CN102830234A - Rapid diagnostic kit for detecting novel marker ST2 of human heart failure - Google Patents
Rapid diagnostic kit for detecting novel marker ST2 of human heart failure Download PDFInfo
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- CN102830234A CN102830234A CN2012102882233A CN201210288223A CN102830234A CN 102830234 A CN102830234 A CN 102830234A CN 2012102882233 A CN2012102882233 A CN 2012102882233A CN 201210288223 A CN201210288223 A CN 201210288223A CN 102830234 A CN102830234 A CN 102830234A
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Abstract
The invention relates to a colloidal gold test strip detection kit for rapid detection of a heart failure marker, i.e., human soluble ST2, in human whole blood, serum and/or plasma, especially in peripheral blood. Based on immunological reaction principles, the kit carries out qualitative or semi-quantitative detection on ST2 in a sample by using a color particle labeled immunochromatographic method. Rapid test strips provided by the invention are successively and mutually overlapped on a base plate, and the test strip is composed of absorbent paper, a sample pad, a cellulose membrane, etc. Color particles are used for coating of an antibody; the test strip comprises a control line and a test line. When the test strip provided by the invention is used for qualitative/quantitative detection on the human heart failure marker human soluble ST2, the advantage of high specificity and sensitivity and simple and convenient operation are obtained. The kit provided by the invention can be used for detection of myocardial injury diseases like human heart failures in clinical practice and scientific research so as to assist in auxiliary diagnosis, guide treatment, prognosis, dynamic monitoring of minimal residual myocardial necrosis (MDR) and the like.
Description
Technical field
The invention belongs to biological technical field, relate to the quick diagnosis detection kit of the depleted mark human soluble of a kind of fast qualitative/detection by quantitative people's whole blood, serum and/or blood plasma central force ST2 concentration.
Background technology
Heart failure is a kind of clinical symptom group of complicacy, is the various cardiopathic serious stages, and its incidence of disease is high, and five-year survival rate and malignant tumour are similar.According to China's 50 tame inpatient of hospital investigation, admission rate in heart failure accounts for 20% of the cardiovascular disease same period, and mortality ratio but accounts for 40%, and the prompting prognosis is serious.With regard to whole Europe, estimate that Symptomatic heart failure patient accounts for the 0.4%-2% of total population, along with the increase at age, morbidity rate in heart failure increases sharply.The main public health problem of the U.S. that become in heart failure.Nearly 5,000,000 people of patient in heart failure suffer from the U.S., annual newly-increased 500,000 people.Annual because of heart failure prescription on individual diagnosis person reaches 1200-1500 ten thousand, total length of stay reaches 6,500,000.
ST2 is one of il-1 (IL-1) receptor family member, strides two kinds of film ST2 (ST2L) and solubility ST2 (sST2), and interleukin-3 3 (IL-33) is the functional ligand of ST2L.After cardiac muscle cell and fibroblast received mechanically stretching, ST2 and IL-33 produce to be increased.In recent years discover that the rising of ST2 level is the same with BNP or NT-proBNP, can reflect the load of heart.
In patients of acute myocardial infarction, 810 routine patients during TIMI 14 studies with TIMI 23 have carried out peripheral blood ST2 level determination, and the result finds that dead patient's peripheral blood ST2 level with the generation heart failure significantly raises in 30 days.The acute dyspnea patient; Januzzi etc. discover 593 routine emergency treatment patients'; The median of patients with heart failure ST2 is higher than non-patients with heart failure, and dead patient ST2 median also is higher than the patient of survival in 1 year, and the rising of ST2 level is the independence and the powerful prediction index of 1 annual death rate.Also have to study and find the chronic heart failure patient, the relevant also generation of predicting sudden cardiac death of the rising of ST2 level with the heart failure order of severity, and the variation of serious patients with heart failure rising ST2 when 2 weeks also is death and the independent prediction index that needs heart transplant.
