CN101504417B - Colloid selenium test paper for semi-quantitative determination of urinary trace albumin - Google Patents
Colloid selenium test paper for semi-quantitative determination of urinary trace albumin Download PDFInfo
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Abstract
The invention provides a test paper for semi-quantitatively detecting colloid selenium in microdose albumin in urine. The test paper can be applied in the fields of early diagnosis of diabetic nephropathy, hypertensive nephropathy and preeclampsia and injury of kidney caused by various poisonous substances. A plastic bottom plate of the test paper is orderly glued with a sampling liquid absorption part, a colloid selenium labeling part, a detection reaction part and a water absorption part, wherein the colloid selenium labeling part is a single-cloning antibody 1 which is coated by a glass fiber membrane, or acetic acid fiber membrane, or nylon membrane and labeled by the colloid selenium; the detection reaction part is a nitrocellulose membrane which is provided with two detection bands and quality control bands, T1 is a single-cloning antibody 2 which is precisely quantitative, and T2 is the single-cloning antibody 2 with a certain concentration; and the quality control bands are coated by antiplague IgG. The invention provides a method for semi-quantitatively detecting the microdose albumin in a urine sample conveniently, visually and quickly.
Description
One, technical field
The present invention is a kind of colloid selenium test paper for semi-quantitative determination of urinary trace albumin, can judge directly quickly and easily whether albumin content is higher than normal level in the urine by this test paper, can be applicable to the early stage auxiliary clinical diagnosis of diabetic nephropathy.The category that belongs to disease vitro detection instrument and equipment.
Two, background technology
Diabetic nephropathy is the chronic kidney complication of diabetes.Diabetic nephropathy generally refers to DGS, and its clinical manifestation is albuminuria, oedema, hypertension or azotemia, and has fasting blood-glucose to increase or diabetic symptom.The no obvious regional disparity of this disease, diabetes I type, II type all can cause.According to statistics, the course of disease is the diabetic of 10-20, and no matter the age size has 50% clinical renal lesions takes place approximately.The diabetic has 5%-15% to die from renal failure approximately.Up to the present the methods for the treatment of of still not having special efficacy.If can diagnose early and treat, developing of prevention renal insufficiency can prolong patient's life-span.
Microdose urine protein (microalbunminuria in 1974, MAU) proposed first and be used for clinical, Mogensen divides interim subclinical transition period with it as diabetic nephropathy (DN) to the eighties in 20th century, thereby MAU becomes the window of DN early diagnosis.MAU is clearly as comprising diabetes (DM), hypertension and other chronic kidney disease patient even general population's cardiovascular complication, kidney prognosis and the dead independent prediction factor in recent years, and is many about the detection of urinary albumin having been classified as people at highest risk's screening indexes in chronic kidney disease and the diabetic nephropathy guide.
(microalbunminuria MAU) refers to that albumin content exceeds healthy people with reference to scope in the urine, but can not detect the variation of this trace with the method for routine microdose urine protein.It is the early stage change of disease that MAU discharges increase, and to the early diagnosis of disease, early treatment has important value and clinical meaning.MAU is the sensitive indicator of prediction diabetic nephropathy, hypertension, angiocardiopathy injury of blood vessel, also can in concealment ephritis and ephritis convalescence urine, occur, and be the important indicator of the early detection injury of kidney of comparison sensitivity.
Microdose urine protein (MAU) be the diabetic nephropathy of generally acknowledging early diagnosis according to one of.The MAU detection method is a lot of at present, radioimmunology, enzyme immunoassay and immune turbidimetry are arranged, but RIA utilizes radioactive isotope as tracer agent, influence health of operators, and serious environment pollution, the instrument that uses is more expensive, and the mensuration flow process of RIA is longer, has limitation in clinical practice; The cost of chemoluminescence method is higher, and reagent and instrument all depend on import; ELISA detects needs instrument, also is not suitable for on-the-spot the detection.
That the electroselenium immunochromatographic method has is quick, highly sensitive, high specificity, good stability, easy and simple to handle, need not any instrument and equipment, the result judges intuitive and reliable, economical and practical characteristics.The object of the present invention is to provide a kind of colloid selenium test paper for semi-quantitative determination of urinary trace albumin, it need not any instrument and equipment, quick, highly sensitive, high specificity, good stability, easy and simple to handle, result judges intuitive and reliable, reduced simultaneously the detection cost effectively, simplified operating process, shorten detection time, had economic and practical characteristics.
