CN104655858A - Fluorescence immunochromatography kit for quantitatively detecting human epididymis secretory protein-4 and preparation method for fluorescence immunochromatography kit - Google Patents
Fluorescence immunochromatography kit for quantitatively detecting human epididymis secretory protein-4 and preparation method for fluorescence immunochromatography kit Download PDFInfo
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Abstract
The invention discloses a fluorescence immunochromatography kit for quantitatively detecting human epididymis secretory protein-4 by taking fluorescent dye as a marker. The fluorescence immunochromatography kit disclosed by the invention realizes fluorescence quantitative detection for the human epididymis secretory protein-4, has the advantages of being good in stability, wide in linear range, good in specificity, accurate to quantify, simple and quick, can be used for simultaneously detecting whole blood, blood serum and plasma samples, and is suitable for hospitals of various levels.
Description
Technical field
The present invention relates to the cancer clinical immunodiagnosis kit field in field of medical examination, be specifically related to the fluorescence immune chromatography kit quantitatively detecting people's epididymal secretory protein-4.
Background technology
Oophoroma betides ovary tissue, so far not yet clearly pathogenetic malignant tumour, and be one of common malignant tumour of female sex organ, its incidence of disease is only lower than cervical carcinoma and carcinoma of corpus uteri, and mortality ratio is relatively high.Oophoroma early clinic atypical symptom, objective, be difficult to distinguish with benign tumor of ovary, the effective ways of shortage early diagnosis, patient often misses the best opportunity for the treatment of.Therefore, one of oophoroma great illness becoming serious threat WomanHealth.
Due to embryonic development and the endocrine function complexity of ovary, preoperative identification of ovarian cancer types is quite difficult.Up to the present, just clinical data statistics both domestic and external, 5 years survival rates of I phase ovarian cancer patients are 90%, and 5 of Patients with Advanced Ovarian Carcinoma years survival rates are only 30%.Therefore, if oophoroma can be detected early, greatly can reduce the mortality ratio of ovarian cancer patients, extend its life span.The diagnosis detecting method of current oophoroma mainly contains two kinds.One is Transvaginal Ultrasound inspection (TUV), can be used for the reproductive organs checking women, comprise uterus, ovary, uterine neck and vagina, current application is comparatively general, but its shortcoming is that it is pernicious or optimum for cannot distinguishing lump, and it mainly relies on the experience of doctor to judge, limitation is very large.Another kind of detection method checks tumor markers CA125, be regarded as " goldstandard " of ovarian cancer diagnosis, but CA125 specificity and susceptibility lower, false positive rate clinically by CA125 diagnosis of ovarian cancer is higher, as breast cancer, liver cancer, cancer of pancreas, carcinoma of urinary bladder, lung cancer may be all positive, in addition, in physiological conditions (as the menstrual period, the gestational period) especially may there is the positive, thus cause the misdiagnosis rate of oophoroma higher, and the positive rate of alone CA125 diagnosis of ovarian cancer is less than 10%, even if combine other detection meanss as ultrasonic examination, positive rate also can only bring up to 20%.To sum up, diagnosis of ovarian cancer still lacks all higher oncobiology index of Sensitivity and Specificity clinically at present.Therefore, in clinical in the urgent need to more responsive effective tumor markers and CA125 complementary.
Application number be 201410164258.5 Chinese invention patent application disclose a kind of preparation method of oophoroma quick diagnosis reagent kit, mainly analyze based on microtiter plate (as 96 orifice plates, 384 orifice plates), highly sensitive, but complicated operation, reaction time is long, and cost is high.If sample size is fewer, then needs to wait for, thus cannot basic hospital be met, comprise demand that is at county level and township hospital.
Summary of the invention
The object of the invention is the deficiency existed for ovarian cancer diagnosis in above-mentioned prior art, provide a kind of basic hospital to Grade A hospital all applicable, highly sensitive, accuracy is strong, easy and simple to handle, can the diagnostic kit of quick diagnosis early metaphase oophoroma.