SaskiaBoisot etc. study 150 acute unstable HF inpatients; (comprise ST2 through using the multiparameter relevant with heart; BNP; NT-proBNP and blood urea nitrogen) analyze the back discovery, mortality ratio when the ST2 level in patient's while in hospital changes measurable 90 days, and this effect is independent of BNP and NT-proBNP.
In a word, human soluble ST2 is proved repeatedly as biomarker and has potential value.The emergency treatment patients serum ST2 level of measuring acute dyspnea or myocardial infarction possibly provide some useful prognosis information for the layering nursing.In addition; The predictive value of ST2 does not receive the influence of age, kidney function damage and body mass index; And the detection of BNP, NT-proBNP receives the influence of these factors usually, and therefore, the mensuration of ST2 combines the mensuration of BNP or NT-proBNP can better improve the accuracy that the heart failure prognosis is judged.
Summary of the invention
The present invention relates to a kind of fast qualitative/detection by quantitative people's whole blood, serum and/or blood plasma, especially the colloidal gold strip detection kit of the depleted mark of peripheral blood central force-human soluble ST2.This test strips can be carried out qualitative/detection by quantitative to people's mark in heart failure-human soluble ST2, has higher specificity and sensitivity, and is simple to operation, also can carry out detection by quantitative with necessary instrument.
Another object of the present invention is the preparation method who has been to provide a kind of people mark in heart failure-human soluble ST2 immunity fast diagnose test paper bar, comprises the preparation of anti-solubility ST2 specific antibody and/or antigen-specific fragment, preparation and the detection method and/or the use of ST2 immunity test strip bar.This test strips is simple to operation, quick, efficient, accurate, and overall process only needs 10 minutes, not disturbed by environmental baseline, and specificity is good, and detection sensitivity is high, and the lowest detection rank can be ng/ml.The present invention is applicable to hospital and emergency treatment unit etc., can realize the fast detecting and/or on-the-spot detection of cardiomyopathieses such as heart failure.
Detection human soluble ST2 immunity colloidal gold test paper strip of the present invention constitutes (see figure 1) successively by sample pad, the gold mark pad that applies Anti-Human's solubility ST2 antibody of colloid gold label, nitrocellulose filter, the adsorptive pads that is coated with Anti-Human's solubility ST2 antibody and IgG at detection line and nature controlling line.At the two ends of this PVC base plate, be respectively equipped with sample pad and absorption pad; Be provided with the nitrocellulose filter detection layers at this PVC base plate middle part; At nitrocellulose filter detection layers and sample pad intersection; Be provided with the gold mark pad that encapsulates golden mark Anti-Human solubility ST2 antibody, be arranged under the sample pad in gold mark pad one end parts, the other end branch is arranged on the detection layers; With the detection layers of sample pad continuity on be provided with detection line and nature controlling line, on this detection line and nature controlling line, be coated with Anti-Human's solubility ST2 antibody and goat-anti IgG respectively.
The pad of described colloid gold label prepares according to following method: after Anti-Human's solubility ST2 antibody and sheep anti-mouse igg are used the gold solution mark respectively, encapsulate on spun glass and promptly get; Wherein said nitrocellulose filter can be with replacements such as nylon membranes.
Described backing can be the holder of various hard, as long as have certain rigidity, the function with appendix or support all can be used for the present invention, for example can be plastic plate (PVC), cardboard etc., is preferably PVC.
The method of application of kit of the present invention and judgment criteria:
(1) detected object: have characteristic illness and/or pectoralgia or uncomfortable, expiratory dyspnea, feel sick, the patient and/or the general population of vomiting, belch, perspiration, palpitaition, dizziness, fatigue, equivocal symptom such as faint.
(2) sample: patient and/or general population's whole blood, serum and/or blood plasma, especially peripheral blood.
(3) detect: sample (whole blood, serum and/or blood plasma, especially peripheral blood) is collected in adds in the sample with inhaling dropper, test paper scale mark (mark) is once immersed in the sample, waited 5-10 minute, sample is by after the absorption fully, but just sentence read result.
