CN101419235A - KIM-1 detection kit and method for making same - Google Patents
KIM-1 detection kit and method for making same Download PDFInfo
- Publication number
- CN101419235A CN101419235A CNA2008100512061A CN200810051206A CN101419235A CN 101419235 A CN101419235 A CN 101419235A CN A2008100512061 A CNA2008100512061 A CN A2008100512061A CN 200810051206 A CN200810051206 A CN 200810051206A CN 101419235 A CN101419235 A CN 101419235A
- Authority
- CN
- China
- Prior art keywords
- kim
- antibody
- detection
- pad
- gold
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a KIM-1 detection reagent kit. Two ends of a pyroxylin film are provided with a gold label pad and an absorption pad respectively; the upper end of the gold label pad is provided with a sample pad; the gold label pad is coated with a purified KIM-1 antibody colloidal gold coupling marker; a detection line is coated with a purified KIM-1 antibody; a quality control line is coated with a normal anti-mouse IgG antibody; and the side of the quality control line is stuck with the absorption pad. The KIM-1 detection reagent kit is prepared through applying an immune colloidal gold technology, is planned to apply in clinic so as to promote wide application of the KIM-1 in clinic, improves the capacity for preventing kidney diseases, rapidly and sensitively detects specific protein, avoids complex operation, needs no special detection instrument, is easy to observe and judge results, and is suitable for various detection environments and various detection requirements. The detection result can be stored for a long period and is convenient to contrast and analyze. The KIM-1 detection reagent kit can be used for immune detection in the level of naked eyes, has extremely small use amount of samples, has no toxicity to operators and no pollution to environment and the like.
Description
Technical field
The present invention relates to a kind of detection method of kidney trouble, a kind of KIM-1 detection kit of using the immune colloidal gold technique development especially is provided, also disclose its preparation method simultaneously, belong to the disease detection technical field.
Background technology
Acute and chronic kidney trouble is clinical common disease, and its M ﹠ M is all very high, seeks susceptibility and specific early stage injury of kidney biomarker, to early diagnosis, treatment with to improve prognosis significant.The kidney injury molecule that the present invention relates to-1 (kidney injury molecule-1, be called for short KIM-1) is a kind of newfound transmembrane glycoprotein, expresses very littlely in normal kidney, increases and express obviously when injury of kidney.Zooscopy shows, the ectodomain fracture afterproduct of KIM-1 can be by discharging in the urine, and it can be used as the biological marker of human renal tubule damage, the situation that the KIM-1 level in the urine of therefore detecting can the Indirect evaluation kidney injury.
The method that detects KIM-1 at present comprises SABC, ELISA, Western blot etc., can be used for scientific research, but is used for clinical a lot of drawbacks that then exist, and as the running program complexity, the time is long etc.
Summary of the invention:
The present invention discloses a kind of KIM-1 detection kit, is used to diagnose kidney trouble.
The present invention also provides the preparation method of KIM-1 detection kit, is applicable to suitability for industrialized production.
KIM-1 detection kit provided by the invention, be respectively equipped with gold mark pad and absorption pad at the two ends of nitrocellulose filter, gold mark pad upper end is a sample pad, gold mark pad is coated with the KIM-1 antibody colloidal gold coupling label of purifying, detection line is coated with the KIM-1 antibody of purifying, nature controlling line is coated with normal anti-mouse IgG antibody, and the nature controlling line side is posted absorption pad.
The preparation method of KIM-1 detection kit of the present invention may further comprise the steps:
The present invention is according to the immunology ultimate principle of antigen-antibody energy specific bond, KIM-1 antibody colloid gold label with purifying, specking is prepared into gold mark pad on glass fibre membrane, the KIM-1 antibody of purifying and normal anti-mouse IgG antibody are coated on detection line place and nature controlling line place on the nitrocellulose filter (NC film) respectively, when containing KIM-1 antigen in the test sample, then in the KIM-1 antibodies of gold mark pad with colloid gold label, and under the effect of absorption pad, permeate swimming forward, combine once more with the KIM-1 antibody on the detection line, macroscopic colour band occurs.
The using method of kit of the present invention is as follows: get the about 0.5ml of urine sample and add in the small test tube, insert test strips then, treat the 1-2 minute clear back of reaction zone observations (see figure 2).1 redness (contrast) precipitation line occurs, be urine examination diagnosis feminine gender; 2 redness (sample and contrast) precipitation line appears, and positive for the urine examination diagnosis, precipitation line does not appear then for invalid.
