CN102207507A - Semi-quantitative detecting test paper of cardiac troponin and preparation method thereof - Google Patents
Semi-quantitative detecting test paper of cardiac troponin and preparation method thereof Download PDFInfo
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Abstract
The invention relates to semi-quantitative detecting test paper of cardiac troponin, mainly comprising five parts: a sample pad, a combination pad, a water sucking pad, a soleplate and a nitric acid fibre film, wherein two parallel antibody capture lines are made on the nitric acid fibre film; one first anti-cardiac troponin I monoclonal antibody with the determined concentration close to the combination pad is used as a detecting line; another goat-anti-rabbit IgG antibody with the determined concentration is used as a standard concentration developing contrast line; the distance between the two lines is 4-7 mm; and the combination pad is sprayed with two substances of a second anti-cardiac troponin I monoclonal antibody and a gold rabbit IgG marked by colloidal gold or colour latex. The quantitative detecting test paper of cardiac troponin in the invention has the advantages of simple preparation method, low cost, convenience for detection and use, and capability of rapidly conveniently semi-quantitatively detecting the content of the cardiac troponin I in the human body blood sample, and is suitable for family application and popularization.
Description
Technical field
The present invention relates to a kind of cardiac troponin half-quantitative detection test paper.
Background technology
Cardiovascular and cerebrovascular disease is first killer of harm humans life and health, incidence trend is also rising year by year, just there is 4.5 hundred million acute myocardial infarctions (AMI) patient in U.S. emergency unit, there are 400,000,000 patients to be diagnosed as acute myocardial infarction, wherein the people of 2-3% really has AMI, and because the diversity of the symptom of AMI, complication is hided and is existed sizable mistaken diagnosis and fail to pinpoint a disease in diagnosis clinical, some myocardial damage is not ignored by doctor and patient because be true to type, and a lot of life dangers appears in not treatment in time.The acute myocardial infarction AMI morbidity is anxious, the mortality ratio height, and the early diagnosis of AMI and treatment in time are extremely important to reducing the AMI mortality ratio.
Troponin is by Troponin I, T, and three kinds of subunits of C are formed, and play important regulating action in muscular movement.After cardiac muscle incurs loss, the cardiac troponin compound is discharged in the blood, in three kinds of subunits, Troponin I (cTnI) is among unique cardiac muscle cell of existence, diagnose the highest serum biochemistry index of AMI sensitivity and specificity up to now, be widely studied determinacy mark as myocardial damage, the document surface, cTnI level in the normal human blood is below 0.5ng/ml, take place in 4-6 hour, to rise after the myocardial infarction, so the cTnI concentration of 0 ~ 4ng/ml is quantitatively extremely important to knowing that doctor's medication and patient go to a doctor.
Cardiac muscle troponin I conventional sense method has the radioimmunoassay method, high performance liquid chromatography, electrochemical methods, protein analyzer, ELISA diagnostic kit and immuno-chromatographic test paper strip etc., these methods have exists radioactive contamination, need professional large-scale instrument, length consuming time needs the professional, can not quantitatively wait shortcoming.Widespread use clinically has certain limitation, is difficult for extensively promoting, and also is not suitable for family and uses.The relevant patent of domestic existing similar cardiac troponin detection method, application number 200310119332.3, name are called the patent of " Rapid Determination Method of human body cardiac muscle troponin I ", the method of a kind of fast measuring cardiac muscle troponin I that it provides, but the complex steps of this method, but also need professional equipment could measure content.Though the patent of application number CN201020185781.3 can be quantitative, but preparation process is numerous and diverse.
Summary of the invention
Technical matters to be solved by this invention be for overcome current colloidal gold immuno-chromatography test paper strip can only be qualitative in actual promoting the use of can not detection by quantitative deficiency, a kind of not specific instrument and professional equipment are provided, with regard to energy rapid evaluation testing result, and testing result carried out the mensuration of sxemiquantitative level, the preparation method of this test paper is provided simultaneously.