(4) detect principle: detection kit of the present invention adopts the concentration of human soluble ST2 albumen in the immunochromatographic method qualitative detection human peripheral.Detection zone at kit is coated with Anti-Human's solubility ST2 antibodies fragment respectively, and the Quality Control district is coated with sheep anti-mouse igg antibody.During detection, be coated with colloid gold label Anti-Human solubility ST2 antibody on the human soluble ST2 albumen in the human peripheral and the kit in advance and combine the formation bond, a color band occurs with/Fab.No matter be to exist and/or low concentration human soluble ST2 antibody in the sample, when liquid level migrated to the Quality Control district that is fixed with sheep anti-mouse igg antibody, the band of a color should be able to appear in the Quality Control district.
If a color detection band appears in detection zone, positive (+) result of expression promptly a color band all occurs in the Quality Control district He in the test section.(note: because the concentration of human soluble ST2 is different in the sample to be checked, thereby the phenomenon of shade possibly appear in the color band in the test section.But within a certain period of time, no matter the color band shade in the test section, all decidable is positive.) show that then the patient has suffered from cardiomyopathieses such as heart failure, and/or have the risk of cardiomyopathieses such as the heart failure of developing into.If have only the Quality Control district color band to occur, and detection zone does not have the color band, explains that then the patient does not suffer from cardiomyopathieses such as heart failure, and/or does not have the risk of cardiomyopathieses such as the heart failure of developing into.Whether if the color band does not appear in the Quality Control district, it is normal etc. to judge again then whether sample size enough maybe need analyze the chromatography process.
The present invention compared with prior art has following advantage: (1) detection speed is fast, and overall process only needs 10 minutes, can realize the detection of single sample or great amount of samples; (2) highly sensitive, lowest detection unit is ng/ml; (3) high specificity and other mark in heart failure do not have cross interference; (4) simple to operation, operating personnel need not through professional training, and by specification gets final product complete operation, is easy to promote the use of; (5), be particularly suitable for on-the-spot the detection without any need for instrument; (6) the side sample range is wide, whole blood, serum and/or blood plasma all can be used as test sample, peripheral blood especially, and required reagent and sample size are few, and sample size is minimum can be to 50-100ul.
Heart failure is the clinical syndrome of a complicacy, and single labelled thing can not reflect all characteristics in heart failure.Human soluble ST2 albumen is as a new label in heart failure, and it has status of equal importance with cardiac muscle troponin I, BNP.Because as if the concentration of BNP can not be explained seriousness in heart failure, so the simultaneous determination of human soluble ST2 albumen and cardiac muscle troponin I and/or BNP, will provide one to judge standard reliably for state of an illness classification in heart failure, treatment.In the heart failure therapy process,, formulate and the adjustment therapeutic strategy except that according to can also be patient's the clinical symptoms according to the level of patient's human soluble ST2 protein level and/or cardiac muscle troponin I level and/or BNP.Therefore the level of human soluble ST2 protein level and/or cardiac muscle troponin I level and/or BNP associating, perhaps human soluble ST2 protein level independence mensuration can provide an objective assessment method for heart failure patient.
Method of the present invention has widely at medical field to be used.For example, this method can be used for general population's examination, comprises the examination by the doctor, comprises hospital, outpatient service institutes and emergency medical department.
Description of drawings
Fig. 1 colloidal gold strip structural representation
Order is followed successively by:
■: sample pad
■: gold mark pad
: backing
: nitrocellulose filter
: thieving paper
Fig. 2 testing result interpretation synoptic diagram
Be followed successively by: positive, negative, invalid, invalid.
Embodiment
Below in conjunction with specific embodiment the present invention is described further, but these embodiment only are used for illustration purpose, scope of the present invention are not constituted any restriction.Those skilled in the art should understand that for, under the prerequisite that does not depart from spirit and scope of the invention, can make amendment or replace, but these modifications and replacement all fall in protection scope of the present invention the details of technical scheme of the present invention and form.
