fixedly have first to add sampling point and second and add sampling point on the described sample pad, described first adds sampling point is arranged on away from the overlapping side of described sample pad and described reaction film, and described second adds sampling point is arranged near the overlapping side of described sample pad and described reaction film.
In a preferred embodiment of the present invention, described detection band is at least one, and described detection is with fixedly has heparin-the platelet factor 4 compound, and the spacing of every described detection band is 5mm.
In a preferred embodiment of the present invention, described heparin-platelet factor 4 compound is respectively platelet factor 4 and heparin dilution to be obtained platelet factor 4 solution and heparin solution with sample diluting liquid, the mass concentration ratio of described platelet factor 4 solution and described heparin solution is 2-5:1, described platelet factor 4 solution and described heparin solution equal-volume are mixed back 4 ℃ spend the night and obtain, described sample diluting liquid is that the pH of 0.01M is 7.2 phosphate buffer.
In a preferred embodiment of the present invention, described quality control band is one, and it is nitrocellulose membrane, cellulose acetate film, nylon membrane or poly tetrafluoroethylene that human IgG, the material of described reaction film are fixedly arranged on the described quality control band.
In a preferred embodiment of the present invention, the fixing human IgG of described quality control band is that to be 7.2 phosphate buffers obtain the concentration adjustment of human IgG the pH with 0.01M for 0.5-1 mg/ml.
In a preferred embodiment of the present invention, described test strips is used for HIT detection of antibodies method, comprises that step is:
(1) sample to be checked is joined described second and add in the sampling point place, cleansing solution is joined first add the washing of sampling point place;
(2) will anti-join described second and add in the sampling point place with fluorescein-labeled two, cleansing solution is joined first add the washing of sampling point place;
(3) place fluorescence detector to detect described test strips.
In a preferred embodiment of the present invention, described cleansing solution is that to contain volume fraction be that 0.5% Tween-20 and pH value are 7.2 0.15M phosphate buffer.
In a preferred embodiment of the present invention, described fluorescein is anthocyanidin Cy series fluorescein or Alexa Fluor series fluorescein.
In a preferred embodiment of the present invention, described two anti-are goat anti-human igg, rabbit anti-human igg or the anti-human IgG of chicken.
The invention has the beneficial effects as follows: immunofluorescence chromatograph test strip of the present invention reaches and is used for HIT detection of antibodies method, the operation of immunofluorescence chromatographic technique is fast and convenient, can realize sxemiquantitative or quantitatively detection in conjunction with detecting instrument, testing process is simple, efficiently, accurately, inexpensive and practical, be not easy omission, not high to operating personnel's technical requirement, can assist clinical early diagnosis to heparin-induced thrombopenia relevant disease, whether severe complications such as quick diagnosis thrombopenia and thromboembolism are to be caused by heparin, and the diagnosis and treatment that the heparin of in time stopping using increased the weight of the state of an illness with avoiding the mistake platelet transfusion play directive function.
Description of drawings
In order to be illustrated more clearly in the technical scheme in the embodiment of the invention, the accompanying drawing of required use is done to introduce simply in will describing embodiment below, apparently, accompanying drawing in describing below only is some embodiments of the present invention, for those of ordinary skills, under the prerequisite of not paying creative work, can also obtain other accompanying drawing according to these accompanying drawings, wherein:
Fig. 1 is the structural representation of immunofluorescence chromatograph test strip one preferred embodiment of HIT antibody test of the present invention;
The mark of each parts is as follows in the accompanying drawing: 1, reaction film, and 2, sample pad, 3, absorption pad, 4, base plate, 5, detect band, 6, quality control band, 7, first adds sampling point, and 8, second adds sampling point.
Embodiment
To the technical scheme in the embodiment of the invention be clearly and completely described below, obviously, described embodiment only is a part of embodiment of the present invention, rather than whole embodiment.Based on the embodiment among the present invention, those of ordinary skills belong to the scope of protection of the invention not making all other embodiment that obtain under the creative work prerequisite.