For solving the problems of the technologies described above, a kind of cardiac troponin half-quantitative detection test paper of the present invention, mainly by sample pad, pad, adsorptive pads, base plate, nitrocellulose membrane five parts are formed, it is characterized in that: draw two parallel antibody capture lines on the described nitrocellulose membrane respectively, one of close pad contains through having determined the first anti-cardiac muscle troponin I monoclonal antibody of concentration, and as detection line, another contains through having determined the goat anti-rabbit igg antibody of concentration, as normal concentration colour developing control line, two lines are at a distance of 4--7mm; Described pad is sprayed with the second anti-cardiac muscle troponin I monoclonal antibody and two kinds of materials of gold mark rabbit igg of collaurum or color latex mark.
Preferably, the adsorptive pads on described nitrocellulose membrane and top, the pad of nitrocellulose membrane and lower end, the handing-over of overlapping respectively between the sample pad of pad and bottom, described overlapping width is 1-4mm.
The gold grain of the second anti-cardiac muscle troponin I monoclonal antibody of used collaurum or color latex mark or latex size are between 20nm-120nm.
Further preferred, adjust the concentration of described goat anti-rabbit igg antibody, anti-cardiac muscle troponin I monoclonal antibody, gold mark cardiac muscle troponin I monoclonal antibody and gold mark rabbit igg respectively, when test sample, when the cardiac muscle troponin I hormone concentration in the sample was 2ng/ml, detection line was identical with the red color depth of normal concentration colour developing control line; When the cardiac muscle troponin I hormone concentration in the sample was lower than 2ng/ml, the redness of detection line was shallower than the redness of normal concentration colour developing control line; When cardiac muscle troponin I hormone concentration in the sample was higher than 2ng/ml, the redness of detection line was deeper than the redness of normal concentration colour developing control line.
A kind of colorimetric card that is used with the described cardiac troponin half-quantitative detection of claim 4 test paper, the red piece that contains multiple red gradient in this colorimetric card, the red piece of every kind of depth is corresponding to the shown redness of cardiac muscle troponin I half-quantitative detection detection paper variable concentrations cardiac muscle troponin I Detection of antigen; By the contrast ratio colour atla, can sxemiquantitative determine the concentration of the cardiac muscle troponin I in the test sample.
Detection method of the present invention is: sample drop 4-5 such as blood are dripped on the sample pad, contain the capillary action of the blood of cardiac muscle troponin I antigen by sample pad, move to the nitrocellulose membrane direction, during through gold-marking binding pad, combine with the anti-cardiac muscle troponin I monoclonal antibody specificity on the gold-marking binding pad, and bond is moved together to nitrocellulose membrane, when on arriving nitrocellulose membrane, being coated with the detection line of anti-cardiac muscle troponin I monoclonal antibody, the combination of double antibodies sandwich specificity takes place, the anti-cardiac muscle troponin I monoclonal antibody of antigen in the sample and golden target is coated on the anti-cardiac muscle troponin I monoclonal antibody of nitrocellulose membrane and fixes, thereby make detection line show redness, the red depth of its detection line and the cardiac muscle troponin I antigen concentration journey direct ratio in the sample of collaurum.Gold mark cardiac muscle troponin I monoclonal antibody that not tested survey line is fixing and cardiac muscle troponin I hormone antigen bond or gold mark cardiac muscle troponin I monoclonal antibody continue along moving on the nitrocellulose membrane, when reaching nature controlling line, gold mark cardiac muscle troponin I monoclonal antibody not with nature controlling line on the goat anti-rabbit igg antibody reaction, but quantitative gold mark rabbit igg and the reaction of nature controlling line goat-anti rabbit, and fixed by nature controlling line, show red, no matter whether contain cardiac muscle troponin I antigen in the sample, or how the concentration of antigen changes, nature controlling line develops the color all the time, and colour is fixed, if nature controlling line does not develop the color, illustrates that test strips is invalid.
