CN103336132A - Detection method of lactoferrin in tears and dedicated colloidal gold detecting card thereof - Google Patents
Detection method of lactoferrin in tears and dedicated colloidal gold detecting card thereof Download PDFInfo
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Abstract
The invention discloses a detection method of lactoferrin in tears and a dedicated colloidal gold detecting card thereof, and belongs to the field of medical diagnostic reagent technology. The lactoferrin rapid colloidal gold detecting card comprises a detecting card and a colloidal gold test strip, and is prepared by following steps: lactoferrins monoclonal antibodies of high sensitivity and specificity are obtained by taking lactoferrins as antigens to immunize mice; the lactoferrin colloidal gold test strip is obtained by using colloidal gold chromatography; and the colloidal gold test strip is fixed in the detecting card through a groove. The content of lactoferrin in tears is detected by using immune-colloidal gold technology, so that detecting cost is low, and detecting accuracy is high.
Description
Technical field
The present invention relates to a kind of method and special-purpose collaurum test card thereof that detects lactoferrin in the tear, is a kind of detection utensil that detects lactoferrin in the tear, belongs to the medical diagnosis technical field.
Background technology
Lactoferrin (lactoferrin is a kind of glycosylated protein LF), belongs to iron-binding protein family, it can reversibility ground in conjunction with two ferric ions, it is to separate from cow's milk the earliest, so gain the name.The about 80kDa of human lactoferrin molecular weight, isoelectric point is about 7.8, is made up of 692 amino acid, is present in the multiple body fluid of human body and the juice as tear, saliva, milk etc.Lactoferrin has the antibacterial functions of wide spectrum, mainly is because lactoferrin can be combined with ferric ion, thereby can the competitive gram-positive bacteria that need to suppress iron, can be combined the anticancer antimicrobial effect of performance in addition with numerous protein and DNA.Lactoferrin has antiviral activity widely, the scope of effect comprises dna virus and RNA virus, its resistance to virus shows in the suction-operated of opposing virus to cell surface, direct and virus or Virus receptors combination, thus eliminate viral invasive ability.In addition, lactoferrin also has anticancer cell, suppresses the function that cholesterol accumulates, suppresses lipid peroxidation.
Lactoferrin is mainly produced by lacrimal secretion in the tear, with tear lysozyme, tear prelbumin and on a small quantity secretion property immunoglobulin (Ig) and growth factor constituted the principal ingredient of albumen in the tear jointly, a protection eye surface is not invaded by foreign object.The angle xerosis of conjunctiva claims dry eyes again, be that any reason causes that the tear membrane stability that the tear quality and quantity is unusual or dynamics causes unusually descends, and with ophthalmic uncomfortable, causing table organization's pathology is the general name of the multiple disease of feature, and the symptom of common scheroma has eyes to have dry and astringent, cusalgia sense, gum more; Eye acid, eye are itched, are kept in dark place and hypopsia etc.The rejuvenation trend of scheroma is obvious in recent years, mainly be because in the modern life, young people's work contacts more and morely with amusement with TV, computer, for a long time in the face of video screen, shortage is in time blinked or is allowed eyes have a rest, and has influenced the lacrimal secretion of eyes.Diagnose the inspection of scheroma to comprise lacrimal secretion experiment, breakup time of tear film, conjunctival epithelium dyeing, marking cytolgical examination, lysozyme content detection, lactoferrin content detection etc. clinically.The content of tear lactoferrin is relatively stable, and the content of lactoferrin is between 1.14 ~ 1.78g/L in the normal tear fluid, and repeatability is better, if lactoferrin content is lower than 0.9g/L in the tear, then diagnosable is scheroma.