Embodiment one:
See also Fig. 1, a kind of immunofluorescence chromatograph test strip is provided, comprise reaction film 1, sample pad 2, absorption pad 3 and base plate 4.Described reaction film 1 is positioned on the described base plate 4, fixedly has on the described reaction film 1 to detect to be with 5 and quality control band 6, and described detection is with 5 to be two, described quality control band 6 is one, the material of described reaction film can be elected nitrocellulose membrane as, cellulose acetate film, nylon membrane or poly tetrafluoroethylene.
Described sample pad 2 and described absorption pad 3 are overlapping with the two side portions of described reaction film 1 respectively, and are positioned on described reaction film 1 and the described base plate 4.Comprise on the described sample pad 2 that first adds sampling point 7 and second and add sampling point 8, described first adds sampling point 7 is arranged on away from the overlapping side of described sample pad 2 and described reaction film 1, and described second adds sampling point 8 is arranged on and approaches the overlapping side of described sample pad 2 and described reaction film 1.
The preparation process of described immunofluorescence chromatograph test strip is:
1) preparation of reaction film
Choose Millipore HF 180 tape backing films as reaction film, it is standby to be cut into 30 * 1.8cm size.Be 7.2 phosphate buffer with the pH value of 0.01M with the concentration adjustment of human IgG be 0.5mg/ml, add volume fraction and be 1% Tween-20, use and draw a film instrument it is sprayed on NC film surface as nature controlling line, drawing a film amount is 0.5 μ l/cm.PH with 0.01M is that 7.2 phosphate buffers are that PBS disposes 20 μ g/ml PF4 and 5 μ g/ml heparin, after both equal-volumes mix, add volume fractions in 4 ℃ of backs of spending the night and be 1% Tween-20, use a stroke film instrument that it is sprayed on NC film surface as detection line, every line is spaced apart 5mm, draw and put into 37 ℃ of baking oven dried overnight immediately after film finishes, room temperature preservation is standby.
2) assembling of test strips
Choose homemade high-quality glass fibre membrane BT100 as the sample pad material, it is standby that it is cut into 30 * 1.8cm size.Choose homemade high-quality thieving paper CH37K, and it is standby that it is cut into 30 * 1.8cm size.Be to paste reaction film earlier on the PVC base plate of 30 * 6cm in specification, the method that sample pad and thieving paper are taked to overlap sticks on the base plate again, and the length of overlap joint is 0.5mm, so that the diffusion of reactant liquor.After assembling, be cut into the wide test strips of 4mm, and be packaged in the plastic casing, be put in the aluminium foil bag, 4 ℃ of lower seals are preserved.
Embodiment two:
Provide a kind of embodiment one described test strips to be used for the method for heparin-induced thrombopenia antibody test:
(1) preparation of fluorescence reaction liquid
To resist human IgG antibody's solution to regulate its concentration with phosphate buffer is 2mg/ml, and pH is 7.5 to 8.0.Get the 1ml antibody-solutions, slowly adding 100 μ l concentration is the cy5 fluorescent dye of 1mg/ml, 4 ℃ down slowly stir 3h after room temperature reaction 1h again, be that 7.4 phosphate buffer is dialysed with pH again, remove unreacted cy5 dyestuff.Reclaim the good fluorescence antibody of coupling after dislysate is finished, after mensuration coupling efficiency and the antibody concentration, under 4 ℃, keep in Dark Place.
(2) detect step
1) it is 18-25 ℃ with test strips and fluorescence reaction liquid balance to room temperature;
2) add 5 μ l sample to be checked to sample pad.
3) adding 15 μ l, to contain volume fraction be that the pH of 0.5% Tween-20 is that 7.2 0.15M PBS is to sample pad.
7) add the anti-human IgG of 5 μ l cy5-to sample pad.
8) repeating step 2) three times.