The present invention also provides the preparation method of above-mentioned cardiac troponin half-quantitative detection test paper, may further comprise the steps:
Select the anti-cardiac muscle troponin I mouse of two strains source property specific monoclonal antibody, promptly anti-cardiac muscle troponin I monoclonal antibody 1 and anti-cardiac muscle troponin I monoclonal antibody 2;
Select anti-cardiac muscle troponin I monoclonal antibody 1 as colloid gold label antibody, use the sodium citrate reducing process to be prepared as the nanogold particle of 20nm ~ 120nm size, centrifugal 10 minutes of 10000RPM/MIN, be prepared as storing solution, with the colloid gold label monoclonal antibody, and adding polyglycol PEG, bovine serum albumin(BSA) BSA makees stabilizing agent;
With the golden labeling antibody 1 of mark mark, spray polyester film or all-glass paper, low temperature drying makes gold-marking binding pad;
Glass fibre membrane is immersed in the solution that phosphate buffer PBS adds surfactant 5 minutes, pulls oven dry about 30 degree again out, make sample pad;
The nitrocellulose membrane that 2cm is wide is bonded at PVC sheet material centre position, and quantitatively sprays anti-cardiac muscle troponin I monoclonal antibody 2 and quantitative two wide bands of 1mm at a distance of 5mm of goat anti-rabbit antibody, low temperature drying 10 hours;
The thieving paper of cutting certain width is pasted the top of nitrocellulose filter on the PVC sheet material and overlapping nitrocellulose membrane 1-3mm;
Gold-marking binding pad is cut into band about 5mm-13mm, pastes the bottom of nitrocellulose membrane on the PVC sheet material, overlapping nitrocellulose membrane 1-3mm;
Sample pad is cut into the wide band of 25mm-30mm, pastes bottom again, handing-over gold-marking binding pad 3mm-5mm at gold-marking binding pad;
Use the adhesive sticker of the band MAX line about 40mm again, be bonded on gold-marking binding pad and the sample pad;
With the big plate that cutting machine will make above, be cut into the band of 2.5mm-6mm, promptly be prepared into described cardiac troponin half-quantitative detection test paper, use the aluminium foil bag hermetically drying standby then.
The preparation method of cardiac troponin detection by quantitative test paper of the present invention is simple, cost is low, detect easy to use, can be fast and convenient, the content of the cardiac muscle troponin I in the semiquantitative Sensitive Detection blood of human body sample, suitable family uses and promotes.
Description of drawings
Below in conjunction with the drawings and specific embodiments technical scheme of the present invention is further described in detail.
Fig. 1 is the front view of cardiac troponin detection by quantitative test paper of the present invention.
Fig. 2 is the side view of cardiac troponin detection by quantitative test paper of the present invention.
The colorimetric card front view that Fig. 3 is used for cardiac troponin detection by quantitative test paper.
Embodiment
Shown in Fig. 1,2, cardiac troponin detection by quantitative test paper of the present invention is mainly by sample pad 01, pad 02, adsorptive pads 06, base plate 07, nitrocellulose membrane 04 5 parts are formed, draw two parallel antibody capture lines on the nitrocellulose membrane 04 respectively, one of close pad contains through having determined the first anti-cardiac muscle troponin I monoclonal antibody of concentration, as detection line 03, another contains through having determined the goat anti-rabbit igg antibody of concentration, as normal concentration colour developing control line 05, be also referred to as nature controlling line, two lines are at a distance of 4--7mm; Be sprayed with the second anti-cardiac muscle troponin I monoclonal antibody and two kinds of materials of gold mark rabbit igg of colloid gold label on the pad 02.The adsorptive pads 06 on nitrocellulose membrane 04 and top, the pad 02 of nitrocellulose membrane 04 and lower end, the handing-over of overlapping respectively between the sample pad 06 of pad 02 and bottom, overlapping width is respectively 1--4mm.The gold grain size of the second anti-cardiac muscle troponin I monoclonal antibody of used colloid gold label is 20nm--120nm.
Adjust the concentration of described goat anti-rabbit igg antibody, anti-cardiac muscle troponin I monoclonal antibody, gold mark cardiac muscle troponin I monoclonal antibody and gold mark rabbit igg respectively, when test sample, when the cardiac muscle troponin I concentration in the sample was 2ng/ml, detection line was identical with the red color depth of normal concentration colour developing control line; When the cardiac muscle troponin I hormone concentration in the sample was lower than 2ng/ml, the redness of detection line was shallower than the redness of normal concentration colour developing control line; When cardiac muscle troponin I concentration in the sample was higher than 2ng/ml, the redness of detection line was deeper than the redness of normal concentration colour developing control line.