The detection method of lactoferrin mainly contains spectrophotometric method and high performance liquid chromatography etc. in the tear.Spectrophotometric method and high performance liquid chromatography are easy and simple to handle, but need the sample size of detection bigger, and the amount of tear sample is considerably less, therefore is not suitable for the detection of lactoferrin in the tear.The present invention a kind ofly has antigenic immunological detection method based on lactoferrin, take immunology competition ratio juris, a kind of quick detection reagent of association colloid gold labelling technique and immunochromatographic method design can detect the lactoferrin in the tear fast specifically.Lactoferrin content in the sample is judged in the colour developing that has or not according to test card sample detection line (T) and nature controlling line (C).The present invention utilizes the lactoferrin antigenicity and a kind of detection method of setting up, has that sample size is few, the advantage of high specificity, and test card is simple to operate simultaneously need not special instrument and equipment fast, is fit to execute-in-place.
Summary of the invention
The object of the invention provides a kind of method and special-purpose collaurum test card thereof that detects the tear lactoferrin.
The special-purpose collaurum test card (seeing accompanying drawing) of lactoferrin content in the detection tear provided by the present invention, it is for arranging test-strips 2 in the test card shell 1 of rectangular flat shell shape, and the test card case surface has the fenestra 3 of detection and well 4; Test-strips 2 is formed by pasting sample pad 8, collaurum film 7, cellulose membrane 6 and absorbing membrane 9 thereon successively by adhesive sticker on the supporting backboard 5; The stacked stickup cellulose membrane 6 in supporting backboard middle part, one endlap of supporting backboard is put and is pasted absorbing membrane 9, the stacked stickup sample pad 8 of the supporting backboard other end, absorbing membrane is inner to overlap with cellulose membrane respectively separately with sample pad the inner, at the clinch of sample pad and cellulose membrane, insert and put between the two and paste one section collaurum film 7; The collaurum film is the glass fibre membrane that contains anti-lactoferrin antibody colloidal gold mark, there is one to detect band and a quality control band on the cellulose membrane, be successively: the detection that contains lactoferrin is with 10, the quality control band 11 that contains anti-rabbit antibody or anti-mouse antibody, test-strips is inserted in the test card shell, sample pad is over against well, and cellulose membrane is over against detecting fenestra.
The toughness material of described supporting backboard 5 for not absorbing water.
Described toughness material is made with hard plastic bar or the cardboard bar that do not absorb water.
Described sample pad 8 is glass fibre membrane, or nylon membrane, PVDF membrane, polyester film.
The pad of collaurum film 7 also is glass fibre membrane, or nylon membrane, PVDF membrane, polyester film.
Described cellulose membrane 6 is nitrocellulose filter, or the carboxylation cellulose membrane.
Described absorbing membrane 9 is the stronger absorbent filter of water-intake capacity or filter paper for oil.
Described lactoferrin gold labeling antibody is the lactoferrin monoclonal antibody of colloid gold label.
Collaurum test card provided by the present invention detects the method for lactoferrin content in the tear, may further comprise the steps:
(1) sample preparation
Fresh tear sample can be directly used in detection, and freezing or refrigeration tear sample needs rewarming to room temperature to detect.
(2) utilize lactoferrin in the above-mentioned collaurum test card test sample
Collaurum test card of the present invention can be qualitative, lactoferrin content in the half-quantitative detection tear sample.Test card is taken out from the test card bag, place room temperature, carry out the sample number into spectrum mark; Draw sample with disposable dropper, splash in the well, 3 in every hole does not make liquid overflow well in the application of sample process; Observations in 5 minutes.
Beneficial effect of the present invention: detect the method for lactoferrin content in the tear with collaurum test card of the present invention, advantage is that to detect required tear sample size few, and easy to use, detection is quick, highly sensitive, the result is accurate.
Description of drawings
Fig. 1 lactoferrin rapid detection card shell and test-strips arrangement figure in the enclosure.1, test card shell, 2, test-strips, 3, detect fenestra, 4, well.
Fig. 2 detects and shows the trace synoptic diagram on the cellulose membrane of strip.6, cellulose membrane, 10, detect band, 11, quality control band.