For solving the problems of the technologies described above, the present invention adopts fluorescence immune chromatography technology, and with people's epididymal secretory protein-4 (human epididymis secretory protein 4, HE4), as Testing index, concrete technical scheme is:
One aspect of the present invention provides a kind of fluorescence immune chromatography kit of quantitative detection people epididymal secretory protein-4, comprise buckle (11), fluorescence immune chromatography test paper bar and damping fluid, wherein, the external shell structure that buckle (11) is fluorescence immune chromatography test paper bar, comprise well (9) and view window (10), as shown in Figure 3.
Fluorescence immune chromatography test paper bar structure as depicted in figs. 1 and 2, comprises sample pad (1), label pad (2), chromatographic film (6), adsorptive pads (7) and base plate (8).When carrying out whole blood sample and detecting, test strips also comprises blood separation membrane (3).Wherein, sample pad (1), label pad (2), blood separation membrane (3), chromatographic film (6) and adsorptive pads (7) are equipped on base plate (8), sample pad (1) is positioned at the below of well (9), and be connected to label pad (2), label pad (2) is connected to chromatographic film (6), chromatographic film (6) is connected to adsorptive pads (7), it is fixed with a quantitative detection line (4) and a nature controlling line (5), preferably the two distance 3mm ~ 8mm, and be positioned at the below of view window (10).When comprising blood separation membrane (3), blood separation membrane (3) is arranged between label pad (2) and chromatographic film (6), now label pad (2) is connected to blood separation membrane (3), blood separation membrane (3) is connected to chromatographic film (6), or blood separation membrane (3) is arranged between sample pad (1) and label pad (2), now sample pad (1) is connected to blood separation membrane (3), blood separation membrane (3) is connected to label pad (2), or blood separation membrane (3) is direct and sample pad (1) merges into same structure.
Label pad (2) is fixed with simultaneously people's epididymal secretory protein-4 specific antibody that fluorescent dye is modified, with the Quality Control molecule that fluorescent dye is modified, the emission wavelength of the fluorescent dye of modified human epididymal secretory protein-4 specific antibody is identical with the emission wavelength of the fluorescent dye modifying Quality Control molecule, and wavelength coverage is 300-1300nm.
Quantitative detection line (4) is fixed with people's epididymal secretory protein-4 specific antibody, the antigenic determinant that this specific antibody identifies is different from the antigenic determinant that people's epididymal secretory protein-4 specific antibody that the fluorescent dye be fixed in label pad (2) is modified identifies.
Nature controlling line (5) is fixed with the biomolecule that can be combined with Quality Control molecular specificity.
Another aspect of the present invention provides the preparation method of the fluorescence immune chromatography kit of above-mentioned quantitative detection people epididymal secretory protein-4, comprises the steps:
Step (1): with specific effect between chemical crosslinking or biomolecule respectively by the surface of conjugated fluorescent dyes to people's epididymal secretory protein-4 specific antibody and Quality Control molecule, obtain people's epididymal secretory protein-4 specific antibody of fluorescent dye modification and the Quality Control molecule of fluorescent dye modification respectively, the emission wavelength of modifying the fluorescent dye of described Quality Control molecule is identical with the emission wavelength of the fluorescent dye of modified human epididymal secretory protein-4 specific antibody, and wavelength coverage is 300 ~ 1300nm;
Step (2): people's epididymal secretory protein-4 specific antibody that fluorescent dye step (1) obtained is modified and the Quality Control molecule that fluorescent dye is modified all are fixed in label pad (2);
Step (3): a quantitative detection line (4) and a nature controlling line (5) are set respectively in chromatographic film (6), wherein, nature controlling line (5) is fixed with the biomolecule that can be combined with Quality Control molecular specificity, quantitative detection line (4) is fixed with people's epididymal secretory protein-4 specific antibody, and the antigenic determinant that this specific antibody identifies is different from the antigenic determinant that people's epididymal secretory protein-4 specific antibody that the fluorescent dye be fixed in label pad (2) is modified identifies;
Step (4): sample pad (1), label pad (2), chromatographic film (6), adsorptive pads (7) and base plate (8) are built into fluorescence immune chromatography test paper bar, when carrying out whole blood sample and detecting, test strips also comprises blood separation membrane (3);
Step (5): fluorescence immune chromatography test paper bar is installed.