The preparation of embodiment 1 colloidal gold diagnosis kit of the present invention
Preparation Anti-Human solubility ST2 monoclonal antibody
Use the human soluble ST2 antibody and/or the Fab immunity mouse inbred lines of purifying respectively, when mice serum produces corresponding antibody, can be with mouse boosting cell and SP2/0 Fusion of Cells.Utilize HAT to select hybridoma, and McAb is detected with EUSA (ELISA) or WB.Using limiting dilution liquid for the hybridoma that detects antibody positive clones; Preserve the positive cell strain simultaneously; Utilize the BALB/c mouse inbred lines to prepare a large amount of monoclonal antibodies; The evaluation of the specific evaluation of antagonist simultaneously, identification epitope, the evaluation and the paired experiment of affinity filter out the best a pair of cell line of effect.The cell line that screens is implanted in the mouse peritoneal again, spent 5-6 days and take out ascites, the monoclonal antibody of utilizing ammonium sulfate precipitation and affinitive layer purification to obtain.
The preparation of colloidal gold strip
The preparation of collaurum
(1) get 3 milliliter of 1% tetra chlorauric acid in 500 milliliters round-bottomed flask with 5 milliliters of micropipettors, the ultrapure water of measuring 297 milliliters also adds in the flask, is mixed with 0.01% tetra chlorauric acid reactant liquor, fully stirring and evenly mixing;
(2) serpentine condenser in the connection is opened condensate water, and is placed on the magnetic force heating stirrer heated and boiled;
(3) put into stirrer, vigorous stirring disposablely then adds 3 milliliters of ammonium citrate solutions rapidly and accurately;
(4) flavous aqueous solution of chloraurate grizzle at first becomes aubergine after about 2 minutes, continues to boil 5 minutes;
(5) close the magnetic force heating stirrer, treat collaurum cooling after, be sub-packed in 500 milliliters the glass reagent bottle, the outside is covered with aluminium foil, and is labelled by regulation;
The collaurum of preparation should present bright aubergine, does not have polymkeric substance and macroscopic deposition; Get in right amount and measure in 530 nano wave length places, ultraviolet absorption value is between 1.1-1.3.
Preparation collaurum bond
(1) go out the total amount of needed protein to be marked according to total with the collaurum of intending mark, every milliliter of colloid gold label 12 micrograms/antibody of cost skill, the antibody amount of mark is 3.6 milligrams;
(2) using the sal tartari of 0.1M or the pH value of 0.1M hydrochloric acid adjusting collaurum is 7.8;
(3) under magnetic stirrer, antibody protein solution is added in the colloidal gold solution, should dropwise add when adding protein, 1 milligram of protein added in about 5 minutes;
(4) antibody and colloidal gold reaction added 5% bovine serum albumin (BSA) after 5 minutes under magnetic stirring apparatus, and making its final concentration is 1%;
After (5) 10 minutes, add 3% PEG2000, to final concentration be 0.3%;
(6) continue reaction perhaps spent the night in 1 hour;
(7) mark is good colloidal gold solution is in 2000r/min, 4 ℃ centrifugal 10 minutes, the sucking-off supernatant discards deposition, to remove big polymkeric substance;
(8) rotating speed of adjusting hydro-extractor is 10000r/min, 4 ℃ centrifugal 30 minutes, abandon supernatant, with 0.01MPBS pH8.2 (the include 1%BSA) dissolving of deposition with original volume, repeated centrifugation three times;
(9) last careful supernatant discarded, deposition is dissolved among the 1/50PBS (including 1%BSA) of original volume, promptly gets immune gold.