The preparation method of cardiac troponin half-quantitative detection test paper may further comprise the steps:
Buy the cardiac muscle troponin I immune mouse, obtain anti-cardiac muscle troponin I mouse source two strains of property specific monoclonal antibody, anti-cardiac muscle troponin I monoclonal antibody 1 and anti-cardiac muscle troponin I monoclonal antibody 2 respectively.Select anti-cardiac muscle troponin I monoclonal antibody 2 as colloid gold label antibody, use the sodium citrate reducing process to prepare 20nm ~ 120nm size, be fit to the nanogold particle of this antibody of mark.Centrifugal 10 minutes of 10000RPM/MIN, the preparation storing solution with the colloid gold label monoclonal antibody, and adds PEG, and BSA makees stabilizing agent.With the golden labeling antibody 1 of mark mark, spray polyester film or all-glass paper.Low temperature drying, the gold-marking binding pad of system.Glass fibre membrane is immersed in the solution that PBS adds surfactant 5 minutes, pulls oven dry about 30 degree again out, make sample pad.The nitrocellulose membrane that 2cm is wide is bonded at PVC sheet material centre position, and quantitatively sprays anti-cardiac muscle troponin I monoclonal antibody 2 and quantitative two wide bands of about 1mm at a distance of 5mm of goat anti-rabbit antibody.Low temperature drying 10 hours.The thieving paper of cutting certain width is pasted on the PVC sheet material, the top of nitrocellulose filter, and overlapping nitrocellulose membrane 1-3mm.Gold-marking binding pad is cut into band about 5mm-13mm, pastes on the PVC sheet material, the bottom of nitrocellulose membrane is about overlapping nitrocellulose membrane 1-3mm.Sample pad is cut into the wide band of 25mm-30mm, pastes bottom again, about handing-over gold-marking binding pad 3mm-5mm at gold-marking binding pad.Use the adhesive sticker of the band MAX line about 40mm again, be bonded on gold-marking binding pad and the sample pad, prevent that it from coming off.Big plate with cutting machine will make above is cut into the band about 2.5mm-6mm, promptly has been prepared into half-quantitative detection human cardiac troponin I test paper strip for semi-quantitative detecting.Standby with the aluminium foil bag hermetically drying.
Before detection Aluminium Foil Package pack is taken out, room temperature is placed about 5min, takes out test paper, and the sample pad bottom is dipped in the sample solution, is no more than the MAX line, and take out 5 seconds, horizontal positioned, 5 minutes visual inspection detection lines and standard nature controlling line.
If the detection line redness is greater than nature controlling line, Troponin I concentration is higher than 2ng/ml in the interpret sample, and the people from source of sample has the possibility of myocardial damage.If the detection line redness is shallower than nature controlling line, the Troponin I concentration in the interpret sample is lower than 2ng/ml.
If wonder the concrete concentration of cardiac muscle troponin I in the sample, can be with the colour developing result of detection line and colour atla contrast, the color lump value that color and detection line are close in the colour atla is the concentration of Troponin I in the sample.Can be quick, convenient, the concrete concentration of Troponin I in the test sample is efficiently estimated and the myocardial damage degree.
The cardiac muscle troponin I semi-quantitative analysis of the present invention preparation detect chromatograph test strip and with the ELISA kit, the serum sample of the heart stalk emergency unit that chooses nine tame hospitals in 157 parts of Wuhan City is detected, the result is as follows:
The cardiac muscle troponin I semi-quantitative analysis detects chromatograph test strip: 142 parts of positives, 15 feminine genders.
ELISA kit: 141 parts of positives, 16 parts of feminine genders.
The cardiac muscle troponin I semi-quantitative analysis detects the chromatograph test strip testing result and the total coincidence rate of ELISA kit testing result is 99.4%.
It should be noted last that, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can make amendment or be equal to replacement technical scheme of the present invention, and not breaking away from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of the claim scope of the present invention.
Claims (6)
1. cardiac troponin half-quantitative detection test paper, mainly by sample pad, pad, adsorptive pads, base plate, nitrocellulose membrane five parts are formed, and it is characterized in that: draw two parallel antibody capture lines on the described nitrocellulose membrane respectively, one of close pad contains through having determined the first anti-cardiac muscle troponin I monoclonal antibody of concentration, as detection line, another contains through having determined the goat anti-rabbit igg antibody of concentration, and as normal concentration colour developing control line, two lines are at a distance of 4--7mm; Described pad is sprayed with the second anti-cardiac muscle troponin I monoclonal antibody and two kinds of materials of gold mark rabbit igg of collaurum or color latex mark.