The present invention adopts specific effect between chemical crosslinking or biomolecule by the surface of conjugated fluorescent dyes to people's epididymal secretory protein-4 specific antibody or the surface of Quality Control molecule, to obtain people's epididymal secretory protein-4 specific antibody of fluorescent dye modification and the Quality Control molecule of fluorescent dye modification respectively.In the present invention, when fluorescent dye surface exists reactive group, it directly can be reacted with specific antibody, not need use chemical cross-linking agent; Otherwise, then need fluorescent dye and people's epididymal secretory protein-4 specific antibody or the coupling of Quality Control molecule phase by chemical cross-linking agent.Wherein, chemical cross-linking agent comprises 1-ethyl-3-(3-DimethylAminopropyl) carbodiimides (EDC), N-hydroxy-succinamide (NHS) and glutaraldehyde etc.
In a preferred embodiment of the invention, adopt fluorescent dye modified human epididymal secretory protein-4 specific antibody or the Quality Control molecule that there are reactive group, step is: dissolved by the fluorescent dye after purifying, then a certain amount of people's epididymal secretory protein-4 specific antibody or Quality Control molecule is added, using damping fluid as reaction medium, react 2 ~ 4 hours, add oxammonium hydrochloride cessation reaction, purify by modes such as chromatogram, chromatographic column or ultrafiltration are centrifugal, obtain people's epididymal secretory protein-4 specific antibody that fluorescent dye modifies or Quality Control molecule.
In order to improve the discrimination of signal and background, in the present invention, the wavelength coverage of fluorescent dye is 300 ~ 1300nm, is preferably 550-800nm.This is because, under ultraviolet irradiation, the fluorescence intensity of chromatographic film, base plate and buckle far can be better than more than 550nm at below 550nm, thus can produce certain impact to the detection of low concentration people epididymal secretory protein-4, therefore preferred emission wavelength is greater than the fluorescent dye of 550nm; In addition, chromatographic film, base plate and buckle are generally extremely weak near infrared region (600 ~ 800nm) fluorescence intensity, therefore, preferred wavelength range be the fluorescent dye of 550-800nm to improve sensitivity further, more preferably fluorescent dye emission wavelength is 665nm.
Fluorescent dye of the present invention comprises organic fluorescent dye and rare earth element fluorescent dye.It is on latex microsphere that fluorescent dye of the present invention can be coupled to, and becomes the form of fluorescent microsphere.Fluorescent dye of the present invention can be the fluorescent dye of single compound or the composite fluorescent dye that is made up of several compound, but preferred single compound fluorescent dye and preferably there is the fluorescent dye of stronger light stability.The fluorescein of fluorescent dye of the present invention such as Alexa Fluro series.
People's epididymal secretory protein-4 specific antibody of the present invention is monoclonal antibody or polyclonal antibody.Quality Control molecule such as rabbit igg, the biomolecule such as goat anti-rabbit igg be combined with Quality Control molecular specificity.
In order to reduce the impact on fluorescent dye fluorescence signal, the present invention adopts the chromatographic film of hypofluorescence, base plate and buckle, and its fluorescent noise is can be very weak being greater than 550nm, thus ensures to obtain high fluorescence signal-to-background ratio, energy good discrimination signal and background, and then improve detection sensitivity.Preferred base plate is white, and surface is with adhesive sticker, and buckle, chromatographic film, base plate and adhesive sticker be not all containing fluorescer.
In the present invention, testing sample can be serum or blood plasma, and now fluorescence immune chromatography test paper bar does not comprise blood separation membrane.Testing sample also can be whole blood, and now fluorescence immune chromatography test paper bar also comprises blood separation membrane, for solidifying, filtration cell.Blood separation membrane can be arranged between label pad and chromatographic film, respectively with label pad and the direct capillary contact of chromatographic film, or be arranged between sample pad and label pad, respectively with sample pad and the direct capillary contact of label pad, also can directly and sample pad merge into same structure, thus sample pad has the effect of sample collection, release and filtration simultaneously.