The detection strip structural representation of Fig. 3 lactoferrin rapid detection card.5, supporting backboard, 6, cellulose membrane, 7, the collaurum film, 8, sample pad, 9, absorbing membrane.
Embodiment
The animal program: with lactoferrin as antigen, 100 μ g/ immunizing dose only is with immune 2 the Balb/c mouse of the fully emulsified pneumoretroperitoneum multi-point injection of isopyknic Freund's complete adjuvant, every 2 ~ 3 weeks again with same dose antigen with incomplete Freund emulsification after immunity 2 ~ 3 times, merge first three day booster immunization once.
Fusion of Cells and cloning: get immune Balb/c mouse boosting cell and myeloma cell SP2/0 and merge in 5 ︰, 1 ratio, adopt indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay that cloning is carried out in positive hole, up to the hybridoma cell strain that obtains the anti-lactoferrin monoclonal antibody of stably excreting.
Cell cryopreservation and recovery: the monoclonal hybridoma strain of getting the lactoferrin that is in exponential phase makes 5 * 10 with cryopreserving liquid
6The cell suspension of individual/mL is sub-packed in frozen pipe, in the medium-term and long-term preservation of liquid nitrogen.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying: select the Balb/c mouse in 10 ages in week, lumbar injection hybridoma suspension, inoculating cell was collected ascites after 7 ~ 10 days, through sad-saturated sulfuric acid amine method and Protein A affinity chromatography purifying.
The preparation of embodiment 2 colloid gold label lactoferrin monoclonal anti nanocrystal composition
The preparation of colloid gold particle: get 1% chlorauric acid solution 1mL, add the chlorauric acid solution that the 99mL ultrapure water becomes final concentration 0.01%, behind the ebuillition of heated, get in the above-mentioned chlorauric acid solution that the disposable rapid adding of 1% trisodium citrate 1.6mL boils, continue to be heated to solution and transfer the black-and-blue shiny red that finally becomes to by faint yellow, continue heating 5 minutes behind the colour stable, the room temperature cooling adds water to original volume.
Preparation and the purifying of gold labeling antibody: use 0.1mol/L K
2CO
3Regulate colloidal gold solution pH to optimal pH, the Ractopamine monoclonal antibody that dropwise adds the optimum protein amount when evenly stirring, all add in 5 minutes, lucifuge continues to stir after 30 minutes, get in the stabilizing solution balance colloidal gold solution of optimum formula, after all adding, 4 ℃ of high speed centrifugations carry out purifying to the protein of mark and concentrate to remove the excess protein that has neither part nor lot in mark, not completely stable colloid gold particle and the various polymkeric substance that may form.
The assembling of embodiment 3 lactoferrin collaurum rapid detection card
Golden labeling antibody is suitably diluted the back be sprayed on pad with the metal spraying machine, 37 ℃ of dryings get the collaurum film.Lactoferrin is diluted to optium concentration with the phosphate buffer of 0.01mol/L pH7.4, is sprayed on the cellulose membrane 6 with spray film instrument, and this is detection line 10; At 5 millimeters sheep anti-mouse iggs of locating to spray optium concentration above detection line, this is nature controlling line 11.After 37 ℃ of dryings, cellulose membrane, collaurum film, sample pad and thieving paper are assembled on the base plate successively, the test strips that is cut into suitable width with automatic cutting machine is packed in the test card shell.
Embodiment 4 lactoferrin collaurum test card detection reaction principles
Add the well of test card when testing sample solution after, solution to be measured spreads to cellulose membrane together by the golden labeling antibody that syphonic effect drives in determinand and the gold-marking binding pad, and finally infiltrates the adsorptive pads end.In diffusion process, if when in the sample determinand being arranged, determinand and golden labeling antibody combination, and then occupied antigen binding site on the golden labeling antibody, stop the combination of detection line on golden labeling antibody and the cellulose membrane (lactoferrin), detection line is not developed the color or develop the color the very weak positive or weak positive (content of lactoferrin reaches finite concentration in the sample) of test sample of namely representing; If when not having testing sample in the sample, golden labeling antibody is met clear red line of detection line demonstration and is namely represented test sample feminine gender (content of lactoferrin does not reach finite concentration in the sample) in moving past journey.Equally, golden labeling antibody also with cellulose membrane on nature controlling line (sheep anti-mouse igg) combination, make nature controlling line show red.The having or do not have and represent the effective or invalid of this test card respectively of nature controlling line color.