Three, summary of the invention
The present invention sets up a kind of colloid selenium test paper of measuring albumin content in the urine by the double antibodies sandwich method.By detection zone colour developing band number is judged whether albumin is higher than normal level in the urine, thereby forewarning function and the early stage auxiliary diagnosis of diabetic nephropathy, hypertension, angiocardiopathy injury of blood vessel disease, concealment ephritis have been realized.
The present invention is pasted with sample liquid absorption portion, electroselenium mark part, detection reaction part and suction part successively on plastic bottom board.
Wherein, described sample liquid absorption portion is filter paper or glass fibre membrane, plays a part during detection to absorb sample solution, is convenient to sample solution and moves up.
Described electroselenium mark part is glass fibre membrane or cellulose acetate film or nylon membrane, and material of mark is MAU monoclonal antibody 1 on it.
Described detection reaction is nitrocellulose membrane partly, has two on it and detects band and quality control band, wherein detects the MAU monoclonal antibody 2 that band T1 is combined with accurate quantification, and T2 is in conjunction with a certain amount of MAU monoclonal antibody 2, and the quality control band bag is by anti-mouse IgG.Judge whether to suffer from diabetic nephropathy etc. according to colour developing band number.
Described suction part is filter paper or glass fibre membrane, is used for the drainage sample solution.
The assembling of test strips: on plastic bottom board, be pasted with sample liquid absorption portion, electroselenium mark part, detection reaction part and suction part successively, namely become microdose urine protein half-quantitative detection test paper.
Principle of the present invention is that bag is marked MAU monoclonal antibody 1 by a certain amount of selenium on the glass fibre membrane, when albumin content is lower than normal amount in the urine, albumin in the urine is at first marked monoclonal antibody 1 combination with selenium, be that albumin+selenium mark monoclonal antibody 1 compound will move to detection band T1 at film in company with urine, combinations take place with the MAU monoclonal antibody 2 of the normal amount of accurate quantification on it, detect band T1 colour developing, detect band T1 colour developing, T2 does not develop the color, selenium mark monoclonal antibody 1 continues to move to quality control band in company with urine at film, quality control band colour developing, after this this negative result.When albumin content is higher than normal amount in the urine, monoclonal antibody 1 combination of albumin and electroselenium mark, be that albumin+selenium mark monoclonal antibody 1 compound will move to detect at film and be with 1 in company with urine, combinations take place with the MAU monoclonal antibody 2 of the normal amount of accurate quantification on it, detect band T1 colour developing, combination can take place with the MAU monoclonal antibody 2 that detects on the band T2 in unnecessary albumin+selenium mark monoclonal antibody 1 compound, detect band T2 colour developing, selenium mark monoclonal antibody 1 continues to move to quality control band in company with urine at film, quality control band colour developing, after this this positive result.
Positive effect of the present invention and effect are: microdose urine protein half-quantitative detection test paper of the present invention has further been realized the more significant half-quantitative detection of clinical detection by the immunochromatography effect of selenium mark reagent.Detect overall process result of determination in 10 minutes, can realize easily that family is detected and on-the-spot the detection, has the specificity height, trace routine is simple, the result accurately and reliably, operating personnel need not professional training, by specification instructs and can obtain testing result, thereby help early diagnosis and the improvement of diabetic nephropathy, can also realize timely monitoring and the tracking of early stage injury of kidney.
Four, description of drawings
Fig. 1 is the front schematic view of structure of the present invention.
The 1st, sample liquid absorption portion, the 2nd, electroselenium mark part, the 3rd, the detection reaction part is by detecting band T1, detecting and be with T2, quality control band C to form; The 4th, the suction part.
Fig. 2 is use presentation of results figure of the present invention.A detects and is with 1 colour developing among Fig. 2, and detect and be with 2 not develop the color, the quality control band colour developing, negative result shows that albuminous content is lower than 20ug/ml in the urine, and is normal.B detects and is with 1 colour developing among Fig. 2, and detect and be with 2 colour developings, the quality control band colour developing, positive result shows that albuminous content is higher than 20ug/ml in the urine, and is ill.Among Fig. 2 c detect be with 1,2 and quality control band all do not develop the color, show that test strips lost efficacy.