Fluorescence immune chromatography kit of the present invention quantitatively detects the people's epididymal secretory protein-4 in testing sample.During detection, join in sample pad by testing sample by well, sample moves along chromatographic film to adsorptive pads direction chromatography.The sample chromatography time is generally 8 ~ 25 minutes, preferably 15 minutes.After chromatography, by Wash buffer solution for cleaning chromatographic film, the time is 3-8 minute, preferably 5 minutes, to reduce background, improves detection sensitivity.Comprise bovine serum albumin(BSA), sucrose and surfactant in Wash damping fluid of the present invention, pH value is 7.0-8.0, and wherein, the concentration of bovine serum albumin(BSA) is 0.05 ~ 2%, and the concentration of sucrose is 1 ~ 15%, and the concentration of surfactant is 0.05 ~ 2%.The preferred polysorbas20 of surfactant, triton x-100, the preferred Tris-HCl damping fluid of damping fluid, phosphate buffer.
Detection fluorescent quantitation instrument of the present invention comprises excitation source module, filtration module, photoelectric conversion module, control analysis module and software systems.Wherein excitation source module comprises light source and beam condensing unit, and this light source is light emitting diode or laser diode, and wavelength is between 600 ~ 700nm.Filtration module comprises optical filter wheel, and this optical filter wheel comprises optical filter not of the same race, to obtain corresponding fluorescence signal.Photoelectric conversion module comprises imageing sensor or photomultiplier.
After chromatography terminates, under light source activation, the fluorescence signal that test strips produces is through filtration module filtering parasitic light and background fluorescence, arrive photoelectric conversion module, obtain digital signal, the fluorescence signal intensity that nature controlling line obtains and the fluorescence signal intensity that detection line obtains have correlativity: if nature controlling line fluorescence signal intensity exceeds the acceptable value of fluorescent quantitation instrument inner setting, illustrate that testing result is invalid; Under the effective prerequisite of testing result, the ratio of the fluorescence signal intensity that detection line obtains and nature controlling line fluorescence signal intensity is higher, represents that the concentration of target detection thing in sample is higher, otherwise lower.
The present invention adopts fluorescent quantitation instrument to detect the standard items of a series of variable concentrations, drawing standard curve.Its standard curve is the relation curve of standard items series concentration (X) and corresponding fluorescence signal intensity (Y), relational expression is Y=y0+bX, fluorescence signal intensity is Y=detection line peak area/nature controlling line peak area, then detect sample, establishing criteria curve obtains the concentration of people's epididymal secretory protein-4 in testing sample.
The principle of work of fluorescence immune chromatography kit of the present invention is: adopt fluorescence immune chromatography technology and double antibody sandwich method principle quantitatively to detect the content of the people's epididymal secretory protein-4 in sample (whole blood, serum or blood plasma).During detection, sample drop is added in loading hole, combines with fluorochrome label thing when sample flows through label pad, and along chromatographic film to adsorptive pads direction capillary moving, flow through the quantitative band in chromatographic film and quality control band respectively.If containing people's epididymal secretory protein-4 in sample, then combine with people's epididymal secretory protein-4 antibody modified by fluorescent dye, when chromatography is to detection line, can be coated in advance the capture antibody of this band catch, thus form double-antibody sandwich compound.Under light source activation, adopt fluorescent quantitation instrument can obtain the fluorescence signal intensity of detection line and nature controlling line, the typical curve obtained according to fluorescent quantitation instrument, and then the concentration containing HE4 in sample can be analyzed.
Major advantage of the present invention is as follows:
1) the present invention adopts organic fluorescent dye or the rare earth element fluorescent dye fluorescent marker as specific antibody, compared with other fluorescent dyes, have that luminous intensity is high, exciting light spectrum width, emission spectrum are narrow, fluorescence lifetime is long, the advantage such as finishing multifunction and good stability.