Detection line and control line encapsulate (will detect antibody sandwich on nitrocellulose filter)
(1) detection line line: respectively Anti-Human's solubility ST2 monoclonal antibody is packed into (albumen trace spray film system) in the Membrane jetter, drawing by the amount of 1ul/cm on the nitrocellulose filter;
(2) Quality Control line: the sheep anti-mouse igg antibody coating buffer is packed into (albumen trace spray film system) in the Membrane jetter, is drawing by the amount of 1ul/cm on the nitrocellulose filter;
(3) encapsulate: 37 ℃ encapsulated 2 hours;
(4) sealing: 37 ℃ were sealed 30 minutes;
(5) drying: the nitrocellulose filter after will encapsulating was put into the vacuum dryer inner drying 20 hours, and airtight preservation is for use.
The pad preparation
(1) opening point film appearance, preheating 30 minutes is done 10 circulations with distilled water;
(2) the OD value of adjustment immunity gold is 30, the immunity gold is added in the plastic containers on the machine left side, and setting program, adjustment 2# shower nozzle discharge rate is 2ul/cm, and immune gold is sprayed on the glass fiber conjugate pad, the position of spray line is in the central authorities of pad;
(3) sprayed pad after, placed 37 ℃ of baking ovens dry 30 minutes;
(4) drying: spun glass behind the mark was put into the vacuum dryer inner drying 20 hours, and it is for use to take out airtight preservation.
The test card preparation
(1) central authorities at plastic bottom board tear the adhesive tape above being overlying on off, stick the nitrocellulose filter that has encapsulated antibody;
(2) tearing wide below the plastic plate central authorities off is 5 millimeters adhesive tape, sticks the pad of spraying collaurum, overlapping 2 millimeters of the nitrocellulose filter of pad front end;
(3) tear off plastic plate bottom wide be 20 millimeters adhesive tape, stick through pretreated sample pad overlapping about 5 millimeters of the front end of sample pad and pad;
(4) the top that tears plastic bottom board off is about 25 millimeters adhesive tape, sticks thieving paper, overlapping about 2 millimeters of thieving paper and nitrocellulose filter;
(5) after assembling was accomplished, with all compactings of each accessory, the result was pasted in inspection, the kilocalorie that assembles is cut into the test strips of 3 mm wides with cutting cutter;
(6) be contained in the test strips that cuts in the test card, promptly get.
The assembling of kit
Be assembled in the box inhaling dropper, sample pipetting volume cup, disposable syringe, the test card for preparing and operation instructions, promptly get kit of the present invention.
Embodiment 2 clinical experimental studies
Sample collection: o'clock 20 routine heart failure patients are carried out empty stomach in morning 7～10 and take out forearm blood, add anti-coagulants such as EDTA and/or heparin immediately, get serum after centrifugal to place-20 ℃ to be equipped with inspection.
Detect and the result:
The detection kit that assembling is accomplished keeps flat, with joining suction pipe draw an amount of peripheral blood, slowly drip 4-5 and drip in the well that detects box and (drip in the middle part of well, accurate as far as possible) with the assurance test result.Under the effect of absorbent material, sample to be checked slowly moves to detection zone and Quality Control district, and the film reaction system starts.No matter have or not human soluble ST2 in the sample to be checked; Place, Quality Control district all should manifest a color band, and this is important criterion, can determine whether to add the peripheral blood of q.s; Whether qualified whether the chromatography process is normal etc., also can be used as simultaneously to judge and detect box a inherent standard.
The result judges:
Whether detect human soluble ST2 to be checked through view window exists.
(1) in the chromatography process; If when not containing human soluble ST2 or its concentration in the peripheral blood sample to be measured and being lower than detection sensitivity; It is the monoclonal antibody generation immune response in the detection band that does not have in the sample on specific antigen and the film; Thereby a color detection band can not appear in the detection zone, a color belt promptly only appears in negative (-) result of expression in the Quality Control district.