Good effect of the present invention is: the first Application immune colloidal gold technique prepares the KIM-1 detection kit, and intend being applied to clinical, to promote KIM-1 in clinical widespread use, improve prevention and control capability to kidney trouble, can be quick, the Sensitive Detection specific protein, avoided complicated operations, need not the special detection instrument, the result is easy to observe and judges, can adapt to multiple testing environment, and multiple detection needs.And testing result can long preservation, is convenient to check analysis.The immune detection that can be used for macroscopic level, the sample consumption is minimum, to operator's avirulence, environmentally safe etc.Therefore use colloidal gold technique development KIM-1 detection kit and have important social meaning and vast market prospect.
Description of drawings
Fig. 1 .KIM-1 pick-up unit synoptic diagram;
1. sample pad; 2. the gold mark fills up; 3. nitrocellulose filter; 4. absorption pad; 5. detection line; 6. nature controlling line.
Fig. 2 .KIM-1 kit testing result is judged.
Fig. 3 is the preparation flow figure of colloid gold particle;
Fig. 4 is the purifying process flow diagram of immune colloid gold.
Embodiment
By following examples the present invention is described for example further, and do not limit the present invention in any way, under the prerequisite that does not deviate from technical solution of the present invention, any change or change that those of ordinary skills that the present invention did are realized easily all will fall within the claim scope of the present invention.
Embodiment 1
According to shown in Figure 1, the KIM-1 detection kit is made up of treated sample pad 1, gold mark pad 2, nitrocellulose filter 3, detection line 5, nature controlling line 6 and absorption pad 4, wherein, gold mark pad 2 is coated with the KIM-1 antibody colloidal gold coupling label of purifying, detection line 5 is coated with the KIM-1 antibody of purifying, nature controlling line 6 is coated with normal anti-mouse IgG antibody, and nature controlling line 6 sides are posted absorption pad 4.
Embodiment 2
1, the preparation of colloid gold particle: get 1% chlorauric acid solution 1ml and add the chlorauric acid solution that the 99ml ultrapure water becomes final concentration 0.01%, behind the ebuillition of heated, get in the 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 5min behind the colour stable, the room temperature cooling replenishes dehydration to original volume (seeing process flow diagram).Observe under transmission electron microscope, visible gold grain size basically identical is evenly distributed.Measure 100 colloid gold particle diameters, the about 20nm of arithmetic average diameter.
The preparation flow figure of colloid gold particle: see Fig. 3.
2, the preparation of immune colloid gold compound: ocular estimate is determined collaurum and KIM-1 antibody usage ratio.Regulate the pH to 8.2 of colloidal gold solution with the solution of potassium carbonate of 0.1mol/L, get 11 clean tube, every pipe packing colloidal gold solution (1ml/ pipe).To (establish control tube in addition by 0.5 μ g～5g) behind the KIM-1 antibody stepwise dilution, order adds mixing in the test tube of a series of 1ml of being equipped with collaurums; Behind the 5min, in 2～11 pipes, add 10% sodium chloride solution 0.1ml respectively, mixing, room temperature leaves standstill the above observations of 2h; No. 1 pipe neither adds antibody and does not also add the chlorination sodium solution, is control tube; No. 11 pipes that do not add antibody also are contrast (seeing Table 1); Do not add the test tube that antibody and addition are not enough to the stable colloid gold, promptly present the coagulation phenomenon by red stain indigo plant, addition meets or exceeds the quantitative test tube of minimum steady and then keeps the red constant of collaurum.Making collaurum red constant and the protein content of the minimum test tube of antibody content is the indispensable protein amount of stablizing the 1ml collaurum with this, also is that minimum steady is quantitative.On the quantitative basis of colloid gold label KIM-1 antibody minimum steady, add the 10% actual institute expense that is the required KIM-1 antibody of stable colloid gold, go out the total amount of needed KIM-1 antibody according to calculation of total in order to the collaurum of mark.Under electromagnetic agitation, standard K IM-1 antibody-solutions is added in the colloidal gold solution, should dropwise add when adding KIM-1 antibody, the about 5min of KIM-1 antibody of 1mg adds; The adding final concentration is 1% bovine serum albumin(BSA) (BSA) under magnetic stirs.