2) buckle, base plate and chromatographic film in kit building block of the present invention all have low fluorescent characteristic when being greater than 550nm, the impact that fluorescent dye fluorescence signal is obtained can be reduced, thus ensure to obtain high fluorescence signal-to-background ratio, and then reach and put forward highly sensitive object.
3) the fluorescence immune chromatography method of quantitative detection people epididymal secretory protein-4 of the present invention is Wash chromatographic technique, non-specific adsorption can be reduced, reduce autofluorescent background, strengthen specific binding, and then raising detection sensitivity, be conducive to accurate quantitative analysis when people's epididymal secretory protein-4 content is extremely low in sample.
4) the inventive method is compared with conventional colloidal gold immunochromatographimethod, have mark good stability, non-specific low, highly sensitive, the range of linearity is wide and the advantage such as quantitatively accurate.
Accompanying drawing explanation
Fig. 1 is the assembling schematic diagram of fluorescence immune chromatography test paper bar, and wherein 1 is sample pad, and 2 is pad, and 3 is blood separation membrane, and 4 is detection line, and 5 is nature controlling line, and 6 is chromatographic film, and 7 is adsorptive pads, and 8 is base plate.
Fig. 2 is fluorescence immune chromatography test paper bar sample test schematic diagram in embodiment 1, and utilizes this kit to test the detected peaks that obtains of sample and Quality Control peak.
Fig. 3 is fluorescence immune chromatography kit schematic diagram, and wherein 11 is buckle, and 9 is well, and 10 is view window, and 4 is detection line, and 5 is nature controlling line.
Fig. 4 is the typical curve of fluorescence immune chromatography kit in embodiment 1, with people's epididymal secretory protein-4 concentration for horizontal ordinate, with fluorescence intensity (detected peaks area ratio Quality Control peak area ratio) for ordinate.
Embodiment
Embodiment 1: with covalent cross-linking mode fluorescent dye modified antibodies, and adopt the quantitative detection of Wash immunochromatography technique to people's epididymal secretory protein-4
1) coupling of fluorescent dye and antibody
Be that the fluorescent dyes rhodamine of 665nm mixes with people's epididymal secretory protein-4 monoclonal antibody of l mg/ml by emission wavelength, room temperature reaction 3h, add 1mol/L oxammonium hydrochloride cessation reaction, and with chromatographic column or chromatographic column separation and purification, obtain people's epididymal secretory protein-4 monoclonal antibody that fluorescent dye is modified.In like manner obtain the rabbit igg (Quality Control molecule) that fluorescent dyes rhodamine is modified.Wherein the fluorescence emission wavelengths of the fluorescent dye of modified human epididymal secretory protein-4 antibody is 665nm with the fluorescence emission wavelengths of the fluorescent dye modifying rabbit igg.
2) structure of kit
Two kinds of fluorochrome label things are mixed with the ratio of 1:1, and add bovine serum albumin(BSA) (content is 1%), sucrose (content is 10%) and surfactant triton x-100 (content is 0.8%), even application is in label pad subsequently, seal after 40 DEG C of dryings, preserve under room temperature.
As shown in Figure 1, assembling quantitatively detects the fluorescence immune chromatography test paper bar of HE4, be made up of sample pad (1), label pad (2), blood separation membrane (3), chromatographic film (6), adsorptive pads (7), be pasted in turn on white base plate (8).Wherein, sample pad is poroid barrier film, and selecting glass fibre, is testing sample collecting region; The rabbit igg that HE4 specific antibody containing fluorescent dye modification in pad and fluorescent dye are modified; Chromatographic film is fixed with quantitative detection line (4) and nature controlling line (5), detection line and nature controlling line be spaced apart 5mm, and detection line (4) is fixed with the specific antibody of another epitope being different from HE4 specific antibody in label pad, nature controlling line (5) is fixed with goat anti-rabbit antibody.After assembling, be cut into required width as requested, and be placed in buckle (11), as shown in Figure 2, add drying agent encapsulation, be jointly configured to fluorescence immune chromatography kit with Wash damping fluid.