(2) if when the concentration of human soluble ST2 is higher than detection threshold in the sample to be checked; Be in the sample human specific ST2 be fixed on film on the detection band in monoclonal antibody combine; Thereby in the detection zone color detection band can appear; Positive (+) result of expression promptly a color band all occurs in the Quality Control district and in the detection zone.(note: because the concentration of human soluble ST2 is different in the sample to be checked, thereby the phenomenon of shade possibly appear in the color band in the test section.But within a certain period of time, no matter the color band shade in the test section, all decidable is positive.)
(3) if the color band does not appear in detection zone and Quality Control district, then testing process failure.Possible is former because operating process is incorrect, kit has gone bad damage or quality defects.
14 routine patients diagnosed in heart failure and 6 routine healthy experimenter's random acquisition peripheral blood samples, the blind check result is following:
Experimental result shows that test strips of the present invention has higher specificity and sensitivity, and test strips and ELISA blind check coincidence rate as a result reach 100%.
Claims (12)
1. the colloidal gold kit of a fast qualitative/detection by quantitative people mark ST2 in heart failure; It is characterized in that: but reagent constituents is the colloidal gold strip of cardiomyopathies such as fast detecting people heart failure, is coated with the anti-solubility ST2 antibodies fragment of colloid gold label on said; Be provided with detection line and nature controlling line, be fixed with anti-solubility ST2 capture antibody or Fab on the detection line; Be fixed with sheep anti-mouse igg on the nature controlling line.
2. novel heart failure detection kit according to claim 1 comprises: plastic gasket, cellulose membrane, gold mark pad, sample pad, absorption pad.
3. the quick diagnosis reagent kit of described detection heart failure is characterized in that, said colored particle can be a kind of colloid gold particle or other silver, iron, magnetic, fuel, latex, fluorescent grain.
4. according to right 1 described kit; It is characterized in that: the anti-ST2 antibody or the Fab of the colloid gold label that encapsulates on the test strips; And Anti-Human's non-fusibility ST2 capture antibody or the Fab fixed on the detection line; Be through immune non-human mammal, the human solubility ST2 that reorganization is separated to from the human cell produces.
5. according to right 1 described kit, it is characterized in that: IgG fixing on the nature controlling line is for discerning the monoclonal antibody of different epi-positions.
6. according to right 1 described kit, it is characterized in that: the experiment sample that is used for fast detecting people mark ST2 in heart failure comprises whole blood, serum or blood plasma, especially peripheral blood.
7. according in the claim 4, described antibody can be monoclonal antibody or or the polyclonal antibody of rabbit, mouse, sheep, horse etc. through fermentation method or immunization preparation.
8. kit according to claim 6; It is characterized in that: when not containing human soluble ST2 or its concentration in the peripheral blood sample to be measured and be lower than detection sensitivity; Can not occur colored a detection in the detection zone and be with, and a color ribbon only in the Quality Control district, occur, negative (-) result of expression.
9. according to right 7 described kits, it is characterized in that: when the concentration of human soluble ST2 in the sample to be checked is higher than detection threshold, can occur a colored band that detects in the detection zone, in the Quality Control district, occur a colored ribbon simultaneously, positive (+) result of expression.
10. according to right 7 described kits, it is characterized in that: because the concentration of human soluble ST2 is different in the sample to be checked, thereby the phenomenon of shade possibly appear in the colored band in the detection zone.But within a certain period of time, no matter the colored band shade in the detection zone, all decidable is positive.
11. according to right 7 described kits, it is characterized in that: when colored band does not appear in the Quality Control district, even colored band appears in detection zone, it is invalid also to declare testing result.
12. according to right 1 described kit; It is characterized in that: colloidal gold strip of the present invention can be used for the myocardial damage property diseases such as detection people heart failure of clinical and scientific research, in order to the dynamic monitoring of auxiliary guiding treatment, prognosis judgement and/or minimal residue myocardial necrosis (MDR) etc.
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| CN107328926A (en) * | 2017-06-29 | 2017-11-07 | 海格德生物科技（深圳）有限公司 | Reagent card, preparation method and the detection method of the multinomial heart failure mark of quick detection |
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