Table 1 is determined collaurum and KIM-1 antibody usage ratio
3, the purifying of immune colloid gold: adopt low temperature supercentrifugation purifying gold mark KIM-1 antibody, to remove in the solution unlabelled KIM-1 antibody and the not collaurum of abundant mark and the various polymkeric substance that in labeling process, may form.Earlier gold is marked KIM-1 antibody under 4 ℃, 3000r/min low-speed centrifugal 40min carefully draws supernatant, discards precipitation; Supernatant is again with 10000r/min4 ℃ of centrifugal 40min, supernatant discarded., spend the night after fully stable to original volume with PBS (including 1%BSA, the 0.02% Sodium azide) dissolution precipitation of 0.01mol/L, pH7.4.With 4 ℃ of centrifugal 40min of 10000r/min, abandon supernatant again.With 1/10, the 4 ℃ of storage standby (seeing process flow diagram) of PBS (including 1%BSA, the 0.02% Sodium azide) dissolution precipitation of 0.01mol/L, pH7.4 to original volume.
The purifying process flow diagram of immune colloid gold: see Fig. 4.
4, the preparation of gold mark pad (in conjunction with discharging pad): colloid gold label KIM-1 antibody is made 1:5,1:10,1:50,1:100,1:500, after the 1:1000 dilution, evenly equivalent is dipped in the plain film of onesize glass fibre and makes gold mark pad.Reach the dilutability of suitable colloidal gold composite of test strips susceptibility requirement, be working concentration.Preserve liquid dilution colloid gold label KIM-1 antibody stoste to working concentration, evenly be dipped in the plain film of glass fibre, sealing is preserved after the vacuum freezedrying.
5, the preparation of solid phase nitrocellulose filter: the experiment reaction zone of nitrocellulose filter, be defined as and detect band, with KIM-1 antibody with the linear bag by in detecting band, distance detecting band 5mm quality control band (C) far away with the linear bag by normal anti-mouse IgG antibody.37 ℃ of dry 2h, 4 ℃ of sealings are preserved.
6, the processing of sample pad: 1% BSA evenly is dipped in the plain film of glass fibre as closed protein matter, and drying at room temperature is standby.
7, absorption pad prepares with the hard absorbent filter.
8, material assembling: will inhale nitrocellulose filter and the hard absorbent filter that urine has been fixed with anti-KIM-1 antibody and anti-mouse IgG antibody with glass fibre, the anti-KIM-1 glass fibre of the golden mark of freeze-drying, respectively and assemble by Fig. 1 on plastic bottom board, accessory is bonding with the available double faced adhesive tape of combining of plastic bottom board or other cohesive material.The cardboard that assembles is by longitudinal shear, and the strip that is cut into width and is 4mm gets final product.
Embodiment 3 test paper using method:
(1) specimen preparation:
Urine: directly test with urine, as muddiness elder generation's centrifuging and taking supernatant or distilled water 1:1 dilution;
(2) operation: get the about 0.5ml of urine sample and add in the small test tube, then sample pad 1 end is inserted test strips, note surpassing sample pad 1, treat that taking-up keeps flat about 1 minute, read the result in 5 minutes.
(3) testing result: referring to Fig. 2,
1 redness (contrast) precipitation line (nature controlling line 6) occurs, be urine examination diagnosis feminine gender;
2 redness (sample and contrast) precipitation line (detection line 5, nature controlling line 6) appears, and positive for the urine examination diagnosis, precipitation line does not appear then for invalid.
Claims (2)
1, a kind of KIM-1 detection kit, be respectively equipped with gold mark pad (2) and absorption pad (4) at the two ends of nitrocellulose filter (3), gold mark pad (2) upper end is sample pad (1), it is characterized in that: gold mark pad (2) is coated with the KIM-1 antibody colloidal gold coupling label of purifying, detection line (5) is coated with the KIM-1 antibody of purifying, nature controlling line (6) is coated with normal anti-mouse IgG antibody, and nature controlling line (6) side is posted absorption pad (4).