Embodiment 5:(Application Example)
Specificity test: test by embodiment 4 described principles, the standard items of other albumen lysozymes, human serum albumins, human transferrin in the tear are made into 10g/L, detect with test card of the present invention, detection line and the equal outlet of nature controlling line (colour developing) on the plain film of display fibers as a result, be that lactoferrin test card and other tear albumen do not have cross reaction, thereby conclude that this test card specificity is good, when detecting, can not be subjected to the interference of other samples.
Susceptibility and uniformity test: test by embodiment 4 described principles, detect 0,0.3,0.6,0.9,1.2,1.5 and the human lactoferrin standard items of 3g/L with collaurum test card of the present invention, repeat 10 times, wherein 0,0.3 and the testing result that detects of the human lactoferrin standard items of 0.6g/L be detection line and the equal outlet of nature controlling line on the cellulose membrane, 0.9,1.2,1.5 and the testing result that detects of the human lactoferrin standard items of 3g/L be only nature controlling line outlet on the cellulose membrane.Therefore the sensitivity of test strip of the present invention is 0.9g/L.Repeat 10 times and detect colour developing degree of depth homogeneous, testing result shows consistent.
Stability test: test card is put into aluminium foil bag pack, adorn simultaneously the drying prescription of being responsible for a task until it is completed in each packaging of aluminium foil bag, room temperature preservation after 12 months every index all meet, namely detect other albumen no cross reactions in the tear, sensitivity reaches 0.9g/L, detect the display depth homogeneous, the reaction result unanimity.
Claims (2)
1. a special-purpose collaurum test card of measuring lactoferrin content in the tear is characterized in that it is in the test card shell (1) of rectangular flat shell shape test-strips (2) to be set, and the test card case surface has detection fenestra (3) and well (4); Test-strips (2) is upward pasted sample pad (8), collaurum film (7), cellulose membrane (6) and absorbing membrane (9) thereon successively by adhesive sticker by supporting backboard (5) and is formed; Supporting backboard (5) middle part stacked stickup cellulose membrane (6), (5) one endlaps of supporting backboard are put and are pasted absorbing membrane (9), supporting backboard (5) the stacked stickup sample pad of the other end (8), absorbing membrane (9) is inner to overlap with cellulose membrane (6) respectively separately with sample pad (8) the inner, at the clinch of sample pad (8) with cellulose membrane (6), insert and put between the two and paste one section collaurum film (7); Collaurum film (7) is for containing the glass fibre membrane of anti-lactoferrin antibody colloidal gold mark, there is one to detect band and a quality control band on the cellulose membrane, be successively: the detection band (10) that contains lactoferrin, the quality control band (11) that contains anti-rabbit antibody or anti-mouse antibody, test-strips (2) is inserted in the test card shell (1), sample pad (8) is over against well (4), and cellulose membrane (6) is over against detecting fenestra (3);
The toughness material of described supporting backboard (5) for not absorbing water, described toughness material is made with hard plastic bar or the cardboard bar that do not absorb water;
Described sample pad (8) is glass fibre membrane, or nylon membrane, marriage vinylidene fluoride film, polyester film;
Described cellulose membrane (6) is nitrocellulose filter, or the carboxylation cellulose membrane;
Described absorbing membrane (9) is the stronger absorbent filter of water-intake capacity or filter paper for oil;
Described lactoferrin gold labeling antibody is the lactoferrin monoclonal antibody of colloid gold label.