2. cardiac troponin half-quantitative detection test paper according to claim 1, it is characterized in that the adsorptive pads on described nitrocellulose membrane and top, the pad of nitrocellulose membrane and lower end, the handing-over of overlapping respectively between the sample pad of pad and bottom, described overlapping width is 1-4mm.
3. cardiac troponin half-quantitative detection test paper according to claim 1 is characterized in that the gold grain of the second anti-cardiac muscle troponin I monoclonal antibody of used collaurum or color latex mark or latex size are between 20nm-120nm.
4. according to the described cardiac troponin half-quantitative detection of one of claim 1-3 test paper, it is characterized in that, adjust the concentration of described goat anti-rabbit igg antibody, anti-cardiac muscle troponin I monoclonal antibody, gold mark cardiac muscle troponin I monoclonal antibody and gold mark rabbit igg respectively, when test sample, when the cardiac muscle troponin I hormone concentration in the sample was 2ng/ml, detection line was identical with the red color depth of normal concentration colour developing control line; When the cardiac muscle troponin I hormone concentration in the sample was lower than 2ng/ml, the redness of detection line was shallower than the redness of normal concentration colour developing control line; When cardiac muscle troponin I hormone concentration in the sample was higher than 2ng/ml, the redness of detection line was deeper than the redness of normal concentration colour developing control line.
5. colorimetric card that is used with the described cardiac troponin half-quantitative detection of claim 4 test paper, the red piece that contains multiple red gradient in this colorimetric card, the red piece of every kind of depth is corresponding to the shown redness of cardiac muscle troponin I half-quantitative detection detection paper variable concentrations cardiac muscle troponin I Detection of antigen; By the contrast ratio colour atla, can sxemiquantitative determine the concentration of the cardiac muscle troponin I in the test sample.
6. the preparation method of a cardiac troponin half-quantitative detection test paper according to claim 5 is characterized in that may further comprise the steps:
Select the anti-cardiac muscle troponin I mouse of two strains source property specific monoclonal antibody, promptly anti-cardiac muscle troponin I monoclonal antibody 1 and anti-cardiac muscle troponin I monoclonal antibody 2;
Select anti-cardiac muscle troponin I monoclonal antibody 1 as colloid gold label antibody, use the sodium citrate reducing process to be prepared as the nanogold particle of 20nm ~ 120nm size, centrifugal 10 minutes of 10000RPM/MIN, be prepared as storing solution, with the colloid gold label monoclonal antibody, and adding polyglycol PEG, bovine serum albumin(BSA) BSA makees stabilizing agent;
The golden labeling antibody 1 that mark is good, spray polyester film or all-glass paper, low temperature drying makes gold-marking binding pad;
Glass fibre membrane is immersed in the solution that phosphate buffer PBS adds surfactant 5 minutes, pulls oven dry about 30 degree again out, make sample pad;
The nitrocellulose membrane that 2cm is wide is bonded at PVC sheet material centre position, and quantitatively sprays anti-cardiac muscle troponin I monoclonal antibody 2 and quantitative two wide bands of 1mm at a distance of 5mm of goat anti-rabbit antibody, low temperature drying 10 hours;
The thieving paper of cutting certain width is pasted the top of nitrocellulose filter on the PVC sheet material and overlapping nitrocellulose membrane 1-3mm;
Gold-marking binding pad is cut into band about 5mm-13mm, pastes the bottom of nitrocellulose membrane on the PVC sheet material, overlapping nitrocellulose membrane 1-3mm;
Sample pad is cut into the wide band of 25mm-30mm, pastes bottom again, handing-over gold-marking binding pad 3mm-5mm at gold-marking binding pad;
Use the adhesive sticker of the band MAX line about 40mm again, be bonded on gold-marking binding pad and the sample pad;
With the big plate that cutting machine will make above, be cut into the band of 2.5mm-6mm, promptly be prepared into described cardiac troponin half-quantitative detection test paper, use the aluminium foil bag hermetically drying standby then.
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