2, the described KIM-1 detectable of claim 1 box preparation method, comprise the steps: KIM-1 antibody colloid gold label with purifying, specking is prepared into gold mark pad (2) on glass fibre membrane (3), the KIM-1 antibody of purifying and normal anti-mouse IgG antibody be coated on respectively detection line (5) on the nitrocellulose filter (3) is located and nature controlling line (6) is located, when containing KIM-1 antigen in the test sample, then in the KIM-1 antibodies of gold mark pad (2) with colloid gold label, and under the effect of absorption pad (4), permeate swimming forward, combine once more with the KIM-1 antibody on the detection line (5), macroscopic colour band occurs.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100512061A CN101419235A (en) | 2008-09-24 | 2008-09-24 | KIM-1 detection kit and method for making same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100512061A CN101419235A (en) | 2008-09-24 | 2008-09-24 | KIM-1 detection kit and method for making same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101419235A true CN101419235A (en) | 2009-04-29 |
Family
ID=40630126
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2008100512061A Pending CN101419235A (en) | 2008-09-24 | 2008-09-24 | KIM-1 detection kit and method for making same |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101419235A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102147410A (en) * | 2010-12-24 | 2011-08-10 | 吉林大学 | Integrin alpha/V/beta3 detection kit and preparation method therefor |
| CN102353782A (en) * | 2010-12-31 | 2012-02-15 | 上海华谷生物技术有限公司 | In vitro diagnostic kit of antihuman kidney injury molecule-1 and its detection method |
| CN104072586A (en) * | 2013-03-29 | 2014-10-01 | 李锐 | Detection reagent and kit of human kidney injury molecule 1 |
| CN104267187A (en) * | 2014-10-15 | 2015-01-07 | 吉林大学 | Preparation and application of test strip for detecting clusterin content of urine |
| CN113125750A (en) * | 2021-04-03 | 2021-07-16 | 江西格朗生物技术有限公司 | Kidney injury molecule 1(Kim-1) detection reagent card |
-
2008
- 2008-09-24 CN CNA2008100512061A patent/CN101419235A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102147410A (en) * | 2010-12-24 | 2011-08-10 | 吉林大学 | Integrin alpha/V/beta3 detection kit and preparation method therefor |
| CN102353782A (en) * | 2010-12-31 | 2012-02-15 | 上海华谷生物技术有限公司 | In vitro diagnostic kit of antihuman kidney injury molecule-1 and its detection method |
| CN104072586A (en) * | 2013-03-29 | 2014-10-01 | 李锐 | Detection reagent and kit of human kidney injury molecule 1 |
| CN104072586B (en) * | 2013-03-29 | 2019-03-01 | 李锐 | The detection reagent and kit of people's kidney injury molecule-1 |
| CN104267187A (en) * | 2014-10-15 | 2015-01-07 | 吉林大学 | Preparation and application of test strip for detecting clusterin content of urine |
| CN113125750A (en) * | 2021-04-03 | 2021-07-16 | 江西格朗生物技术有限公司 | Kidney injury molecule 1(Kim-1) detection reagent card |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2007282856B2 (en) | A test strip for lateral flow assays | |
| CN201886025U (en) | Immunochromatography strip for rapidly and quantitatively detecting procalcitonin | |
| CN105445449A (en) | Apparatus for rapidly and semi-quantitatively detecting uric acid in saliva, and making method thereof | |
| CN104931696B (en) | Immunochromatographiassays assays kit, immunochromatographiassays assays device and immunochromatographiassays assays method | |
| EP3598131B1 (en) | Immunochromatographic test piece for extracting and measuring sugar chain antigen, which is capable of preventing non-specific reaction | |
| JP5430995B2 (en) | Assay method and assay device | |
| CN101419235A (en) | KIM-1 detection kit and method for making same | |
| CN204330778U (en) | A kind of alkaline phosphatase detects immunity test strip fast | |
| CN103743901A (en) | Colloidal gold test strip for detecting canine parvovirus | |
| CN105021823A (en) | Immunochromatographic analysis device, immunochromatographic analysis method, and immunochromatographic analysis kit | |
| JP3757171B2 (en) | Extraction method of microbial antigen | |
| CN102830226A (en) | Highly sensitive immunochromatography method and immunochromatography kit | |
| CN105606808A (en) | Group A/B streptococcus colloidal gold immunochromatograohic assay detection device and detection method thereof | |
| CN108761075A (en) | A kind of deoxynivalenol quantifies rapid detection card and its detection method | |
| AU2003244594B2 (en) | Pretreatment kit for saliva and pretreatment method for saliva | |
| CN102226811A (en) | Preparation method of diagnostic test paper for detecting H-FABP (heart-type fatty acid binding protein) | |
| JP5011244B2 (en) | Test substance detection method | |
| CN107389918A (en) | A kind of immunochromatographic method test paper assembled using chitin/chitosan tunica fibrosa as colloidal label pad and preparation method thereof | |
| CN110988365A (en) | Quantitative immune colloidal gold detection card and kit for cystatin C, microalbuminuria and urinary creatinine | |
| JP4980944B2 (en) | Immunological measurement method | |
| JP2010071827A (en) | Assay method | |
| CN101587122A (en) | Swine Hemagglutinating encephalomyelitis virus colloidal gold antigen detecting test paper and preparing method thereof | |
| CN104730248A (en) | Rapid diagnosis and detection test paper card for detecting swine fever as well as preparation and application method of rapid diagnosis and detection test paper card | |
| CN201397332Y (en) | Colloidal gold test strip for rapidly detecting swine influenza virus | |
| CN101520459B (en) | Rabbit hemorrhagic disease virus colloidal gold test strip |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20090429 |