9) place fluorescence signal to read instrument test strips, read fluorescence signal and measure.
The above only is embodiments of the invention; be not so limit claim of the present invention; every equivalent structure or equivalent flow process conversion that utilizes description of the present invention to do; or directly or indirectly be used in other relevant technical field, all in like manner be included in the scope of patent protection of the present invention.
Claims (9)
1. immunofluorescence chromatograph test strip, it is characterized in that, comprise reaction film, sample pad, absorption pad and base plate, described reaction film is positioned on the described base plate, the band of detection and quality control band are fixedly arranged on the described reaction film, described sample pad and described absorption pad are overlapping with the two side portions of described reaction film respectively, and be positioned on described reaction film and the described base plate, fixedly have first to add sampling point and second and add sampling point on the described sample pad, described first adds sampling point is arranged on away from the overlapping side of described sample pad and described reaction film, and described second adds sampling point is arranged near the overlapping side of described sample pad and described reaction film.
2. immunofluorescence chromatograph test strip according to claim 1 is characterized in that, described detection band is at least one, and described detection is with fixedly has heparin-the platelet factor 4 compound, and the spacing of every described detection band is 5mm.
3. immunofluorescence chromatograph test strip according to claim 2, it is characterized in that, described heparin-platelet factor 4 compound is respectively platelet factor 4 and heparin dilution to be obtained platelet factor 4 solution and heparin solution with sample diluting liquid, the mass concentration ratio of described platelet factor 4 solution and described heparin solution is 2-5:1, described platelet factor 4 solution and described heparin solution equal-volume are mixed back 4 ℃ spend the night and obtain, described sample diluting liquid is that the pH of 0.01M is 7.2 phosphate buffer.
4. immunofluorescence chromatograph test strip according to claim 1 is characterized in that, described quality control band is one, and it is nitrocellulose membrane, cellulose acetate film, nylon membrane or poly tetrafluoroethylene that human IgG, the material of described reaction film are fixedly arranged on the described quality control band.
5. immunofluorescence chromatograph test strip according to claim 4 is characterized in that, the fixing human IgG of described quality control band is that to be 7.2 phosphate buffers obtain the concentration adjustment of human IgG the pH with 0.01M for 0.5-1 mg/ml.
6. test strips according to claim 1 is used for HIT detection of antibodies method, it is characterized in that, comprises that step is:
(1) sample to be checked is joined described second and add in the sampling point place, cleansing solution is joined first add the washing of sampling point place;
(2) will anti-join described second and add in the sampling point place with fluorescein-labeled two, cleansing solution is joined first add the washing of sampling point place;
(3) place fluorescence detector to detect described test strips.
7. detection method according to claim 6 is characterized in that, described cleansing solution is that to contain volume fraction be that 0.5% Tween-20 and pH value are 7.2 0.15M phosphate buffer.
8. detection method according to claim 6 is characterized in that, described fluorescein is anthocyanidin Cy series fluorescein or Alexa Fluor series fluorescein.
9. detection method according to claim 6 is characterized in that, described two anti-are goat anti-human igg, rabbit anti-human igg or the anti-human IgG of chicken.
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| CN108267591A (en) * | 2017-01-03 | 2018-07-10 | 普迈德（北京）科技有限公司 | A kind of immune microsphere chromatographs quick HIT antibody tests test paper |
| CN109030816A (en) * | 2017-06-09 | 2018-12-18 | 西门子医学诊断产品有限责任公司 | For diagnosing the activation experiments of heparin-induced thrombopenia |
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| CN108267591A (en) * | 2017-01-03 | 2018-07-10 | 普迈德（北京）科技有限公司 | A kind of immune microsphere chromatographs quick HIT antibody tests test paper |
| CN109030816A (en) * | 2017-06-09 | 2018-12-18 | 西门子医学诊断产品有限责任公司 | For diagnosing the activation experiments of heparin-induced thrombopenia |
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Application publication date: 20131009 |