2. detect the method for lactoferrin content in the tear with the described collaurum test card of claim 1, it is characterized in that may further comprise the steps:
(1) sample preparation
Fresh tear sample can be directly used in detection, and freezing or refrigeration tear sample needs rewarming to room temperature to detect;
(2) utilize lactoferrin in the described collaurum test card test sample
Described collaurum test card can be qualitative, lactoferrin content in the half-quantitative detection tear sample; Test card is taken out from the test card bag, place room temperature, carry out the sample number into spectrum mark; Draw sample with disposable dropper, splash in the well, 3 in every hole does not make liquid overflow well in the application of sample process; Observations in 5 minutes.
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Cited By (9)
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| CN107509721A (en) * | 2016-06-15 | 2017-12-26 | 北京师范大学 | A kind of method for preserving tear |
| CN107709991A (en) * | 2015-10-05 | 2018-02-16 | 广东盛泽康华生物医药有限公司 | Method and apparatus for diagnosing ocular inflammation and scheroma |
| CN107735026A (en) * | 2015-05-01 | 2018-02-23 | 黛尔格诺斯蒂尔有限公司 | The method for measuring tear component in ocular fluid samples |
| CN109068971A (en) * | 2016-01-14 | 2018-12-21 | 黛尔格诺斯蒂尔有限公司 | The method for measuring the tear component in ocular fluid samples |
| CN109124692A (en) * | 2018-07-17 | 2019-01-04 | 北京大学第三医院 | Non-invasive materials, storage and the transportation resources of ocular sample |
| CN110068688A (en) * | 2019-05-22 | 2019-07-30 | 福建农林大学 | Flow measurement chromatography detection card is immunized in lactoferrin competition law nano flower in a kind of cow's milk |
| CN113721013A (en) * | 2021-11-02 | 2021-11-30 | 首都医科大学附属北京朝阳医院 | Eye surface liquid collecting and detecting device |
| CN115561458A (en) * | 2022-07-08 | 2023-01-03 | 华中科技大学同济医学院附属协和医院 | Colloidal gold rapid detection card for diagnosing biliary tract occlusion and preparation method thereof |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107735026A (en) * | 2015-05-01 | 2018-02-23 | 黛尔格诺斯蒂尔有限公司 | The method for measuring tear component in ocular fluid samples |
| NL2017209A (en) * | 2015-07-30 | 2017-02-02 | Chinese Acad Inspection & Quarantine | Test strip for double-antibody sandwich colloidal golddetection against human lactoferrin |
| CN107709991A (en) * | 2015-10-05 | 2018-02-16 | 广东盛泽康华生物医药有限公司 | Method and apparatus for diagnosing ocular inflammation and scheroma |
| CN109068971A (en) * | 2016-01-14 | 2018-12-21 | 黛尔格诺斯蒂尔有限公司 | The method for measuring the tear component in ocular fluid samples |
| CN107509721A (en) * | 2016-06-15 | 2017-12-26 | 北京师范大学 | A kind of method for preserving tear |
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| CN110068688A (en) * | 2019-05-22 | 2019-07-30 | 福建农林大学 | Flow measurement chromatography detection card is immunized in lactoferrin competition law nano flower in a kind of cow's milk |
| CN110068688B (en) * | 2019-05-22 | 2020-08-14 | 福建农林大学 | Lactoferrin competition method nanoflower immunity detection flow chromatography detection card in cow's milk |
| CN113721013A (en) * | 2021-11-02 | 2021-11-30 | 首都医科大学附属北京朝阳医院 | Eye surface liquid collecting and detecting device |
| CN115561458A (en) * | 2022-07-08 | 2023-01-03 | 华中科技大学同济医学院附属协和医院 | Colloidal gold rapid detection card for diagnosing biliary tract occlusion and preparation method thereof |
| CN115561458B (en) * | 2022-07-08 | 2023-12-05 | 华中科技大学同济医学院附属协和医院 | Colloidal gold rapid detection card for diagnosing biliary tract occlusion and preparation method thereof |
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Application publication date: 20131002 |