3) detection of sample
A) HE4 antigen standard normal human serum is formulated as 0ng/mL and 1000ng/mL concentration respectively as dilution;
B) by step a) in the HE4 standard solution of two kinds of concentration mix according to the ratio of 10000:0,9999:1,9990:10,9900:100,7500:2500,5000:5000 and 0:10000 successively;
C) respectively by step b) in preparation serum solution (100 μ L) drip in well (9), forward chromatography reaction 15min;
D) Wash damping fluid is prepared, pH value is 7.5, buffer system is the phosphate of 20mM, add bovine serum albumin(BSA) (concentration is 1%), sucrose (concentration is 10%) and surfactant triton x-100 (concentration is 0.8%), add 50 μ L at sample well, leave standstill 5min.
E) be placed in fluorescent quantitation instrument and obtain fluorescence signal intensity, and draw corresponding typical curve, refer to Fig. 4;
F) with step c) and steps d) operation, detect testing sample, luminoscope detected peaks is shown in Fig. 3, after chromatography terminates, be placed in fluorescent quantitation instrument and obtain fluorescence signal intensity, and according to step e) in typical curve analyze the content of HE4 in this sample;
G) output detections report.
4) interpretation of result
Result shows, its lowest detection is limited to 0.05ng/mL, is minimumly quantitatively limited to 0.1ng/mL, and batch in and batch between repeatability all better, coefficient R 2>0.99, has reference value to the early diagnosis of oophoroma.
Claims (10)
1. quantitatively detect a fluorescence immune chromatography kit for people's epididymal secretory protein-4, comprise buckle (11), fluorescence immune chromatography test paper bar and Wash damping fluid, it is characterized in that:
The external shell structure that buckle (11) is fluorescence immune chromatography test paper bar, comprises well (9) and view window (10);
Fluorescence immune chromatography test paper bar comprises sample pad (1), label pad (2), chromatographic film (6), adsorptive pads (7) and base plate (8), sample pad (1), label pad (2), chromatographic film (6) and adsorptive pads (7) are equipped on base plate (8), sample pad (1) is positioned at the below of well (9), and be connected to label pad (2), label pad (2) is connected to chromatographic film (6), chromatographic film (6) is connected to adsorptive pads (7), chromatographic film (6) is fixed with a quantitative detection line (4) and a nature controlling line (5), detection line (4) and nature controlling line (5) are positioned at the below of view window (10),
Label pad (2) is fixed with simultaneously people's epididymal secretory protein-4 specific antibody that fluorescent dye is modified, with the Quality Control molecule that fluorescent dye is modified, the emission wavelength of the fluorescent dye of modified human epididymal secretory protein-4 specific antibody is identical with the emission wavelength of the fluorescent dye modifying Quality Control molecule, and wavelength coverage is 300-1300nm;
Quantitative detection line (4) is fixed with people's epididymal secretory protein-4 specific antibody, the antigenic determinant that this specific antibody identifies is different from the antigenic determinant that people's epididymal secretory protein-4 specific antibody that the fluorescent dye be fixed in label pad (2) is modified identifies; And
Nature controlling line (5) is fixed with the biomolecule that can be combined with Quality Control molecular specificity.
2. kit according to claim 1, it is characterized in that, described fluorescence immune chromatography test paper bar also comprises blood separation membrane (3), it is arranged between label pad (2) and chromatographic film (6), now label pad (2) is connected to blood separation membrane (3), and blood separation membrane (3) is connected to chromatographic film (6); Or be arranged between sample pad (1) and label pad (2), now sample pad (1) is connected to blood separation membrane (3), and blood separation membrane (3) is connected to label pad (2); Or direct and sample pad (1) merges into same structure.
3. kit according to claim 1, is characterized in that, described fluorescent dye is organic fluorescent dye or rare earth element fluorescent dye, and emission wavelength is 550-800nm, preferred 665nm.