Five, embodiment
Embodiment one microdose urine protein MONOCLONAL ANTIBODIES SPECIFIC FOR
With human serum albumins (U.S. Sigma product) immune mouse.Choosing tires meets the requirements of mouse boosting cell and murine myeloma cell hybridization fusion, preparation hybridoma.Select positive high hybridoma and carry out repeatedly subclone, up to the cell line that filters out energy stably excreting MAU monoclonal antibody.
Employing inoculation hybridoma to the method for mouse peritoneal induces ascites, extracts ascites and be purified to obtain monoclonal antibody 1,2, proves at antigenic determinants different on the antigen.
The preparation of embodiment two electroseleniums
In reactor, add 10% shitosan, add ascorbic acid, selenous acid, ascorbic acid behind the 2min successively, dripped selenous acid, ascorbic acid at interval on the 4th minute from the reaction beginning, carry out spectral scan then, be advisable to occur maximum absorption band between 500～600nm, it is standby to collect product.
Preparation and the assembling of embodiment three half-quantitative detection MAU colloid gold test papers
1. sample liquid absorption portion is handled:
Filter paper or all-glass paper are immersed 2min among the PBS of 0.1mol/l pH7.4, take out, 80 ℃ of oven dry or alternate manner drying, sample liquid absorption portion.
2. the preparation of electroselenium mark part and processing:
With electroselenium and MAU monoclonal antibody 1 mixing by a certain percentage, make it form MAU monoclonal antibody 1 Dan standing grain continuous and meandering pretty an ancient spear covered with clouds
2.1 the pre-service of MAU monoclonal antibody 1 to be marked
In advance at 0.005mol/lNaCl, 4 ℃ of dialysed overnight are removed unnecessary electrolyte in the pH7.0 solution with MAU monoclonal antibody 1, and 4 ℃ of centrifugal 60min of 6500～13000r/min then remove precipitation, adjust protein concentration and namely can be used for mark to 1mg/ml.
Optimize the experiment condition of electroselenium mark MAU monoclonal antibody 1:
Adjust the pH value: with 0.1molK2CO3 MAU monoclonal antibody 1 solution is transferred to 5.0; Determine the optimum dose ratio of MAU monoclonal antibody 1 by photoelectric colorimetry: prepare the equal-volume monoclonal antibody solution (1ml) of a series of variable concentrations, add respectively in the 5ml electroselenium, rapidly mixing, then, each adds 1ml 10%NaCl solution, shakes up, and leaves standstill to survey each pipe behind the 5min.According to the size of colloid gold particle, OD measures between 520～580nm, is ordinate with the OD value, and the protein consumption is that horizontal ordinate is made a curve, and the protein consumption of getting that place that curve is close with transverse axis at first is the suitableeest stable quantity.
2.2 the preparation of antibody selenium probe
After being determined, the optimal pH of the suitableeest stable quantity of monoclonal antibody and mark just can carry out mark.Concrete steps are as follows:
Go out the total amount of needed monoclonal antibody to be marked according to the calculation of total in order to the electroselenium of mark; Under electromagnetic agitation, monoclonal antibody solution is added in the electroselenium solution, the about 5min of the protein of 1mg adds; Adding 5% bovine serum albumin(BSA) (BSA) under magnetic stirs, to make its final concentration be 1%; Continue to stir 10min.The solution of making is MAU monoclonal antibody 1 solution of electroselenium mark, and measurement volumes is standby.
2.3 the purifying of antibody selenium probe
Electroselenium mark MAU monoclonal antibody 1 solution with putting into sucrose or silica gel, is concentrated to 1/10 of original volume, adds to propylene glucosan S-400 chromatography with the centrifugal 20min sucking-off of 1500r/min supernatant again and lean on purifying; With 0.02mol/l pH8.2TBS wash-out, the liquid when collecting the color peak value, 4 ℃ of preservations are standby.
2.4 electroselenium mark MAU monoclonal antibody 1 section processes
Electroselenium mark MAU monoclonal antibody 1 solution that purifying is good is poured in the groove, and all-glass paper is immersed 1min, takes out, and drying at room temperature or vacuum freeze drying are the electroselenium mark part.
3. detection reaction section processes
Glutaraldehyde solution with 0.8% soaks nitrocellulose membrane 30min, takes out 37 ℃ of oven dry, be with T1 and T2 by 2 monoclonal antibodies 2 as detecting with flush coater spraying bag on it, T1 is the monoclonal antibody 2 of accurate quantification, and the quality control band bag is the detection reaction part by anti-mouse IgG.
4. suction section processes:
After the filter paper drying at room temperature, be the suction part.
5. the assembling of half-quantitative detection MAU colloid selenium test paper
On plastic bottom board, paste sample liquid absorption portion, electroselenium mark part, detection reaction part and suction part successively, be half-quantitative detection MAU colloid selenium test paper.
The service instruction of embodiment four colloid selenium test papers
Sampling: collect urina sanguinis and get final product.
Detect: the end that test paper is had an arrow mark immerses and is equipped with in the container of urine, and liquid level surpasses MAX line labeling, has approximately to take out after 5 seconds to keep flat visual inspection result after 5～10 minutes.
Observe: if detect band T1 colour developing, T2 does not develop the color, and the quality control band colour developing proves that then the albumin in the sample has not surpassed limit value, and is negative.If detect band T1 colour developing, the T2 colour developing, the quality control band colour developing proves that then the albumin in the sample has surpassed limit value, and is positive.If detect be with 1,2 and quality control band all do not develop the color, then prove the test strips actual effect.
Claims (2)
1. colloid selenium test paper for semi-quantitative determination of urinary trace albumin, it comprises plastic bottom board, sample liquid absorption portion, the electroselenium mark part, detection reaction part and suction part, described electroselenium mark part is made of glass fibre membrane or cellulose acetate film or nylon membrane, bag is by the MAU monoclonal antibody 1 of electroselenium mark on the film, the detection reaction part is made of nitrocellulose membrane, have two and detect band and quality control band, detect the MAU monoclonal antibody 2 that band T1 is combined with accurate quantification, detect band T2 and be combined with certain density monoclonal antibody 2, the quality control band bag is by anti-mouse IgG, it is characterized in that: the MAU monoclonal antibody 1 of selenium mark is prepared by following method: under electromagnetic agitation, monoclonal antibody solution is added in the electroselenium solution, the about 5min of the protein of 1mg adds, and adding 5% bovine serum albumin(BSA) under magnetic stirs, to make its final concentration be 1%; Continue to stir 10min, the solution of making is MAU monoclonal antibody 1 solution of electroselenium mark, and measurement volumes is standby.
2. according to right 1 described a kind of colloid selenium test paper for semi-quantitative determination of urinary trace albumin, it is characterized in that: when albumin content is lower than normal amount in the detected urine, monoclonal antibody 1 combination of albumin and electroselenium mark, be that albumin+selenium mark monoclonal antibody 1 compound will move to detection band T1 at film in company with urine, combinations take place with the MAU monoclonal antibody 2 of the normal amount of accurate quantification on it, detect band T1 colour developing, T2 does not develop the color, selenium mark monoclonal antibody 1 continues to move to quality control band in company with urine at film, the quality control band colour developing, this negative result.When albumin content is higher than normal amount in the urine, monoclonal antibody 1 combination of albumin and electroselenium mark, be that albumin+selenium mark monoclonal antibody 1 compound will move to detection band T1 at film in company with urine, combinations take place with the MAU monoclonal antibody 2 of the normal amount of accurate quantification on it, detect band T1 colour developing, combination can take place with the MAU monoclonal antibody 2 that detects on the band T2 in unnecessary albumin+selenium mark monoclonal antibody 1 compound, detect band T2 colour developing, selenium mark monoclonal antibody 1 continues to move to quality control band in company with urine at film, the quality control band colour developing, this positive result.
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| CN102192980B (en) * | 2010-03-08 | 2014-04-09 | 苏州浩欧博生物医药有限公司 | Nonmetallic colloidal particle immune analysis reagent and method |
| CN103525695B (en) * | 2012-07-04 | 2015-05-20 | 长沙中生众捷生物技术有限公司 | Portable kidney function detecting system |
| CN104807999A (en) * | 2014-01-28 | 2015-07-29 | 兰州大学 | Electroselenium rapid detection test strip |
| CN106443012B (en) * | 2016-09-12 | 2018-11-06 | 三诺生物传感股份有限公司 | A kind of test strips and preparation method thereof and the application in microdose urine protein and β2-microglobulin joint-detection |
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| CN101004419A (en) * | 2007-01-25 | 2007-07-25 | 兰州大学 | Colloid selenium test paper for detecting pulpy kidney, and type of pathogenesis bacteria |
| CN101140286A (en) * | 2007-10-16 | 2008-03-12 | 李红玉 | Colloid selenium test paper of semi-quantitative determination oxidized low density lipoprotein |
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