4. kit according to claim 1, is characterized in that, described chromatographic film, base plate and the buckle fluorescence when being greater than 550nm is very weak or not containing fluorescer, preferred base plate is white, and surface is with adhesive sticker, and both are not all containing fluorescer.
5. kit according to claim 1, it is characterized in that, described Wash damping fluid comprises bovine serum albumin(BSA), sucrose and surfactant, pH value is 7.0-8.0, wherein, the concentration of bovine serum albumin(BSA) is 0.05 ~ 2%, and the concentration of sucrose is 1 ~ 15%, and the concentration of surfactant is 0.05 ~ 2%.
6. the preparation method of the fluorescence immune chromatography kit of quantitative detection people epididymal secretory protein-4 according to claim 1, is characterized in that, comprise the steps:
Step (1): with specific effect between chemical crosslinking or biomolecule respectively by the surface of conjugated fluorescent dyes to people's epididymal secretory protein-4 specific antibody and Quality Control molecule, obtain people's epididymal secretory protein-4 specific antibody of fluorescent dye modification and the Quality Control molecule of fluorescent dye modification respectively, the emission wavelength of modifying the fluorescent dye of described Quality Control molecule is identical with the emission wavelength of the fluorescent dye of modified human epididymal secretory protein-4 specific antibody, and wavelength coverage is 300 ~ 1300nm;
Step (2): people's epididymal secretory protein-4 specific antibody that fluorescent dye step (1) obtained is modified and the Quality Control molecule that fluorescent dye is modified all are fixed in label pad (2);
Step (3): a quantitative detection line (4) and a nature controlling line (5) are set respectively in chromatographic film (6), wherein, nature controlling line (5) is fixed with the biomolecule that can be combined with Quality Control molecular specificity, quantitative detection line (4) is fixed with people's epididymal secretory protein-4 specific antibody, and the antigenic determinant that this specific antibody identifies is different from the antigenic determinant that people's epididymal secretory protein-4 specific antibody that the fluorescent dye be fixed in label pad (2) is modified identifies;
Step (4): sample pad (1), label pad (2), chromatographic film (6), adsorptive pads (7) and base plate (8) are built into fluorescence immune chromatography test paper bar;
Step (5): fluorescence immune chromatography test paper bar is installed.
7. method according to claim 6, it is characterized in that, also comprise the step that blood separation membrane (3) are set, blood separation membrane (3) is arranged between label pad (2) and chromatographic film (6), now label pad (2) is connected to blood separation membrane (3), and blood separation membrane (3) is connected to chromatographic film (6); Or be arranged between sample pad (1) and label pad (2), now sample pad (1) is connected to blood separation membrane (3), and blood separation membrane (3) is connected to label pad (2); Or direct and sample pad (1) merges into same structure.
8. method according to claim 6, is characterized in that, described fluorescent dye is organic fluorescent dye or rare earth element fluorescent dye, and emission wavelength is 550-800nm, preferred 665nm.
9. method according to claim 6, is characterized in that, described chromatographic film, base plate and the buckle fluorescence when being greater than 550nm is very weak or not containing fluorescer, preferred base plate is white, and surface is with adhesive sticker, and both are not all containing fluorescer.
10. method according to claim 6, it is characterized in that, also comprise the step of preparation Wash damping fluid, Wash damping fluid comprises bovine serum albumin(BSA), sucrose and surfactant, pH value is 7.0-8.0, and wherein, the concentration of bovine serum albumin(BSA) is 0.05 ~ 2%, the concentration of sucrose is 1 ~ 15%, and the concentration of surfactant is 0.05 ~ 2%.
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| CN111198264A (en) * | 2020-02-12 | 2020-05-26 | 北京大弘生物技术有限公司 | System for quantitatively detecting heavy metal cadmium and preparation method thereof |
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| CN111198264B (en) * | 2020-02-12 | 2023-09-29 | 武汉睿奇生物工程有限公司 | System for quantitatively detecting heavy metal cadmium and preparation method thereof |
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